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A173T/A332S
increased electron transfer (1.2fold)
A173T/F414L
increased electron transfer (1.2fold), 70% decrease in O2 sensitivity
A173V/A332S/F414I/V560T
increased electron transfer (6.4fold), decrease in O2 sensitivity
A332S/V560T
increased electron transfer (1.2fold), 70% decrease in O2 sensitivity
A449C
-
site-directed mutagenesis, the mutation results in almost completely diminished activity compared to the wild-type enzyme
E84C
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site-directed mutagenesis, the mutation does not affect enzyme activity. Attachment of gold nanoparticles to the purified proteins leads to an immediate and dramatic decrease in activity
F414Y
increased electron transfer
H172K
site-directed mutagenesis, mutant H172K shows increased thermosensitivity compared to the wild-type enzyme
H172K/H220D
site-directed mutagenesis, mutant H172K/H220D does not show significant differences in thermal stability but about 70% increased initial activity compared to the wild-type enzyme
H220D
site-directed mutagenesis, mutant H220D shows increased thermosensitivity and reduced activity compared to the wild-type enzyme
H447C
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site-directed mutagenesis, the mutation does not affect enzyme activity. Attachment of gold nanoparticles to the purified proteins leads to an immediate and dramatic decrease in activity
I94V/T30S
increased O2 sensitivity, increased electron transfer (1.9fold)
L500D
site-directed mutagenesis, inactive mutant
N2Y/K13E/T30V/I94V/K152R
site-directed mutagenesis of mutant M12, pH optimum and sugar specificity of M12 mutant of GOx is similar to the wild-type enzyme, while thermostability is slightly decreased. Mutant M12 GOx expressed in Pichia pastoris shows three times higher activity compared to wild-type GOx towards redox mediators like N,N-dimethyl-nitroso-aniline used for glucose strips manufacturing. Mutant M12 GOx remains very specific for glucose but has higher activity for galactose compared to wild-type GOx
Q124R/L569E
site-directed mutagenesis, the mutation has no significant effect on stability but causes a twofold increase of the enzyme's specific activity
Q469K
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
Q90R
site-directed mutagenesis, the mutant shows increased sensitivity to thermal denaturation, with R1 and R2 values 60% and 80% lower than wild-type enzyme respectively
Q90R/Y509E/T554M
the triple mutant is a glucose oxidase with high stability
S307C
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site-directed mutagenesis, the mutation does not affect enzyme activity. Attachment of gold nanoparticles to the purified proteins leads to an immediate and dramatic decrease in activity
T110A
the mutant enzyme displays 12.3fold reduced O2 consumption
T110S
increased electron transfer
T110S/T34V
increased electron transfer
T110S/V20Y
increased O2 sensitivity
T30V/I94V/A162T
2.9fold increase in kcat/Km, decrease in t1/2(60°C) by 1.5°C
T30V/I94V/A162T/R537K/M556V
4.0fol2.6fold increase in kcat/Km, increase in t1/2(60°C) by 5.25°C
T554M
random mutagenesis, the mutation generates a sulfur-pi interaction, the mutant shows 60% reduced activity and 40% increased thermal stability compared to the wild-type enzyme
T56V/T132S/C521S
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site-directed mutagenesis, the mutant shows improved catalytic efficiency, mutation C521S does not alter enzyme activity, but the attachment of AuNPs to the native free thiol is prevented
V20Y
increased electron transfer
Y435C
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site-directed mutagenesis, the mutation does not affect enzyme activity. Attachment of gold nanoparticles to the purified proteins leads to an immediate and dramatic decrease in activity
Y509E
site-directed mutagenesis, the mutation does not cause a significant change in the thermal stability of the enzyme, but causes increased enzyme activity compared to the wild-type enzyme
H220D
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site-directed mutagenesis, mutant H220D shows increased thermosensitivity and reduced activity compared to the wild-type enzyme
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Q124R/L569E
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site-directed mutagenesis, the mutation has no significant effect on stability but causes a twofold increase of the enzyme's specific activity
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Q90R/Y509E
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site-directed mutagenesis, the mutation does not cause a significant change in the thermal stability of the enzyme, but causes increased enzyme activity compared to the wild-type enzyme
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T554M
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random mutagenesis, the mutation generates a sulfur-pi interaction, the mutant shows 60% reduced activity and 40% increased thermal stability compared to the wild-type enzyme
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F418A
12.6fold increase in apparent Km value
H520A
the enzyme variant is almost completely inactive
H520V
the enzyme variant is almost completely inactive
H563A
the enzyme variant is completely inactive
H563V
the enzyme variant is completely inactive
K19E
-
site-directed mutagenesis
K23E
-
site-directed mutagenesis
K260E.
