1.1.1.1 alcohol dehydrogenase biotechnology possible usage of the enzyme in bioindustrial processes and as biosensor 1.1.1.6 glycerol dehydrogenase biotechnology production of 1,2-propanediol in yeast 1.1.1.6 glycerol dehydrogenase biotechnology GlyDH is active with immobilized N6-CM-NAD+, suggesting that N6-CM-NAD+ can be immobilized on an electrode to allow TmGlyDH activity in a system that reoxidizes the cofactor electrocatalytically, development of a bioelectrocatalytic reactor 1.1.1.8 glycerol-3-phosphate dehydrogenase (NAD+) biotechnology deletion of the NAD+-dependent glycerol-3-phosphate dehydrogenase gene in an industrial ethanol-producing strain and expression of either the non-phosphorylating NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase from Bacillus cereus, strain AG2A, or the NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase GAPDH from Kluyveromyces lactis, strain AG2B, in the deletion strain. Recombinant strain AG2A exhibits a 48.70% decrease in glycerol production and a 7.60% increase in ethanol yield relative to the amount of substrate consumed, while recombinant strain AG2B exhibits a 52.90% decrease in glycerol production and a 7.34% increase in ethanol yield relative to the amount of substrate consumed, compared with the wild-type strain. The maximum specific growth rates of the recombinant AG2A and AG2B are higher than that of the gpd2 deletion strain and are indistinguishable compared with the wild-type strain in anaerobic batch fermentations 1.1.1.9 D-xylulose reductase biotechnology pretreatment of sugarcane bagasse hydrolysate to eliminate toxic compounds unsuitable for use as growth medium in xylitol production. Optimization of adsorption time, type of acid used, concentration and charcoal leads to a high ratio of xylose reductase, EC1.1.1.21, to xylitol dehydrogenase, EC1.1.1.9, of 4.5 1.1.1.9 D-xylulose reductase biotechnology strain overexpressing enzyme has improved xylitol productivity, production of up to 57g/l xylitol from 225 g/l D-arabitol, via D-xylulose 1.1.1.9 D-xylulose reductase biotechnology cells previously grown in sugar cane bagasse hemicellulosic hydrolysate are effective in enhancing xylitol production by keeping the xylose reductase (EC 1.1.1.21) activity at high levels, reducing the xylitol dehydrogenase (EC 1.1.1.9) activity and increasing xylitol volumetric productivity (26.5%) with respect to the inoculum cultivated in semidefined medium.Therefore, inoculum adaptation to sugar cane bagasse hemicellulosic hydrolysate is an important strategy to improve xylitol productivity 1.1.1.9 D-xylulose reductase biotechnology the productivity and yield of xylitol fermentation by the XYL2-disrupted mutant are remarkably enhanced by screening suitable cosubstrates and optimizing the process 1.1.1.11 D-arabinitol 4-dehydrogenase biotechnology the gene can be expressed in agronomic plants to withstand abiotic stresses 1.1.1.21 aldose reductase biotechnology use of enzyme in production of xylitol from bagasse hydrolysate, enzyme activity is higher in medium containing acetic acid than in control medium 1.1.1.21 aldose reductase biotechnology ALDRXV4 gene from Xerophyta viscosa is a potential candidate for developing stress-tolerant crop plants 1.1.1.27 L-lactate dehydrogenase biotechnology a chimeric bifunctional enzyme composing of galactose dehydrogenase from Pseudomonas fluorescens and lactate dehydrogenase from Bacillus stearothermophilus is successfully constructed. The chimeric enzyme is able to recycle NAD with a continuous production of lactate without any externally added NADH 1.1.1.27 L-lactate dehydrogenase biotechnology genetic tools for use in Clostridium thermocellum that allow creation of unmarked mutations while using a replicating plasmid. The strategy employs counter-selections developed from the native C. thermocellum hpt gene and the Thermoanaerobacterium saccharolyticum tdk gene and is used to delete the genes for both lactate dehydrogenase (Ldh) and phosphotransacetylase (Pta) 1.1.1.28 D-lactate dehydrogenase biotechnology construction of a metabolically engineered Saccharomyces cerevisiae that produces D-lactic acid efficiently. Two copies of the D-lactate dehydrogenase gene from Leuconostoc mesenteroides subsp. mesenteroides strain NBRC3426 are introduced into the genome. The D-lactate production reaches 61.5 g/l, the amount of glucose being transformed into D-lactic acid is 53.0% under non-neutralizing conditions. The D-lactic acid is of extremely high optical purity of 99.9% or higher 1.1.1.34 hydroxymethylglutaryl-CoA reductase (NADPH) biotechnology overexpression of HMG1 is the most effective among all other genes in both hosts Saccharomyces cerevisiae ATCC 200589 and ATCC 76625 for prenyl alcohol production 1.1.1.36 acetoacetyl-CoA reductase biotechnology construction an evaluation of a polyhydroxybutyrate production system using Zea mays chloroplasts expressing the enzyme from Alcaligenes eutrophus 1.1.1.37 malate dehydrogenase biotechnology MDH is widely used in coenzyme regeneration, antigen immunoassays and bioreactors 1.1.1.40 malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+) biotechnology malic enzyme is a pivotal regulator in lipid accumulation in green microalga Chlorella pyrenoidosa, and presents a breakthrough of generating ideal algal strains for algal nutrition and biofuels 1.1.1.42 isocitrate dehydrogenase (NADP+) biotechnology the icdA gene is a potentially valuable tool for modulating citric acid production by metabolic engineering 1.1.1.44 phosphogluconate dehydrogenase (NADP+-dependent, decarboxylating) biotechnology immobilization of 6PDGH on ASMNPs can be an effective way for its biotechnological and biosensor applications 1.1.1.47 glucose 1-dehydrogenase [NAD(P)+] biotechnology larger scale production of NAD(P)H in bioreactors by usage of the enzyme, a thermostable enzyme is advantageous 1.1.1.47 glucose 1-dehydrogenase [NAD(P)+] biotechnology azoreductase and glucose 1-dehydrogenase are coupled for both continuous generation of the cofactor NADH and azo dye removal. The results show that 85% maximum relative activity of azoreductase in an integrated enzyme system is obtained at the conditions: 1 U azoreductase: 10 U glucose 1-dehydrogenase, 250 mM glucose, 1.0 mM NAD+ and 150 microM methyl red 1.1.1.47 glucose 1-dehydrogenase [NAD(P)+] biotechnology Escherichia coli transformants are prepared coexpressing the yeast reductase YOL151W and Bacillus GDH for the production of Ethyl (R, S)-4-chloro-3-hydroxybutanoate 1.1.1.47 glucose 1-dehydrogenase [NAD(P)+] biotechnology (±)-ethyl mandelate are important intermediates in the synthesis of numerous pharmaceuticals. Efficient routes for the production of these derivatives are highly desirable. A co-immobilization strategy is developed to overcome the issue of NADPH demand in the short-chain dehydrogenase/reductase (SDR) catalytic process. The SDR from Thermus thermophilus HB8 and the NAD(P)-dependent glucose dehydrogenase (GDH) from Thermoplasma acidophilum DSM 1728 are co-immobilized on silica gel. This dual-system offers an efficient route for the biosynthesis of (+/-)-ethyl mandelate 1.1.1.47 glucose 1-dehydrogenase [NAD(P)+] biotechnology co-immobilization of ketoreductase (KRED) and glucose dehydrogenase (GDH) on highly cross-linked agarose (sepharose) via affnity interaction between His-tagged enzymes (six histidine residues on the N-terminus of the protein) and agarose matrix charged with nickel (Ni2+ ions). Immobilized enzymes are applied in a set of biotransformation reactions in repeated batch flow-reactor mode. Immobilization reduces the requirement for cofactor (NADP+) and allows the use of higher substrate concentration in comparison with free enzymes 1.1.1.47 glucose 1-dehydrogenase [NAD(P)+] biotechnology enzymatic reduction of the nicotinamide biomimetic cofactors 1-phenethyl-1,4-dihydropyridine-3-carboxamide using glucose dehydrogenase mutant I192T/V306I provides a regeneration system for artificial cofactors. The I192T/V306I mutant enzyme shows 10fold higher activity with 1-phenethyl-1,4-dihydropyridine-3-carboxamide compared with the wild-type enzyme. Using this engineered variant in combination with an enoate reductase from Thermus scotoductus results in an enzyme-coupled regeneration process for biomimetic cofactor without ribonucleotide or ribonucleotide analogue and full conversion of 10 mM 2-methylbut-2-enal with 1-phenethyl-1,4-dihydropyridine-3-carboxamide as cofactor 1.1.1.47 glucose 1-dehydrogenase [NAD(P)+] biotechnology production of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate, an important chiral intermediate for the synthesis of rosuvastatin, using carbonyl reductase coupled with glucose dehydrogenase. A recombinant Escherichia coli strain harboring carbonyl reductase R9M and glucose dehydrogenase is constructed with high carbonyl reduction activity and cofactor regeneration efficiency. The recombinant Escherichia coli cells are applied for the efficient production of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate with a substrate conversion of 98.8%, a yield of 95.6% and an enantiomeric excess of more than 99.0% under 350 g/l of tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate after 12 h reaction. A substrate fed-batch strategy is further employed to increase the substrate concentration to 400 g/l resulting in an enhanced product yield to 98.5% after 12 h reaction in a 1 l bioreactor. Meanwhile, the space-time yield is 1182.3 g/l*day 1.1.1.47 glucose 1-dehydrogenase [NAD(P)+] biotechnology the robust stability of the enzyme makes it an attractive participant for cofactor regeneration on practical applications, especially for the catalysis implemented in acidic pH and high temperature 1.1.1.48 D-galactose 1-dehydrogenase biotechnology design and synthesis by protein molecular modelling and ligand docking of several chimeric mimodye-ligands comprising a NAD-pseudomimetic moiety of anthraquinone diaminobenzosulfonic acid and a galactosyl-mimetic moiety of 2-amino-2-deoxygalactose or shikimic acid for usage as tailored ligands in selective affinity chromatography during enzyme purification/production, immobilization on a gel resin, overview 1.1.1.50 3alpha-hydroxysteroid 3-dehydrogenase (Si-specific) biotechnology transfection of cells with plasmids encoding a 3alpha-hydroxysteroid dehydrogenase-Del1 deposition domain fusion protein. The Del1 deposition domain immobilizes the enzyme in the extracellular matrix without interfering with its enzymatic activity. Extracellular matrix conditioned by cells transfected with 3alpha-hydroxysteroid dehydrogenase-Del1 deposition domain fusion significantly suppresses the growth of otherwise untreated LNCaP cells 1.1.1.67 mannitol 2-dehydrogenase biotechnology recombinant Escherichia coli expressing the enzyme from Leuconostoc pseudomesenteroides expressing strong catalytic activity of an NADH-dependent reduction of D-fructose to D-mannitol in cell extracts of the recombinant Escherichia coli strain can be utilized as an efficient biocatalyst for D-mannitol formation 1.1.1.81 hydroxypyruvate reductase biotechnology conditional and specific down-regulation of farnesyltransferase in canola using the AtHPR1 promoter driving an RNAi construct results in yield protection against drought stress in the field 1.1.1.90 aryl-alcohol dehydrogenase biotechnology biotechnological production of vanillin 1.1.1.95 phosphoglycerate dehydrogenase biotechnology metabolic engineering of Corynebacterium glutamicum for L-serine production by enzyme overexpression 1.1.1.118 glucose 1-dehydrogenase (NAD+) biotechnology immobilization of GDH1 on the surface of a graphite felt electrode, construction and optimization of an electrochemical bioreactor, co-immobilization of 3,4-dihydroxybenzaldehyde as mediator allows the system to operate at 0.2 V and increases both the activity (2.4-times) and the stability of the immobilized enzyme by 2.2-times, the immobilized enzyme is termed IMGDH1 1.1.1.122 D-threo-aldose 1-dehydrogenase biotechnology the purified alpha1,2-fucosidase and L-fucose dehydrogenase have sufficiently high activities in phosphate-buffered saline (pH 7.0) at 37 °C, making it possible to develop a one-pot method for the quantitative determination of 2'-fucosyllactose in fermentation samples. The application of this method is more convenient for quantifying 2'-fucosyllactose in a variety of samples that may be obtained from different phases of the biotechnological production of this oligosaccharide. The method is useful for simple and rapid screening of active variants during the development of any industrially important microbial strain producing 2'-fucosyllactose 1.1.1.138 mannitol 2-dehydrogenase (NADP+) biotechnology optimization of culture conditions for production of mannitol, best conditions give 213 g/l mannitol from 250 g/l fructose 1.1.1.149 20alpha-hydroxysteroid dehydrogenase biotechnology a process is developed that that allows the production of 20alpha- dihydrodydrogesterone at technical scale (several grams of 20a-DHD per week and fermenter). Genetic improvement of the production strain, an increase of substrate solubility by addition of ß-cyclodextrin, and the development of a sophisticated high-cell density fermentation at pilot scale are employed. By usage of the exemplary substrate progesterone, it is hsown that this innovative fission yeast-based whole cell biotransformation process is transferable to the conversion of other AKR1C1 substrates without special adaptation 1.1.1.149 20alpha-hydroxysteroid dehydrogenase biotechnology an aldo-keto reductases-dependent whole-cell biotransformation process is established that can be used for production of human aldo-keto reductases metabolites on a large scale 1.1.1.194 coniferyl-alcohol dehydrogenase biotechnology the recombinant Rhodococcus opacus strain PD630, expressing the coniferyl alcohol dehydrogenase from Rhodococcus sp. strain HR199, together with the coniferyl aldehyde dehydrogenase, and the vanillyl alcohol oxidase, the latter from Penicillium simplicissimus strain CBS, is able to produce vanillin from ferulic acid and eugenol 1.1.1.195 cinnamyl-alcohol dehydrogenase biotechnology the spruce CAD promoter represents a valuable tool for research and biotechnology applications related to xylem and wood 1.1.1.216 farnesol dehydrogenase (NADP+) biotechnology recombinant farnesol dehydrogenase may provide a useful molecular tool in manipulating juvenile hormone biosynthesis to generate transgenic plants for pest control 1.1.1.227 (-)-borneol dehydrogenase biotechnology the gene encoding the borneol dhdrogenase is a target for metabolic engineering for improvement of essential oil production 1.1.1.244 methanol dehydrogenase biotechnology metabolic engineering of Escherichia coli for high yield production of succinic acid driven by methanol. When methanol assimilation module is introduced into succinic acid producing Escherichia coli by employing the NAD-dependent methanol dehydrogenase from Bacillus methanolicus and ribulose monophosphate pathway from different donor organisms, succinic acid yield is increased from 0.91 g/g to 0.98 g/g with methanol as an auxiliary substrate under the anaerobic fermentation 1.1.1.255 mannitol dehydrogenase biotechnology constitutive expression of enzyme in Nicotiana tabacum confers significantly enhanced resistance to Alternaria alternata, but not to Cercospora nicotianae 1.1.1.267 1-deoxy-D-xylulose-5-phosphate reductoisomerase biotechnology DXR plays a role in the methyl-D-erythritol 4-phosphate pathway, which is responsible for the biosynthesis of a substantial number of natural compounds of biological and biotechnological importance and is considered as a target to develop new herbicides and antimicrobial drugs 1.1.1.274 2,5-didehydrogluconate reductase (2-dehydro-D-gluconate-forming) biotechnology enzyme is a target for the construction of a NADH-utilizing mutant strain in the industrial production of vitamin C 1.1.1.275 (+)-trans-carveol dehydrogenase biotechnology applicability of strains with high enzyme content or recombinant overproducing strains for production of (+)-carvone, which is used as a flavor compound 1.1.1.301 D-arabitol-phosphate dehydrogenase biotechnology expression of the D-arabitol phosphate dehydrogenase gene of Enterococcus avium in the D-ribulose- and D-xylulose-producing strain results in a strain of Bacillus subtilis capable of converting D-glucose to D-arabitol with a high yield (28%) and little by-product formation 1.1.1.312 2-hydroxy-4-carboxymuconate semialdehyde hemiacetal dehydrogenase biotechnology production of 2-pyrone-4,6-dicarboxylic acid from protocatechuate as a precursor for biopolymers 1.1.1.312 2-hydroxy-4-carboxymuconate semialdehyde hemiacetal dehydrogenase biotechnology engineering plants with the proposed de-novo PDC pathway provides an avenue to enrich biomass with a value-added co-product while simultaneously improving biomass quality for the supply of fermentable sugars. Implementing this strategy into bioenergy crops has the potential to support existing microbial fermentation approaches that exploit lignocellulosic biomass feedstocks for PDC production 1.1.3.2 L-lactate oxidase biotechnology coupling of mutant S218C in 94% yield to maleimide-activated methoxypoly(ethylene glycol) 5000. PEGylation causes about 30% small decrease in the specific activity of the S218C mutant, and it does not change the protein stability 1.1.3.4 glucose oxidase biotechnology the enzyme encapsulated in the liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine is a useful biocatalyst for the prolonged glucose oxidation 1.1.3.4 glucose oxidase biotechnology GOX is the most widely used enzyme for the development of electrochemical glucose biosensors and biofuel cell in physiological conditions 1.1.3.4 glucose oxidase biotechnology transgenic expression of glucose oxidase may be deployed to improve cold tolerance potential of higher plants 1.1.3.4 glucose oxidase biotechnology bacteriostatic agent. The combination of different concentrations of glucose oxidase and glucose could significantly inhibit the growth of Agrobacterium and Escherichia coli in logarithmic phase during the fermentation process 1.1.3.4 glucose oxidase biotechnology the enzyme is used for a number of applications in biotechnology and clinical diagnostics 1.1.3.6 cholesterol oxidase biotechnology biocatalysis, industrial steroid drug production, steroid production as diagnostic tool 1.1.3.6 cholesterol oxidase biotechnology Cholesterol oxidase has two major biotechnological applications, i.e. in the determination of serum (and food) cholesterol levels and as biocatalyst providing valuable intermediates for industrial steroid drug production 1.1.3.9 galactose oxidase biotechnology technology: creating hybrid-system by immobilization of GOase on gold electrode, technology enables creation of biosensors and biofuel cells and studying electrochemically the catalytic mechanism of reactions for which free radicals and electron-transfer reactions are involved 1.1.3.10 pyranose oxidase biotechnology studies on biosensors and biofuel cell anodes 1.1.3.10 pyranose oxidase biotechnology studies on stabilization of enzymatic activity by immobilization 1.1.3.10 pyranose oxidase biotechnology biocatalyst for carbohydrate transformations toward higher-value products 1.1.3.10 pyranose oxidase biotechnology P2Ox is a biocatalyst with high potential for biotransformations of carbohydrates and in synthetic carbohydrate chemistry. P2Ox is useful as bioelement in biofuel cells, replacing glucose oxidase 1.1.3.10 pyranose oxidase biotechnology enzyme P2O is a useful biocatalyst in several biotechnological applications, including biotransformation of carbohydrates such as D-glucose and D-galactose to generate 2-oxo-sugars that can be further reduced at the C1 position to yield D-fructose and D-tagatose, respectively 1.1.3.13 alcohol oxidase biotechnology in the core promoter and 5' untranslated region of the gene, mutations in the TATA box motif, regions downstream of the transcription start site or next to the start codon in the 5' UTR have a significant effect on expression. Mutations in most other regions are tolerated. These neutral core promoter positions, not affecting expression, can be exploited to introduce extrinsic sequence elements such as cloning sites and bacterial promoters 1.1.3.17 choline oxidase biotechnology the immobilized enzyme is used in amperometric biosensors 1.1.3.17 choline oxidase biotechnology development and application of enzyme-based gas sensor 1.1.3.17 choline oxidase biotechnology development and applications of biosensors 1.1.3.17 choline oxidase biotechnology development of inhibition biosensors for pesticide determination, butyrylcholinesterase/choline oxidase enzyme electrode and tyrosine electrode compared 1.1.3.21 glycerol-3-phosphate oxidase biotechnology the enzyme is used as triglyceride biosensor when immobilized on a pencil graphite electrode 1.1.3.47 5-(hydroxymethyl)furfural oxidase biotechnology development of a facile gene shuffling approach to rapidly combine stabilizing mutations in a one-pot reaction. This allows the identification of the optimal combination of several beneficial mutations. The approach quickly discriminates stable and active multi-site variants, making it a very useful addition to FRESCO (framework for rapid enzyme stabilization by computational libraries) method 1.1.5.2 glucose 1-dehydrogenase (PQQ, quinone) biotechnology bioengineering of water-soluble isozyme PQQGDH-B production at industrial level 1.1.5.2 glucose 1-dehydrogenase (PQQ, quinone) biotechnology engineering of the soluble enzyme GDH-B to enable the electron transfer to the electrode in absence of artificial electron mediator by mimicking the domain structure of the quinohemoprotein ethanol dehydrogenase from Comamonas testosteroni, which is composed of a PQQ-containing catalytic domain and a cytochrome c domain 1.1.5.2 glucose 1-dehydrogenase (PQQ, quinone) biotechnology engineering PQQ glucose dehydrogenase with improved substrate specificity 1.1.5.2 glucose 1-dehydrogenase (PQQ, quinone) biotechnology enzyme has a great potential for application as glucose sensor constituent 1.1.5.2 glucose 1-dehydrogenase (PQQ, quinone) biotechnology optimization of an expression system using Pichia pastoris for use in industrial level production 1.1.5.2 glucose 1-dehydrogenase (PQQ, quinone) biotechnology surface charge engineering of the enzyme for optimization of downstream processing in large scale enzyme production 1.1.5.2 glucose 1-dehydrogenase (PQQ, quinone) biotechnology the enzyme is used for glucose biosensor diagnosis 1.1.5.2 glucose 1-dehydrogenase (PQQ, quinone) biotechnology a Glucose sensitive biosensor containing GDH immobilized on Prussian blue (PB)-modified graphite electrode is designed. Properties of the biosensor are investigated in the cathodic and anodic response detection regions. It is shown, that anodic response of the biosensor is sum of two signals: direct electron transport from reduced pyrroloquinoline quinine to the electrode and by formation of the pyrroloquinoline quinone-oxygen-Prussion blue-carbon ternary complex. Cathodic response of the biosensor is based on the oxidation of the reduced pyrroloquinoline quinone by Prussian blue-oxygen-Prussian blue complex. Electrochemical regeneration of the enzyme does not produce free hydrogen peroxide 1.1.5.2 glucose 1-dehydrogenase (PQQ, quinone) biotechnology application as biosensor 1.1.5.B3 quinone dependent L-lactate dehydrogenase biotechnology the PQQ-dependent lactate dehydrogenase from Gluconobacter is capable of direct electron transfer at carbon and gold electrodes, which makes it useful for application in biosensors or biofuel cells, overview 1.1.5.4 malate dehydrogenase (quinone) biotechnology the disruption of the mqo gene results in increased L-lysine production. The mutation supports industrial levels of L-lysine production in Corynebacterium glutamicum 1.1.5.9 glucose 1-dehydrogenase (FAD, quinone) biotechnology construction of a direct electron transfer bioanode composed of FADGDH and a single-walled carbon nanotube. The bioanode exhibits a large anodic current due to the enzymatic reaction (1 mA/cm) at ambient temperature and works at 70°C for 12 h 1.1.5.14 fructose 5-dehydrogenase biotechnology an automated, enzymatic insulin assay is developed. Principle: Fructose is produced by the action of inulinase on inulin present in the sample. The resulting fructose reacts with D-fructose dehydrogenase in the presence of the oxidized form of 1-methoxy-5-methylphenazinium methylsulfate (1-m-PMS) to produce the reduced form of 1-m-PMS. Reduced 1-m-PMS acts on dissolved oxygen to produce hydrogen peroxide, which, through the action of peroxidase, oxidatively condenses N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine and 4-aminoantipyrine to transform them into quinoneimine dye. The absorbance of the quinoneimine dye is measured spectrophotometrically to determine the concentration of inulin in the sample. The new enzymatic assay offers a more convenient and more accurate measurement of inulin and may be suitable for routine procedures by automated analyzers in clinical laboratories 1.1.5.14 fructose 5-dehydrogenase biotechnology multi-walled carbon nanotubes synthesized on platinum plate (MWCNTs/Pt) electrode are immediately immersed into solutions of FDH to immobilize the enzyme onto electrode surfaces. Thereafter, a well-defined catalytic oxidation current based on FDH is observed from ca. -0.15V, which is close to the redox potential of heme c as a prosthetic group of FDH. From an analysis of a plot of the catalytic current versus substrate, the calibration range for the fructose concentration is up to ca. 40 mmol/dm3, and the apparent Michaelis-Menten constant is evaluated to be 11 mmol/l. The obtained results are useful in applications to prepare the third-generation biosensors and other future bioelectrochemical devices 1.1.5.14 fructose 5-dehydrogenase biotechnology using fructose dehydrogenase-catalyzed conversion of d-fructose to 5-ketofructose, followed by quantitation of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] formazan production by direct spectrophotometry, an assay to measure serum fructose concentration is developed. The fructose dehydrogenase-based enzymatic assay correlates highly with gas chromatography-mass spectroscopic analysis of serum fructose. The assay is highly specific, exhibits no cross-reactivity with other sugars and is easy to perform 1.1.9.1 alcohol dehydrogenase (azurin) biotechnology co-immobilization of enzyme with redox polymer poly(vinylpyridine) complex functionalized with osmium bis(bipyridine) chloride on an electrode. The enzyme electrode readily oxidizes primary alcohols and secondary alcohols with maximum current densities varying between 0.43 and 0.98 A per m2 depending on the substrate and the operation temperature. The enzyme electrode is enantioselective in the oxidation of secondary alcohols. A strong preference is observed for the (S)-2-alcohols, the enantioselectivity increases with increasing chain length. The enantiomeric ratio E increases from 13 for (R,S)-2-butanol to approximately 80 for (R,S)-2-heptanol and (R,S)-2-octanol 1.1.99.18 cellobiose dehydrogenase (acceptor) biotechnology the Pichia expression system is well suited for high-level production of recombinant enzyme 1.1.99.18 cellobiose dehydrogenase (acceptor) biotechnology biosensors with a cellulosic carrier containing self-assembled nanocomposites of CDH and other enzymes allow the determination of 100 nm dopamine 1.1.99.18 cellobiose dehydrogenase (acceptor) biotechnology cellobiose dehydrogenase is a promising enzyme for the development of biosensors and biofuel cells 1.1.99.18 cellobiose dehydrogenase (acceptor) biotechnology class II cellobiose dehydrogenases are potential candidates for glucose biosensors and biofuel cell anodes 1.1.99.29 pyranose dehydrogenase (acceptor) biotechnology PDH could be a very interesting alternative to pyranose oxidase for applications in biotechnology or biofuel cells, electrical wiring of the enzyme bound to graphite rod electrodes is studied 1.1.99.29 pyranose dehydrogenase (acceptor) biotechnology PDH is an alternative to pyranose oxidase for applications in biotechnology or biofuel cells, electrical wiring of the enzyme bound to graphite rod electrodes is studied 1.1.99.29 pyranose dehydrogenase (acceptor) biotechnology PDH can be very efficiently wired with osmium polymers having E°' values close to that of PDH 1.1.99.30 2-oxo-acid reductase biotechnology preparative scale production of pyruvate from (R)-lactate in an enzyme-membrane reactor with coupled electrochemical regeneration of the artificial mediator anthraquinone-2,6-disulfonate, process modeling and calculation 1.1.99.35 soluble quinoprotein glucose dehydrogenase biotechnology application as biosensor. Application for glucose sensing. s-GDH can be applied for the ultrasensitive detection of PQQ down to picomolar concentrations 1.2.1.8 betaine-aldehyde dehydrogenase biotechnology BADH overexpression in maize is beneficial for drought tolerance and the three transgenic maize lines can be used for further breeding experiments. The agronomic traits of transgenic maize are not affected by the overexpression of BADH 1.2.1.8 betaine-aldehyde dehydrogenase biotechnology overexpression of AMADHs with high BADH activities is an important strategy to genetically engineer Solanaceae crop plants, such as tomato and tobacco, to produe glycine betaine 1.2.1.11 aspartate-semialdehyde dehydrogenase biotechnology development of lysine-overproducing strains 1.2.1.11 aspartate-semialdehyde dehydrogenase biotechnology the modofied enzyme with altered substrate specificity using NAD(H) is preferred in biotechnological production of amino acids due to lower costs and higher stability 1.2.1.12 glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) biotechnology molecular evolution or metabolic engineering protocols can exploit substrate channeling of D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and L-lactate dehydrogenase (LDH) for metabolic flux control by fine-tuning substrate-binding affinity for the key enzymes in the