a two-step catalytic mechanism: Asp192 is the catalytic nucleophile, Glu232 is the catalytic acid-base, and Asp290 functions as a transition state stabilizer. By forming a strong hydrogen bond with the hydroxyl groups at C2 and C3 of the glucosyl residue being transferred, the anionic side chain of Asp290 is suggested to provide selective stabilization to oxocarbenium ion-like transition states flanking the covalent alpha-glucosyl enzyme intermediate
establishing of a resveratrol glycosylation method using the enzyme and IL AMMOENG 101 as the most effective cosolvent, solubility at pH 6.5 and 60°C, in the presence of 20% of different cosolvents and 1 M sucrose, overview
because alpha-glucopyranosyl arsenate decomposes hydrolytically in a non-enzymatic reaction, the overall arsenolysis of sucrose is essentially irreversible
sucrose phosphorylase catalyzes three types of overall reaction: glucosyl transfer to and from phosphate, hydrolysis, and transglucosylation. Arsenate can replace phosphate as glucosyl acceptor substrate, other glucosyl acceptors are caffeic acid, benzoic acid, acetic acid, and formic acid
the wild-type enzyme shows poor activity with flavonoids or stilbenoids, e.g. resveratrol, (+)-catechin and (-)-epicatechin, while the compounds are substrates of the enzyme mutant Q345F
the wild-type enzyme shows poor activity with flavonoids or stilbenoids, e.g. resveratrol, (+)-catechin and (-)-epicatechin, while the compounds are substrates of the enzyme mutant Q345F
assay method with production of alpha-D-glucose-1-phosphate is coupled to the reduction of NAD+ in the presence of phosphoglucomutase and glucose-6-phosphate dehydrogenase
sucrose phosphorylase is likely to serve a catabolic function in vivo, fueling the energy metabolism of the cell with Glc1P and D-fructose produced from sucrose
mapping of the acceptor site of the enzyme by saturation mutagenesis and screening, overview. Residues Arg135, Leu343, and Tyr344 contribute to the specificity for phosphate, residues Tyr132 and Asp342 contribute to the specificity for D-fructose, and residues Pro134, Tyr196, His234, Gln345 contribute to the specificity for both. Alternative acceptors that are glycosylated rather efficiently (e.g. D-arabitol) interact with the same residues as fructose, whereas poor acceptors like pyridoxine do not seem to make any specific interactions with the enzyme
an enzyme monomer consists of 4 domains: A, B, B', and C, domain A comprises the (beta/alpha)8-barrel including the active site, domain B' is involved in modulation of substrate acces via the substrate channel
an enzyme monomer consists of 4 domains: A, B, B', and C, domain A comprises the (beta/alpha)8-barrel including the active site, domain B' is involved in modulation of substrate acces via the substrate channel
recombinant enzyme mutant Q345F, in the presence of sucrose, and in complex with glucose, X-ray diffraction structure determination and analysis at 2.7 A resolution, PDB ID 5C8B
recombinant enzyme, hanging drop vapour diffusion method, 0.0025 ml protein solution, containing 0.5-1.0 mg/ml protein, 10 mM Tris-HCl, pH 7.1, is mixed with eual volume of precipitant solution containing 27% w/v PEG 4000, 0.1 M Tris-HCl, pH 8.5, and 0.1 sodium acetate, at 25°C, 3-14 days, X-ray diffraction structure determination and analysis at 1.77 A resolution
site-directed mutagenesis, the mutation allows efficient glucosylation of resveratrol, (+)-catechin and (-)-epicatechin in yields of up to 97% whereas the wild-type enzyme favours sucrose hydrolysis. The crystal structure of the variant reveals a widened access channel with a hydrophobic aromatic surface that is likely to contribute to the improved activity towards aromatic acceptors. The generation of this channel can be explained in terms of a cascade of structural changes arising from the Q345F exchange, structural changes in the active site of BaSP Q345F, modeling, overview
formation of a glutaraldehyde cross-linked enzyme aggregate for high improvement of the enzyme's stability at 60°C, molecular imprinting of the cross-linked enzyme aggregate with a suitable substrate, i.e. glycerol, involving enzyme precipitation by tert-butyl alcohol can 2fold increase the transglucosylation activity, stability and specificity of the modified enzyme, method, overview. The modified enzyme is more useful as industrial biocatalyst than the native enzyme
immobilization of the enzyme by cross-linking leads to a 17 degree higher temperature tolerance compared to the soluble enzyme from Bifidobacterium adolescentis, overview
increasing the thermostability of sucrose phosphorylase by a combination of sequence- and structure-based mutagenesis, substitution of the most flexible residues with amino acids that occur more frequently at the corresponding positions in related sequences, and substitutions to promote electrostatic interactions
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene sucP, cloning from genomic library, DNA and amino acid sequence determination and analysis, determination of promotor region, expression in Escherichia coli
heterologous expression in a food-grade Bacillus subtilis strain. Efficiently secreted into the extracellular medium in the absence of a signal peptide. After culturing the recombinant strain in a 3-l bioreactor, crude enzyme yield and activity reach 7.5 g/l and 5.3 U/ml, respectively
the enzyme is useful as transglucosylation catalyst for synthesis of alpha-D-glucosides as industrial fine chemicals, overview. The enzyme is also used in the industrial process for production of 2-O-(alpha-D-glucopyranosyl)-sn-glycerol as active ingredient of cosmetic formulations
sucrose phosphorylase is a promising biocatalyst for the production of special sugars and glycoconjugates, but its transglycosylation activity rarely exceeds the competing hydrolytic reaction
sucrose phosphorylase can glycosylate a variety of small molecules using sucrose as cheap ut efficient donor substrate. The immobilized enzyme is optimized due to a higher temperature tolerance compared to the soluble enzyme from Bifidobacterium adolescentis, overview
effective kojibiose synthesis i as performed using the crude enzyme solution from the supernatant of fermentation broth of Bacillus subtilis expressing the enzyme from Bifidobacterium adolescentis, providing a basis for potential industrial-scale preparation of kojibiose. Kojibiose is prebiotic element, a low-calorie sweetener and exhibits antitoxic activity, thereby promoting the emergence of new drugs