-
site-directed mutagenesis
K424I
-
site-directed mutagenesis, the mutation does not significantly affect the enzyme activity
K48/50E
-
site-directed mutagenesis
Q184E
-
site-directed mutagenesis
Q75E
-
site-directed mutagenesis
R516K
the mutant enzyme whose side chain forms only one hydrogen bond with the 3-OH group of beta-D-glucose, exhibits an 80fold higher apparent Km (513 mM) but a Vmax only 70% lower than the wild type
R516Q
the complete elimination of a hydrogen-bond interaction between residue 516 and the 3-OH group of beta-D-glucose through the substitution R516Q effects a 120fold increase in the apparent Km for glucose (to 733 mM) and a decrease in the Vmax to 1/30
S114A/F355L
increased electron transfer, 88% decrease in O2 sensitivity
V464A/K424E
2.4fold increase in electron transfer, 95% decrease in O2 sensitivity
V564S
1.1fold increase in electron transfer, 88% decrease in O2 sensitivity
F418A
-
12.6fold increase in apparent Km value
-
H520A
-
the enzyme variant is almost completely inactive
-
R516K
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the mutant enzyme whose side chain forms only one hydrogen bond with the 3-OH group of beta-D-glucose, exhibits an 80fold higher apparent Km (513 mM) but a Vmax only 70% lower than the wild type
-
R516Q
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the complete elimination of a hydrogen-bond interaction between residue 516 and the 3-OH group of beta-D-glucose through the substitution R516Q effects a 120fold increase in the apparent Km for glucose (to 733 mM) and a decrease in the Vmax to 1/30
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H447K
site-directed mutagenesis, introduction of two symmetrical, intermolecular salt bridges at the dimer interface, between K447 and D70
H447K
site-directed mutagenesis, the shows similar initial activity but higher thermal sensitivity compared to the wild-type enzyme
L569E
site-directed mutagenesis, the mutant shows about 50% increased initial activity compared to the wild-type enzyme
L569E
site-directed mutagenesis, the thermal stability of the mutant is similar to the wild-type enzyme, but the initial activity is increased compared to the wild-type enzyme
Q345K
site-directed mutagenesis, introduction of the mutation to create a salt bridge with D177
Q345K
site-directed mutagenesis, the mutant shows highly reduced thermal stability and about 50% increased initial activity compared to the wild-type enzyme
Q469K/L500D
site-directed mutagenesis, the mutant shows strongly reduced activity compared to the wild-type enzyme
Q469K/L500D
site-directed mutagenesis, the thermal stability of the mutant is similar to the wild-type enzyme, but the initial activity is reduced compared to the wild-type enzyme
Q90R/Y509E
site-directed mutagenesis, the mutation does not cause a significant change in the thermal stability of the enzyme, but causes increased enzyme activity compared to the wild-type enzyme
Q90R/Y509E
site-directed mutagenesis, the mutation introduces a new salt bridge near the interphase of the dimeric protein structure, the mutation does not cause a significant change in the thermal stability of the enzyme, but causes increased enzyme activity compared to the wild-type enzyme
T30S/I94V
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
T30S/I94V
site-directed mutagenesis, a thermoresistant mutant
T56V/T132S
-
site-directed mutagenesis, the mutant shows improved catalytic efficiency. The protein has three native cysteines, of which two are involved in a disulfide bond and the third is a free cysteine, Cys 521
T56V/T132S
mutant enzyme displays better catalytic properties than the native enzyme
Y169C/A211C
compared with wild-type enzyme, the half-life of the mutant, at 40 °C increases approximately 48fold. The kcat and catalytic efficiency are enhanced 0.7fold and 1.6fold, respectively
Y169C/A211C
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compared with wild-type enzyme, the half-life of the mutant, at 40 °C increases approximately 48fold. The kcat and catalytic efficiency are enhanced 0.7fold and 1.6fold, respectively
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G423D
-
site-directed mutagenesis, the mutant shows no activity compared to the wild-type enzyme
G423D
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site-directed mutagenesis, the mutants containing the mutation G423D leads to quadruple mutants that are not able to reconstitute. The mutant enzymes displays a dramatic decrease in activity compared to thré wild-type enzyme
K424E
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site-directed mutagenesis, the single mutation results in a significant increase in the current density which becomes 2.4 fold higher than the current obtained for the wild-type