competing reaction paths 1.2.1.44 cinnamoyl-CoA reductase biotechnology CCR downregulation may become a successful strategy to improve biomass processing if the variability in down-regulation and the yield penalty can be overcome 1.2.1.44 cinnamoyl-CoA reductase biotechnology potential exploitation of rationally engineered forms of CCR and CAD2 for the targeted modification of monolignol composition in transgenic plants 1.2.1.46 formaldehyde dehydrogenase biotechnology development of novel formaldehyde-selective amperometric biosensors based on immobilized NAD+- and glutathione dependent formaldehyde dehydrogenase with high selectivity to formaldehyde and a low cross-sensitivity to other substances, the laboratory prototype of the sensor is applied for FA testing in some real samples of pharmaceutical (formidron), disinfectant (descoton forte) and industrial product (formalin) 1.2.1.50 long-chain acyl-protein thioester reductase biotechnology production of jojoba plant wax esters 1.2.1.67 vanillin dehydrogenase biotechnology Amycolatopsis sp. ATCC 39116 vdh mutant represents an optimized and industrially applicable platform for biotechnological production of natural vanillin 1.2.1.68 coniferyl-aldehyde dehydrogenase biotechnology biotransformation of eugenol to ferulic acid by a recombinant strain of Ralstonia eutropha H16. The gene calB, encoding coniferyl aldehyde dehydrogenase, and ehyAB and calA encoding eugenol hydroxylase and coniferyl alcohol dehydrogenase, respectively, are amplified and combined to construct a catabolic gene cassette. This gene cassette is cloned in the broad-host-range vector pBBR1-JO2 and transferred to Ralstonia eutropha H16. A recombinant strain of Ralstonia eutropha H16 harboring this plasmid expresses functionally active eugenol hydroxylase, coniferyl alcohol dehydrogenase, and coniferyl aldehyde dehydrogenase. Cells of Ralstonia eutropha H16 from the late-exponential growth phase are used asa biocatalysts for the biotransformation of eugenol to ferulic acid. A maximum conversion rate of 2.9 mM of eugenol per h per liter of culture is achieved with a yield of 93.8 mol% of ferulic acid from eugenol within 20 h, without further optimization 1.2.1.68 coniferyl-aldehyde dehydrogenase biotechnology highly efficient two-step biotransformation of eugenol to ferulic acid and further conversion to vanillin in recombinant strains of Escherichia coli. Maximum production rate for ferulic acid at large scale is 14.4 mM per h per liter of culture, yield of 93.3% with respect to the added amount of eugenol 1.2.1.70 glutamyl-tRNA reductase biotechnology recombinant Escherichia coli allows efficient production of 5-aminolevulinic acid directly from glucose 1.2.1.75 malonyl-CoA reductase (malonate semialdehyde-forming) biotechnology the crystallographic data indicate how to construct a bispecific cofactor binding site and to engineer a malonyl-CoA into methylmalonyl-CoA reductase for polyester building block production 1.2.4.1 pyruvate dehydrogenase (acetyl-transferring) biotechnology active expression of enzyme from non-halophilic Zymomonas mobilis in the haloarchaeon Haloferax volcanii with no difference in the secondary structure. Post-transcriptional mechanisms in the stationary phase appear to limit the amount of recombinant protein expressed 1.2.7.1 pyruvate synthase biotechnology pyruvate:ferredoxin oxidoreductase PFR1 and [Fe-Fe]-hydrogenase HYDA1 of Chlamydomonas can be coupled for pyruvate-dependent H2 production 1.3.1.8 acyl-CoA dehydrogenase (NADP+) biotechnology optimization of oil-based extended fermentation of recombinant Streptomyces cinnamonensis, expressing the enzyme from Streptomyces collinus, is used to provide methylmalonyl-CoA precursors for monensin biosynthesis, overview 1.3.1.31 2-enoate reductase biotechnology enoate reductase(ER)-functionalized poplar powder(FPP) and glucose-6-phosphate dehydrogenase(GDH)-FPP enable the continuous conversion of 4-(4-methoxyphenyl)-3-buten-2-one with NAD+ recycling. The immobilization strategy is simple and inexpensive and exploits a method for the immobilization and application of enoate reductase and its cofactor recycling system 1.3.1.32 maleylacetate reductase biotechnology Sphingobium chlorophenolicum has assembled new catabolic pathways to degrade pentachlorophenol and use the ring-cleavage products as their carbon sources 1.3.3.5 bilirubin oxidase biotechnology the enzyme functions effectively as the biocathode of a H2/O2 biofuel cell. It is immmobilized as a multiple layer in a cationic polymer (poly-L-lysine) matrix on an electrode surface. The BOD-modified electrode catalyzes four-electron reduction of O2 to water without any mediator, to produce a diffusion-controlled voltammogram for the O2 reduction in a quiescent solution. Construction of such a multiple enzyme layer is useful for increasing the current density even in direct electron transfer-type bioelectrocatalysis 1.3.3.5 bilirubin oxidase biotechnology the „wired" enzyme is superior to pure platinum as a electrocatalyst of the four-electron electroreduction of O2 to water. The "wired" bilirubin oxidase-coated carbon cathode operates for more than 1 week at 37°C in a glucose-containing physiological buffer solution. Key application would be in a glucose-O2 biofuel cell. The cathode is short-lived in serum, losing its electrocatalytic activity in a few hours. The damaging serum component is a product of the reaction of urate and dissolved oxygen. Exclusion of urate, by application of Nafion(TM) film in the cathode, improves the stability in serum 1.3.3.6 acyl-CoA oxidase biotechnology potential depolluting agent by degradation of oils 1.3.3.6 acyl-CoA oxidase biotechnology several biotechnological applications: production of metabolites, such as citrate, secretion of proteins, degradation of fatty acids 1.3.3.11 pyrroloquinoline-quinone synthase biotechnology cofactor engineering of PQQ in Gluconobacter oxydans is beneficial for enhancing the production of quinoprotein-related products 1.3.5.1 succinate dehydrogenase biotechnology a new host-vector system for Mortierella alpina 1S-4, zygomycetes, on the basis of self-cloning for the industrial application of Mortierella transformants is developed. Transformants expressing the Escherichia coli uidA gene encoding beta-glucuronidase by using the mutant H243L as the selectable marker (leading to to carboxin resistance) 1.3.7.4 phytochromobilin:ferredoxin oxidoreductase biotechnology transposon-based directed tagging strategy using maize Ds element generates a wide diversity of tagged and non-tagged alleles that can be used to generate allelic series or deletion of clustered genes 1.3.7.4 phytochromobilin:ferredoxin oxidoreductase biotechnology the flooding (rf) mutation identified may provide a target for biotechnological renovation of tomato germplasm in future breeding 1.4.1.1 alanine dehydrogenase biotechnology heterologous expression of the Bacillus subtilis AlaDH in Lactococcus lactis using the promoter of lactate dehydrogenase from Streptococcus thermophilus leads to a better alanine production in the recombinant strain 1.4.1.2 glutamate dehydrogenase biotechnology a strategy to control flocculation is investigated using dimorphic yeast, Benjaminiella poitrasii as a model. Parent form of this yeast (Y) exhibit faster flocculation (11.1 min) than the monomorphic yeast form mutant Y-5 (12.6 min). Flocculation of both Y and Y-5 can be altered by supplementing either substrates or inhibitor of NAD-glutamate dehydrogenase (NAD-GDH) in the growth media. The rate of flocculation is promoted by alpha-ketoglutarate or isophthalic acid and decelerated by glutamate with a statistically significant inverse correlation to corresponding NAD-GDH levels. This opens up new possibilities of using NAD-GDH modulating agents to control flocculation in fermentations for easier downstream processing 1.4.1.2 glutamate dehydrogenase biotechnology method describes immobilization of enzymes by the maximum amount of subunits and rigidification of the enzyme subunits involved in the immobilization 1.4.1.2 glutamate dehydrogenase biotechnology the rate of flocculation is promoted by a-ketoglutarate or isophthalic acid and decelerated by glutamate with a statistically significant inverse correlation to corresponding NAD-GDH levels. These interesting findings open up new possibilities of using NAD-GDH modulating agents to control flocculation in fermentations for easier downstream processing 1.4.1.4 glutamate dehydrogenase (NADP+) biotechnology enzyme TrGDH is a promising candidate gene for maintaining or improving yields in crop plants via genetic engineering 1.4.1.9 leucine dehydrogenase biotechnology an efficient stereospecific enzymatic synthesis of L-valine, L-leucine, L-norvaline, L-norleucine and L-isoleucine from the corresponding alpha-keto acids by coupling the reactions catalysed by leucine dehydrogenase and glucose dehydrogenase/galactose mutarotase. Giving high yields of L-amino acids, the procedure is economical and easy to perform and to monitor at a synthetically useful scale (1-10 g) 1.4.1.16 diaminopimelate dehydrogenase biotechnology high thermostability and relaxed substrate profile of Symbiobacterium thermophilum meso-DAPDH warrant it as an excellent starting enzyme for creating effective D-amino acid dehydrogenases by protein engineering 1.4.1.20 phenylalanine dehydrogenase biotechnology application of the immobilised mutant enzyme N145A that is remarkably robust, even in the presence of high concentrations of polar or non-polar organic solvents such as acetone, methanol, n-hexane, toluene and methylene chloride in the synthesis of p-NO2-phenylalanine from the poorly water-soluble p-NO2-phenylpyruvic acid. 100% stereoselectivity 1.4.1.20 phenylalanine dehydrogenase biotechnology use of fed-batch cultivation for achieving high cell densities for the pilot-scale production of the recombinant phenylalanine dehydrogenase 1.4.3.3 D-amino-acid oxidase biotechnology DAAO can be used to synthesize cephalosporin antibiotics 1.4.3.3 D-amino-acid oxidase biotechnology the biotechnological applications of the enzyme range from biocatalysis to convert cephalosporin C into 7-amino cephalosporanic acid to gene therapy for tumor treatment 1.4.3.3 D-amino-acid oxidase biotechnology the enzyme is used as a biocatalyst in cephalosporin C conversion on industrial scale 1.4.3.10 putrescine oxidase biotechnology potential of putrescine oxidase for the bioproduction of N-heterocycles from cadaverine. Complete biotransformation of cadaverine is observed in whole cells at physiological conditions 1.4.3.11 L-glutamate oxidase biotechnology an amperometric microbiosensor for real time monitoring L-glutamate release in neural tissue, based on enzymatic oxidation catalyzed by the L-glutamate oxidase is developed. By means of a sol-gel coating method, L-glutamate oxidase is entrapped in a biocompatible gel layer that provides a benign environment and retains enzyme activity on the surface of Pt microelectrode. Prior to gel layer formation, a modification on the surface of Pt microelectrode with poly(phenylene diamine) enables the microbiosensor screen majority of common potential interfering substances existing in physiological samples. The resulting L-glutamate microbiosensors are characterized by a fast response, high sensitivity, favourable selectivity and excellent long-term stability 1.4.3.11 L-glutamate oxidase biotechnology application of L-glutamate oxidase with catalase (KatE) to whole-cell systems for glutaric acid production in Escherichia coli. The 2-oxoglutarate regeneration system has potential for improving production in various aminotransferase systems 1.4.3.11 L-glutamate oxidase biotechnology engineering of L-glutamate oxidase has great potentials to enhance the industrial production of 2-oxoglutarate. A whole-cell biocatalyst for 2-oxoglutarate production is developed by co-expression of both S280T/H533L mutant and KatE catalase. S280T/H533L mutant has high maximal velocity (Vmax: 0.2313 mM/mg/min) and the low Km-value of 2.7 mM. Randomized ribosome binding site (RBS) sequences are introduced to generate vectors with varying expression levels of S280T/H533L and KatE, and two optimized coexpression strains are obtained after screening. The 2-oxoglutarate production reaches a maximum titer of 181.9 g/l after 12 h conversation using the optimized whole-cell biocatalyst, with a molar conversion rate of substrate higher than 86.3% in the absence of exogenous catalase, while the molar conversion rate of substrate using the wild-type biocatalyst is less than 30% 1.4.3.11 L-glutamate oxidase biotechnology to simplify technological processes and reduce production costs, cascade biocatalysis for 2-oxoglutarate production is constructed by simultaneously expressing L-glutamate oxidase (LGOX) from Streptomyces viridosporus and KatG from Escherichia coli W3110 in Escherichia coli BL21 (DE3). In vivo cascade biocatalysis is constructed and optimized by promoter engineering to finely control the coexpression of LGOX and KatG, thus resulting in a significant increase in 2-oxoglutarate concentration and its conversion rate with no catalase addition 1.4.3.16 L-aspartate oxidase biotechnology StLASPO represents an appropriate biocatalyst for the resolution of racemic solutions of D,L-aspartate and a well-suited protein scaffold to evolve a L-amino acid oxidase activity by protein engineering 1.4.3.22 diamine oxidase biotechnology supramolecular tandem assays exploit the dynamic binding of a fluorescent dye with a macrocyclic host in competition with the binding of the substrate and product. Two examples of enzymatic reactions were investigated: the hydrolysis of arginine to ornithine catalyzed by arginase and the oxidation of cadaverine to 5-aminopentanal by diamine oxidase, in which the substrates have a higher affinity to the macrocycle than the products (substrate-selective assays). The depletion of the substrate allows the fluorescent dye to enter the macrocycle in the course of the enzymatic reaction, which leads to the desired fluorescence response. For arginase, p-sulfonatocalix[4]arene is used as the macrocycle, and for diamine oxidase, cucurbit[7]uril (CB7) is used. An additional reporter pair, namely cucurbit[7]uril (CB7)/acridine orange (AO) is applied and the potential of tandem assays for inhibitor screening is demonstrated 1.5.1.3 dihydrofolate reductase biotechnology in vivo screening system to select for functionally active proteins with increased solubility. Fusion of enzyme to green fluorescent protein as reporter for solubility 1.5.1.3 dihydrofolate reductase biotechnology method for screening combinatorial or other libraries of enzyme based on affinities of the inhibitors with the enzyme 1.5.1.3 dihydrofolate reductase biotechnology method for screening combinatorial or other libraries of Plasmodium falciparum enzyme based on affinities of the inhibitors with the enzyme 1.5.1.39 FMN reductase [NAD(P)H] biotechnology enzyme-catalyzed cofactor regeneration is a significant approach to avoid large quantities consumption of oxidized cofactor, which is vital in a variety of bioconversion reactions. NADH: FMN oxidoreductase is an ideal regenerating enzyme because innocuous molecular oxygen is required as an oxidant. But the by-product H2O2 limits its further applications at the industrial scale, therefore, mutants with improved features are constructed 1.6.1.1 NAD(P)+ transhydrogenase (Si-specific) biotechnology enzymatic NADH production system in reverse micelles using a bacterial glycerol dehydrogenase. The present system is further extended to NADPH production in reverse micelles by coupling with a bacterial soluble transhydrogenase that catalyses the conversion of NADP+ to NADPH using NADH. Glycerol dehydrogenase and soluble transhydrogenase have potential for use in redox cofactor recycling in reverse micelles, which allows the use of catalytic quantities of NAD(P)H in organic media 1.6.1.1 NAD(P)+ transhydrogenase (Si-specific) biotechnology Escherichia coli strain is transformed with a two plasmid system, one encoding the udhA gene and the other one encoding the phb operon. The functionality of this particular system is successfully demonstrated in PHB production experiments. Both productivity and yield of PHB can be increased when NADPH availability is increased 1.6.2.4 NADPH-hemoprotein reductase biotechnology the enzyme is displayed on the cell surface of Escherichia coli, creating a whole-cell biocatalyst for oxidoreduction of various substrates 1.6.5.2 NAD(P)H dehydrogenase (quinone) biotechnology the flavoprotein WrbA from Escherichia coli represents a new family of multimeric flavodoxin-like proteins implicated in cell protection against oxidative stress, WrbA has NAD(P)H: quinone reductase activity, forms multimers and binds FMN only weakly 1.8.3.7 formylglycine-generating enzyme biotechnology bioconjugation chemistry, formylglycine-generating enzymes catalyze the site-specific oxidation of a cysteine residue to the aldehyde-containing amino acid Ca-formylglycine (FGly). This noncanonical residue can be generated within any desired target protein and can subsequently be used for bioorthogonal conjugation reactions 1.8.3.7 formylglycine-generating enzyme biotechnology site-specific bioconjugation. Formylglycine-generating enzymes allow to posttranslationally introduce the amino acid Calpha-formylglycine (FGly) into recombinant proteins, starting from cysteine or serine residues within distinct consensus motifs 1.8.3.7 formylglycine-generating enzyme biotechnology site-specific conjugation strategy for dual antibody-drug conjugates using aerobic formylglycine-generating enzymes 1.8.3.7 formylglycine-generating enzyme biotechnology the enzyme is an enabling biotechnology tool due to the robust utility of the aldehyde product as a bioconjugation handle in recombinant proteins 1.8.4.11 peptide-methionine (S)-S-oxide reductase biotechnology enzyme is a target for modification of redox-dependent regulation 1.8.4.12 peptide-methionine (R)-S-oxide reductase biotechnology enzyme is a target for modification of redox-dependent regulation 1.10.3.2 laccase biotechnology expression of non-fused enzyme and hydrophobin-enzyme fusion protein in Trichoderma reesei, intracellular accumulation and degradation of fusion protein, production of non-fused enzyme at up to 920 mg per l of fed-batch culture, purification from culture supernatant 1.10.3.2 laccase biotechnology five SvLAC genes (SvLAC9, SvLAC13, SvLAC15, SvLAC50, and SvLAC52) fulfill the criteria established to identify lignin-related candidates. They are strong candidates to be involved in lignin polymerization in Setaria viridis and might be good targets for lignin bioengineering strategies 1.10.3.2 laccase biotechnology robust catalytic efficiency in the presence of organic solvents suggest its industrial and biotechnological application potentials for the sustainable development of green chemistry 1.10.3.2 laccase biotechnology the purified enzyme displays greater efficiency in Remazol Brilliant Blue R decolourization (90%) in absence of redox mediator, an important property for biotechnological applications 1.11.1.5 cytochrome-c peroxidase biotechnology cytochrome c peroxidase as a platform to develop specific peroxygenation catalysts 1.11.1.6 catalase biotechnology development of simple methods for production and purification of catalases, determination of adsorption capacity and effects upon binding on enzyme activity of different minerals, binding capacities and activities at different pH/pI, one of the most promising adsorbent is hydroxylapatite, overview 1.11.1.6 catalase biotechnology wheat grass detoxifying substance in production or cultivation of Paramecium on wheat grass powder inoculated with Klebsiella pneumoniae 1.11.1.10 chloride peroxidase biotechnology CPO is used as a versatile biological catalyst 1.11.1.13 manganese peroxidase biotechnology biotechnological applications require large amounts of low-cost enzymes, one of the appropriate approaches for this is to utilize the potential of lignocellulosic wastes, some of which may contain significant concentrations of soluble carbohydrates and inducers of enzyme synthesis, ensuring efficient production of ligninolytic enzymes 1.11.1.14 lignin peroxidase biotechnology Pleurotus ostreatus is a good candidate for scale-up ligninolytic enzyme production 1.12.7.2 ferredoxin hydrogenase biotechnology practical application in solar energy bioconversion 1.13.11.1 catechol 1,2-dioxygenase biotechnology product is precursor of the industrially important compound adipic acid 1.13.11.9 2,5-dihydroxypyridine 5,6-dioxygenase biotechnology the enzyme catalyzes one step in a new process of detoxification/biotransformation of N-heterocyclic aromatic compounds 1.13.11.12 linoleate 13S-lipoxygenase biotechnology the tomloxD gene encoding the enzyme has potential applications in engineering cropping plants that are resistant to biotic and/or abiotic stress factors 1.13.11.49 chlorite O2-lyase biotechnology degradation of benzene from anoxic polluted soil with chlorate 1.13.12.2 lysine 2-monooxygenase biotechnology immobilization of L-lysine-2-monooxygenase on an electrode surface, via polymerization of polyvinyl alcohol, provides a biosensor that detects L-lysine concentrations down to 0.01 mM 1.13.12.2 lysine 2-monooxygenase biotechnology immobilization on silica gel provides a flow-through analyzer for concentrations between 5.5 and 55 mM L-lysine at pH 8.2, it retains 50% activity after two months 1.13.12.5 Renilla-type luciferase biotechnology expression of native gene and commercial synthetic gene, optimized for expression, in several cell lines and in mouse. Use of synthetic gene as primary reporter gene with high sensitivity in living rodents 1.13.12.5 Renilla-type luciferase biotechnology use of enzyme as a reporter is dependent on the promotor driving its expression, the presence of co-transfected transgenes, and the androgen responsiveness of the cell line used 1.13.12.5 Renilla-type luciferase biotechnology use of native coelenterazine and its derivatives –e, -f, -h, as substrates for use in cell culture and living animals 1.13.12.5 Renilla-type luciferase biotechnology popular reporter enzyme for gene expression and biosensor applications 1.13.12.5 Renilla-type luciferase biotechnology split luciferase complementation is applied to study dynamic protein-protein interactions in live bacteria. Nonspecific inhibition of Rluc activity by small molecule effectors compromises the utility of this technique in measuring dynamic protein-protein interactions 1.13.12.5 Renilla-type luciferase biotechnology an advanced Fc-binding probe, FcUni-RLuc, is produced and functionally assayed for labelling IgGs. The Fc antibody binding sequence HWRGWV is fused to Renilla luciferase, and the purified probe is employed for bioluminescence enzyme-linked immunoabsorbance assay of Her2 positive cells 1.13.12.6 Cypridina-luciferin 2-monooxygenase biotechnology use of enzyme as a potent secreted reporter 1.13.12.7 firefly luciferase biotechnology imaging technology 1.13.12.7 firefly luciferase biotechnology molecular biology studies with luciferase as reproter gene, bioimaging 1.13.12.7 firefly luciferase biotechnology extensive and advantageous application of this enzyme in biotechnology is restricted due to its low thermal stability 1.13.12.18 dinoflagellate luciferase biotechnology the dinoflagellate luciferase gene is an efficient marker of gene expression in mammalian cells 1.13.12.19 2-oxoglutarate dioxygenase (ethene-forming) biotechnology different cultivation factors on ethylene formation in Saccharomyces cerevisiae expressing the EFE in continuous cultures is investigated. Main finding is that oxygen availability is crucial for ethylene production. By employing three different nitrogen sources it is shown that the nitrogen source available can both improve and impair the ethylene productivity 1.13.12.19 2-oxoglutarate dioxygenase (ethene-forming) biotechnology EFE is a promising biotechnology target because the expression of a single gene is sufficient for ethylene production in the absence of toxic intermediates 1.14.11.17 taurine dioxygenase biotechnology model system for non-heme iron oxygenases 1.14.11.26 deacetoxycephalosporin-C hydroxylase biotechnology production of beta-lactam antibiotics 1.14.11.66 [histone H3]-trimethyl-L-lysine9 demethylase biotechnology continual removal of H3K9 promoter methylation by Jmjd2 demethylases represents a novel mechanism ensuring transcriptional competence and stability of the pluripotent cell identity 1.14.13.22 cyclohexanone monooxygenase biotechnology biocatalysis system for Baeyer-Villiger oxidations, the average specific oxidation rate and product molar yield based on reaction substrate reaches 0.15 g/g dry cells/h (21.9 micromol/min/g of dry cells), at high cell densities (20 g dry cells/l) the specific product formation rate is lower with 0.12 g/g dry cells/h and 17.5 micromol/min/g of dry cells (probably due to low availability of the energy source glucose), though absolute yield is 2fold higher 1.14.13.25 methane monooxygenase (soluble) biotechnology high particulate methane monooxygenase activity may contribute to the synthesis of poly-beta-hydroxybutyrate in the cell, which may be used to improve the yield of poly-beta-hydroxybutyrate in methanotrophs 1.14.13.25 methane monooxygenase (soluble) biotechnology high particulate methane monooxygenase activity may contribute to the synthesis of poly-beta-hydroxybutyrate in the cell, which may be used to improve the yield of poly-beta-hydroxybutyrate in methanotrophs. Poly-beta-hydroxybutyrate content of strain OB3b can reach the highest level in the shortest time as compared to other methanotrophs. Nutrients deficiency condition is beneficial for strain IMV3011 to synthesize PHB whereas it is not beneficial for other strains 1.14.13.25 methane monooxygenase (soluble) biotechnology methanol can be employed to produce large amounts of Methylosinus trichosporium biomass containing sMMO. Enzyme expression can be maintained during growth on methanol which may aid in the development of sMMO-based industrial and environmental processes 1.14.13.25 methane monooxygenase (soluble) biotechnology method by which sMMO can be produced by strain OB3b while growing on methanol in copper-containing medium 1.14.13.146 taxoid 14beta-hydroxylase biotechnology an antisense suppression approach, repressing the expression of the taxoid 14beta-hydroxlyase gene in yew cell cultures, is useful to inhibit the expression of other important genes in side-route of Taxol pathway and this may diverts the flow of taxadiene mainly towards Taxol 1.14.13.231 tetracycline 11a-monooxygenase biotechnology protocol for the nuclear transformation of Chlamydomonas reinhardtii using tetX as a selectable marker that confers stable resistance to tetracycline up to 100 microg/ml. TetX may be used to transform Chlamydomonas reinhardtii chloroplasts, related microalgae and other aerobic organisms sensitive to any tetracycline antibiotic 1.14.14.1 unspecific monooxygenase biotechnology cytochrome P450 monooxygenase as a tool for metabolizing of herbicides in plants 1.14.14.1 unspecific monooxygenase biotechnology EUI and the GA metabolism pathway are useful targets for increasing the agronomic value of crops 1.14.14.1 unspecific monooxygenase biotechnology enzymatic activity of P450SMO makes it an attractive biocatalyst for asymmetric synthesis of enantiopure sulfoxides 1.14.14.1 unspecific monooxygenase biotechnology most Cree anti-diabetic plant ethanolic extracts have the potential to affect CYP2C- and 3A4-mediated metabolism, and have the potential to affect the bioavailability and pharmacokinetics of conventional and traditional medicines during concomitant use, thus there is a potential risk of interactions if these traditional medicines are used with conventional therapeutic products, but several extracts may also have the potential to pharmacoenhance the activity of some medicines 1.14.14.1 unspecific monooxygenase biotechnology scanning chimeragenesis can be a useful method for producing new enzymatic products from CYP102A1 and may be used as a new systematic tool for changing substrate selectivity and regiospecificity among any two cytochromes P450 that have a common substrate, to study the interaction between enzymes and the substrate or to create new chimeric proteins for pharmaceutical and industrial uses 1.14.14.3 bacterial luciferase biotechnology establishment and evaluation of the enzyme used in a luciferase-based reporter system, pPL2lux, harboring the listerial secA and hlyA promoters translationally fused to luxABCDE, overview 1.14.14.3 bacterial luciferase biotechnology the enzyme and cyanine fluorescent protein are useful dual reporters for the quantitative analysis of the effects of n-dodecyltrimethylammonium bromide on whole cells and intracellular proteins of Pseudomonas putida 1.14.14.16 steroid 21-monooxygenase biotechnology the CYP21 expression model system using resting Schizosaccharomyces pombe cells can be used for biotransformations 1.14.14.17 squalene monooxygenase biotechnology genetic manipulation of the ERG1 gene is a promising tool for increasing squalene production in yeast 1.14.14.36 tyrosine N-monooxygenase biotechnology engineering of the dhurrin pathway from Sorghum bicolor into the chloroplasts of Nicotiana tabacum. The entire pathway can be introduced into the chloroplast by integrating membrane-bound cytochrome P450 enzymes CYP79A1, CYP71E1, and soluble glucosyltransferase UGT85B1 into a neutral site of the Nicotiana tabacum chloroplast genome. The two P450s and the UGT85B1 are functional when expressed in the chloroplasts and convert endogenous tyrosine into dhurrin using electrons derived directly from the photosynthetic electron transport chain, without the need for the presence of an NADPH-dependent P450 oxidoreductase. The dhurrin produced in the engineered plants amounts to 0.1–0.2% of leaf dry weight compared to 6% in sorghum 1.14.14.36 tyrosine N-monooxygenase biotechnology in vitro reconstitution of the entire dhurrin biosynthetic pathway from tyrosine is accomplished by the insertion of CYP79 (tyrosine N-hydroxylase), P450ox, and NADPH-P450 oxidoreductase in lipid micelles in the presence of uridine diphosphate glucose glucosyltransferase 1.14.14.37 4-hydroxyphenylacetaldehyde oxime monooxygenase biotechnology engineering of the dhurrin pathway from Sorghum bicolor into the chloroplasts of Nicotiana tabacum. The entire pathway can be introduced into the chloroplast by integrating membrane-bound cytochrome P450 enzymes CYP79A1, CYP71E1, and soluble glucosyltransferase UGT85B1 into a neutral site of the Nicotiana tabacum chloroplast genome. The two P450s and the UGT85B1 are functional when expressed in the chloroplasts and convert endogenous tyrosine into dhurrin using electrons derived directly from the photosynthetic electron transport chain, without the need for the presence of an NADPH-dependent P450 oxidoreductase. The dhurrin produced in the engineered plants amounts to 0.1-0.2% of leaf dry weight compared to 6% in sorghum 1.14.14.37 4-hydroxyphenylacetaldehyde oxime monooxygenase biotechnology in vitro reconstitution of the entire dhurrin biosynthetic pathway from tyrosine is accomplished by the insertion of CYP79 (tyrosine N-hydroxylase), P450ox, and NADPH-P450 oxidoreductase in lipid micelles in the presence of uridine diphosphate glucose glucosyltransferase 1.14.14.86 ent-kaurene monooxygenase biotechnology P2 may provide a tool to investigate the regulation of GA metabolism for plant growth and development via affection of ent-kaurene oxidase 1.14.14.102 N-methylcoclaurine 3'-monooxygenase biotechnology the enzyme provides practical means to genetically engineer valuable secondary metabolites in this important medicinal plant 1.14.15.1 camphor 5-monooxygenase biotechnology bioengineered Escherichia coli cells possess a heterologous self-sufficient P450 catalytic system that may have advantages in terms of low cost and high yield for the production of fine chemicals 1.14.15.6 cholesterol monooxygenase (side-chain-cleaving) biotechnology the development of a cholesterol biosensor based on screen-printed electrodes modified with multi-walled carbon nanotubes and with the cytochromes P450scc may ensure a high sensitivity. Role of the nanotubes in mediating electron transfer to the cytochrome P450scc is verified as further improved with respect to the case of rhodium-graphite electrodes modified by the use of gold nanoparticles 1.14.18.1 tyrosinase biotechnology prepared CSG1.0 possesses macropores which permit fluid to pass through, and micropores in the skeleton of macropores which increase the specific surface area for ligands to immobilize on. These hybrid membranes could be used to immobilize ligands for affinity sorption. Advanced application of the hybrid membranes can be developed for application in tissue engineering, enzyme immobilization, catalysts support, fuel cells, etc. 1.14.18.1 tyrosinase biotechnology the produced enzyme has similar properties as the one produced in the native Trichoderma reesei host and expression in the Pichia pastoris provides good opportunities for future protein engineering, screening and functional studies of this important class of enzymes 1.14.19.2 stearoyl-[acyl-carrier-protein] 9-desaturase biotechnology random mutagenesis and mutational analysis allows for the achievement of high seed stearic acid content with no associated negative agronomic characteristics, raandom mutagenesis as a rheostat for agronomically important traits 1.14.19.35 sn-2 acyl-lipid omega-3 desaturase (ferredoxin) biotechnology potential of exploiting FAD overexpression as a tool to ameliorate drought tolerance in plants 1.14.20.1 deacetoxycephalosporin-C synthase biotechnology production of beta-lactam antibiotics 1.14.20.5 flavone synthase I biotechnology product of the combined activity of flavone synthase and flavone 7-O-methyltransferase produces genkwanin (7-O-methyl apigenin) which exhibits antibacterial activity against Vibrio cholerae and Enterococcus faecalis, and anti-inflammatory activity, direct plant extracts result in limited amounts 1.15.1.1 superoxide dismutase biotechnology transfection of the Ctsod gene may have great potential for the genetic improvement of salt tolerance in plants 1.16.3.1 ferroxidase biotechnology expression system is developped producing about 2 mg of purified Bacillus sp. strain PL-12 Mn(II) oxidase per liter of Escherichia coli culture in 5 days 1.16.3.1 ferroxidase biotechnology compartmentalized iron oxide biomineralization by the enzyme yields uniform nanoparticles strictly determined by the sizes of the compartments, allowing customization for highly diverse nanotechnological applications 1.17.1.4 xanthine dehydrogenase biotechnology the enzyme can be useful in biotechnlogical applications requiring special conditions, e.g. extreme pH values 1.17.1.9 formate dehydrogenase biotechnology overexpression of the NAD+-dependent formate dehydrogenase in Escherichia coli doubles maximum yield of NADH from 2 to 4 mol NADH/mol glucose consumed 1.17.1.9 formate dehydrogenase biotechnology immobilization of enzyme on highly activated glyoxyl agarose, optimization protocol. Optimized enzyme retains 50% of the offered activity and becomes 50times more stable at high temperature and neutral pH. Optimal temperature increases by 10°C at pH 4.5. Immobilized enzyme accepts dextran-NAD+ as a substrate, use on ultra-filtration reactors to catalyze the recycling of NAD+ 1.17.1.9 formate dehydrogenase biotechnology study on stability of recombinant enzyme in homogeneous aequeous solution, at gas-liquid interfaces in a bubble column, and under shear stress. Level of enzyme stability in solution also depends on the enzyme variant employed. Mutants C23S and C23S/C262A perform much better than wild-type in quiescent solution and reactors with little mechanical stress. Mutant C23S/C262A is less stable than wild-type at high stress levels in the Couette viscometer or the bubble column 1.17.1.9 formate dehydrogenase biotechnology use of enzyme for regeneration of NADH. Coexpression of alpha-haloketone resistant enzyme mutants and a carbonyl reductase from Kluyveromyces aestuarii for production of ethyl-(S)-4-chloro-3-hydroxybutanoate with optical purity of product of 99% enantiomeric excess, from ethyl-4-chloroacetoacetate 1.17.1.9 formate dehydrogenase biotechnology FDH from Candida boidinii is an important biocatalyst for the regeneration of the cofactor NADH in industrial enzyme-catalyzed reductions 1.17.3.2 xanthine oxidase biotechnology construction of amperometric biosensors based in xanthine oxidase which has been immobilized by covalent binding to gold electrodes modified with dithiobis-N-succinimidyl propionate. Redox dyes thionine and methylene blue work well as electron acceptors for reduced enzyme 1.17.99.2 ethylbenzene hydroxylase biotechnology application of artificial neural networks for prediction of reaction kinetics 1.18.1.2 ferredoxin-NADP+ reductase biotechnology enzyme can be an electron source in biotechnological applications 1.20.1.1 phosphonate dehydrogenase biotechnology application of mutant Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/Q132R/V71I/E130K/I313L/A325V/A176R for regeneration of NADPH in xylose reductase-catalyzed xylitol synthesis and alcohol dehydrogenase-catalyzed (R)-phenylethanol synthesis. comparison of enzyme with commercial Pseudomonas sp. formate dehydrogenase. Mutant Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/Q132R/V71I/E130K/I313L/A325V/A176R shows higher substrate conversion and higher total turnover numbers for NADP+ than formate dehydrogenase 1.20.1.1 phosphonate dehydrogenase biotechnology evaluation of enzyme mutants for regeneration of reduced cofactors NADH and NADPH in industrial processes and comparison with Candida boidinii formate dehydrogenase 1.20.1.1 phosphonate dehydrogenase biotechnology improvement of enzyme for use in regeneration of NADH and NADPH, application in bioconversion of trimethylpyruvate to L-tert-leucine as model reaction 1.20.1.1 phosphonate dehydrogenase biotechnology use of enzyme mutant E175A/A176R for regeneration of NADH and NADPH. Model system converting xylose into xylitol by NADP-dependent xylose reductase and comparison of regeneration of NADPH by enzyme mutant and by Pseudomonas sp. formate dehydrogenase 1.21.3.1 isopenicillin-N synthase biotechnology production of beta-lactam antibiotics 1.21.3.10 hercynylcysteine S-oxide synthase biotechnology the Neurospora crassa ergothioneine biosynthetic pathway may be a more suitable platform than the mycobacterial one for ergothioneine production through metabolic engineering because the ergothioneine and glutathione biosynthetic pathways are uncoupled and they do not compete with each other anymore in Neurospora crassa 1.97.1.1 chlorate reductase biotechnology using a mass spectrometry (MS)-based proteomics approach signature peptides derived from chlorite dismutase and perchlorate reductase (subunit A and B) are identified as biomarkers of perchlorate presence and biodegradation. The biomarker peptides are detected at perchlorate concentrations as low as 0.1 mM and at different time-points in both pure cultures and within perchlorate-reducing environmental enrichment consortia. This technique can be a useful for monitoring bioremediation processes of other anthropogenic environmental contaminants with known metabolic pathways 2.1.1.37 DNA (cytosine-5-)-methyltransferase biotechnology using a truncated and highly active form of human DNMT1 and a designed hemimethylated DNA substrate, a robust, efficient, and economical fluorescence assay is developped suitable for in vitro high-throughput screening of DNMT1 2.1.1.77 protein-L-isoaspartate(D-aspartate) O-methyltransferase biotechnology useful in biotechnical applications 2.1.1.95 tocopherol C-methyltransferase biotechnology overexpression of enzyme in Latuca sativa results in higher enzyme activity and the conversion of the gamma-tocopherol pool to alpha-tocopherol in transgenic lettuce 2.1.1.100 protein-S-isoprenylcysteine O-methyltransferase biotechnology the enzyme is a target for metabolic engineering of crop species for drought tolerance by targeted alterations in isoprenylcysteine methylation 2.1.1.140 (S)-coclaurine-N-methyltransferase biotechnology CNMT should be quite useful for biotransformation of the intermediates of alkaloid biosynthesis to N-methyltransferred products 2.1.1.143 24-methylenesterol C-methyltransferase biotechnology SMT2-1 overexpression leads to changes of phytosterol content and the ratio of campesterol to sitosterol in fiber cell. At the rapid elongation stage of fiber cell, total phytosterol and sitosterol contents are increased while campesterol content is decreased in transgenic fibers. The ratio of campesterol to sitosterol declines strikingly. The transgenic fibers are shorter and thicker than control fibers. Exogenous application of sitosterol or campesterol inhibits control fiber cell elongation in cotton ovule culture system in vitro. Campesterol treatment partially rescues fiber elongation in overexpressing plants 2.1.1.156 glycine/sarcosine N-methyltransferase biotechnology enzyme can be used in betaine production for improvement of stress tolerance of commercially important microbes in agriculture and industry, and for nutritial improvement of transgenic crop plants, that do not produce betaine naturally 2.1.1.157 sarcosine/dimethylglycine N-methyltransferase biotechnology enzyme can be used in betaine production for improvement of stress tolerance of commercially important microbes in agriculture and industry, and for nutritial improvement of transgenic crop plants, that do not produce betaine naturally 2.1.1.159 theobromine synthase biotechnology large-scale production of transgenic enzyme-deficient Coffea arabica and Camellia sinensis plants are a practical possibilty for production of decaffeinated coffee or tea 2.1.1.160 caffeine synthase biotechnology large-scale production of transgenic enzyme-deficient Coffea arabica and Camellia sinensis plants are a practical possibilty for production of decaffeinated coffee or tea 2.1.1.240 trans-resveratrol di-O-methyltransferase biotechnology large-scale production of plant metabolites via microbial approaches is a promising alternative to chemical synthesis and extraction from plant sources Development of an Escherichia coli system containing an artificial biosynthetic pathway, involving the enzyme, that produces methylated resveratrol analogues, such as pinostilbene (3,4'-dihydroxy-5-methoxystilbene), 3,5-dihydroxy-4'-methoxystilbene, 3,4'-dimethoxy-5-hydroxystilbene, and 3,5,4'-trimethoxystilbene, from simple carbon sources 2.1.1.278 indole-3-acetate O-methyltransferase biotechnology potential use of reducing IAA methyltransferase activity as a method to enhance reproductive success in plants 2.1.2.1 glycine hydroxymethyltransferase biotechnology the AlkS/PalkB-expression system is shown as an efficient tool for the production of recombinant serine hydroxymethyltransferase in Escherichia coli fed-batch fermentations 2.1.3.3 ornithine carbamoyltransferase biotechnology HepG2 is an immortalized human hepatoma cell line that has been used for research into bioartificial liver systems. However, a low level of ammonia detoxification is its biggest drawback. Stable overexpression of arginase I and ornithine transcarbamylase in HepG2 cells improves its ammonia detoxification 2.2.1.1 transketolase biotechnology improvement of biocatalytic processes using transketolase over prolonged reaction times will need to address the formation of cofactor-associated intermediate state 2.2.1.1 transketolase biotechnology substrate specificity of transketolase for the donor substrate is broader than expected. Possibility of detecting wild-type transketolase activity in vitro from a L-tyrosine derivative bearing a D-threo ketose, based on the release of L-tyrosine. For cells both auxotrophic for L-tyrosine and expressing transketolase, it shall be possible to carry out this assay in vivo. This strategy may offer the first stereospecific selection test for transketolase mutants 2.2.1.6 acetolactate synthase biotechnology construction of a vector system for chloroplast transformation with acetolactate synthase, generation of a series of Arabidopsis thaliana mutated acetolactate synthase genes and introduction of constructs with the aminoglycoside 3'-adenyltransferase gene into the Nicotiana tabacum chloroplast genome by particle bombardment 2.2.1.6 acetolactate synthase biotechnology the reaction specificity of acetolactate synthase from Thermus thermophilus can be redirected to catalyze acetaldehyde formation to develop a thermophilic pyruvate decarboxylase. Quadruple mutant Y35N/K139R/V172A/H474R shows 3.1fold higher acetaldehyde-forming activity than the wild-type mainly because of H474R amino acid substitution, which likely generates two new hydrogen bonds near the thiamine diphosphate-binding site 2.2.1.7 1-deoxy-D-xylulose-5-phosphate synthase biotechnology increase production level of CoQ10 by coexpression of decaprenyl diphosphate synthase and 1-deoxy-D-xylulose 5-phosphate synthase isolated from Rhizobium radiobacter ATCC 4718 in recombinant Escherichia coli is shown 2.3.1.4 glucosamine-phosphate N-acetyltransferase biotechnology metabolic engineering of Escherichia coli for industrial production of glucosamine and N-acetylglucosamine by overexpression of glucosamine synthase and glucosamine 6-phosphate N-acetyltransferase and inactivation of catabolic genes 2.3.1.8 phosphate acetyltransferase biotechnology genetic tools for use in Clostridium thermocellum that allow creation of unmarked mutations while using a replicating plasmid. The strategy employs counter-selections developed from the native Clostridium thermocellum hpt gene and the Thermoanaerobacterium saccharolyticum tdk gene and is used to delete the genes for both lactate dehydrogenase (Ldh) and phosphotransacetylase (Pta) 2.3.1.15 glycerol-3-phosphate 1-O-acyltransferase biotechnology PpGPAT9 may be another genetic resource to enhance storage oil yields from oilseed crops 2.3.1.19 phosphate butyryltransferase biotechnology use of enzyme for in vitro biosynthesis of poly(hydroxyalkanoic acid) 2.3.1.20 diacylglycerol O-acyltransferase biotechnology overexpression of mutated DGAT1 in Arabidopsis can be used to enhance oil content 2.3.1.20 diacylglycerol O-acyltransferase biotechnology combining the overexpression of TAG biosynthetic genes, DGAT1 and GPD1, appears to be a positive strategy to achieve a synergistic effect on the flux through the TAG synthesis pathway, and thereby further increase the oil yield of Camelina sativa, application of genetic engineering approaches to boost the metabolic flux of carbon into seed oils 2.3.1.20 diacylglycerol O-acyltransferase biotechnology evaluation of the possibility of using the enzyme in biotechnological approaches where a reduction of polyunsaturated fatty acids in the oil is desired 2.3.1.20 diacylglycerol O-acyltransferase biotechnology the enzymes catalyzing the terminal steps of triacylglycerol (TAG) formation, DGAT and PDAT play crucial roles in determining the flux of carbon into seed TAG and thus have been considered as the key targets for engineering oil production 2.3.1.20 diacylglycerol O-acyltransferase biotechnology WS/DGAT might have possible additional specificities, making it highly attractive for biotechnological applications such as biodiesel production 2.3.1.28 chloramphenicol O-acetyltransferase biotechnology monitoring of starter (Leuconostoc mesenteroides DRC) growth in lactic acid-fermented Kimchi (salted Chinese cabbage) for predicting starter predominance during fermentation by stable transformation of the chloramphenicol acetyltransferase gene into the chromosomal DNA with a transposon vector 2.3.1.28 chloramphenicol O-acetyltransferase biotechnology the development of the chloramphenicol acetyltransferase gene cat as a new selectable marker for plastid transformation is reported. By selecting for chloramphenicol resistance, tobacco chloroplast transformants are readily obtained. Transplastomic lines quickly reach the homoplasmic state, accumulate the chloramphenicol acetyltransferase enzyme to high levels and transmit their plastid transgenes maternally into the next generation 2.3.1.37 5-aminolevulinate synthase biotechnology Propionibacterium acidipropionici TISTR442 produce the highest amount of 5-aminolevulinic acid (ALA) when cultivated in medium supplemented with glycine at 18 g/l. This optimal condition for ALA production via the addition of glycine provides an easy and low-cost technique for themass cultivation of Propionibacterium acidipropionici 2.3.1.39 [acyl-carrier-protein] S-malonyltransferase biotechnology the enzyme is active as part of a recombinant engineered modular enzyme complex system for modified and designed polyketid synthesis, overview 2.3.1.46 homoserine O-succinyltransferase biotechnology enzyme methionine biosynthesis deregulation mutant strains are useful for construction/production of L-methionine excreting strains 2.3.1.54 formate C-acetyltransferase biotechnology D-lactate production for the use in biopolymer production as biodegradable alternative for oil-derived plastics 2.3.1.74 chalcone synthase biotechnology Agrobacterium-mediated infection of Petunia hybrida plants with tobacco rattle virus bearing fragments of Petunia genes results in systemic infection and virus-induced gene silencing of the homologous host genes. Infection with TRV containing a chalcone synthase fragment results in silencing of anthocyanin production in infected flowers. Value of virus-induced gene silencing with tandem constructs containing CHS as reporter and a target gene as a tool for examining the function of floral-associated genes 2.3.1.84 alcohol O-acetyltransferase biotechnology a low-cost fermentation process for isoamyl acetate biosynthesis by overexpressing the yeast alcohol acetyl-transferase AFT1 in Escherichia coli is developed 2.3.1.84 alcohol O-acetyltransferase biotechnology a low-cost fermentation process for isoamyl acetate biosynthesis by overexpressing the yeast alcohol acetyl-transferase AFT2 in Escherichia coli is developed 2.3.1.94 6-deoxyerythronolide-B synthase biotechnology a derivative of Escherichia coli is genetically engineered to produce 6-deoxyerythronolide B, the macrocyclic core of the antibiotic erythromycin 2.3.1.94 6-deoxyerythronolide-B synthase biotechnology a new Escherichia coli stain, YW9, is created, featuring a plasmid-free heterologous pathway for the production of the polyketide product 6-deoxyerythronolide B 2.3.1.94 6-deoxyerythronolide-B synthase biotechnology polyketide synthases synthesize the polyketide cores of biologically active compounds, including natural products that have become important pharmaceuticals, like erythromycin 2.3.1.122 trehalose O-mycolyltransferase biotechnology an enzyme assay using the natural substrate trehalose dimycolate is developed. The colorimetric assay is based on the quantification of glucose from the degradation of trehalose, which is the product from catalytic activity of antigen 85A. This assay is suitable for robust high-throughput screening (HTS) for compound library screening against mycolyltransferase. The assay has a very low coefficient of variance (0.04) in 96-well plates and shows a Z' factor of 0.67-0.73, indicating the robustness of the assay 2.3.1.150 Salutaridinol 7-O-acetyltransferase biotechnology increases in pharmaceutical alkaloid content of potential economic significance have been achieved, RNAi suppression is a powerful way to obtain products that poppy does not normally accumulate 2.3.1.158 phospholipid:diacylglycerol acyltransferase biotechnology the enzymes catalyzing the terminal steps of triacylglycerol (TAG) formation, DGAT and PDAT play crucial roles in determining the flux of carbon into seed TAG and thus have been considered as the key targets for engineering oil production 2.3.1.164 isopenicillin-N N-acyltransferase biotechnology Penicillium chrysogenum npe6 lacking isopenicillin N acyltransferase activity is an excellent host for production of different beta-lactam antibiotics 2.3.1.165 6-methylsalicylic-acid synthase biotechnology production of unnatural polyketides e.g. 4-hydroxy-6-methyl-2-pyrone in E. coli and yeast after heterologous expression of the enzyme 2.3.1.165 6-methylsalicylic-acid synthase biotechnology functional heterologous expression of enzyme requires the presence of a 4’-phosphoantetheinyl transferase for activation. Coexpression of both enzymes results in production of 6-methylsalicylic acid in good yields 2.3.1.165 6-methylsalicylic-acid synthase biotechnology polyketides are a group of natural products that have gained much interest due to their use as antibiotics, cholesterol lowering agents, immunosuppressors and other drugs, it is therefor of general interest to transfer polyketide synthase genes into heterologous hosts that can overproduce the corresponding polyketides 2.3.1.165 6-methylsalicylic-acid synthase biotechnology production of 6-methylsalicylic acid which is used as human pharmaceutical 2.3.1.165 6-methylsalicylic-acid synthase biotechnology biotechnological de novo production of m-cresol from sugar in complex yeast extract-peptone medium with the yeast Saccharomyces cerevisiae. A heterologous pathway based on the decarboxylation of the polyketide 6-methylsalicylic acid is introduced into a CEN.PK yeast strain. Overexpression of codon-optimized 6-methylsalicylic acid synthase from Penicillium patulum together with activating phosphopantetheinyl transferase npgA from Aspergillus nidulans results in up to 367 mg/l 6-methylsalicylic acid production. Additional genomic integration of the genes have a strongly promoting effect and 6-methylsalicylic acid titers reach more than 2 g/l. Simultaneous expression of 6-methylsalicylic acid decarboxylase patG from Aspergillus clavatus leads to the complete conversion of 6-methylsalicylic acid and production of up to 589 mg/L m-cresol 2.3.1.167 10-deacetylbaccatin III 10-O-acetyltransferase biotechnology optimizing the semi-biosynthetic method in bacteria potentially provides a practical means of developing commercial-scale production of baccatin III analogs, which serve as key intermediates in the semi-synthesis of second-generation taxols 2.3.1.175 deacetylcephalosporin-C acetyltransferase biotechnology Penicillium chrysogenum npe6 lacking isopenicillin N acyltransferase activity is an excellent host for production of different beta-lactam antibiotics 2.3.1.175 deacetylcephalosporin-C acetyltransferase biotechnology studies of the enzyme will be useful in gaining a full view of the possibilities for overcoming the acetyl transfer bottleneck and for the future development of cephalosporin antibiotics 2.3.1.179 beta-ketoacyl-[acyl-carrier-protein] synthase II biotechnology modulating KASII activity is sufficient to convert the composition of a temperate seed oil into that of a palm-like tropical oil, overview 2.3.1.183 phosphinothricin acetyltransferase biotechnology an improved transformation method for biocontrol agent, Beauveria bassiana, is developed 2.3.1.183 phosphinothricin acetyltransferase biotechnology creation of herbicide resistant plants 2.3.1.183 phosphinothricin acetyltransferase biotechnology the bar gene is introduced into the cork oak genome to create resistance to the broad-spectrum herbicide phosphinothricin, commercially named Basta, Roundup Ready or Finale 2.3.1.194 acetoacetyl-CoA synthase biotechnology the enzyme represents a potential target to increase the flux through the mevalonate pathway 2.3.1.304 poly[(R)-3-hydroxyalkanoate] polymerase biotechnology recombinant protein production and purification from Escherichia coli is often accompanied with expensive and complicated procedures, especially for therapeutic proteins. By using an intein cleavable polyhydroxyalkanoate synthase fusion, recombinant proteins can be first produced and sequestered on a natural resin, the polyhydroxyalkanoate (PHA) inclusions, then separated from contaminating host proteins via simple PHA bead isolation steps, and finally purified by specific release into the soluble fraction induced by a pH reduction 2.3.2.13 protein-glutamine gamma-glutamyltransferase biotechnology after fermentation in presence of enzyme, wheat dough has higher resistance to stretching and lower extensibility than control, dough contains more of the smallest and less large air bubbles. Enzyme improves formation of protein network in bread baked from normal or organic flour but at higher dosage causes uneven ditribution 2.3.2.13 protein-glutamine gamma-glutamyltransferase biotechnology reaction product of putrescine-pectin conjugate and soy flour protein may be used foredible films with low water vapor permeability and improved mechanical properties 2.3.2.13 protein-glutamine gamma-glutamyltransferase biotechnology developments in mTGase engineering together with its role in biomedical applications including biomaterial fabrication for tissue engineering and biotherapeutics, overview 2.3.2.13 protein-glutamine gamma-glutamyltransferase biotechnology enzyme TGZ treatment effectively improves the textural properties of milk protein concentrate (MPC) gel at strength level and water-holding capacity. Optimal texture of MPC gel is achieved after TGZ treatment using 2 U/g TGZ for 2 h at 35°C and pH 7.0, method evaluation and optimization, overview 2.3.2.13 protein-glutamine gamma-glutamyltransferase biotechnology in production of homogeneous antibody-drug conjugates, the enzyme is useful for site-specific conjugation of glutamine-based acyl donor substrates and drugs to native and engineered lysines in human immunoglobulins by microbial transglutaminase, overview 2.3.2.13 protein-glutamine gamma-glutamyltransferase biotechnology microbial transglutaminase (mTG) is used as a crosslinking agent in the preparation of gelatin sponges. The physical properties of the materials are evaluated by measuring their material porosity, water absorption, and elastic modulus. The stability of the sponges are assessed via hydrolysis and enzymolysis, overview. To evaluate the cell compatibility of them TG crosslinked gelatin sponges (mTG sponges), adipose-derived stromal stem cells are cultured and inoculated into the scaffold. Cell proliferation and viability are measured using alamarBlue assay and LIVE/DEAD fluorescence staining, respectively. Cell adhesion on the sponges is observed by scanning electron microscopy. mTG sponges have uniform pore size, high porosity and water absorption, and good mechanical properties. In subcutaneous implantation (in Sprague-Dawley rats), the material is partially degraded in the first month and completely absorbed in the third month. Cell experiments show evident cell proliferation and high viability. The cells grow vigorously and adhered tightly to the sponge. In conclusion, mTG sponge has good biocompatibility and can be used in tissue engineering and regenerative medicine 2.3.2.13 protein-glutamine gamma-glutamyltransferase biotechnology the microbial transglutaminase is used for biotechnological and biomedical engineering, protein engineering by post-translational modification towards the generation of multifunctional conjugates. Biotechnological applications, detailed overview. Transglutaminase-mediated surface immobilization, a widely-used technique to increase stability of labile and cost-intensive enzymes and enable their reuse 2.3.2.13 protein-glutamine gamma-glutamyltransferase biotechnology use of MTG to site-specifically and covalently immobilize a substrate peptide-tagged protein, e.g. BirA, to a support, i.e. amine-modified magnetic microspheres (MMS) 2.3.2.15 glutathione gamma-glutamylcysteinyltransferase biotechnology plastid targeting of PCS 2.3.2.15 glutathione gamma-glutamylcysteinyltransferase biotechnology SrPCS1 has potential applications in genetic engineering of plants for enhancing heavy metal tolerance and phytoremediation of contaminated soils 2.3.2.15 glutathione gamma-glutamylcysteinyltransferase biotechnology SrPCS3 has potential applications in genetic engineering of plants for enhancing heavy metal tolerance and phytoremediation of contaminated soils 2.3.2.15 glutathione gamma-glutamylcysteinyltransferase biotechnology