K424E
increased electron transfer (2.4fold), 20% decrease in O2 sensitivity
additional information
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preparation of surface variants that contain artificial polymer poylethylene glycol. All surface modifications of glucose oxidase beyond that of the wild-type enzyme give rise to altered behavior for hydrogen transfer in the active site such that the kinetic isotope effect becomes more temperature-dependent upon perturbation
additional information
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engineering of glucose oxidase by site-specific attachment of a maleimide-modified gold nanoparticle to the enzyme for enabling direct electrical communication between the conjugated enzyme and an electrode required for using the enzyme as biosensor, evaluation, overview
additional information
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enzyme adsorption on different particles with homogeneous or nanostructured surfaces and coated with different compounds, i.e. 11-amino-1-undecanethiol, 12-mercaptododecanoic acid, 1-dodecanethiol, and 11-(1H-pyrol-11-(1H-pyrol-1-yl)undecane-1-thiol), only 9% of the activity of the native protein is preserved on 11-(1H-pyrol-11-(1H-pyrol-1-yl)undecane-1-thiol), but the substrate affinity of the adsorbed GOx is best on 11-(1H-pyrol-11-(1H-pyrol-1-yl)undecane-1-thiol) where its catalytic activity is worst, secondary structure of thhe enzyme is altered compared to enzyme in solution, overview
additional information
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laccase and glucose oxidase in poly(ethyleneimine) microcapsules for immobilization in paper, activity, conformation and thermal stability, overview. The KM for GOx does not change after microencapsulation. Microencapsulation improves the thermal stability of GOx at temperatures up to 60°C due to stabilization of its active conformation but reduces the thermal stability of laccase because of the increased coordination between PEI and copper atoms in the enzyme's active site
additional information
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macroporous silica foam is used as a nanoreactor to co-confine glucose oxidase and horseradish peroxidase with enzymatic cascade reactions, which act in tandem inside nanoreactors, for oxidation of glucose and 3,3',5,5'-tetramethylbenzidine, the catalytic activity of the co-confined enzymes is reduced, but stabilities of co-confined enzymes in denaturing agents, such as guanidinium chloride (GdmCl) and urea, are higher than those of free enzymes in solution compared to that of free enzymes in solution at room temperature. Adsorption amounts of glucose oxidase and horseradish peroxidase into macropores under different conditions, overview
additional information
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modulation of calibration parameters of biosensors, in which glucose oxidase is used for biorecognition, in the presence of different chlorides by following the transient phase dynamics ofoxygen concentration with an oxygen optrode, mechanism, overview. the maximum calculated signal change was amplifiedfor about 20% in the presence of sodium and magnesium chlorides. The value of the kinetic parameter decreases along with the addition of salts and increases only at sodium chloride concentrations over 0.5 mM, MgCl2 causes a 1.3fold essential increase of the maximum signal change parameter A in a salt concentration, ranging from 0.1 to 0.4 M. AlCl3 inhibits the enzyme at 5 mM, and at higher salt concentrations over 0.1 M, the catalytic activity is completely inhibited
additional information
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PEGylation of GOx provides stability against denaturation or hydrolytic cleavage, glycosylation site-targeted PEGylation of glucose oxidase retains native enzymatic activity, bioconjugate's potential of the enzyme in an optical biosensing assay, overview. The bioconjugate is entrapped within a poly(2-hydroxyethyl methacrylate) hydrogel containing an oxygen-sensitive phosphor, and the construct is shown to respond approximately linearly over the physiologically-relevant glucose range, overview
additional information