enhanced biosynthesis of cadmium(II)-selenide (CdS) nanoparticles (NPs) through Arabidopsis thaliana phytochelatin synthase-modified Escherichia coli with fluorescence effect in detection of pyrogallol and gallic acid. CdS NPs act as a classical II-VI semiconductor with various interesting properties and incomparable advantages. Enzyme PCS can effectively capture and enrich Cd2+ so that the generation and subsequent application process of CdS NPs will be greatly improved. A direct detection method based on such CdS NPs is established for determination of pyrogallol and gallic acid, two organic molecules with multiple advantages and wide applications. The method provides an excellent alternative to traditional chemosynthesis and quantitative analysis, which also displays great potential for technological improvement towards green synthesis and application. Method development and evaluation, detailed overview 2.3.3.14 homocitrate synthase biotechnology the alteration of homocitrate synthase activity can be a useful strategy for improving sustained photobiological hydrogen production in cyanobacteria. Greater sustained H2 production and higher nitrogenase activities of the DELTAhupL DELTAnifV1 mutant culture grown under air 2.3.3.21 (R)-citramalate synthase biotechnology fermentation of C5 and C6 sugars to ethanols and other metabolites under thermophilic conditions 2.4.1.1 glycogen phosphorylase biotechnology enzyme is a useful marker in the regulation of of glycogen metabolism and reproduction of Crassostrea gigas 2.4.1.1 glycogen phosphorylase biotechnology study on kinetics of inactivation and aggregation at 0.7 M guanidine hydrochloride. Osmolytes trimethylamine-N-oxide and betaine exhibit the highest protective efficacy against phosphorylase b inactivation. Test system for the study of the effects of macromolecular crowding induced by osmolytes on aggregation of proteins 2.4.1.4 amylosucrase biotechnology the enzyme fused to a starch-binding domain (SBD) is introduced in two potato genetic backgrounds to synthesize starch granules with altered composition, and thereby to broaden starch applications. The modified larger starches not only have great benefit to the potato starch industry by reducing losses during starch isolation, but also have an advantage in many food applications such as frozen food due to its extremely high freeze-thaw stability. Modified starches show a higher digestibility after alpha-amylase treatment 2.4.1.4 amylosucrase biotechnology treatment of pre-gelatinized rice and barley starches with amylosucrase from Neisseria polysaccharea is a potential way of replacing commercial resistant starch production 2.4.1.4 amylosucrase biotechnology amylosucrase has great potential in the biotechnology and food industries, due to its multifunctional enzyme activities. It can synthesize alpha-1,4-glucans, like amylose, from sucrose as a sole substrate. It can also utilize various other molecules as acceptors. In addition, amylosucrase produces sucrose isomers such as turanose and trehalulose. It also efficiently synthesizes modified starch with increased ratios of slow digestive starch and resistant starch, and glucosylated functional compounds with increased water solubility and stability. It produces turnaose more efficiently than other carbohydrate-active enzymes. Amylose synthesized by amylosucrase forms microparticles and these can be utilized as biocompatible materials with various bio-applications, including drug delivery, chromatography, and bioanalytical sciences 2.4.1.4 amylosucrase biotechnology arbutin as a safe hydroquinone derivative is one of most important skin-whitening ingredients including beta-arbutin and alpha-arbutin. The batch-feeding whole-cell biocatalysis by Amy-1 is a promising technology for alpha-arbutin production with enhanced yield and molar conversion rate 2.4.1.5 dextransucrase biotechnology potential application of the C-terminal enzyme domain GBD-7 as an affinity tag onto cheap resins like for rapid purification of dextrans 2.4.1.8 maltose phosphorylase biotechnology enzyme based biosensor for phosphate 2.4.1.8 maltose phosphorylase biotechnology production of crystalline trehalose from maltose with immobilized enzyme 2.4.1.8 maltose phosphorylase biotechnology production of trehalose from starch in industrial scale 2.4.1.9 inulosucrase biotechnology enzyme is used for inulin synthesis 2.4.1.18 1,4-alpha-glucan branching enzyme biotechnology RNAi technology is applied to suppress the expression of starch branching enzyme IIa and IIb and to increase amylose content in maize endosperm, and stably inherit high-amylose maize lines 2.4.1.18 1,4-alpha-glucan branching enzyme biotechnology RNAi technology is applied to suppress the expression of starch branching enzyme IIa and IIb and to increase amylose content in maize endosperm, and stably inherit high-amylose maize lines. Transgenic maize lines with amylose content of up to 55.89% are produced, which avoid the significant decreases in starch content and grain yield that occur in high-amylose starch branching enzyme IIb gene mutant 2.4.1.25 4-alpha-glucanotransferase biotechnology industrial production of cycloamylase with mutant enzyme Y54G, which shows high cyclization activity and low hydrolytic activity 2.4.1.62 ganglioside galactosyltransferase biotechnology the galactosylation of IFNalpha2b[Tn] with an engineered variant of CgtB clearly demonstrates that a bacterial glycosyltransferase is able to glycosylate a human protein in vitro. This is the first time that the direct glycosylation of a human protein by a bacterial glycosyltransferase is reported 2.4.1.85 cyanohydrin beta-glucosyltransferase biotechnology integration of genes CYP79A1, CYP71E1, and UGT85B1 in Nicotiana tabacum chloroplast genome and functional expression, the enzymes convert endogenous tyrosine into dhurrin using electrons derived directly from the photosynthetic electron transport chain, without the need for the presence of an NADPH-dependent P450 oxidoreductase. The dhurrin produced in the engineered plants amounted to 0.1-0.2% of leaf dry weight compared to 6% in the origin Sorghum bicolor. Plant P450s involved in the synthesis of economically important compounds can be engineered into the thylakoid membrane of chloroplasts, and their full catalytic cycle can be driven directly by photosynthesis-derived electrons 2.4.1.90 N-acetyllactosamine synthase biotechnology enzyme can be useful in the glycosylation of cytokines, enzymes or other glycosylated compounds modifying their functions for use in medical therapies 2.4.1.91 flavonol 3-O-glucosyltransferase biotechnology the increase of enzyme expression due to high C/N ration in growth medium can be used for generation of plants with an optimized flavonoid/anthocyanin content or desirable organ coloration 2.4.1.101 alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase biotechnology immobilization of the enzyme as maltose-binding fusion protein on an amylose resin for production of high-mannose type oligosaccharides 2.4.1.101 alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase biotechnology production of glycoprotein therapeutics in recombinant Saccharomyces cerevisiae adapted to production of hybrid- and complex-type carbohydrates 2.4.1.101 alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase biotechnology the fusion of the human beta 1,4-galacosyltransferase with the targeting domain of Nicotiana N-acetylglucosaminyltransferase I does not increase the level of beta 1,4-galactosylation in alfalfa, the expression in alfalfa strongly reduces the biosynthesis of Lewis a glycoepitope responsible for plant N-glycan immunogenicity in mammals 2.4.1.152 4-galactosyl-N-acetylglucosaminide 3-alpha-L-fucosyltransferase biotechnology a baculoviral expression system of FucT-IX appears to be a promising strategy for overproduction as compared to overproduction in Escherichia coli or mammalian cells 2.4.1.153 UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminyltransferase biotechnology use of enzyme gene in combination with tunicamycin as a selection marker for transformation of Arabidopsis, identification of transformants at a very early stage post germinantion 2.4.1.155 alpha-1,6-mannosyl-glycoprotein 6-beta-N-acetylglucosaminyltransferase biotechnology transfection of sense cDNA of enzyme results in increase in the N-acetylglucosamine-beta1,6-mannose-alpha1,3-branch of epidermal growth factor receptor. Transfection with antisense CDNA of enzyme rsults in reverse reaction. Altered expression of enzyme will change the glycan structure and function of epidermal growth factor receptor, which may modify downstream signal transduction 2.4.1.211 1,3-beta-galactosyl-N-acetylhexosamine phosphorylase biotechnology LNBP is cultured with sucrose phosphorylase, UDP-glucose hexose 1-phosphate uridylyltransferase, and UDP-glucose 4-epimerase to produce beta-D-galactopyranosyl-1,3-N-acetyl-D-glucosamine using sucrose as substrate in a 10l reaction mixture, 500 mmol beta-D-galactopyranosyl-1,3-N-acetyl-D-glucosamine are produced after 600 h, beta-D-galactopyranosyl-1,3-N-acetyl-D-glucosamine can be used as bifidus factor in human milk 2.4.1.212 hyaluronan synthase biotechnology optimization of the recombinant enzyme expression in Escherichia coli for large scale production of hyaluronan polymers for usage in basic studies, and for biotechnological creation of functional carbohydrates in medical purposes, engineering of produced product chain length 2.4.1.227 undecaprenyldiphospho-muramoylpentapeptide beta-N-acetylglucosaminyltransferase biotechnology MurG is interesting to evaluate as a potential antibiotic target, as it has no counterpart in mammalian cells 2.4.2.8 hypoxanthine phosphoribosyltransferase biotechnology genetic tools for use in Clostridium thermocellum that allow creation of unmarked mutations while using a replicating plasmid. The strategy employs counter-selections developed from the native Clostridium thermocellum hpt gene and the Thermoanaerobacterium saccharolyticum tdk gene and is used to delete the genes for both lactate dehydrogenase (Ldh) and phosphotransacetylase (Pta) 2.4.2.24 1,4-beta-D-xylan synthase biotechnology enzyme activity is hardly affected by addition of organic solvents 2.4.2.38 glycoprotein 2-beta-D-xylosyltransferase biotechnology the plant is useful for expression of human monoclonal antibodies free of zoonotic pathogens, co-expression of RNAi to block beta-1,2-xylosyltransferase and alpha-1,3-fucosyltransferase prevents unwanted plant specific N-glycosylation of the recombinant antibody proteins, overview 2.4.2.38 glycoprotein 2-beta-D-xylosyltransferase biotechnology glycoengineering: humanized N-glycosylation in plants by knock-down of plant-specific N-glycan maturation enzymes, pharmaceutical glycoprotein production in plant 2.4.2.38 glycoprotein 2-beta-D-xylosyltransferase biotechnology pharmaceutical protein production in plant, glycoengineering: humanized N-glycosylation in plants by knock-down of plant-specific N-glycan maturation enzymes 2.4.3.1 beta-galactoside alpha-(2,6)-sialyltransferase biotechnology recombinant enzyme may be used for in vitro synthesis of oligosaccharides 2.4.3.3 alpha-N-acetylgalactosaminide alpha-2,6-sialyltransferase biotechnology Escherichia coli system may be used as a starting point for the evolution of sialyltransferases with better expression characteristics or altered donor/acceptor specificities 2.4.3.5 galactosyldiacylglycerol alpha-2,3-sialyltransferase biotechnology use for chemoenzymatic synthesis of 13C-labeled sialyloligosaccharide 2.4.99.18 dolichyl-diphosphooligosaccharide-protein glycotransferase biotechnology promising candidate for glycoengineering, the development of glycan-based vaccines and therapeutics 2.5.1.15 dihydropteroate synthase biotechnology the Bacillus anthracis DHPS pterin-binding pocket is analysed using five docking programs (FlexX, Surflex, Glide, GOLD, and DOCK) and nine scoring functions using pose selection/scoring and enrichment studies. Pose selection and scoring use the 7-amino-3-(1-carboxyethyl)-1-methyl-pyrimido (4,5-c)- pyridazine-4,5(1H; 6H)-dione (AMPPD) co-crystal structure as the source structure. RMSD calculations are used to determine how well specific docking/scoring combinations pose and score the ligand in the pterin site. Surflex with Surflex-Score and Glide with GlideScore are the best overall performers for use in virtual screening against the DHPS target, with neither combination showing statistically significant superiority over the other in enrichment studies or pose selection. Post-docking ligand relaxation and consensus scoring does not improve overall enrichment 2.5.1.16 spermidine synthase biotechnology the FSPD1 gene is considered useful for gene transfer technology aiming at improving environmental stress tolerance of sweet potato 2.5.1.18 glutathione transferase biotechnology commercial fusion partner using for enhancing the solubility of recombinant proteins 2.5.1.19 3-phosphoshikimate 1-carboxyvinyltransferase biotechnology conferring glyphosate tolerance in transgenic crop plants 2.5.1.19 3-phosphoshikimate 1-carboxyvinyltransferase biotechnology glyphosate tolerance in bacteria 2.5.1.19 3-phosphoshikimate 1-carboxyvinyltransferase biotechnology strain PCC6803 DnaE intein N-terminal and C-terminal splicing domains can reconstitute EPSPS activities and glyphosate resistance in plant 2.5.1.29 geranylgeranyl diphosphate synthase biotechnology reconstruction of the isoprenoid pathway in Escherichia coli. To engineer a host that has the capability to supply geranylgeranyl diphosphate, a common precursor of isoprenoids, isopentenyl diphosphate isomerase (encoded by idi) from Escherichia coli and geranylgeranyl diphosphate synthase (encoded by gps) from Archaeoglobus fulgidus are cloned and overexpressed. The latter is shown to be a multifunctional enzyme converting dimethylallyl diphosphate to geranylgeranyl diphosphate. These two genes and the gene cluster (crtBIYZW) of the marine bacterium Agrobacterium aurantiacum are introduced into Escherichia coli to produce astaxanthin, an orange pigment and antioxidant. The metabolically engineered strain produces astaxanthin at a very high rate 2.5.1.32 15-cis-phytoene synthase biotechnology creation of marker-free transgenic plants 2.5.1.32 15-cis-phytoene synthase biotechnology developement nutritional plants enriched with carotenoids 2.5.1.34 4-dimethylallyltryptophan synthase biotechnology the combination of the two dimethylallyltryptophan synthases FgaPT2 and 7-DMATS (EC 2.5.1.34 and EC 2.5.1.80) can be successfully used for chemoenzymatic synthesis of the diprenylated derivatives. The potential of recombinant enzymes from secondary metabolite biosynthesis as promising tools for the production of designed compounds is demonstrated 2.5.1.47 cysteine synthase biotechnology transgenic plants expressing serine acetyltransferase and cysteine synthase can mitigate detrimental effects of cadmium toxicity, perhaps by efficiently producing and accumulating sulfuric compounds 2.5.1.54 3-deoxy-7-phosphoheptulonate synthase biotechnology increased production of aromatic amino acids in E. coli mutants with modified phosphoenolpyruvate metabolism and enhanced transketolase activity 2.5.1.54 3-deoxy-7-phosphoheptulonate synthase biotechnology E. coli strains, overproducing the enzyme, excrete it to the medium, which can also be used as a bioindicator for enhanced carbon commitment into the pathway 2.5.1.54 3-deoxy-7-phosphoheptulonate synthase biotechnology fruit-specific manipulation of the conversion of primary to specialized metabolism, e.g. by expressing 3-deoxy-7-phosphoheptulonate synthase in tomato fruits, is an attractive approach for improving fruit aroma and flavour qualities as well as discovering novel fruit-specialized metabolites. Metabolic profiling of transgenic tomato plants expressing a bacterial feedback-insensitive AroG gene, overview 2.5.1.63 adenosyl-fluoride synthase biotechnology fluorochemical production (18F-labelled organic compounds) for medicinal chemistry research (positron emission tomography), when enzyme acts together with other enzymes in the fluorometabolite production (purine nucleoside phosphorylase, isomerase, aldolase, enzyme generating fluoroacetaldehyde in a retro-aldol reaction, fluoroacetaldehyde dehydrogenase or pyridoxal phosphate-dependent enzyme) to generate fluoroacetaldehyde and 4-fluorothreonine 2.5.1.63 adenosyl-fluoride synthase biotechnology production of radiolabelled nucleosides as tracers for cancer cell uptake studies via positron emission tomography 2.5.1.63 adenosyl-fluoride synthase biotechnology the rate of the enzymatic fluorination reaction can be enhanced by using ionic liquids and immobilizing the enzyme with a water-absorbing polymer which stabilizes the enzyme, 1-octyl-3-methylimidazolium hexafluorophosphate and 1-hexyl-3-methylimidazolium hexafluorophosphate raise the conversion yield 2.4times or 1.6times compared to Tris-HCl buffer, pH 8.0, 1-hexyl-3-methylimidazolium tetrafluoroborate, 1-octyl-3-methylimidazolium tetrafluoroborate, 1-octyl-3-methylimidazolium hexafluorophosphate, and 1-hexyl-3-methylimidazolium hexafluorophosphate increase total conversation yields 2.2times to 4times more in immobilized compared to non-immobilized enzyme 2.5.1.63 adenosyl-fluoride synthase biotechnology the enzyme is used for [18F]-radiolabelling of bioactive peptides, overview 2.5.1.75 tRNA dimethylallyltransferase biotechnology production of ornamental crops with large flowers and crop species with larger fruit 2.5.1.78 6,7-dimethyl-8-ribityllumazine synthase biotechnology a circularly permuted variant of lumazine synthase affords versatile building blocks for the construction of nanocompartments that can be easily produced, tailored, and diversified. The topologically altered protein self-assembles into spherical and tubular cage structures with morphologies that can be controlled by the length of the linker connecting the native termini. Permutated lumazine synthase proteins integrate into wild-type and other engineered lumazine synthase assemblies by coproduction in Escherichia coli to form patchwork cages. This coassembly strategy enables encapsulation of guest proteins in the lumen, modification of the exterior through genetic fusion, and tuning of the size and electrostatics of the compartments 2.5.1.78 6,7-dimethyl-8-ribityllumazine synthase biotechnology the C-terminal tail of ribiflavin synthase can act as an encapsulation tag capable of targeting other proteins to the lumazine synthase capsid interior. Fusion of to either the last 11 or the last 32 amino acids of riboflavin synthase, yields variant GFP11 or GFP32, respectively. After purification, lumazine synthase capsids that have been coproduced in bacteria with GFP11 and GFP32 are 15- and 6fold more fluorescent, respectively. GFP11 is localized within the lumazine synthase capsid. Fusing the last 11 amino acids of riboflavin synthase to the C-terminus of the Abrin A chain also leads to its encapsulation by lumazine synthase at a level similar to that of GFP11. Mild changes in pH and buffer identity trigger dissociation of the GFP11 guest 2.5.1.80 7-dimethylallyltryptophan synthase biotechnology the combination of the two dimethylallyltryptophan synthases FgaPT2 and 7-DMATS (EC 2.5.1.34 and EC 2.5.1.80) can be successfully used for chemoenzymatic synthesis of the diprenylated derivatives. The potential of recombinant enzymes from secondary metabolite biosynthesis as promising tools for the production of designed compounds is demonstrated 2.6.1.1 aspartate transaminase biotechnology an enzymatic method for the synthesis of the amino acid Phe is developed. AAT from porcine heart is immobilized by covalent attachment and entrapment, and the resulting immobilized preparations are compared to the soluble enzyme both in terms of stability and reaction efficiency 2.6.1.11 acetylornithine transaminase biotechnology the pCR2.1-argD complementation plasmid is stably maintained in the argD(1000)::Tn5 transposon mutant growing in host tissues without any antibiotic selection. The pCR2.1-argD complementation plasmid can be useful for the expression of genes, markers, and reporters in Erwinia amylovora growing in planta, without concern about losing the plasmid over time 2.6.1.13 ornithine aminotransferase biotechnology genetic engineering of plants for increased production of the osmoprotectant proline, transgenic plants overexpressing OAT display enhanced tolerance to salt and drought due to increased proline content 2.6.1.18 beta-alanine-pyruvate transaminase biotechnology an alternative deracemization method for the efficient production of L-homoalanine using D-amino acid oxidase (vide infra) and omega-TA is proposed 2.6.1.18 beta-alanine-pyruvate transaminase biotechnology recombinant Escherichia coli cells containing overexpressed omega-TA activity are immobilized by entrapment in LentiKats and the effect of permeabilization on free cells and their immobilized counterparts is analyzed. Cells are applied in the synthesis of the aromatic amines 3-amino-1-phenylbutane and 1-phenylethylamine. The best results are obtained with CTAB 0.1% for permeabilization which results in an increase in reaction rate by 40% compared to the whole cells. The synthesis of 1-phenylethylamine is carried out using isopropyl amine and L-alanine as amino donors. Using whole cell biocatalysis, the reaction with isopropyl amine is one order of magnitude faster than with L-alanine 2.6.1.36 L-lysine 6-transaminase biotechnology production of beta-lactam antibiotics 2.6.1.62 adenosylmethionine-8-amino-7-oxononanoate transaminase biotechnology evaluation of biotin overproduction, biotin production by Bacillus subtilis fermentation can be optimized by addition of exogenous lysine or mutational deregulation of lysine in the producing strain 2.6.1.85 aminodeoxychorismate synthase biotechnology conversion of aminodeoxychorismate synthase into anthranilate synthase employing a bioinformatics method for predicting mutations required to functionally interconvert homologous enzymes. Complementation of an anthranilate synthase-deficient strain of Escherichia coli grown on minimal medium leads to several aminodeoxychorismate synthase mutants that allow growth in 6 days compared to 2 days for wild-type anthranilate synthase. The purified mutant enzymes catalyze the conversion of chorismate to anthranilate at rates that are about 50% of the rate of wild-type aminodeoxychorismate synthase-catalyzed conversion of chorismate to aminodeoxychorismate. The residues mutated do not contact the substrate 2.6.1.86 2-amino-4-deoxychorismate synthase biotechnology conversion of aminodeoxychorismate synthase into anthranilate synthase employing a bioinformatics method for predicting mutations required to functionally interconvert homologous enzymes. Complementation of an anthranilate synthase-deficient strain of Escherichia coli grown on minimal medium leads to several aminodeoxychorismate synthase mutants that allow growth in 6 days compared to 2 days for wild-type anthranilate synthase. The purified mutant enzymes catalyze the conversion of chorismate to anthranilate at rates that are about 50% of the rate of wild-type aminodeoxychorismate synthase-catalyzed conversion of chorismate to aminodeoxychorismate. The residues mutated do not contact the substrate 2.7.1.11 6-phosphofructokinase biotechnology design of strains with improved antibiotic production 2.7.1.16 ribulokinase biotechnology improvement of a bacterial L-arabinose utilization pathway consisting of L-arabinose isomerase from Bacillus subtilis and L-ribulokinase and L-ribulose-5-phosphate 4-epimerase from Escherichia coli after expression of the corresponding genes in Saccharomyces cerevisiae. These improvements make up a new starting point for the construction of more-efficient industrial L-arabinose-fermenting yeast strains by evolutionary engineering 2.7.1.17 xylulokinase biotechnology putative use of lignocellulosic biomass as feedstock for the chemical industry 2.7.1.32 choline kinase biotechnology enzyme based assay for choline content of feed 2.7.1.48 uridine/cytidine kinase biotechnology overexpression of gene udk encoding uridine/cytidine kinase interferes with T7 bacteriophage growth. This inhibition can be overcome by inhibition of host RNA polymerase by overexpression of gene 2 or by treatment with rifampicin. General model for the requirement of host RNA polymerase inhibition 2.7.1.78 polynucleotide 5'-hydroxyl-kinase biotechnology application in DNA and RNA sequencing 2.7.1.86 NADH kinase biotechnology NADH kinase can be employed as an effective metabolic manipulation target to improve poly-3-hydroxybutyrate synthesis 2.7.1.86 NADH kinase biotechnology recombinant protein overproduction often results in oxidative stress, causing deviations from the optimal redox cofactor regeneration balance. This becomes one of the limiting factors in obtaining high levels of heterologous protein production. Overexpression of NADH kinase enzyme from Saccharomyces cerevisiae (Pos5) in the cytosol of Pichia pastoris results in a significant improvement in the production of the model protein 2.7.1.119 hygromycin-B 7''-O-kinase biotechnology use the hygromycin phosphotransferase gene (hpt) as a selective marker gene for tracking plastid transformation in rice (Oryza sativa) 2.7.1.163 hygromycin B 4-O-kinase biotechnology the enzyme can be used as selective marker gene product in production of transgenic plants 2.7.1.163 hygromycin B 4-O-kinase biotechnology the enzyme is widely used as selective marker gene product in production of engineered crops, e.g. rice 2.7.1.163 hygromycin B 4-O-kinase biotechnology mediates hygromycin resistance 2.7.1.163 hygromycin B 4-O-kinase biotechnology resistance against hyromycin B mediated by transformation of the hph gene 2.7.1.163 hygromycin B 4-O-kinase biotechnology used as selectable marker gene 2.7.1.176 UDP-N-acetylglucosamine kinase biotechnology lethal expression of bacterial toxin-antitoxin system toxins in eukaryotic microalgae, which can form the basis of a novel method for harvesting of microalgal cellular contents. Chlorella vulgaris is a eukaryotic microalga with potential for the production of biofuels. Its thick and rigid cell wall is an impediment to cost-effective, large-scale harvesting of biofuels from these cells 2.7.2.7 butyrate kinase biotechnology optimization for biotechnological production of polyhydroxyalkanoic acids and polythioethers 2.7.2.11 glutamate 5-kinase biotechnology an artificial bifunctional enzyme, gamma-glutamyl kinase/gamma-glutamyl phosphate reductase, improves NaCl tolerance when expressed in Escherichia coli 2.7.2.11 glutamate 5-kinase biotechnology the D154N mutation results in a prominant increase in cell viability after freezing at -20°C compared to the viability of the cells harboring the wild-type PRO1 gene, method for breeding novel freeze-tolerant yeast strains 2.7.2.15 propionate kinase biotechnology in order to realise an efficient production of 1-propanol through amino acid biosynthetic pathway using glucose or glycerol as a carbon source L-threonine overproducing strain, TH20 is employed as a base strain and engineered to establish a novel pathway leading to the formation of1-propanol under aerobic condition. The engineered strain, PRO2, harbouring a plasmid overexpressing the atoDA, adhEmut, thrABC, ackA and cimA genes is able to produce more than 10 g/L of 1-propanol from glucose or glycerol in aerobic fed-batch fermentation 2.7.3.2 creatine kinase biotechnology use as biomarker of sperm cell membrane degradation 2.7.3.2 creatine kinase biotechnology stability of immobilized enzyme 2.7.4.1 ATP-polyphosphate phosphotransferase biotechnology ATP supply for synthesis of D-amino acid dipeptides 2.7.4.1 ATP-polyphosphate phosphotransferase biotechnology polyphosphate kinases use inexpensive and stable polyphosphate as a phosphate donor and phosphorylate nucleoside 5'-monophosphate as well as 5'-diphosphates. This makes them of special interest for application in ATP regeneration systems, which can be efficiently coupled to ATP-consuming enzymes in environmentally friendly and sustainable biotechnological processes 2.7.4.25 (d)CMP kinase biotechnology CMP kinase and actetate kinase in a whole cell-biocatalysis to obtain CTP 2.7.7.7 DNA-directed DNA polymerase biotechnology thermostable polymerase used in PCR 2.7.7.7 DNA-directed DNA polymerase biotechnology short-patch compartmentalized self-replication will be a powerful strategy for the generation of polymerases with altered substrate specificity for applications in nano- and biotechnology and in the enzymatic synthesis of antisense and RNAi probes 2.7.7.7 DNA-directed DNA polymerase biotechnology DNA polymerase plays prominent roles in numerous biotechnologies. The use of diphosphate substrates has the potential to make practical the incorporation of expensive analogs, such as isotopically labeled or chemically modified nucleotides, eliminating the need for challenging triphosphate syntheses. This feature of DNA polymerases may also provide a method for detecting nucleotides used in high-throughput DNA sequencing 2.7.7.19 polynucleotide adenylyltransferase biotechnology assay in microtiter format 2.7.7.B22 transposase biotechnology application of a hyperactive transposase variant to generate mutants with integrated genes for the expression of the superfolder green fluorescent protein gene or a 2-ketodecarboxylase gene in Acidithiobacillus ferrooxidans, which enables the production and secretion of isobutyric acid. An inverse PCR method identifies the insertion sites of the 2-ketodecarboxylase gene, i.e. functional exogenous metabolic genes have been chromosomally integrated 2.7.7.27 glucose-1-phosphate adenylyltransferase biotechnology the results suggest that the sweetpotato ibAGP1 promoter and its transit peptide are a strong foreign gene expression system that can be used for molecular farming in potato plants 2.7.7.31 DNA nucleotidylexotransferase biotechnology use in production of synthetic homo- and heteropolymers, N-acetylation or N-alkylation of derivatives of dNTPs 2.7.7.31 DNA nucleotidylexotransferase biotechnology efficient production of recombinant enzyme in E. coli 2.7.7.50 mRNA guanylyltransferase biotechnology enzyme can be used as a tool for specific 5'-end-labeling of mRNA 2.7.7.60 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase biotechnology high-throughput methods for the screening of 2C-methyl-D-erythritol synthase IspC protein, 4-diphosphocytidyl-2C-methyl-D-erythritol synthase IspD protein, 4-diphosphocytidyl-2Cmethyl-D-erythritol kinase IspE protein, and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase IspF protein, against large compound libraries. Assays use up to three auxiliary enzymes monitored at 340 and all robust 2.7.7.65 diguanylate cyclase biotechnology immobilization of etDGC in sol-gel, a silica matrix that is known to encapsulate biological macromolecules via non-covalent entrapment, is described. The sol-gel matrix preserves the enzymatic activity of the enzyme, and thus, could be a viable method for DGC protein immobilization and c-di-GMP production 2.7.8.2 diacylglycerol cholinephosphotransferase biotechnology targeting of unusual fatty acids to triacylglycerol synthesis and their exclusion from membrane lipids are not achieved on the basis of the diacylglycerol substrate specificities of enzyme 2.7.8.7 holo-[acyl-carrier-protein] synthase biotechnology mutant ACPs will be valuable in dissecting the structure-function relationships of ACP and its participation in synthesis and regulation of fatty acid, phospholipids, and other products of medical and biotechnological importance 2.7.8.7 holo-[acyl-carrier-protein] synthase biotechnology identification of short peptide tags of 12 residues as efficient substrate for site-specific protein labeling catalyzed by enzyme 2.7.8.7 holo-[acyl-carrier-protein] synthase biotechnology biotechnological de novo production of m-cresol from sugar in complex yeast extract-peptone medium with the yeast Saccharomyces cerevisiae. A heterologous pathway based on the decarboxylation of the polyketide 6-methylsalicylic acid is introduced into a CEN.PK yeast strain. Overexpression of codon-optimized 6-methylsalicylic acid synthase from Penicillium patulum together with activating phosphopantetheinyl transferase npgA from Aspergillus nidulans results in up to 367 mg/l 6-methylsalicylic acid production. Additional genomic integration of the genes have a strongly promoting effect and 6-methylsalicylic acid titers reach more than 2 g/l. Simultaneous expression of 6-methylsalicylic acid decarboxylase patG from Aspergillus clavatus leads to the complete conversion of 6-methylsalicylic acid and production of up to 589 mg/L m-cresol 2.7.8.15 UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase biotechnology combination of GPT and tunicamycin is a potential selectable marker system for potato transformation, overview 2.7.9.4 alpha-glucan, water dikinase biotechnology the starch-phosphorylating enzymes are attractive candidates for the control of flux through starch degradation 2.7.11.15 beta-adrenergic-receptor kinase biotechnology engineering of a GRK2 mutant sensitive to a specific inhibitor 2.8.3.1 propionate CoA-transferase biotechnology establishing of an on-site monitoring of volatile fatty acids VFA, such as propionate, biosensing system using recombinant propionate CoA transferase and recombinant acyl-CoA oxidase, this system produces hydrogen peroxide in the presence of acetyl-CoA, oxygen, and VFA substrates, which can be quantified by colorimetric methods using peroxidase and dye reagents, e.g., 4-aminobenzoic acid plus 4-aminoantipyrine, overview 2.8.3.1 propionate CoA-transferase biotechnology propionate-biosensor, using propionate coenzyme A transferase from Clostridium propionicum and short-chain acyl-CoA oxidase from Arabidopsis thaliana as enzyme electrodes 2.8.3.11 citramalate CoA-transferase biotechnology production process of enantiomeric pure (S)-(+)-citramalic acid from itaconic acid 3.1.1.1 carboxylesterase biotechnology engineered enzyme can be used to produce the enantiomer (S)-ketoprofen for pharmacological or pharmaceutical use 3.1.1.1 carboxylesterase biotechnology enzyme is an important biocatalyst for the preparation of a wide range of selectively protected and enantiomerically pure compounds 3.1.1.1 carboxylesterase biotechnology rational protein engineering for direct evolution of suitable enantioselective biocatalysts for synthesis of chiral substances 3.1.1.1 carboxylesterase biotechnology the enzyme can be used as a biocatalyst at suboptimal temperatures through addition of DMSO as an activator 3.1.1.1 carboxylesterase biotechnology due to its high thermal stability the enzyme is a very attractive enzyme for biotechnological purposes 3.1.1.3 triacylglycerol lipase biotechnology the enzyme can be used for hydrolysis and synthesis of various esters, mutagenic modification and optimization of Candida rugosa isozymes for enantioselective, substrate-specific biocatalysis, improvement of thermostability, enantioselectivity, and substrate specificity, possible reactions are hydrolysis, direct esterification, acidolysis, alcoholysis, ester-interchange, and glycerolysis, overview 3.1.1.3 triacylglycerol lipase biotechnology the alkaline lipase is stable and active in organic solvents and a water-restricted environment and has a great potential as a biotechnological tool e.g. in organosynthetic reactions in water-restricted medium or in control and prevention of metalworking fluid putrification in the metal industry 3.1.1.3 triacylglycerol lipase biotechnology biocatalytic surfactant production, for example the synthesis of myristyl myristate. 3.1.1.4 phospholipase A2 biotechnology the enzyme is active and stable at higher temperatures, which suggests its great potential in biotechnological applications 3.1.1.7 acetylcholinesterase biotechnology enzyme immobilised on a nanostructured Langmuir-Blodgett proteo-glycolipidic bilayer, directly interfaced with an efficient optical device. Direct investigation of the kinetic behaviour of enzyme 3.1.1.8 cholinesterase biotechnology ChE activity from Palaemon serratus eyes seems to be a reliable and sensitive biomarker for organophosphate insecticides even when organisms are simultaneously exposed to mercury 3.1.1.14 chlorophyllase biotechnology encapsulation of the enzyme in micelles of different media within alginate hydrogels increases the enzyme activity in e.g. Tris buffer or hexane, extent of enhancement of the partition coefficient depends on the amount and hydrophobicity of the components intrduced into alginate, affecting the hydrophobic-hydrophilic balance of the gel 3.1.1.20 tannase biotechnology immobilization of the enzyme by microencapsulation with a coacervate calcium alginate membrane surrounding a liquid core improves the thermal and pH stability significantly 3.1.1.20 tannase biotechnology production of the enzyme for industrial purposes 3.1.1.20 tannase biotechnology production of the enzyme for industrial purposes, usage in quality improvement in the production of beer, wine 3.1.1.20 tannase biotechnology hydrolysis of tannic acid by enzyme immobilized on alginate beads, enzyme retains about 85% of initial activity and is active after extensive reuse 3.1.1.20 tannase biotechnology immobilization of enzyme by microencapsulation with a coacervate calcium alginate membrane surrounding a liquid core. Yield is 36.8% of initial enzyme activity, pH stability and thermal stability improve after microencapsulation. Enzyme can be used for up to 15 runs 3.1.1.20 tannase biotechnology maximum production of enzyme by growth on coffee husk, supplemented with 0.6% tannic acid, and 50% w/v moisture 3.1.1.20 tannase biotechnology production of enzyme by Aspergillus growing on fourfold diluted olive mill waste water is stable during more than 30 h and correlates with about 70% degradation of phenolic compounds present in the waste 3.1.1.20 tannase biotechnology synthesis both of ellagic acid and enzyme is maximal between 48 h and 72 h of growth of microorganism, at around 28 to 35°C and at about 5 g/l tannin 3.1.1.20 tannase biotechnology synthesis of enzyme in fed-batch culture, at pH 5.0, achieves 7000 IU/l 3.1.1.68 xylono-1,4-lactonase biotechnology considerable interest exists in utilizing the hemicellulose biomass for fine chemical production by converting xylose microbially to xylonic acid, other proposed uses of xylonic acid are in textile bleaching or in electroplating 3.1.1.73 feruloyl esterase biotechnology ability of the enzyme to be active in alkaline pH may be advantageous in biotechnological applications and especially in the treatment of alkaline woodpulp 3.1.1.73 feruloyl esterase biotechnology chimeric enzyme FaeA/Aspergillus kawachii family 42 carbohydrate-binding module as an innovative enzymatic tool for biotechnological applications and biotransformation of plant biomass 3.1.1.73 feruloyl esterase biotechnology enhancement of FAE activity in the small intestine and the colon by using orally ingested microencapsulated FAE-producing lactic acid bacteria. Microencapsulation renders the potentially beneficial product of FAE de-esterification, namely ferulic acid, more bioavailable while at the same time avoiding the problems associated with oral administration of free bacterial cells. The released ferulic acid by FAE may well prove to have several chemopreventive effects in chronic diseases. This approach may also be useful in the industrial production of ferulic acid for use in the food industry 3.1.1.74 cutinase biotechnology adsorption of enzyme onto the surface of poly(methyl methacrylate) latex particles. Up to 50% decrease in specific activity at pH-values 4.5 and 5.2. Almost no inactivation upon adsorption at pH 7.0 and 9.2. 60% increase in maximum adsorption with temperature raising from 25 to 50°C 3.1.1.74 cutinase biotechnology immobilization of enzyme on sodium form of zeolite Y, half-life 590 days at 30°C. Immobilization on zolite A, halft-life of 54 days at 30°C. Half-lives after immobilization on Alumina and Accurel-PA6 are 109 and 10 days, resp. Higher temperatures induce a remarkable stability loss in all preparations. At 30°C, enzymatic activities obtained wit the immobilization on zeolite A are the highest ones 3.1.1.74 cutinase biotechnology study on enzyme encapsulated in sol-gel matrices prepared with alkyl-alkoxysilane precursors of different chain length. Specific activity of entrapped enzyme is comparable to enzyme immobilized on zeolite Y, with incorporation of different additives bringing about an enhancement of enzyme activity and operational stability 3.1.1.74 cutinase biotechnology use of enzyme in a membrane reactor in presence of 1-hexanol, operational half-life of 674 days 3.1.1.74 cutinase biotechnology engineering new cutinase-inspired biocatalysts with tailor-made properties 3.1.1.74 cutinase biotechnology high-level secretion of cutinase in Pichia pastoris may be a promising alternative to many expression systems previously used for the large-scale production of cutinase in Saccharomyces cerevisiae as well as Escherichia coli. The functional expression of a large amount of extracellular cutinase offers the opportunity for developing an efficient high-throughput screening procedure for the improvement of enzymatic property and the development of novel biocatalysis of cutinase 3.1.1.75 poly(3-hydroxybutyrate) depolymerase biotechnology the optimum production of PHB depolymerase is observed at pH 8 and 7, at 45°C, 1% substrate concentration and in the presence of lactose as an additional carbon source 3.1.1.81 quorum-quenching N-acyl-homoserine lactonase biotechnology bacteria harboring the qsdA gene interfere very efficiently with quorum-sensing-regulated functions, demonstrating that qsdA is a valuable tool for developing quorum-quenching procedures 3.1.1.81 quorum-quenching N-acyl-homoserine lactonase biotechnology extreme temperature and pH resistance, resistance to proteases, wide substratum spectrum, and high specific activity enable the application of this QQ enzyme in a wide range of biotechnological applications 3.1.1.117 (4-O-methyl)-D-glucuronate---lignin esterase biotechnology glucuronoyl esterases are interesting candidates for biotechnological applications in plant biomass processing and genetic modification of plants 3.1.1.117 (4-O-methyl)-D-glucuronate---lignin esterase biotechnology glucuronoyl esterases are microbial enzymes with potential to cleave the ester bonds between lignin alcohols and xylan-bound 4-O-methyl-D-glucuronic acid in plant cell walls. This activity renders glucuronoyl esterases attractive research targets for biotechnological applications. One of the factors impeding the progress in glucuronoyl esterases research is the lack of suitable substrates. A facile preparation is described of methyl esters of chromogenic 4-nitrophenyl and 5-bromo-4-chloro-3-indolyl beta-D-glucuronides for qualitative and quantitative glucuronoyl esterase assay coupled with beta-glucuronidase as the auxiliary enzyme. The indolyl derivative affording a blue indigo-type product is suitable for rapid and sensitive assay of glucuronoyl esterase in commercial preparations as well as for high throughput screening of microorganisms and genomic and metagenomic libraries 3.1.2.2 palmitoyl-CoA hydrolase biotechnology enzyme is involved in fatty acid biosynthesis and may be a good target for improvement of special oil production in transgenic plants 3.1.2.2 palmitoyl-CoA hydrolase biotechnology enzyme can be used for bioengineering of transgenic plants to produce oils with desired properties 3.1.2.6 hydroxyacylglutathione hydrolase biotechnology overexpression of glyoxalase II, plants are able to grow, flower, and set normal viable seeds in presence of 5 mM ZnCl2, without any penalty. Enzyme overexpression may also confer tolerance to cadmium or lead. Major sink for metal accumulation are roots. Double transgenics expressing both glyoxalase I and II perform better than single-gene transformants. Under high zinc conditions, transgenic plants show restricted methylglyoxal accumulation and less lipid peroxidation 3.1.2.20 acyl-CoA hydrolase biotechnology enzyme is involved in fatty acid biosynthesis and may be a good target for improvement of special oil production in transgenic plants 3.1.3.1 alkaline phosphatase biotechnology the chemical modification by chemically modifying aliphatic or amino groups using tetracarboxy-benzophenone derivatives can be utilized to increase the thermostability of psychrophilic and mesophilic enzymes while retaining their high intrinsic activity or increasinf their activity 3.1.3.1 alkaline phosphatase biotechnology the chemical modification by chemically modifying aliphatic or amino groups using tetracarboxy-benzophenone derivatives can be utilized to increase the thermostability of psychrophilic and mesophilic enzymes while retaining their high intrinsic activity or increasing their activity 3.1.3.1 alkaline phosphatase biotechnology alkaline phosphatase from Escherichia coli is immobilized by copolymerization with resorcinol. The phosphatase-polyresorcinol complex synthesized retains about 74% of the original enzymatic activity. On addition to soil, free enzyme is completely inactivated in 4 days, whereas the phosphatase-polyresorcinol complex is comparatively stable.Barley seed coated with the immobilized enzyme exhibits higher rhizosphere phosphatase activity. Under pot culture conditions, an increase in the soil inorganic phosphorus is detected when the seed is encapsulated with the phosphatase-polyresorcinol complex, and a positive influence on biomass and inorganic phosphorus concentration of shoot is observed 3.1.3.1 alkaline phosphatase biotechnology an assay is developed for determination of the activity of the ALP using the effect of enhancement of fluorescence of the europium(III)-tetracycline 3:1 complex (Eu(3)TC). Its luminescence, peaking at 616 nm if excited at 405 nm, is enhanced by a factor of 2.5 in the presence of phosphate. Phosphate coordinates to Eu(3)TC and enhances its luminescence intensity as a result of the displacement of water from the inner coordination sphere of the central metal. The assay is performed in a time-resolved (gated) mode, which is shown to yield larger signal changes than steady-state measurement of fluorescence. The limit of detection for ALP is 4 micromol/l. Based on this scheme, a model assay for theophylline as inhibitor for ALP is developed with a linear range from 14 to 68 micromol/l of theophylline 3.1.3.1 alkaline phosphatase biotechnology the data demonstrate the potential utility of genetically engineering PhoK for the bioprecipitation of uranium from alkaline solutions 3.1.3.1 alkaline phosphatase biotechnology a simple and fast dynamically coated capillary electrophoretic method is developed for the characterization and inhibition studies of alkaline phosphatases 3.1.3.2 acid phosphatase biotechnology immobilization of the purified enzyme on glutaraldehyde-activated aminopropyl controlled-pore glass activates the enzyme, assay automatization, system evaluation, overview 3.1.3.2 acid phosphatase biotechnology improved stability and catalytic kinetics of acid phosphatase immobilized on composite beads of chitosan and activated clay as a model system for enzyme immobilization optimization 3.1.3.2 acid phosphatase biotechnology improvement of enzymic reaction by intercalated clay surfaces with free and immobilized enzyme, overview 3.1.3.7 3'(2'),5'-bisphosphate nucleotidase biotechnology GhHL1 is a functional and good candidate gene that might be used to improve salt tolerance in plants 3.1.3.8 3-phytase biotechnology a 20fold increase in total root phytase activity in transgenic lines expressing Aspergillus niger phytase results in improved phosphorus nutrition, such that the growth and phosphorus content of the plants is equivalent to control plants supplied with inorganic phosphate. Use of gene technology to improve the ability of plants to utilize accumulated forms of soil organic phosphorus 3.1.3.8 3-phytase biotechnology the complete hydrolysis of phytate by the enzyme, which is proposed on the basis of its capability to cleave any phosphate group of phytate, is a highly desired property for the biotechnological application of the enzyme 3.1.3.8 3-phytase biotechnology potential of using yeast as a phytase carrier in the gastrointestinal tract. The enzyme may be a 3-phytase, EC 3.1.3.8, or a 6-phytase, EC 3.1.3.26. The product of the hydrolysis of myo-inositol hexakisphosphate (i.e. myo-inositol 1,2,3,4,5-pentakisphosphate) or myo-inositol 1,3,4,5,6-pentakisphosphate has not been identified 3.1.3.8 3-phytase biotechnology production of phytase in laboratory-scale fermenter. Maintaining an acidic environment (pH 1.5-1.8) in the fermentation broth after the initial buildup of cell mass along with proper fragmentation of filamentous fungi results in significant improvement in phytase productivity 3.1.3.8 3-phytase biotechnology a cross-linked enzyme aggregate (CLEA) of 3-phytase is synthesised, which is incubated with vanadate and tested as a biocatalyst in the asymmetric sulfoxidation of thioanisole using hydrogen peroxide as the oxidant. The results show that the 3-phytase-CLEA demonstrates a similar efficiency (ca. 95% conversion) and asymmetric induction (ca. 60%) as the free enzyme. Moreover, the 3-phytase-CLEA can be reused at least three times without significant loss of activity 3.1.3.8 3-phytase biotechnology to evaluate the ability of EDTA to improve phytate P utilization and the possible synergistic effect between EDTA and microbial phytase an experiment is conducted using 360 Ross 308 broiler chicks. The experiment is carried out using a completely randomized design. Four replicate of 15 chicks per each are fed dietary treatments. Phytase supplementation of P-deficient diets significantly improves weight gain and feed efficiency, but it has no effect on feed consumption. Microbial phytase supplementation significantly decreases alkaline phosphatase concentration. Results obtained suggest no synergistic effect between phytase and EDTA in broiler chicks 3.1.3.8 3-phytase biotechnology Pichia pastoris containing cell-surface phytase releases phosphorus from feedstuff at a level similar to secreted phytase 3.1.3.8 3-phytase biotechnology synthesis of a modified phytase gene with 1256 bp in length with optimal codons for expression in Pichia pastoris. A Pichia pastoris strain that expresses the modified phytase gene phyA-mod shows a 50% increase in phytase activity level 3.1.3.11 fructose-bisphosphatase biotechnology photosynthesis-elevated transplastomic plants expressing FBP/SBPase promote growth and productivity of approximately 1.8fold as compared with wild-type plants, suggesting that the effective packaging of multigenes, FBP/SBPase and a gene involved in value-added traits into plastid genomes simultaneously provide further improvement of the potential for plastid transformation and more efficient production of foreign proteins, such as edible vaccines, pharmaceuticals and antibodies, in tobacco leaves 3.1.3.12 trehalose-phosphatase biotechnology The transformation of rice plants with a gene that encodes a bifunctional fusion enzyme of trehalose-6-phosphate synthase and – phosphatase from Escherischia coli increases the trehalose levels in these plants, shows no visible growth inhibition. The production of trehalose in these plants results in increased tolerance to drought, salt, and cold stresses. 3.1.3.21 glycerol-1-phosphatase biotechnology production of 1,3-propanediol from glucose in engineered Klebsiella pneumoniae strain 3.1.3.22 mannitol-1-phosphatase biotechnology engineering of D-mannitol production in Lactococcus lactis by overexpression of recombinant mannitol 1-phosphatase and mannitol 1-phosphate dehydrogenase from Eimeria tenella and Lactobacillus plantarum leading to 50% increased glucose-to-mannitol conversion, overview 3.1.3.22 mannitol-1-phosphatase biotechnology the enzyme is a target for engineering to produce salt-tolerant transgenic horticultural crop plants 3.1.3.26 4-phytase biotechnology a 20fold increase in total root phytase activity in transgenic lines expressing Aspergillus niger phytase results in improved phosphorus nutrition, such that the growth and phosphorus content of the plants is equivalent to control plants supplied with inorganic phosphate. Use of gene technology to improve the ability of plants to utilize accumulated forms of soil organic phosphorus 3.1.3.26 4-phytase biotechnology the complete hydrolysis of phytate by the enzyme, which is proposed on the basis of its capability to cleave any phosphate group of phytate, is a highly desired property for the biotechnological application of the enzyme 3.1.3.26 4-phytase biotechnology potential of using yeast as a phytase carrier in the gastrointestinal tract. The enzyme may be a 3-phytase, EC 3.1.3.8, or a 6-phytase, EC 3.1.3.26. The product of the hydrolysis of myo-inositol hexakisphosphate i.e. myo-inositol 1,2,3,4,5-pentakisphosphate or myo-inositol 1,3,4,5,6-pentakisphosphate has not been identified 3.1.3.26 4-phytase biotechnology production of phytase in laboratory-scale fermenter. Maintaining an acidic environment (pH 1.5-1.8) in the fermentation broth after the initial buildup of cell mass along with proper fragmentation of filamentous fungi results in significant improvement in phytase productivity 3.1.3.26 4-phytase biotechnology high levels of stable phytase is expressed in the culture medium of transgenic Medicago truncatula cell suspension cultures 3.1.3.26 4-phytase biotechnology in vitro digestibility tests show recombinantly expressed phytase is at least as efficient as commercial phytase for hydrolyzing phytate in corn-based animal feed and is therefore suitable sources of phytase supplement 3.1.3.26 4-phytase biotechnology phytase from Bacillus subtilis (168phyA) is constitutively expressed in tobacco and Arabidopsis to generate transgenic plants capable of utilizing exogenous phytate. In tobacco, phytase activities in transgenic leaf and root extracts are seven to eight times higher than those in wild-type extracts; whereas, the extracellular phytase activities of transgenic plants are enhanced by four to six times. Similar results are observed from the transgenic Arabidopsis. These results may offer a new perspective on mobilizing soil phytate into inorganic phosphate for plant uptake 3.1.3.26 4-phytase biotechnology phytase from Bacillus subtilis is introduced into the cytoplasm of tobacco cells that results in equilibrium shift of inositol biosynthesis pathway, thereby making more phosphate available for primary metabolism. The transgenic line exhibit phenotypic changes like increased flowering, lower seed IP6/IP5 ratio, and enhanced growth under phosphate starvation conditions compared to wild type 3.1.3.26 4-phytase biotechnology phytase is very suitable to be used in animal feed particularly in common carp feed because of its optimum pH with excellent thermal stability. Bacillus phytase supplementation of 300 U/kg can gain the same result as that of 1000 U/kg supplementation of acidic phytase and neutral phytase supplementation of 1000 U/kg can replace the inorganic phosphorus supplement. A combination of Bacillus phytases and other acidic phytases might induce a more effective hydrolysis of phytate in both the stomach and small intestine of animals in terms of the pH of the animal gastrointestinal tract 3.1.3.26 4-phytase biotechnology ten bean cultivars are evaluated for variability in phytate, phenolic, and mineral contents, phytase activity, and antioxidant properties to elucidate the relationship of these components. Multivariate data analysis performed on 22 components analyzed in this study using principal component analysis and cluster methods demonstrate that differences in phytase, antioxidant activity, mineral contents, and bioavailability are much larger within market class than among bean cultivars 3.1.3.26 4-phytase biotechnology Yersinia rohdei phytase is an attractive additive to animal feed 3.1.3.35 thymidylate 5'-phosphatase biotechnology production of thymidine by expression of enzyme in a Brevibacteruium helvolum mutant resistant to fluorouracil, hydroxyurea, trimethoprim 3.1.3.37 sedoheptulose-bisphosphatase biotechnology antisense transgenic plants, in mature, fully expanded leaves, enzyme activity is closely related with photosynthetic capacity, in youngest leaves, photosynthetic rates are close to or higher than those of wild type plants, decreased enzymic activity also leads to reduction in carbohydrate levels, particularly in starch 3.1.3.37 sedoheptulose-bisphosphatase biotechnology bifunctional fructose-1,6/seduheptulose-1,7-bisphosphatase expressed in Nicotiana tabacum, plants show enhanced photosynthetic efficiency and growth characteristics as well as higher photosynthetic CO2 fixation and more final dry matter 3.1.3.62 multiple inositol-polyphosphate phosphatase biotechnology recombinant avian MINPP (0.7 micromol/mg protein/min) is the most active phytase found to date in any animal cell 3.1.4.1 phosphodiesterase I biotechnology study on substrate specificity and structural requirements using synthetic oligonucleotide derivatives suitable for labeling of nucleic acids 3.1.4.1 phosphodiesterase I biotechnology use of enzyme for direct detection of certain tandem DNA damage 3.1.4.2 glycerophosphocholine phosphodiesterase biotechnology efficient selection system for the identification of phosphotriesterase mutants with enhanced catalytic properties for selected organophosphate substrates 3.1.4.4 phospholipase D biotechnology use of enzyme for production of the alkylphosphate ester with anticancer activity, octadecylphospho-L-serine 3.1.4.4 phospholipase D biotechnology the feature of the enzyme, to be highly active in moderate pH and temperature, makes PLD684 easy to handle and thus substantiate its potential biocatalytic application in the industrial scale 3.1.4.41 sphingomyelin phosphodiesterase D biotechnology use of enzyme as probe of membrane sphingomyelin 3.1.4.45 N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase biotechnology the uncovering enzyme is generated as soluble enzyme and so can be used to conduct the in vitro phosphorylation of purified recombinant lysosomal enzymes 3.1.8.1 aryldialkylphosphatase biotechnology chemo-enzymatic procedure for chiral synthesis of soman analogues, use of mutants to reverse stereoselectivity 3.1.8.1 aryldialkylphosphatase biotechnology future biotechnological developments of PTE as a detoxifying enzyme 3.1.21.3 type I site-specific deoxyribonuclease biotechnology potential uses for EcoR124I as a nanoactuator within a biosensor in the field of bionanotechnology. It may be used as a molecular dynamo that can measure events from single molecules of DNA, providing a highly sensitive biosensor for detecting events that interrupt translocation events 3.1.21.4 type II site-specific deoxyribonuclease biotechnology evolvement of mutant enzymes with altered DNA cleavage specificities by application of an in vivo positive and negative selection system that applies evolutionary pressure either to favor the cleavage of a desired target sequence or to disfavor the cleavage of a nontarget sequence 3.1.21.4 type II site-specific deoxyribonuclease biotechnology generation of cleavage specificities of restriction endonucleases by swapping putative target recognition domains between the type IIB enzymes AloI, PpiI from Pseudomonas putida, and TstI from Thermus scotoductus. Individual target recognition domains recognize distinct parts of the bipartite DNA targets of these enzymes and are interchangeable. Engineering of a functional type IIB restriction endonuclease having previously undescribed DNA specificity and application in generation of type II enzymes with predetermined specificity 3.1.21.7 deoxyribonuclease V biotechnology method of linear amplification of DNA, allows 100fold amplification of target molecules 3.1.26.5 ribonuclease P biotechnology RNA-mediated RNA cleavage events are being increasingly exploited to disrupt RNA function, an important objective in post-genomic biology. RNase P, a ribonucleoprotein enzyme that catalyzes the removal of 5'-leaders from precursor tRNAs, has previously been utilized for sequence-specific