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construction of a nanodevice coupled with an integrated real-time detection system for evaluation of the function of biomolecules in biological processes, and enzymatic reaction kinetics occurring at the confined space or interface. A nanochannel-enzyme system in which the enzymatic reaction is coupled with an electrochemical method is constructed. The model system is established by covalently linking glucose oxidase (GOD) onto the inner wall of the nanochannels of the porous anodic alumina (PAA)membrane. For enzyme assembling, the PAA membranes are first treated with silane to form epoxy groups modified inner surface of PAA nanochannels. Then GOD is assembled onto the membrane and the inner wall of the nanochannels through a ring-opening reaction. An gold disc is attached at the end of the nanochannel of the PAA membrane as the working electrode for detection of H2O2 product of enzymatic reaction. The effects of ionic strength, amount of immobilized enzyme and pore diameter of the nanochannels on the enzymatic reaction kinetics are analysed, method evaluation, overview
additional information
construction of enzyme mutant B11 with a C-terminal fusion with Saccharomyces cerevisiae Aga2 protein, the fusion proteins display on the surface of yeast EBY100 cells and show 2fold increased activity compared to the wild-type enzyme at pH 5.5 Aga2-GOx fusion proteins in the yeast cell wall can also be used as immobilized catalysts for the production of gluconic acid. The yeast surface display is developed for the directed evolution of antibodies in Saccharomyces cerevisiae, and involves the fusion of antibody variable domains to Aga2p, the adhesion subunit of the yeast agglutinin protein. Aga2p binds via disulfide bonds to the membrane protein Aga1p, which is embedded in the membrane via a glycosylphosphatidylinositol (GPI) anchor. The Aga2-antibody fusion gene is cloned in the vector pCTCON, whereas the Aga1p gene is integrated into the yeast genome, but both are under the control of galactose-inducible promoters. The surface display system is used for the directed evolution of horseradish peroxidase and expression of GOx for applications in biofuel cells. The kcat of the wild-type and B11 fusion enzymes are 1.65fold and 1.30fold lower than of the non-fusion enzymes, respectively, and the Km values of the wild-type and B11 fusion enzymes are 1.52fold and 1.74fold higher than of the non-fusion enzymes, respectively
additional information
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construction of enzyme mutant B11 with a C-terminal fusion with Saccharomyces cerevisiae Aga2 protein, the fusion proteins display on the surface of yeast EBY100 cells and show 2fold increased activity compared to the wild-type enzyme at pH 5.5 Aga2-GOx fusion proteins in the yeast cell wall can also be used as immobilized catalysts for the production of gluconic acid. The yeast surface display is developed for the directed evolution of antibodies in Saccharomyces cerevisiae, and involves the fusion of antibody variable domains to Aga2p, the adhesion subunit of the yeast agglutinin protein. Aga2p binds via disulfide bonds to the membrane protein Aga1p, which is embedded in the membrane via a glycosylphosphatidylinositol (GPI) anchor. The Aga2-antibody fusion gene is cloned in the vector pCTCON, whereas the Aga1p gene is integrated into the yeast genome, but both are under the control of galactose-inducible promoters. The surface display system is used for the directed evolution of horseradish peroxidase and expression of GOx for applications in biofuel cells. The kcat of the wild-type and B11 fusion enzymes are 1.65fold and 1.30fold lower than of the non-fusion enzymes, respectively, and the Km values of the wild-type and B11 fusion enzymes are 1.52fold and 1.74fold higher than of the non-fusion enzymes, respectively
additional information
glucose oxidase is chemically modified to increase the stability of GOx using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and sodium benzoate or aniline. The modification forms an amide bond between benzoate and lysines or aniline with glutamate and aspartate residues. The labeling of primary amines (lysines and the N-terminus) by benzoate is measured through a trinitrobenzene sulfonic acid (TNBS) assay
additional information
glucose oxidase is immobilized on mesoporous SBA-15 silica and two mesocellular foams (MCF) characterized by similar surface area and pore volumes but different pore/cell dimensions, covalent grafting of the enzyme through amide bonds, overview. The immobilized protein activity is significantly higher for the mesocellular foam with both cells and windows size larger than the enzyme dimensions. Enzyme GOx exhibits higher thermal stability when immobilized on the mesocellular foam compared to thefree enzyme
additional information