cleavage of cellular RNAs 3.1.26.5 ribonuclease P biotechnology RNase P of Escherichia coli contains a catalytic RNA subunit (M1 RNA) that can be engineered to cleave tRNA-like substrates and other target RNAs, including specific mRNAs. The enzyme can be expressed in infected human U373MG cells or fibroblasts, or in murine PA317 cells, and therein be used for inhibition of targeting and cleavage of host RNA by Human cytomegalovirus enzyme, HCMV strain AD169, through blocking substrate mRNA expression, methods of using engineered RNase P catalytic RNA for in vitro and in vivo in trans-cleavage of target viral mRNA, overview. customized M1GS RNA and full-length RNase P are effective in cleaving both viral and cellular mRNAs and blocking their expression in cultured cells 3.1.31.1 micrococcal nuclease biotechnology co-expression of Staphylococcal nuclease in Escherichia coli to reduce the viscosity of the bioprocess feedstock through auto-hydrolysis of nucleic acids, viscosity is an important physical property of the process stream and a significant factor in the optimization of various downstream processing unit operations including cell disruption, clarification, filtration, and chromatography 3.2.1.1 alpha-amylase biotechnology the thermotolerant, glucose tolerant maltooligosaccharide-forming alpha-amylase is potent for biotechnological application 3.2.1.1 alpha-amylase biotechnology coexpressed in a construct in Corynebacterium glutamicum, combined with Escherichia coli K-12 lysine decarboxylase for an one-step production of cadaverine, from soluble starch and the yield of cadaverine is 23.4 mM after 21 h. Construction of the Escherichia coli-Corynebacterium glutamicum shuttle vector, producing lysine decarboxylase under the control of the high constitutive expression promotor, and transformed this vector into Corynebaterium glutamicum. The engineered Corynebacterium glutamicum expresses both lysine decarboxlase and alpha-amylase, which retains their activity 3.2.1.1 alpha-amylase biotechnology improvement of the thermal stability of alpha-amylase by combinatorial coevolving-site saturation mutagenesis (CCSM), in which the functionally correlated variation sites of proteins are chosen as the hotspot sites to construct focused mutant libraries. Method leads to identification of beneficial mutation sites, and enhances the thermal stability of wild-type alpha-amylase Amy7C by 8°C 3.2.1.3 glucan 1,4-alpha-glucosidase biotechnology immobilization of the enzyme on alginate beads for large-scale hydrolysis of starch in a fluidized bed of enzyme-alginate particles, method development, comparison to packed and batch mode, overview 3.2.1.3 glucan 1,4-alpha-glucosidase biotechnology improvement of the yeast enzyme for starch degradation in biotechnological applications by introduction of the starch binding domain from the glucoamylase of Aspergillus niger, chimeric enzyme in Saccharomyces cerevisiae strain Y428, overview 3.2.1.3 glucan 1,4-alpha-glucosidase biotechnology the enzyme is potentially useful in improvement of industrial starch processing by eliminating the need to adjust both pH and temperature 3.2.1.3 glucan 1,4-alpha-glucosidase biotechnology the enzyme might be useful in biotechnological processes 3.2.1.3 glucan 1,4-alpha-glucosidase biotechnology enzyme immobilization on polyacrylamide gel results in an enzyme with increases thermostability for use in biocatalysis 3.2.1.3 glucan 1,4-alpha-glucosidase biotechnology the enzyme from commercial preparation is immobilized by sorption on a carbon support Sibunit, starch and dextrin hydrolysis kinetic parameters of glucoamylase, including the rate constant of thermal inactivation, show that immobilization of the enzyme results in a 1000fold increase in enzyme stability in comparison to the dissolved enzyme, presence of the dextrin substrate has a stabilizing effect, increase in dextrin concentration to 53% increases the thermostability of the immobilized enzyme, the immobilized-enzyme biocatalyst for starch saccharification has a high operational stability, half-inactivation time at 60°C exceeds 30 days 3.2.1.3 glucan 1,4-alpha-glucosidase biotechnology the enzyme immobilized on foamed glass covered with the catalytic filament carbon layer is highly active and stable, the effect of the carbon layer synthesized on the surface of aluminum oxide on the properties of biocatalysts shows that the glucoamylase adsorbed on the carbon-containing mesoporous ny-aluminum oxide exhibits a greater activity than the glucoamylase adsorbed on the macroporous alpha-aluminum oxide, kinetics, overview 3.2.1.4 cellulase biotechnology Production of immobilized enzyme on Sepabeads EC-BU (hydrophobic interactions are the driving force for the adsorption of the enzyme to the carrier) 3.2.1.4 cellulase biotechnology considering its thermostable, alkali-stable, halostable and organic solvent-tolerant properties, the enzyme might be potentially useful for future applications in biotechnological processes 3.2.1.4 cellulase biotechnology recombination of the catalytic domains of three glycoside hydrolase family 48 bacterial cellulases (Cel48), i.e. Clostridium cellulolyticum CelF, Clostridium stercorarium CelY, and Clostridium thermocellum CelS, to create a diverse library of Cel48 enzymes with an average of 106 mutations from the closest native enzyme. The library is based on the Clostridium thermocellum CelS architecture, which consists of a 70-kDa catalytic domain connected to the organism's respective dockerin domain. Large variations in properties such as the functional temperature range, stability, and specific activity on crystalline cellulose are found. Functional status and stability are predictable from simple linear models of the sequence-property data. Recombined protein fragments contribute additively to these properties in a given chimera 3.2.1.6 endo-1,3(4)-beta-glucanase biotechnology construction of a transgenic yeast, Saccharomyces cerevisiae, expressing the enzyme, results in a food-grade yeast that has the potential to improve the brewing properties of beer 3.2.1.8 endo-1,4-beta-xylanase biotechnology increasingly used in a number of biotechnological processes, including bread-making, gluten-starch separation, improving nutritional properties of animal feed, and the bleaching of cellulose pulp in paper manufacturing 3.2.1.11 dextranase biotechnology bioconversion, modification of alternan 3.2.1.14 chitinase biotechnology the enzyme is a good candidate for application in the production of protoplasts of algal cells 3.2.1.14 chitinase biotechnology the enzyme is used for the biotechnological production of specific chitosan oligomers and for the characterization of chitosan polymers via enzymatic fingerprinting 3.2.1.20 alpha-glucosidase biotechnology expression of the enzyme in rice plants can transduce enhanced resistance against sheath blight pathogen Rhizoctonia solani by inactivation of the RS toxin 3.2.1.21 beta-glucosidase biotechnology immobilization of the enzyme on gelatin, overview, the very stable gelatin-immobilized enzyme can be used in continuous synthesis of beta-glucosides by transglucosylation 3.2.1.21 beta-glucosidase biotechnology the enzyme might be useful as biocatalyst in the synthesis of glyco-conjugates, overview 3.2.1.21 beta-glucosidase biotechnology production of isoflavone aglycones by the enzyme. Isoform BGL1 shows broad substrate specificity to various isoflavone glycosides, the residual ratio is reached to 6.2% of the total amount of isoflavone glycosides and the hydrolysis reaction is almost finished within 48 h 3.2.1.21 beta-glucosidase biotechnology the beta-glucosidase has a potential for biotechnological applications in the bioconversion of lignocellulosic materials 3.2.1.21 beta-glucosidase biotechnology the bifunctional beta-glucosidase/xylosidase can be used in simultaneous saccharification of cellulose and xylan into fermenantable glucose and xylose 3.2.1.22 alpha-galactosidase biotechnology Concavalin A-alpha-galactosidase complex entrapped in calcium alginate and crosslinked Concavalin A-alpha-galactosidase complex entrapped calcium alginate retain 74 and 61% activity, respectively. Crosslinked Concavalin A-alpha-galactosidase entrapped complex exhibit enhanced thermostability and show 62% of activity (38%) after 360 min at 65°C. Entrapped crosslinked Concavalin A-alpha-galactosidase complex preparation is superior in the continuous hydrolysis of oligosaccharides in soymilk by batch processes compared to the other entrapped preparations 3.2.1.23 beta-galactosidase biotechnology the enzyme is suitable for obtaining fermentable sugars from whey wastes 3.2.1.23 beta-galactosidase biotechnology single enzyme molecule assays performed on the native enzyme as well as recombinant enzyme with and without His-tag reveal significant differences in the average combined turnover numbers indicating that in vivo and in vitro produced enzymes are not identical and the presence of a C-terminal His-tag may alter the activity of beta-galactosidase 3.2.1.28 alpha,alpha-trehalase biotechnology immobilization of the crude enzyme on chitin resulting in stable, specific, and reusable reactors for application in other biotechnological processes, the immobilized enzyme shows no significant loss of activity being reused 10times and stored at 10°C, in 50 mM sodium maleate, pH 6.0, for 55 days 3.2.1.31 beta-glucuronidase biotechnology comparison of Escherichia coli and Staphylococcus sp. RLH1 beta-glucuronidase as gene fusion markers in plant transformation experiments. The Staphylococcus enzyme shows higher catalytic activity and increased accessible surface area of active site residues compared with the Escherichia coli protein 3.2.1.B34 Sulfolobus acidocaldarius beta-glycosidase biotechnology construction of a series of Sulfolobus-Escherichia coli shuttle vectors based on the small multicopy plasmid pRN1 from Sulfolobus islandicus. The shuttle vectors do not integrate into the genome and do not rearrange. They allow functional overexpression of genes, and the beta-glycosidase (lacS) gene of ulfolobus solfataricus could function as selectable marker in Suldfolobus solfataricus 3.2.1.35 hyaluronoglucosaminidase biotechnology a fluorescent substrate (FRET-HA) to quantitatively assess hyaluronidase activity is developed 3.2.1.35 hyaluronoglucosaminidase biotechnology novel products in hyaluronan digested by commercially available bovine testicular hyaluronidase are found which originate from a novel enzyme contaminat in the BTH 3.2.1.37 xylan 1,4-beta-xylosidase biotechnology coexpression of enzyme with Trichoderma reesei xylanase II in Saccharomyces cerevisiae allows for degradation of birchwood xylan to D-xylose 3.2.1.37 xylan 1,4-beta-xylosidase biotechnology use of strain for bioconversion of xylan-rich plant wastes to value-added products 3.2.1.37 xylan 1,4-beta-xylosidase biotechnology use of stable transgenic Medicago truncatula plants as an expression system for purification and characterization of proteins 3.2.1.37 xylan 1,4-beta-xylosidase biotechnology the bifunctional beta-glucosidase/xylosidase can be used in simultaneous saccharification of cellulose ans xylan into fermenantable glucose and xylose 3.2.1.37 xylan 1,4-beta-xylosidase biotechnology a method for bioconversion of 3-O-beta-D-xylopyranosyl-6-O-beta-D-glucopyranosyl-cycloastragenol into cycloastragenol is optimized. A green and efficient biotransformation method is established for 3-O-beta-D-xylopyranosyl-6-O-beta-D-glucopyranosyl-cycloastragenol using beta-glucosidase Dth3 and beta-xylosidase Xln-DT 3.2.1.37 xylan 1,4-beta-xylosidase biotechnology the beta-xylosidase when combined with xylanase shows positive effect on xylan hydrolysis. The higher amount of glucose and xylose generated from sugarcane bagasse hydrolysis using the commercial cocktail Multifect CL supplemented with beta-xylosidase demonstrates that this enzyme has potential to be used as a supplement for commercial cocktails to improve the yield of xylose and glucose release, and this is of great importance for the production of second generation ethanol 3.2.1.39 glucan endo-1,3-beta-D-glucosidase biotechnology enzyme inhibits growth of phytopathogen Scerotium rolfsii with 50’% effective dose value of 0.0027 mg/ml 3.2.1.39 glucan endo-1,3-beta-D-glucosidase biotechnology enzyme is able to damage cell-wall structures of phytopathogenic fungi Pythium aphanidermatum and Rhizoctonic solani Ag-4 3.2.1.39 glucan endo-1,3-beta-D-glucosidase biotechnology beta-1,3-glucanases may be useful in fungal transformations or other biotechnological applications where low-temperature cell wall disintegration is preferred 3.2.1.39 glucan endo-1,3-beta-D-glucosidase biotechnology BglS27 is a good candidate for utilization in biotechnological applications such as plant protection, feed, and food preservation 3.2.1.40 alpha-L-rhamnosidase biotechnology enzyme efficiently releases monoterpenols from an aroma precursor from muscat grape juice 3.2.1.40 alpha-L-rhamnosidase biotechnology potential use as a biocatalyst for diverse biotechnological applications 3.2.1.41 pullulanase biotechnology use of enzyme entrapped in calcium alginate beads for hydrolysis of starch 3.2.1.45 glucosylceramidase biotechnology the measurement of ABG activities in dry blood spots using a tandem mass spectrometry is suitable for high-throughput analysis of at-risk individuals and potentially for newborn screening for Gaucher disease 3.2.1.49 alpha-N-acetylgalactosaminidase biotechnology use of a modified alpha-N-acetylgalactosaminidase in the development of enzyme replacement therapy for Fabry disease 3.2.1.54 cyclomaltodextrinase biotechnology Archaeoglobus fulgidus utilizes an unusual pathway of starch degradation involving cyclodextrins as intermediates, extracellular cyclodextrins are transported into the cell and linearized via a CDase 3.2.1.54 cyclomaltodextrinase biotechnology chemical or molecular operones can be used in refolding of various aggregate-prone proteins of commercial and medical importance after overexpression in Escherichia coli 3.2.1.55 non-reducing end alpha-L-arabinofuranosidase biotechnology degradation 3.2.1.55 non-reducing end alpha-L-arabinofuranosidase biotechnology degradation of lignocellulose, hemicellulose and pectin 3.2.1.63 1,2-alpha-L-fucosidase biotechnology the purified alpha1,2-fucosidase and L-fucose dehydrogenase have sufficiently high activities in phosphate-buffered saline (pH 7.0) at 37 °C, making it possible to develop a one-pot method for the quantitative determination of 2'-fucosyllactose in fermentation samples. The application of this method is more convenient for quantifying 2'-fucosyllactose in a variety of samples that may be obtained from different phases of the biotechnological production of this oligosaccharide. The method is useful for simple and rapid screening of active variants during the development of any industrially important microbial strain producing 2'-fucosyllactose 3.2.1.70 glucan 1,6-alpha-glucosidase biotechnology isolation of a strain producing large amounts of enzyme and able to grow on starch, potential use of strain in shrimp feed production 3.2.1.74 glucan 1,4-beta-glucosidase biotechnology hydrolysis of lignocellulosic materials 3.2.1.75 glucan endo-1,6-beta-glucosidase biotechnology conditions for synthesis of enzyme in batch culture 3.2.1.75 glucan endo-1,6-beta-glucosidase biotechnology maximal overproduction of enzyme is achieved in buffered medium where pH-induced aspartyl proteases are absent or when nitrogen sources such as yeast extract are substrate for these proteases 3.2.1.75 glucan endo-1,6-beta-glucosidase biotechnology use of enzyme in analysis of yeast cell wall protein 3.2.1.78 mannan endo-1,4-beta-mannosidase biotechnology bio-bleaching for kraft pulp production 3.2.1.78 mannan endo-1,4-beta-mannosidase biotechnology optimization of mannanase gene for expression in Pichia pastoris by substitution of 258 nucleotides with their corresponding counterparts according to the codon usage in Pichia pastoris, which has no change on the beta-mannanase amino acid sequence. Compared to the activity of wild-type, the expression enzyme of the optimized beta-mannanase gene acquires approximately 35% more activity 3.2.1.84 glucan 1,3-alpha-glucosidase biotechnology transgenic plants might have improved resistance to fungal pathogens 3.2.1.91 cellulose 1,4-beta-cellobiosidase (non-reducing end) biotechnology fusion of the C-terminus of Caulobacter crescentus surface (S)-layer protein RsaA with the beta-1,4-glycanase Cex from the cellulolytic bacterium Cellulomonas fimi yielding a robust, catalytically active product, biocatalyst system evaluation 3.2.1.114 mannosyl-oligosaccharide 1,3-1,6-alpha-mannosidase biotechnology the Lec36 cell line will be useful for expressing therapeutic glycoproteins with hybrid-type glycans and as a sensitive host for detecting mutations in human MAN2A1 causing type II congenital dyserythropoietic anemia 3.2.1.120 oligoxyloglucan beta-glycosidase biotechnology use of fungal and plant enzyme during liquefaction of raw and blanched apple 3.2.1.120 oligoxyloglucan beta-glycosidase biotechnology method for rapidly identifying six of the most commonly found xyloglucan oligosaccharide units using purified enzyme 3.2.1.129 endo-alpha-sialidase biotechnology degradation of non-toxic modified polysialic acid hydrogel scaffold in neuro-regenerative tissue engineering (4.26 microgram enzyme + 39 mg hydrogel), in phosphate buffered saline (400 microl, pH 7.4), at 37°C, degradation speed 2-11 days depending on cross-linker amount (0.6, 0.8, 2 equivalents diepoxyoctane, no activity with 3 equivalents diepoxyoctane), hydrogel was coated with collagen I, poly-L-lysine/collagen I, or diluted matrigel for neurite formation in PC12 cells 3.2.1.129 endo-alpha-sialidase biotechnology degradation of non-toxic modified polysialic acid hydrogel scaffold in neuro-regenerative tissue engineering: no degradation in 12 days with 1 microg/ml active enzyme + 105 cubic mm hydrogel in phosphate buffered saline (pH 7.4), at room temperature, increase to 4 microg/ml at end of week 2 initiates degradation, total degradation after 4 weeks, hydrogel was coated with poly-L-lysine, poly-L-ornithine-laminin or collagen for neurite formation in neonatal and adult rat Schwann cells, neural rat stem cells, and dorsal root ganglionic cells from rats 3.2.1.132 chitosanase biotechnology chitosanase is used as a tool for the biotechnological transformation of chitosan 3.2.1.133 glucan 1,4-alpha-maltohydrolase biotechnology natural design of the transglycosylation activtiy of the enzyme at high concentrations of acceptor sugar molecules may now be altered for biotechnological applications to produce branched oligosaccharides with higher efficiency 3.2.1.133 glucan 1,4-alpha-maltohydrolase biotechnology producing enzymes with modified substrate specificity, hydrolysis, and transglycosilation activities, as a way to produce specific functional carbohydrate materials 3.2.1.133 glucan 1,4-alpha-maltohydrolase biotechnology maltogenic alpha-amylase from Bacillus stearothermophilus immobilized onto poly(urethane urea) microparticles shows high storage and thermal stability and reusability for starch hydrolysis. It has a great potential for biotechnology 3.2.1.135 neopullulanase biotechnology thermostable debranching enzyme for biotechnology 3.2.1.147 thioglucosidase biotechnology Aspergillus sp. NR463U4 maintains constant myrosinase production for 8 months. High production and prolonged stability of myrosinase demonstrates that this mutant could be a candidate for industrial application 3.2.1.156 oligosaccharide reducing-end xylanase biotechnology a novel method for producing a glycosynthase from an inverting glycoside hydrolase by mutating a residue that holds the nucleophilic water molecule (Y198) with the general base residue while keeping the general base residue intact 3.2.1.168 hesperidin 6-O-alpha-L-rhamnosyl-beta-D-glucosidase biotechnology bulk biotransformations, the hydrolytic and transglycosidic activity of alpha-rhamnosyl-beta-glucosidase has potential use for industrial processing of plant-based food and the products of the transglycosylation products could play a role as starting materials for the development of new drugs. The immobilization allows the kinetic control of the process 3.2.1.176 cellulose 1,4-beta-cellobiosidase (reducing end) biotechnology recombination of the catalytic domains of three glycoside hydrolase family 48 bacterial cellulases (Cel48), i.e. Clostridium cellulolyticum CelF, Clostridium stercorarium CelY, and Clostridium thermocellum CelS, to create a diverse library of Cel48 enzymes with an average of 106 mutations from the closest native enzyme. The library is based on the Clostridium thermocellum CelS architecture, which consists of a 70-kDa catalytic domain connected to the organism's respective dockerin domain. Large variations in properties such as the functional temperature range, stability, and specific activity on crystalline cellulose are found. Functional status and stability are predictable from simple linear models of the sequence-property data. Recombined protein fragments contribute additively to these properties in a given chimera 3.2.1.176 cellulose 1,4-beta-cellobiosidase (reducing end) biotechnology recombination of the catalytic domains of three glycoside hydrolase family 48 bacterial cellulases (Cel48), i.e. Clostridium cellulolyticum CelF, Clostridium stercorarium CelY, and Clostridium thermocellum CelS, to create a diverse library of Cel48 enzymes with an average of 106 mutations from the closest native enzyme. The library is based on the Clostridium thermocellum CelS architecture, which consists of a 70-kDa catalytic domain connected to the organism's respective dockerin domain. Two of the most stabilizing blocks are predicted to be from the parent CelS at blocks located in the C-terminus of the catalytic domain, close to where the dockerin attaches. Two of the highly stable chimeras also hydrolyze more cellulose than the most active parental enzyme 3.2.1.177 alpha-D-xyloside xylohydrolase biotechnology saccharification of cell wall polymers 3.2.1.183 UDP-N-acetylglucosamine 2-epimerase (hydrolysing) biotechnology a GNE-deficient HEK293 cell line proves its potential for the production of glycoproteins with modified sialylation, gaining therapeutic and diagnostic glycoproteins, and efficient application of metabolic oligosaccharide engineering 3.2.2.22 rRNA N-glycosylase biotechnology combined expression of a barley class II chitinase and type I ribosome inactivating protein in transgenic Brassica juncea provides protection against Alternaria brassicae 3.2.2.22 rRNA N-glycosylase biotechnology ricin is a prototype for the construction of chimeric molecules, called immunotoxins, based on the structure of the A-B toxins. An application of the ricin B as a carrier is the fusion and expression with different viral antigens used for vaccination therapies. A fusion protein combining the genes for endotoxin of Bacillus thuringiensis with the ricin B chain, and transgenic rice and maize plants expressing the fusion protein are more toxic to insects than plants containing the toxin gene alone 3.2.2.22 rRNA N-glycosylase biotechnology saporin conjugated to oxytocin is an effective neurotoxin for in vivo elimination of cells that express oxytocin receptors and is potentially useful to analyze central nervous system mechanisms that involve the action of oxytocin on food intake and other physiological processes 3.2.2.22 rRNA N-glycosylase biotechnology saporin is fused to ubiquitin to allow a rapid degradation of the toxin via the ubiquitin-proteasome system in non-target cells. Saporin is the preferred toxin for constructing anti-neuronal immunotoxins and neuropeptide-toxin conjugates. The saporin gene is also used as a tool for suicide gene therapy 3.4.11.5 prolyl aminopeptidase biotechnology potential use of the enzyme in the transgenic breeding to improve heavy metal resistance in crop species 3.4.11.24 aminopeptidase S biotechnology enzyme is attractive for diverse applications, e.g. the processing of recombinant DNA proteins and fusion protein production due to its heat stability, high activity, and small size 3.4.11.24 aminopeptidase S biotechnology the enzyme is useful in many biotechnological applications e.g. in processing of recombinant DNA, proteins, and fusion protein products 3.4.13.9 Xaa-Pro dipeptidase biotechnology the enzyme is of particular interest because it can be used in many biotechnological applications 3.4.16.6 carboxypeptidase D biotechnology Kex-1-C611 is proposed as a convenient biotechnological reagent for the cleavage of fusion proteins 3.4.17.1 carboxypeptidase A biotechnology multi-block, surfactant copolymers are suitable for applications in which refolding of denaturated or misfolded proteins and suppression of aggregation are important objects 3.4.17.1 carboxypeptidase A biotechnology a strategy is shown for the expression of MeCPA-His6PR in the cytosol of Escherichia coli and a relatively simple procedure to purify it to homogeneity. The bacterial system yields about 0.5 mg of pure enzyme per liter of cell culture and is more convenient and less expensive than is the production of MeCPA in insect cells 3.4.17.10 carboxypeptidase E biotechnology carboxypeptidase E is used in an in vitro system for the production of bioactive peptides 3.4.21.1 chymotrypsin biotechnology dependence of activity on pH and temperature makes chymotrypsin II a biotechnological alternative for food processing when low temperatures are needed like in fish ripening, fish sauce production, fish protein hydrolysate production and other emerging processes. This enzyme together with other proteases from sardine viscera may aid in the enzymatic treatment of stick-water, in which a reduction in viscosity is required for further processing of effluent 3.4.21.B3 duodenase biotechnology comparative evaluation of specificity of different proteases can give rise to methods employing new proteases in biotechnology 3.4.21.4 trypsin biotechnology enzyme molecules are associated with the outer protein layer of rotavirus virions propagated in cell culture medium containing the enzyme. Enzyme is present only in triple-layer particles, not in double-layer particles. Enzyme associated with virions is inactive, activity is recovered only when the outer capsid is solubilized. Incorporation of trypsin into rotavirus particles may enhance its infectivity 3.4.21.4 trypsin biotechnology recombinant Streptomyces erythraeus trypsin is fundamental to the use for proteomics applications 3.4.21.5 thrombin biotechnology fibrin is a biopolymer that has been used in a variety of biomaterial, cell delivery and tissue engineering applications, the enzyme thrombin catalyzes the formation of fibrin microfibrils, which form a three-dimensional mesh in which cells can be directly embedded at the time of gel formation, method development, overview 3.4.21.5 thrombin biotechnology usage of human thrombin in a fibrin glue, method development, thrombin initiates clotting and cross-linking of fibrin from cryoprecipitate to produce an entirely autologous fibrin glue, overview 3.4.21.9 enteropeptidase biotechnology EK is immobilised on hexamethylamino Sepabeads or on amino-modified paramagnetic microspheres. 50% of activity remains after immobilisation 3.4.21.9 enteropeptidase biotechnology purification of 6.8 mg bioactive enzyme from 1l fermentation broth 3.4.21.9 enteropeptidase biotechnology study presents a simple and cost-effective procedure for a large-scale production 3.4.21.9 enteropeptidase biotechnology enteropeptidase is a serine protease used in different biotechnological applications. For many applications the smaller light chain can be used to avoid the expression of the rather large holoenzyme 3.4.21.21 coagulation factor VIIa biotechnology to overcome the defect for high-level expression of the functional recombinant coagulation FVII in Sf9 cell this method involves simultaneous expression of both human c-carboxylase and human FVII genes in the host leading to a high-level expression of functional recombinant factor VII 3.4.21.35 tissue kallikrein biotechnology the chicken oviduct-specific transient expression system can produce relatively high level of authentic recombinant enzyme with a potential for further development for therapeutic use 3.4.21.B48 kumamolysin biotechnology attractive candidate for industrial-scale biopeptide production under thermoacidophilic conditions 3.4.21.50 lysyl endopeptidase biotechnology Lys C is used for protein digestion to yield at least two labeling sites with a mass difference of 4 Da. Dimethyl multiplex labelled peptides are analysed by MALDI-TOF. This technique enables investigating time course or dosage dependence of protein expressions. 