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in situ RAFT polymerization of four different monomers including acrylic acid (AA), methyl acrylate (MA), poly (ethylene glycol) acrylate (PEG-A) and tert-butyl acrylate (TBA) are polymerized directly on the surface of enzyme GOx to afford GOx-poly (PEG-A)(GOx-PPEG-A), GOx-poly(MA)(GOx-PMA), GOx-poly(AA)(GOx-PAA), and GOx-poly(TBA)(GOx-PTBA) conjugates, respectively. PAA and PPEG-A represent the hydrophilic polymers, while PMA and PTBA stand for the hydrophobic ones. Higher bioactivity is obtained for GOx modified with hydrophilic polymers compared with that modified with hydrophobic ones. All the tested polymers can enhance the stability of the GOx, while the hydrophobic GOx-polymers conjugates exhibit much better stability than the hydrophilic ones. Method overview
additional information
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the enzyme adopts a stable secondary conformation with some degree of freedom at active sites under acidic-neutral pH values, when either free in solution or immobilized on Nafion. Immobilization on Nafion actually increases the amount of active enzyme (Vmax) and affinity for glucose (inversely proportional to Km) at pH 6.0
additional information
usage of a strategy that combined random and rational approaches to isolate uncharacterized mutations of Aspergillus niger glucose oxidase with improved properties. GOX library construction in Saccharomyces cerevisiae and random mutagenesis and screening for mutants with improved thermal stability
additional information
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usage of a strategy that combined random and rational approaches to isolate uncharacterized mutations of Aspergillus niger glucose oxidase with improved properties. GOX library construction in Saccharomyces cerevisiae and random mutagenesis and screening for mutants with improved thermal stability
additional information
mutant glucose oxidase (B11-GOx) is obtained from directed protein evolution and wild-type enzyme. Higher glucose oxidation currents are obtained from B11-GOx both in solution and polymer electrodes compared to wild type enzyme. Improved electrocatalytic activity towards electrochemical oxidation of glucose from the mutant enzyme. The enzyme electrode with the mutant enzyme B11-GOx shows a faster electron transfer indicating a better electronic interaction with the polymer mediator
additional information
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usage of a strategy that combined random and rational approaches to isolate uncharacterized mutations of Aspergillus niger glucose oxidase with improved properties. GOX library construction in Saccharomyces cerevisiae and random mutagenesis and screening for mutants with improved thermal stability
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additional information
the removal of aromatic or bulky residues at positions 73, 418 or 430 result in decreases in the maximum rates of glucose oxidation to less than 1/90
additional information
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the removal of aromatic or bulky residues at positions 73, 418 or 430 result in decreases in the maximum rates of glucose oxidation to less than 1/90
additional information
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for use on electrode surfaces, the key amino acid at the entrance of the active site of glucose oxidase from Penicilium amagasakiense, Lys424, Gln75, Gln184, and Gly423, are redesign by nonactive site mutations, leading to enzymatic anodes with 2.4fold higher current densities, making the biosensor more effective. 424 is the key position
additional information
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the removal of aromatic or bulky residues at positions 73, 418 or 430 result in decreases in the maximum rates of glucose oxidation to less than 1/90
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additional information
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construction of a Penicillium funiculosum highly active strain 46.1 from parental strain BIM F-15 as a producer of extracellular GOx by induced mutagenesis technique. The GOx from Penicillium funiculosum strain 46.1 differs from GOx purified from the parent strain BIM F-15 by reduced Michaelis constant, higher efficiency of glucose oxidation, pH dependence, and thermal stability, but it has similar thermal optimum. The enzyme-encoding gene has no special mutation
additional information
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construction of a Penicillium funiculosum highly active strain 46.1 from parental strain BIM F-15 as a producer of extracellular GOx by induced mutagenesis technique. The GOx from Penicillium funiculosum strain 46.1 differs from GOx purified from the parent strain BIM F-15 by reduced Michaelis constant, higher efficiency of glucose oxidation, pH dependence, and thermal stability, but it has similar thermal optimum. The enzyme-encoding gene has no special mutation
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