3.4.21.50 lysyl endopeptidase biotechnology Lys C is used to incorporate two 18O atoms into the carboxyl termini of peptides for proteomics 3.4.21.53 Endopeptidase La biotechnology protein synthesis in Escherichia coli, enzyme and protease Clp participate in the physiological disintegration of cytoplasmic inclusion bodies, their absence minimizing the protein removal up to 40%. Clp takes the major and enzyme a minor role in processing of aggregation-prone proteins and also of polypeptides physiologically released from inclusion bodies 3.4.21.62 Subtilisin biotechnology subtilisin-30 kDa gamma-glutamyl transpeptidase complex exhibits better catalytic properties and can be exploited in various biotechnological applications 3.4.21.62 Subtilisin biotechnology subtilisin-like serine protease has a great advantage over other proteases in high resistance to heat, denaturants, detergents and chelating agents and therefore has great potential for application in biotechnology fields 3.4.21.64 peptidase K biotechnology proteinase K is successfully applied for the activation of purified pro-recombinant transglutaminase either as free or immobilized enzyme and the free enzyme is also applicable directly in the crude cell extract of Escherichia coli. Proteinase K enables a simple two-step activation/purification procedure resulting in protease-free and almost pure transglutaminase preparations 3.4.21.64 peptidase K biotechnology DNase I and proteinase K eliminate DNA from injured or dead bacteria but not from living bacteria in microbial reference systems and natural drinking water biofilms for subsequent molecular biology analyses, method evaluation, overview 3.4.21.66 Thermitase biotechnology computational procedure OptGraft for placing a novel binding pocket onto a protein structure so as its geometry is minimally perturbed. Transfer of a calcium-binding pocket from thermitase protein, PDB entry 1thm, into the first domain of rat CD2 protein, PDB entry 1hng results in new proteins that all exhibit high affinities for terbium and can selectively bind calcium over magnesium 3.4.21.79 granzyme B biotechnology engineering of mutant enzyme suitable for cleavage of fusion proteins 3.4.21.96 Lactocepin biotechnology conversion of lactocepin substrate binding regions by allele exchange can effectively alter lactocepin specificity in industrial strains of Lactococcus lactis without significantly affecting other important strain properties. The methodology can be used to alter lactocepin specificity in commercial starter cultures with a propensity for bitter flavour defect 3.4.21.106 hepsin biotechnology the aim of this study is to establish a cell line that directly secrets rFVIIa into cell culture medium: Factor VII and hepsin cDNAs are isolated from HepG2 cell line and cloned into pcDNA3-1 vector. The constructs are co-trasfected to CHO cell line. A cell line that permanently expresses recombinant factor VII (rFVII) and hepsin is established. FVIIa protein is secreted to medium of CHO cells co-transfected with pcNDA3-1-FVII and pcNDA3-1-hepsin. A three- to fourfold decrease in clotting time is observed when human FVII-depleted plasma is used in combination with human thromboplastin in the presence of rFVII, confirming the biological activity of rFVII. A cell line is established expressing FVIIa using genetic engineering methods 3.4.21.107 peptidase Do biotechnology htrA mutants show improved expression of envelope-associated proteins 3.4.21.111 aqualysin 1 biotechnology a maltose biding protein (MBP)-fused proaqualysin I expression plasmid is developed in which MBP is attached to the N-terminus of proaqualysin I. MBP appears effectively to suppress the folding-promoting activity of the N-terminal propeptide when the bacteria are grown at 30°C, leading to a massive accumulation of fusion aqualysin I precursor. The precursor is converted efficiently to mature aqualysin I by heat treatment at 70°C. By analyzing the product it is confirmed that aqualysin I is initially expressed as a whole fusion protein and then processed autocatalytically 3.4.21.118 kallikrein 8 biotechnology enzyme might contribute to the remodeling of extracellular components after decidualization 3.4.22.1 cathepsin B biotechnology inhibition of cathepsin B greatly improves the developmental competence of bovine oocytes and increases the number of high-quality embryos 3.4.22.2 papain biotechnology immobilization of papain and dehydration by n-propanol in low-water media at pH 6.4 and 25°C in 150 mM sodium phosphate buffer, stability is increased by solid state cysteine, overview 3.4.22.2 papain biotechnology papain immobilization on magnetic composite microspheres at pH 8.0 and 30°C, quantification and kinetics, overview, the immobilized enzyme exhibits better environmentally adaptability and reusability than the soluble enzyme 3.4.22.2 papain biotechnology the enzyme plays a key role in biotechnology and has a range of important applications in cell isolation, leather, cosmetic, textiles, detergents, food, and pharmaceutical industries 3.4.22.2 papain biotechnology evaluation of toxic and mutagenic potential of papain and its potential antioxidant activity against induced-H2O2 oxidative stress in Escherichia coli strains by cytotoxicity assay, growth inhibition test, WP2-mutoxitest and plasmid-DNA treatment, and agarose gel electrophoresis. Papain exhibits negative results for all tests 3.4.22.2 papain biotechnology immobilisation of papain on gold nanorods enhances enzyme stability and efficiency, opening new opportunities for biotechnological applications 3.4.22.14 actinidain biotechnology actinidin might be utilized to eliminate the milk fat globule membranes (MFGM) protein residues from cream and its derivatives 3.4.22.25 glycyl endopeptidase biotechnology plant protease proregions have a potential as regulators of cysteine proteinases in biotechnological systems and to target proteases of pests 3.4.22.34 Legumain biotechnology studies on vacuolar targeting, targeting motif of legumain as a potential tool for plant engineering analyzed 3.4.22.37 gingipain R biotechnology a naive camel nanobody library is constructed and phage display is used to select one nanobody toward RgpB with picomolar affinity. The nanobody is highly specific for RgpB given that it does not bind to the homologous gingipain HRgpA, indicating the presence of a binding epitope within the immunoglobulin-like domain of RgpB. RgpB can be used as a specific biomarker for Porphyromonas gingivalis infection 3.4.22.50 V-cath endopeptidase biotechnology this CPD-BmMNPV bacmid system provides rapid protein production in silkworms and can be used for the production of recombinant eukaryotic proteins without proteolytic degradation 3.4.22.68 Ulp1 peptidase biotechnology substrate-trapping Ulp1(3)(C580S) interacts robustly with human SUMO1, SUMO2 and SUMO2 chains, making it a potentially useful tool for the analysis and purification of SUMO-modified proteins 3.4.22.70 sortase A biotechnology the bacterial transpeptidase sortase A is a well-established tool in protein chemistry and catalyzes the chemoselective ligation of peptides and proteins 3.4.23.49 omptin biotechnology engineering of enzyme variants with targeted, high substrate specificity 3.4.23.49 omptin biotechnology utilization of outer-membrane endoprotease OmpT variants as processing enzymes for production of peptides from designer fusion proteins, e.g. useful in motilin production, overview 3.4.23.49 omptin biotechnology OmpT protease could be a possible factor responsible for reducing the expression of GFP at 37°C for wildtype GFP clone in K12 hosts like DH5alpha, JM109, LE 392 3.4.24.B24 L-alanine-D-glutamate endopeptidase biotechnology bacteriophage peptidoglycan-hydrolyzing enzymes have found several applications in biotechnology and medicine, e.g. for surface-decontamination purposes, for biopreservation of food and feed and as potential topical antimicrobials 3.4.24.26 pseudolysin biotechnology A2 protease is usable for shrimp waste deproteinization in the process of chitin preparation, percent of protein removal after 3 h hydrolysis at 40°C with an enzyme/substrate ratio of 5 U/mg protein is about 75%. A2 proteolytic preparation also demonstrates powerful depilating capabilities of hair removal from bovine skin 3.4.24.32 beta-Lytic metalloendopeptidase biotechnology extracellular digestion of biofilms by Lysobacter gummosus depends on multiple bacteriolytic and proteolytic enzymes, which can be exploited for biofilm control, a following gel filtration step highly reduces enzyme activity 3.4.24.34 neutrophil collagenase biotechnology human MMP-8 is coupled to epoxy activated silica matrix in an immobilized enzyme reactor which is used for the online screening of known MMP-8 inhibitors in zonal chromatography and inhibition experiments 3.4.25.1 proteasome endopeptidase complex biotechnology the 20S proteasome could be an as yet not identified bottleneck in the expression and secretion of some heterologous proteins by Streptomyces lividans. Optimization of the production of specific heterologous proteins is likely to require engineered Streptomyces lividans strains, in which the proteasome has been removed 3.5.1.1 asparaginase biotechnology development of a MCE method (micellar electrokinetic electrophoresis) that is sufficiently sensitive and selective for the separation of amino amides and determination of enzyme kinetic constants of L-Asnase 3.5.1.1 asparaginase biotechnology statistically based experimental design to maximize the production of glutaminase-free L-asparaginase. The individual optimum levels of initial pH of the medium, temperature, rpm of shaking incubator, and inoculum size are found to be 6.90, 29.8°C, 157 rpm, and 2.61% (v/v), respectively, for the production of L-asparaginase. After physical process parameters optimization, the production and productivity of L-asparaginase is enhanced by 26.39% (specific activity) and 10.19%, respectively. Maximization of L-asparaginase production is achieved at 12 h under optimal levels of physical process parameters in shake flask level 3.5.1.3 omega-amidase biotechnology development of a method to synthesize 2-oxoglutaramate which is not commercially available and development of an enzyme assay method to measure the hydrolysis of 2-oxoglutaramate and 2-oxosuccinamate in a 96-well plate format 3.5.1.4 amidase biotechnology the enzyme is useful in biocatalytic nitrile hydrolysis in a system together with nitrile hydratase, EC 4.2.1.84 3.5.1.5 urease biotechnology immobilization of urease on arylamine glass beads results in improved thermal, storage and operational stability. Because of inertness of support and stability of immobilized urease, the preparation can find applications in artificial kidney and urea estimation in biological fluids 3.5.1.5 urease biotechnology in food industry immobilisation of acid urease on an inert carrier has the potential advantages of significant cost savings, improved stability or resistance to shear or inhibitory compound inactivation. Purified acid urease preparation is covalently immobilised onto biocompatible porous chitosan beads of different size. The kinetics of urea degradation in a model wine solution using this biocatalyst is of the pseudofirst order with respect to the urea concentration in the liquid bulk, the apparent pseudo-first order kinetic rate constant ranging from about two thirds to one fifth of that pertaining to free acid urease 3.5.1.5 urease biotechnology soybean (Glycine max) urease is immobilized on alginate and chitosan beads and shows improved stability. This could have a potential role in haemodialysis machines 3.5.1.70 aculeacin-A deacylase biotechnology recombinant enzyme covalently immobilized onto several epoxy-activated supports in order to obtain a robust biocatalyst to be used in industrial bioreactors. The best biocatalyst is obtained by attaching the enzyme on Sepabeads EC-EP5 3.5.1.77 N-carbamoyl-D-amino-acid hydrolase biotechnology immobilization of the evolved carbamoylase offers a promising way for the efficient production of D-hydroxyphenylglycine from D-L-hydroxyphenylhydantoin 3.5.1.77 N-carbamoyl-D-amino-acid hydrolase biotechnology the isolated DCase enzyme, with improved thermostability and solubility, is expected to be used in the development of a fully enzymatic process for the industrial production of D-amino acids 3.5.1.81 N-Acyl-D-amino-acid deacylase biotechnology D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 is used for the biotechnological production of D-amino acid from the racemic mixture of N-acyl-DL-amino acids 3.5.1.87 N-carbamoyl-L-amino-acid hydrolase biotechnology production of optically pure L-amino acids by an enzymatic method named hydantoinase process 3.5.1.88 peptide deformylase biotechnology to avoid incomplete deformylation for proteins overexressed in Escherichia coli 3.5.1.88 peptide deformylase biotechnology peptide deformylase is a target protein for developing antibacterial drugs against Xanthomonas oryzae pv. oryzae 3.5.1.93 glutaryl-7-aminocephalosporanic-acid acylase biotechnology a biological and colometric method is evaluated for determination of cephalosporin acylase product in bacteria 3.5.1.93 glutaryl-7-aminocephalosporanic-acid acylase biotechnology with an aim to increase the yield of 7-aminocephalosporanic acid production, a stepwise strategy, statistical medium is applied for optimizing the medium composition for the production of CPC acylase 3.5.1.97 acyl-homoserine-lactone acylase biotechnology the enzyme can be used in quenching quorum sensing 3.5.1.97 acyl-homoserine-lactone acylase biotechnology the enzyme is useful as a biosensor 3.5.4.1 cytosine deaminase biotechnology a chitosan-entrapped cytosine deaminase nanocomposite is developped. Sustained release of cytosine deaminase from the nanocomposite up to one week depicts its potential implication in prodrug inducted enzyme therapy 3.5.4.6 AMP deaminase biotechnology multicopy integrants of crt genes and co-expression of AMP deaminase improve lycopene production in Yarrowia lipolytica. It is possible to make use of the obtained strains to meet the industrial demand of lycopene production on the basis of further genetic and process optimization 3.5.4.16 GTP cyclohydrolase I biotechnology construction of transgenic tomato plants expressing GCHI for engineering of the peteridine branch of folate synthesis in Lycopersicon esculentum by folate biofortification, overview 3.5.4.16 GTP cyclohydrolase I biotechnology creation of a phenylalanine sink by transgenic overexpression of phenylalnine hydroxylase and GTP cyclohydrolase I in the skin to reduce high phenylalanine concentrations in the blood in phenylketonuria metabolic disease 3.5.4.23 blasticidin-S deaminase biotechnology the enzyme is a selection marker for genetic transformation of the diatom Phaeodactylum tricornutum. An easy handle system of antibiotic selection with blasticidin-S and the resistance gene bsr in Phaeodactylum tricornutum is reported. Blasticidin-S works by blocking protein translation and therefore it is not expected to affect the genomic DNA of transformed cells. Especially for reverse genetics studies via genome editing, when phenotypes should be determined based on cell-linages with as less off-target genetic changes as possible, utilization of this antibiotic is a major improvement. Additionally, blasticidin-S selection can be used in combination with nourseothricin- and potentially also with Zeocin-resistant cells. This extension of the genetic toolbox can be useful for further molecular characterization or biotechnological application of the model diatom Phaeodactylum tricornutum and possibly for other species of diatoms 3.5.5.5 Arylacetonitrilase biotechnology preparation of optically pure carboxylic acids 3.5.5.7 Aliphatic nitrilase biotechnology enzyme can be recombinantly produced in high yield and at mild reaction conditions, the robust enzyme can be a suitable biocatalyst for industrial applications 3.7.1.2 fumarylacetoacetase biotechnology participation in the synthesis of vitamin E and other tocopherols 3.8.1.3 haloacetate dehalogenase biotechnology since heat-stable dehalogenases can be used for detoxification of halogenated aliphatic compounds, PH0459 is a useful target for biotechnological research 3.8.1.5 haloalkane dehalogenase biotechnology enzyme, covalently immobilized on a polyethylenimine impregnated gamma-alumina support with an optimal loading of 70-75 mg/g and a maximal loading of 156 mg/g, retains more than 40% of its maximal activity, unaltered pH dependency compared to the native enzyme, thermostability and resistance towards inactivation by organic solvents of the immobilized enzyme are improved by an order of magnitude 3.8.1.5 haloalkane dehalogenase biotechnology haloalkane hydrolysis by lyophilized cells in solid/gas biofilter and in the aqueous phase. Comparison of substrates 3.8.1.5 haloalkane dehalogenase biotechnology hydrolysis of haloalkanes by lyophilized cells in solid-gas biofilter and aqueous phase. For both systems, pH 9.0 and 40°C are the best conditions. In the aqueous phase, cells are less sensitive to variation in pH -value than in gas phase 3.8.1.5 haloalkane dehalogenase biotechnology hydrolysis of haloalkanes by lyophilized cells in solid-gas biofilter, analysis of reaction with 1-chlorobutane. Activity and stability of cells depends on the quantitiy of HCl produced. Triethylamine is used as a volatile buffer that controls the local pH-value and the dehalogenization state of enzyme. Cells broken by lysozyme are more stable than intact cells. Initial reaction rate of 4.5 micromol per min and mg of cell is observed 3.8.1.5 haloalkane dehalogenase biotechnology hydrolytic dehalogenation of 1-chlorobutane in a non-conventional gas phase system under a controlled water thermodynamic activity and in aqueous phase. Comparison of Rhodococcus erythropolis and Xanthobacter autotrophicus 3.8.1.5 haloalkane dehalogenase biotechnology hydrolytic dehalogenation of 1-chlorobutane in a non-conventional gas phase system under a controlled water thermodynamic activity and in aqueous phase. Maximal transformation capacity of 1.4 g of 1-chlorobutane per day with 1 g of Xanthobacter autotrophicus lyophilized cells. Comparison of Rhodococcus erythropolis and Xanthobacter autotrophicus 3.8.1.8 atrazine chlorohydrolase biotechnology bioremediation 3.13.1.4 3-sulfinopropanoyl-CoA desulfinase biotechnology the enzyme is part of the 3,3'-dithiopropionate degradation pathway. The elucidation of this pathway and the identification of the genes could provide an strategy to engineer strains suitable for biotechnological production of polythioesters 4.1.1.1 pyruvate decarboxylase biotechnology the reaction specificity of acetolactate synthase from Thermus thermophilus can be redirected to catalyze acetaldehyde formation to develop a thermophilic pyruvate decarboxylase. Quadruple mutant Y35N/K139R/V172A/H474R shows 3.1fold higher acetaldehyde-forming activity than the wild-type mainly because of H474R amino acid substitution, which likely generates two new hydrogen bonds near the thiamine diphosphate-binding site 4.1.1.2 oxalate decarboxylase biotechnology determination of oxalic acid in food and biological samples like urine, plasma, serum, wort and beer 4.1.1.7 benzoylformate decarboxylase biotechnology Production of immobilized enzyme on Sepabeads EC-EA by C-C coupling 4.1.1.23 orotidine-5'-phosphate decarboxylase biotechnology to develop a marker recycling system, the orotidine-5'-monophosphate decarboxylase gene of Rhodosporidium toruloides is isolated 4.1.1.31 phosphoenolpyruvate carboxylase biotechnology phosphoenolpyruvate carboxylase and Lactococcus lactis pyruvate carboxylase are overexpressed in Escherichia coli concurrently to improve the production of succinate. This coexpression system is also applied to mutant strains of Escherichia coli strategically designed by inactivating the competing pathways of succinate formation 4.1.1.31 phosphoenolpyruvate carboxylase biotechnology elevated acetate concentrations have an inhibitory effect on growth rate and recombinant protein yield, and thus elimination of acetate formation is an important aim towards industrial production of recombinant proteins 4.1.1.39 ribulose-bisphosphate carboxylase biotechnology it is examined if recombinant protein accumulation can be enhanced by genetically fusing the recombinant reporter protein, luciferase, to the carboxy-terminal end of an abundant endogenous protein, the large subunit of ribulose bisphosphate carboxylase. A native protein-processing site from preferredoxin (preFd) is placed between the Rubisco LSU and luciferase coding regions in the fusion protein construct. Results demonstrate the utility of using fusion proteins to enhance recombinant protein accumulation in algal chloroplasts, and also show that engineered proteolytic processing sites can be used to liberate the exogenous protein from the endogenous fusion partner, allowing for the purification of the intended mature protein 4.1.1.39 ribulose-bisphosphate carboxylase biotechnology Rubisco appears as suitable objective for biotechnological optimization of hydrogen production because of its relevance controlling the hydrogenase main competitor electron sink (the Calvin-Benson cycle), as well as starch accumulation and photorespiratory oxygen consumption 4.1.1.39 ribulose-bisphosphate carboxylase biotechnology RuBisCO protein readily forms a network with a very high gel strength, but upon deformation it has a brittle character (low critical strain, low fracture strain). RuBisCO exhibits high potential as a functional ingredient for the design of new textures at low protein concentration 4.1.1.50 adenosylmethionine decarboxylase biotechnology overexpression of enzyme is sufficient for accumulation of spermidine in leaves and spermidine and spermine in seeds 4.1.1.52 6-methylsalicylate decarboxylase biotechnology biotechnological de novo production of m-cresol from sugar in complex yeast extract-peptone medium with the yeast Saccharomyces cerevisiae. A heterologous pathway based on the decarboxylation of the polyketide 6-methylsalicylic acid is introduced into a CEN.PK yeast strain. Overexpression of codon-optimized 6-methylsalicylic acid synthase from Penicillium patulum together with activating phosphopantetheinyl transferase npgA from Aspergillus nidulans results in up to 367 mg/l 6-methylsalicylic acid production. Additional genomic integration of the genes have a strongly promoting effect and 6-methylsalicylic acid titers reach more than 2 g/l. Simultaneous expression of 6-methylsalicylic acid decarboxylase patG from Aspergillus clavatus leads to the complete conversion of 6-methylsalicylic acid and production of up to 589 mg/L m-cresol 4.1.1.61 4-hydroxybenzoate decarboxylase biotechnology use of enzyme for conversion of phenol into 4-hydroxybenzoic acid 4.1.1.76 arylmalonate decarboxylase biotechnology enzyme is of biotechnological interest for its use in the synthesis of fine chemicals 4.1.1.123 phenyl-phosphate phosphatase/carboxylase biotechnology the enzyme system may be useful for biotechnical phenol carboxylation 4.1.2.4 deoxyribose-phosphate aldolase biotechnology DERA has the potential to resist high concentrations of acetaldehyde and may serve as an industrial catalyst 4.1.2.4 deoxyribose-phosphate aldolase biotechnology efficient process for the production of thermophilic DERA designed from the point of view of recombinant enzyme concentration and productivity, which is economically and technically viable and can be used as a guide for production of other synthetically useful enzymes 4.1.2.5 L-threonine aldolase biotechnology development of a growth-dependent selection system for identification of L-threonine aldolases in Pseudomonas putida KT2440. The functionality is demonstrated with different growth studies by introducing recombinant, nonnative The functionality was demonstrated with different growth studies by introducing recombinant, nonnative threonine aldolases into the selection strain, restoring its deficiency in growth in minimal medium supplemented with DL-threo-beta-phenylserine as sole carbon source 4.1.2.9 phosphoketolase biotechnology expression of bacterial phosphoketolase in Saccharomyces cerevisiae (that does not demonstrate efficient phosphoketolase activity naturally) can efficiently divert intracellular carbon flux toward C2-synthesis, thus showing potential to be used in metabolic engineering strategies aimed to increase yields of acetyl-CoA derived compounds 4.1.2.9 phosphoketolase biotechnology pathway engineering advances in a high-potential alternative route, the phosphoketolase pathway, which facilitates bypass of pyruvate decarboxylation and enables complete carbon conservation in bioprocesses targeting pentose phosphate pathway and/or acetyl-CoA-derived products 4.1.2.22 fructose-6-phosphate phosphoketolase biotechnology expression of bacterial phosphoketolase in Saccharomyces cerevisiae (that does not demonstrate efficient phosphoketolase activity naturally) can efficiently divert intracellular carbon flux toward C2-synthesis, thus showing potential to be used in metabolic engineering strategies aimed to increase yields of acetyl-CoA derived compounds 4.1.2.48 low-specificity L-threonine aldolase biotechnology a continuous bioconversion system for L-threo-3,4-dihydroxyphenylserine production is developed that uses whole-cell biocatalyst of recombinant Escherichia coli expressing L-TA genes cloned from Streptomyces avelmitilis MA-4680. Maximum conversion rates are observed at 2 M glycine, 145 mM 3,4-dihydroxybenzaldehyde, 0.75% Triton-X, 5 g Escherichia coli cells/l, pH 6.5 and 10°C. In the optimized condition, overall productivity is 8 g/l 4.1.3.3 N-acetylneuraminate lyase biotechnology coupled bioconversion for preparation of N-acetyl-D-neuraminic acid using immobilized N-acetyl-D-glucosamine-2-epimerase and N-acetyl-D-neuraminic acid lyase, a two-step enzymatic system involving immobilized both enzymes is used for the conversion of GlcNAc to NeuAc in a single reactor, optimum ratio is 3-6.25 U/ml of N-acetyl-D-glucosamine-2-epimerase and 12.5-25 U/ml of N-acetyl-D-neuraminic acid lyase 4.1.3.3 N-acetylneuraminate lyase biotechnology efficient method for N-acetyl-D-neuraminic acid production using coupled bacterial cells with a safe temperature-induced system, a precursor for producing many pharmaceutical drugs such as zanamivir which have been used in clinical trials to treat and prevent the infection with influenza virus, such as the avian influenza virus H5N1 and the current 2009 H1N1 4.1.3.3 N-acetylneuraminate lyase biotechnology chemoenzymatic synthesis of N-acetyl-D-neuraminic acid from N-acetyl-D-glucosamine and pyruvate 4.1.3.3 N-acetylneuraminate lyase biotechnology cross-linked enzyme aggregates 4.1.3.27 anthranilate synthase biotechnology development of a microbial system for the environmentally-compatible synthesis of anthranilate generated by metabolic engineering of trpD gene from strain W3110 trpD9923 4.1.3.27 anthranilate synthase biotechnology development of ASA2 as a chloroplast selective marker, which is important not only for biosafety reasons, but also for practical reason due to the scarcity of primary selective markers available 4.1.3.27 anthranilate synthase biotechnology Oryza sativacalli overexpressing OASA1D:OASA1D is a system for the production of significant amounts of pharmacologically useful indole alkaloids in rice 4.1.99.1 tryptophanase biotechnology encapsulation of enzyme in wet nanoporous silica gels to selectively stabilize tertiary and quarternary protein conformations and to develop bioreactors and biosensors 4.1.99.2 tyrosine phenol-lyase biotechnology encapsulation of enzyme in wet nanoporous silica gels to selectively stabilize tertiary and quarternary protein conformations and to develop bioreactors and biosensors 4.1.99.2 tyrosine phenol-lyase biotechnology tyrosine phenol lyase modifies and synthesizes natural and non-natural amino acids, synthesizes precursor of a large number of relevant compounds and plays an important role in phenolic waste treatment 4.1.99.3 deoxyribodipyrimidine photo-lyase biotechnology a new class of photolyases with specificity for cyclobutane pyrimidine dimers in ssDNA is defined. Members of these branch are found in bacteria, plants, and animals, and are designated Cry-DASH, because of the lack of significant photorepair activity on dsDNA 4.1.99.3 deoxyribodipyrimidine photo-lyase biotechnology it should be possible to perform wavelength tuning of the Thermus photolyase by using artificial flavin chromophores and photolyase variants whose antenna chromophore-binding sites are reengineered by molecular modeling and mutagenesis 4.2.1.3 aconitate hydratase biotechnology molecular chaperones GroEL/GroES are co-expressed with soluble, biologically active recombinant aconitase in Escherichia coli by cultivation in a bioreactor at different temperatures under optimized conditions. The yield of functional aconitase is enhanced, either in presence of co-expressed GroEL/ES or at low temperature cultivation. The outcome from the chaperone assisted folding of aconitase is more pronounced at lower temperature 4.2.1.20 tryptophan synthase biotechnology enzyme is a target for structure-based design of herbicides 4.2.1.30 glycerol dehydratase biotechnology development of an economical and eco-friendly biological process for the production of 1,3-propanediol by an operon harboring the dhaB1, dhaB2, and yqhD genes, from renewable resources 4.2.1.47 GDP-mannose 4,6-dehydratase biotechnology repression of the GMD gene is thus very useful for deleting immunogenic total fucose residues and facilitating the production of pharmaceutical glycoproteins in plants 4.2.1.66 cyanide hydratase biotechnology searching for enzymes in the bioremediation of cyanide-containing waste 4.2.1.81 D(-)-tartrate dehydratase biotechnology constitutive production of enzyme for selective cleavage of racemic tartaric acid and quantitative detection of D(-)-tartrate 4.2.1.104 cyanase biotechnology analysis of strain characteristics for biotechnological application, detoxification of cyanide- or thiocyanate-containing soils and industrial effluents 4.2.1.108 ectoine synthase biotechnology inducible recombinant enzyme expression in Escherichia coli confers the capability to grow on high concentration of osmolytes by increasing the osmotolerance via L-ectoine production 4.2.1.108 ectoine synthase biotechnology ectoine has importance in dermopharmacy as anti ageing agents in skin creams, as components of shampoo, for oral care and as adjuvants for vaccines 4.2.1.119 enoyl-CoA hydratase 2 biotechnology recombinant 46 kDa hydratase 2 survives in a purified form under storage, thus being the first protein of this type amenable to application as a tool in metabolic studies 4.2.1.127 linalool dehydratase biotechnology possible biotechnological applications of Ldi, in particular, are industrial butadiene and isoprene production from renewable sources 4.2.1.134 very-long-chain (3R)-3-hydroxyacyl-CoA dehydratase biotechnology the enzyme is a potential target for biotechnological optimization of the production of docosahexenoic acid (DHA) in the Aurantiochytrium sp. thraustochytrid strain PKUSW7 4.2.1.135 UDP-N-acetylglucosamine 4,6-dehydratase (configuration-retaining) biotechnology assay targets enzymes involved in the biosynthesis of the unusual bacterial sugar diNAcBac and the transfer of diNAcBac-phosphate to UndP. This multienzyme assay, together with the established assays for the individual enzymes, can be used to screen for inhibitors, and may be used to evaluate substrate flux along the inhibited pathway. This assay is optimized for maximum sensitivity to inhibition of PglF, PglE, PglD, and PglC by balancing the enzyme concentrations such that each is partially rate determining 4.2.2.2 pectate lyase biotechnology optimization of fermentation conditions 4.2.2.2 pectate lyase biotechnology the enzyme can be used for bioscouring of cotton 4.2.2.2 pectate lyase biotechnology the pectinase preparation from Bacillus macerans can compete with the commercial preparations for the process of cotton fabric boil off 4.2.2.2 pectate lyase biotechnology a combined (enzymatic and chemical) process using a Bacillus pumilus strain (DKS1), isolated from the soil, is used to degum ramie bast fibres. Results indicate the process provides an economical and eco-friendly method for the small scale as well as large-scale degumming of decorticated ramie fibre. Results are of importance for the textile as well as paper industry 4.2.2.2 pectate lyase biotechnology application of a commercial pectinase for a range of concentrations and treatment times creates pectin-free textiles with low wax content. Assessment of physicochemical properties such as, wettability, whiteness index, polymerization degree, crystallinity index, color depth, as well as low-stress mechanical properties, proves that bioscouring can be as much efficient as the conventional alkaline treatment 4.2.2.2 pectate lyase biotechnology enhancing PGL production by controlling the optimal ratio provides an alternative approach to enhance heterologous protein production with Pichia pastoris 4.2.2.2 pectate lyase biotechnology parameters for maximum production of PGL by yeast strain Debaryomyces nepalensis in bioreactor are determined: optimal levels of pH, aeration and agitation rate is found to be 7.0, 300 rpm and 1 vvm, respectively. Combined feeding of inducer (lemon peel) and carbon source (galactose) at 12 h is the best strategy for enhanced production of PGL. The production is increased by 1.8fold and productivity 1.4fold for PGL when compared to batch culture 4.2.2.3 mannuronate-specific alginate lyase biotechnology use of alginate lyase for engineering of alginate polymers for applications in various industrial, agricultural, and medical fields 4.2.2.3 mannuronate-specific alginate lyase biotechnology most of Pseudomonas strains have copious alginates enclosing the bacteria cells, which makes it difficult to transfer exogenous DNA into the cells during transformation. Pretreatment of Pseudomonas sp. QDA with alginate lyase before electroporation increases transformation efficiency approximately 10000fold than without pretreatment of alginate lyase, and a high transformation efficiency nearly the same as that of alginate production deficient mutant is obtained. Among the alginate lyases tested, AL2 is the most effective enzyme for pretreatment. This electroporation procedure is also efficient for Pseudomonas aeruginosa FRD1 (mucoid) and PAO1 (non-mucoid) 4.2.2.3 mannuronate-specific alginate lyase biotechnology a new highly specific and sensitive capillary electrophoresis method for the determination of the total alginic acid (AA) content in pharmaceutical formulations is described by means of capillary electrophoresis at 230 nm after treatment with alginate lyase and separation of unsaturated products, DELTA-oligomers (DELTA-HexA-[HexA]n), in particular, DP3 (DELTA-HexA-HexA-HexA) and DP4 (DELTAHexA-HexA-HexA-HexA). The capillary electrophoresis method is applied to the determination of AA content of both solid and liquid formulations that also contain antacid ingredients, mainly aluminium, sodium and potassium bicarbonate, and emulsifying and flavouring agents 4.2.2.3 mannuronate-specific alginate lyase biotechnology the algino-oligosaccharides show an elicitor activity stimulating the accumulation of phytoalexin and inducing phenylalanine ammonia lyase in soybean cotyledon, and antimicrobial activity on Pseudomonas aeruginosa 4.2.2.3 mannuronate-specific alginate lyase biotechnology the bacterium A7 is shown to be alginate lyase-producing in genus Gracilibacillus and effective in degrading alginate to oligosaccharides in wakame during composting process 4.2.2.3 mannuronate-specific alginate lyase biotechnology controlled mono-PEGylation of A1-III alginate lyase mutant A53C produces a conjugate with wild type levels of activity. The PEGylated mutant exhibits enhanced solution phase kinetics with bacterial alginate. In vitro binding studies with both enzyme-specific antibodies, from immunized New Zealand white rabbits, and a single chain antibody library, derived from a human volunteer show that the PEGylated enzyme is substantially less immunoreactive. More than 90% of adherent, mucoid, Pseudomonas aeruginosa biofilms are removed from abiotic surfaces following a one h treatment with the PEGylated mutant, whereas the wild type enzyme removes only 75% of biofilms in parallel studies 4.2.2.14 glucuronan lyase biotechnology a glucuronan lyase is immobilized on a monolithic Convective Interaction Media disk. Degradations of three glucuronans with various O-acetylation degrees is investigated and compared with degradations using free enzyme. The immobilized glucuronan lyase is inhibited by the O-acetylation degree like the free enzyme. 1H NMR analyses are used to study the O-acetylation degree of oligoglucuronans and demonstrate that the average degrees of polymerization are inclusive between 4 and 13 after 24 h of degradation. This first immobilization of a glucuronan lyase constitutes a tool to produce oligoglucuronans 4.2.2.16 levan fructotransferase (DFA-IV-forming) biotechnology production of Arthrobacter levan fructotransferase from recombinant Escherichia coli at high levels via secretion directed by a novel N-terminal motif, TMITNSSSVP. A large amount of extracellular recombinant levan fructotransferase can be produced with high productivity through cost-effective processes 4.2.2.18 inulin fructotransferase (DFA-III-forming) biotechnology improved enzyme for large scale production of low-calorie sweet food additive 4.2.2.18 inulin fructotransferase (DFA-III-forming) biotechnology large scale production of sweet food additive DFA III via recombinant expression in E. coli, genetic engineering of the enzyme for enlarged thermotolerance, immobilization of the enzyme on alginate beads 4.2.2.20 chondroitin-sulfate-ABC endolyase biotechnology bond strength of two etch-and-rinse adhesives to chondroitinase ABC treated dentin is investigated. Human extracted molars are treated with chondroitinase ABC. Increased mean values of microtensile bond strength and reduced nanoleakage expression are shown for both adhesives after chondroitinase ABC treatment of the dentin surface. This study supports the hypothesis that adhesion can be enhanced by removal of chondroitin 4/6 sulfate and dermatan sulfate, probably due to a reduced amount of water content and enlarged interfibrillar spaces 4.2.2.21 chondroitin-sulfate-ABC exolyase biotechnology preservation of ChABC activity during release by immobilizing ChABC in chitosan nerve conduits and encapsulating ChABC in poly(DL-lactic acid) microspheres using an appropriate stabilizer. Immobilizing ChABC in nerve conduitss markedly improves its stability. The activity of ChABC that is immobilized in chitosan nerve conduits by ionic bonding is 0.07 U/mg. 48% of this activity is retained at 48 h after immobilization. Poly(DL-lactic acid) microspheres, fabricated by the double emulsion method, are applied as carriers in the controlled release of ChABC. Stabilizers, including nanogold of 10 nm, polylysine of Mw 500-2000 and polylysine of Mw 20000-30000, are added to microspheres to maintain the activity of ChABC. Polylysine stabilizes ChABC most effectively. The ChABC activity is 0.0162 U/ml after seven days of release 4.2.3.6 trichodiene synthase biotechnology mutants D98E, D99E, D102E generate varying proportions of anomalous sesquiterpenes 4.2.3.25 S-linalool synthase biotechnology the enzyme can be used to modify the flavor/nuritional value of vegetables, e.g. tomato fruits, by enzyme expression in transgenic plants 4.2.3.25 S-linalool synthase biotechnology biotechnological potential of the engineered wine yeast to modify the sensorial qualities of wine 4.2.99.18 DNA-(apurinic or apyrimidinic site) lyase biotechnology is frequently used in gene technology due to its strong exonucleolytic activity 4.2.99.21 isochorismate lyase biotechnology alternative computational rational approach to improve the secondary catalytic activity of enzymes, taking as a test case the IPL enzyme. The approach is based on the use of molecular dynamic simulations employing hybrid quantum mechanics/molecular mechanics methods that allow describing breaking and forming bonds 4.3.1.1 aspartate ammonia-lyase biotechnology overview on commercial applications 4.3.1.1 aspartate ammonia-lyase biotechnology enhancement of recombinant protein production in Escherichia coli by coproduction of aspartase. The excretion of acetate by the aerobic growth of Escherichia coli on glucose is a manifestation of imbalanced flux between glycolysis and the tricarboxylic acid (TCA) cycle. This may restrict the production of recombinant proteins in E. coli, due to the limited amounts of precursor metabolites produced in TCA cycle. To approach this issue, an extra supply of intermediate metabolites in TCA cycle is made by conversion of aspartate to fumarate, a reaction mediated by the activity of L-aspartate ammonia-lyase 4.3.1.1 aspartate ammonia-lyase biotechnology putative and attractive enzyme for the enantioselective synthesis of N-substituted aspartic acids 4.3.1.1 aspartate ammonia-lyase biotechnology a complete biocatalytic process to synthesize high concentrations of L-aspartate catalyzed by aspartase from Bacillus sp. YM55-1 (AspB) is established using an immobilized enzyme in three different supports. MANA-agarose derivative could be selected as the most suitable biocatalyst for the synthesis of Asp due to the simplicity of the method and performance 4.3.1.2 methylaspartate ammonia-lyase biotechnology Fusobacterium varium can simultaneously utilize both glucose and L-glutamate as energy sources, intracellular concentrations of methylaspartate ammonia-lyase is elevated when the bacterium is cultured in media supplemented with excess L-glutamate 4.3.1.12 ornithine cyclodeaminase biotechnology expression of ocd from Pseudomonas putida in an ornithine overproducing platform strain with deletions of argR and argF (ORN1) from Corynebacterium glutamicum results in proline production with yields up to 0.31 g proline/g glucose 4.3.1.14 3-Aminobutyryl-CoA ammonia-lyase biotechnology lysine fermentation pathway 4.3.1.24 phenylalanine ammonia-lyase biotechnology compared to the free enzyme, the PAL-CLEA exhibit increased stability of the enzyme against various deactivating conditions such as pH, temperature, denaturants, and organic solvents and show higher storage stability than its soluble counterpart. Additionally, PAL-CLEAs can be recycled at least for 12 consecutive batch reactions without dramatic activity loss, increases the commercial potential of PAL for synthesis of L-phenylalanine 4.3.1.24 phenylalanine ammonia-lyase biotechnology the enzyme can be used for the development of dietary foods and biotechnological products for patients with phenylketonuria 4.3.3.2 strictosidine synthase biotechnology modulating the substrate specificity of strictosidine synthase is a critical step toward indole alkaloid pathway engineering to yield nonnatural alkaloids 4.3.3.2 strictosidine synthase biotechnology redesigning the substrate specificity of strictosidine synthase is an important step toward re-engineering the TIA pathway to produce products with novel or improved biological properties 4.3.3.7 4-hydroxy-tetrahydrodipicolinate synthase biotechnology enzyme is a target for herbicide and anti-microbial action 4.4.1.1 cystathionine gamma-lyase biotechnology use of strain as adjunct starter in cheese making 4.4.1.4 alliin lyase biotechnology lachrymatory factor synthase and alliinase function in tandem, with the alliinase furnishing the sulfenic acid substrate on which the lachrymatory factor synthase acts. The lachrymatory factor synthase modulates the formation of biologically active thiosulfinates that are downstream of the alliinase in a manner dependent upon the relative concentrations of the lachrymatory factor synthase and the alliinase. These observations suggest that manipulation of lachrymatory factor synthase-to-alliinase ratios in plants displaying this system may provide a means by which to rationally modify organosulfur small molecule profiles to obtain desired flavor and/or odor signatures, or increase the presence of desirable biologically active small molecules 4.5.1.1 DDT-dehydrochlorinase biotechnology positive correlation of enzyme activity and resistance against DDT 4.5.1.1 DDT-dehydrochlorinase biotechnology positive correlation of enzyme activity and resistance against DDT, and also between enzyme activity and GST activity 4.5.1.1 DDT-dehydrochlorinase biotechnology positive correlation of enzyme activity and resistance against DDT, non-correlation between enzyme activity and GST activity 4.6.1.18 pancreatic ribonuclease biotechnology synthesis of molecularly imprinted polymers from the monomers styren and polyethyleneglycol 400 dimethacylate with high rebinding efficiency of RNase A to polymer. Polymers show high selectivity for RNase A and high stability 4.6.1.19 ribonuclease T2 biotechnology isolate could be a novel renewable source of DNase-free RNase enzyme 4.6.1.22 Bacillus subtilis ribonuclease biotechnology bacisubin is an antifungal protein 4.6.1.24 ribonuclease T1 biotechnology the RNase G mutation could be applied in the breeding of producer strains of pyruvate and its derivatives such as valine 4.8.1.2 aliphatic aldoxime dehydratase biotechnology nitrile compounds are important intermediates in some industrial processes to produce nylon and acrylic fibers, insecticides, and pharmaceuticals. A more environmentally benign process of aldoxime dehydration is needed, for which a biological dehydration of aldoxime is a possible candidate 4.98.1.1 protoporphyrin ferrochelatase biotechnology co-expression with ferrochelatase along with the addition of a small amount of delta-aminolevulinic acid, is sufficient to produce fully incorporated heme protein. This method is applicable for both Cys-ligated and His-ligated heme proteins 4.99.1.2 alkylmercury lyase biotechnology coexpression of enzyme and mercuric reductase in Arabidopsis thaliana leads to growth on 50fold higher methylmercury concentrations than wild-type plants 4.99.1.2 alkylmercury lyase biotechnology engineering of enzyme as to be targeted for accumulation in the endoplasmic reticulum and for secretion to the cell wall rendering the expressing plants resistant to organic mercury 5.1.1.1 alanine racemase biotechnology use of enzyme gene as a promoter-screening tool for identification of conditional promoters in Lactobacillus plantarum. Screen for clones capable of complementing the D-alanine auxotroph phenotype of enzyme deletion mutant in media containing enzyme inhibitor D-cycloserine 5.1.1.5 lysine racemase biotechnology enzyme is a novel non-antibiotic selectable marker for plant transformation 5.1.1.10 amino-acid racemase biotechnology since commercially available D-Trp is chemically synthesized and expensive a mutant BAR protein might represent an effective method to synthesize D-Trp 5.1.1.10 amino-acid racemase biotechnology an enzyme lyophilisate of amino acid racemase from Pseudomonas putida is used for in situ racemization. Crystallization experiments accompanied by enzymatic racemization lead to a significant increase of crystallized L-Asn 5.1.1.17 isopenicillin-N epimerase biotechnology The results indicate that Penecillium chrysogenum can be used as heterologous host for the production of deacetylcephalosporin C 5.1.1.17 isopenicillin-N epimerase biotechnology production of beta-lactam antibiotics 5.1.2.2 mandelate racemase biotechnology the demonstrated application of a membrane bioreactor will be a useful method for large-scale dynamic kinetic resulution (DKR) of mandelic acid and for possible other bioconversions in organic media 5.1.3.1 ribulose-phosphate 3-epimerase biotechnology efficient utilization of the available glucose and xylose in the lignocellulosic hydrolysates is the important issue for economic cellulosic ethanol production. Simultaneous utilization of xylose is realized by the coupling of glucose metabolism and xylose utilization through RPE1 deletion in xylose-utilizing Saccharomyces cerevisiae 5.1.3.8 N-acylglucosamine 2-epimerase biotechnology coupled bioconversion for preparation of N-acetyl-D-neuraminic acid using immobilized N-acetyl-D-glucosamine-2-epimerase and N-acetyl-D-neuraminic acid lyase, a two-step enzymatic system involving immobilized both enzymes is used for the conversion of GlcNAc to NeuAc in a single reactor, optimum ratio is 3-6.25 U/ml of N-acetyl-D-glucosamine-2-epimerase and 12.5-25 U/ml of N-acetyl-D-neuraminic acid lyase 5.1.3.8 N-acylglucosamine 2-epimerase biotechnology efficient method for N-acetyl-D-neuraminic acid production using coupled bacterial cells with a safe temperature-induced system, a precursor for producing many pharmaceutical drugs such as zanamivir which have been used in clinical trials to treat and prevent the infection with influenza virus, such as the avian influenza virus H5N1 and the current 2009 H1N1 5.1.3.14 UDP-N-acetylglucosamine 2-epimerase (non-hydrolysing) biotechnology production of erythropoietin (EPO) in Chinese Hamster Ovary (CHO) cells 5.2.1.5 linoleate isomerase biotechnology the enzyme can be useful for biocatalysis of linoleic acid to conjugated linoleic acid 5.3.1.5 xylose isomerase biotechnology putative use of lignocellulosic biomass as feedstock for the chemical industry 5.3.1.8 mannose-6-phosphate isomerase biotechnology effective use of enzyme gene as selectable marker gene for transformation of embryogenic calli of Carica papaya 5.3.1.8 mannose-6-phosphate isomerase biotechnology selection system for transformation of onion using enzyme gene and Agrobacterium. Transformation rates are around 25% 5.3.1.8 mannose-6-phosphate isomerase biotechnology use of enzyme as selectable marker for transformation of Oriza sativa immature embryo via Agrobacterium 5.3.1.8 mannose-6-phosphate isomerase biotechnology use of enzyme gene as selectable marker for transformation of Penniseum glaucum. Enzyme gene is a superior selectable marker for improving transformation efficiencies when compared to antibiotic or herbicide selectable marker genes 5.3.1.8 mannose-6-phosphate isomerase biotechnology the pmi/mannose selection system is highly efficient for producing transgenic Oncidium Gower Ramsey without using antibiotics or herbicides 5.3.1.24 phosphoribosylanthranilate isomerase biotechnology the work provides a method of exchanging variably sized loops within the (beta/alpha)8 fold, affording a novel starting point for the screening of novel activities as well as modest diversions from an original activity 5.3.3.2 isopentenyl-diphosphate DELTA-isomerase biotechnology an Escherichia coli strain is engineered for isoprenoid ether lipid biosynthesis through digeranylgeranylglycerylphosphate, DGGGP 5.3.3.2 isopentenyl-diphosphate DELTA-isomerase biotechnology overexpression of SlIPI in engineered Escherichia coli can promote the biosynthesis of beta-carotene in bacteria, the result provides a foundation for terpenoid metabolic engineering using the SlIPI gene 5.3.3.2 isopentenyl-diphosphate DELTA-isomerase biotechnology the prenyl alcohol production by Escherichia coli transformants with overexpression of isoprenoid biosynthesis genes is examined 5.3.3.B2 linoleate (10E,12Z)-isomerase biotechnology the enzyme shows the ability to produce fatty acid components of vegetable oils with novel physiological activities in crops 5.3.4.1 protein disulfide-isomerase biotechnology overexpression of Plasmodium falciparum PDI isozymes A and B for production of a disulfide-rich transmission-blocking vaccine candidate Pfs25 in Pichia pastoris, the expression level is enhance by co-expression of the endogenous Pichia pastoris enzyme in Pfs25 3fold, production method evaluation 5.3.4.1 protein disulfide-isomerase biotechnology overexpression of Plasmodium falciparum PDI isozymes A and B for production of a disulfide-rich transmission-blocking vaccine candidate Pfs25 in Pichia pastoris, the expression level is enhanced by co-expression of the endogenous Pichia pastoris enzyme in Pfs25 clone 3fold, production method evaluation 5.3.99.6 allene-oxide cyclase biotechnology constitutive overexpression of enzyme, 53fold increase of 12-oxo-phytodienoic acid methyl ester in stamen, 51fold increase of jasmonic acid in buds and 7.5fold increase in sepals. Increase in jasmonates and octadecanoids is accompanied by decreased levels of free lipid hydro(per)oxy compounds 5.4.2.7 phosphopentomutase biotechnology the Escherichia coli expressing Bacillus sphaericus phosphopentomutase is an excellent catalyst as to production of 2'-deoxyribonucleoside in the presence of acetaldehyde and phosphorylated compounds 5.4.2.8 phosphomannomutase biotechnology enzymatic synthesis of TMP and 2-deoxy-6-phosphate glucose can successfully produce large amount of TDP-2-deoxy-glucose in one-pot by employing phosphomannomutase 5.4.2.8 phosphomannomutase biotechnology the double mutant lacking the PMI and PMM genes produces 8-deoxyamphoteronolides in good yields along with trace levels of glycosylated amphotericins, with further genetic engineering these mutants may activate alternative hexoses as GDP-sugars for transfer to aglycones in vivo 5.5.1.4 inositol-3-phosphate synthase biotechnology the enzyme is a possible target for genetic modification of maize plants to get low phytic acid containing variants 6.1.1.1 tyrosine-tRNA ligase biotechnology incorporation of unusual specific amino acids into proteins in in vitro translation systems by mutant enzyme with altered substrate specificity. e.g. mutant Y32Q/D158A 6.1.1.1 tyrosine-tRNA ligase biotechnology site-specific incorporation of 3-iodo-L-tyrosine into proteins in a cell-free system for use in specialized in vitro translation systems 6.1.1.1 tyrosine-tRNA ligase biotechnology use of mutant Y43G for specialized protein synthesis as a carrier for additional amino acids and derivatives for use in e.g. crystal structure determination by X-ray diffraction 6.1.1.1 tyrosine-tRNA ligase biotechnology a mutant Methanococcus jannaschii tyrosyl amber suppressor tRNA, Tyr MjtRNA CUR/tyrosyl-tRNA synthetase (MjTyrRS) pair is developed to uniquely incorporate phenylselenocysteine in response to the amber TAG codon in Escherichia coli. After being efficiently converted into dehydroalanine under mild conditions, Michael addition reactions with the corresponding thiols can be used to synthesize N-methyl- and N-acetyl-lysine analogues 6.1.1.1 tyrosine-tRNA ligase biotechnology Escherichia coli-based cell-free system for the production of proteins with a non-natural amino acid incorporated site-specifically is described. A mutant Methanococcus jannaschii tyrosyl-tRNA synthetase (mTyrRS) and tRNATyr pair are used as orthogonal elements. The mTyrRS experienced proteolysis and modified protein yields improves with higher synthetase addition (200-300 mg/mL) 6.1.1.1 tyrosine-tRNA ligase biotechnology a rapid, straightforward, one plasmid dual positive/negative selection system for the evolution of aminoacyl-tRNA synthetases with altered specifities in Escherichia coli is developed 6.1.1.1 tyrosine-tRNA ligase biotechnology the present engineering allows Escherichia coli TyrRS variants for non-natural amino acids to be developed in Escherichia coli, for use in both eukaryotic and bacterial cells for genetic code expansion 6.1.1.2 tryptophan-tRNA ligase biotechnology EcTrpRS is used as a model system for in silicio docking studies with various Trp analogs using the FlexX-Pharm strategy. Relative binding energies for Trp analogs in TrpRS are calculated that correlate well with their translational activities in Escherichia coli. FlexX-Pharm predicted the binding sites of fluoro-, amino-, hydroxyl- and aza-containing Trp analogs within 1.5 A of Trp in the homology model of EcTrpRS 6.1.1.20 phenylalanine-tRNA ligase biotechnology design of an enzyme variant which incorporates aryl ketones into proteins 6.1.1.20 phenylalanine-tRNA ligase biotechnology misacylation of suppressor tRNAPhe CUA by the PheRS mutant A294G with 4-iodo-L-phenylalanine might be useful in application in protein engineering since an aryl iodide tag on proteins can be used for site-specific functionalization of proteins, used for cell-free protein synthesis as a stoichiometric reagent, overview 6.1.1.27 O-phospho-L-serine-tRNA ligase biotechnology the mutant SepRS-tRNA pairs may be useful for translational incorporation of O-phosphoserine into proteins in response to the stop codons UGA and UAG, so that it could ligate O-phosphoserine to a suppressor tRNA for genetic-code expansion 6.2.1.12 4-coumarate-CoA ligase biotechnology viability of a flavonoid network to utilize acrylic acid analogues and describe the combinatorial mutasynthesis of novel unnatural flavonoids using recombinant Saccharomyces cerevisiae, overview 6.3.1.20 lipoate-protein ligase biotechnology the enzyme lipoic acid ligase (LplA) can be used for PRIME (probe incorporation mediated by enzymes) labeling, a rapid and specific fluorescent labeling method of the protein of interest by LplA 6.3.2.2 glutamate-cysteine ligase biotechnology a protein transduction approach whereby recombinant GCL protein can be rapidly and directly transferred into cells when coupled to the HIV TAT protein transduction domain. The TAT-GCL fusion proteins are capable of heterodimerization and formation of functional GCL holoenzyme in vitro. Exposure of Hepa-1c1c7 cells to the TAT-GCL fusion proteins results in the time- and dose-dependent transduction of both GCL subunits and increased cellular GCL activity and glutathione levels. A heterodimerization-competent, enzymatically deficient GCLC-TAT mutant was also generated in an attempt to create a dominant-negative suppressor of GCL 6.3.2.3 glutathione synthase biotechnology overexpression of the enzyme in transgenic plants offers a promising strategy for the production of plants with superior heavy-metal phytoremediation capacity 6.3.2.12 dihydrofolate synthase biotechnology production of active dihidrofolate synthase in milligram scale 6.3.2.26 N-(5-amino-5-carboxypentanoyl)-L-cysteinyl-D-valine synthase biotechnology production of beta-lactam antibiotics 6.3.4.16 carbamoyl-phosphate synthase (ammonia) biotechnology ammonia elimination as functional marker in hepatocyte cultivation and zonation in a bioreactor, and construction of a bioartificial liver, overview 6.3.5.6 asparaginyl-tRNA synthase (glutamine-hydrolysing) biotechnology inhibitor represents a lead scaffold to discover and develop antifilarial drugs 6.4.1.1 pyruvate carboxylase biotechnology coexpression of recombinant pyruvate coarboxylase in BHK-21 cells improves the production of human erythropoietin in a continuously perfused bioreactor 6.4.1.1 pyruvate carboxylase biotechnology FLAG-tagged human pyruvate carboxylase is introduced into a dihydrofolate-deficient CHO cell line DG44. Through the expression of the human pyruvate carboxylase enzyme, lactate formation in CHO cell culture can be efficiently reduced. This effect of expression of the human pyruvate carboxylase is observed not only in adherent batch culture using the serum-containing medium but in the serum-free suspension fed-batch culture as well, demonstrating its potential use to extend the culture longevity of CHO cell culture, which often shows a significant accumulation of lactate 6.4.1.2 acetyl-CoA carboxylase biotechnology the enzyme is a target for development of herbicides 6.5.1.2 DNA ligase (NAD+) biotechnology LigN is unique amongst DNA ligase enzymes in displaying maximal DNA strand joining activity at above 3 M salt levels. As such the LigN enzyme has potential both as a novel tool for biotechnology and as a model enzyme for studying the adaptation of proteins to high intracellular salt levels 7.2.2.3 P-type Na+ transporter biotechnology expression of triple hemagglutinin-tagged enzyme in Nicotiana tabacum confers increased NaCl and LiCl tolerance to cells. Under moderate slat stress, enzyme expression results in accumulation of less Na+, Li+ and K+ than in wild-type 7.2.2.10 P-type Ca2+ transporter biotechnology the ability to manipulate metal transporters, such as by altering substrate specificity, is an essential step in developing genetically engineered plants that can be used for phytoremediation strategies for specific metals 7.2.2.14 P-type Mg2+ transporter biotechnology Overexpression of enzyme in Escherichia coli results in formation of inclusion bodies. Co-expression of DnaK/DnaJ prevents inclusion bodies and leads to the integration of more enzyme into the membrane. Co-expression of GroEL/GroES, Ffh/4.5S-RNA or SecA are less effective 7.2.2.21 Cd2+-exporting ATPase biotechnology expression of enzyme in Saccharomyces cerevisiae strikingly decreases Cd2+ tolerance of yeast cells. Yeast expressing the non-functional mutant D398A can grow on selective medium containing up to 0.1 mM Cd2+, while those expressing the intact enzyme cannot grow in presence of 0.001 mM Cd2+. Enzyme is localized in the endoplasmic reticulum, so hypersensitivity to Cd2+ is due to Cd2+ accumulation in the reticulum lumen. Zn2+ does not protect cells against Cd2+ poisoning 7.2.2.22 P-type Mn2+ transporter biotechnology the ability to manipulate metal transporters, such as by altering substrate specificity, is an essential step in developing genetically engineered plants that can be used for phytoremediation strategies for specific metals 7.4.2.8 protein-secreting ATPase biotechnology glutathione S-transferase-enzyme fusion protein functions analogously to native protein. Overexpression of regulatory protein YscL impairs secretion