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H75N
mutation decreases the binding of methyl red and nitrofurazone and has no effect on the bining of NADPH
K109A
K109 might only be involved in the binding of the 2'-phosphate group of NADPH and have no effect on the binding of NADH
K109H
K109 might only be involved in the binding of the 2'-phosphate group of NADPH and have no effect on the binding of NADH
Y74W
mutation decreases the binding of methyl red and nitrofurazone and has no effect on the bining of NADPH
A123F
36% of wild-type activity
D184G
complete loss of activity
E16G
46% of wild-type activity
F127G
complete loss of activity
L59G
10% of wild-type activity
N106A
3% of wild-type activity
N121A
170% of wild-type activity
R18G
9% of wild-type activity
R21G
382% of wild-type activity
R66A
47% of wild-type activity
V122Y
14% of wild-type activity
W105A
-
mutant, complete loss of both affinity for FMN and enzyme activity
W105F
-
mutant, lower Vmax value, decrease 30.6fold
W105G
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mutant, complete loss of both affinity for FMN and enzyme activity
W105H
-
mutant, lower Vmax value, decrease 8.2fold
W105Q
-
mutant, lower Vmax value, decrease 68.2fold
W105Y
-
mutant, substitution does not significantly decrease the Vmax of the enzyme, 22% reduction
W62A
35% of wild-type activity
Y129G
complete loss of activity
F162A
Phe-162 is chosen because it is predicted to participate in the substrate binding on top of the isoalloxazine ring, as observed in the AzoR-inhibitor structure
R59A
the results indicate that Arg-59 decides the substrate specificity of AzoR
R59G
site-directed mutagenesis, the interaction of FMN with substrates methyl red and methyl orange changes from van der Waals interaction in the wild-type to hydrogen bonding in the mutant, the association constants decrease
Y120A
Tyr-120 is chosen because it is predicted to participate in the substrate binding on top of the isoalloxazine ring, as observed in the AzoR-inhibitor structure
L49C/D108C
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the mutant enzyme shows increased thermal stability at 50°C (increased half-life from 12.6 min in wild type to 26.66 min in the mutant enzyme). The mutant enzyme can also tolerate 5% (w/v) NaCl and retains 30% of original activity after 24 h incubation
Y131F
the mutant shows increased specific activity with methyl red and reduced specific activity with balsalazide compared to the wild type enzyme
Q192R
-
site-directed mutagenesis, analysis of initial activity and thermostability (at 55°C, 60 min) relative to parental mutant variant B1G6
Q192R/A46P/V159A
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site-directed mutagenesis, analysis of initial activity and thermostability (at 55°C, 90 min) relative to parental mutant variant 16B7
Q192R/A46P/V159A/A48P
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site-directed mutagenesis, analysis of initial activity and thermostability (at 60°C, 45 min) relative to parental mutant variant 2A1
Q192R/A46P/V159A/C129S
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site-directed mutagenesis, analysis of initial activity and thermostability (at 60°C, 45 min) relative to parental mutant variant 23C10
Q192R/A46P/V159A/C129S/A178D/A31S/K74E/A88G/L143Q
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site-directed mutagenesis, analysis of initial activity and thermostability (at 85°C, 150 min) relative to parental mutant variant 2F11
Q192R/A46P/V159A/C129S/A178D/A77T/F98L/N131D
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site-directed mutagenesis, analysis of initial activity and thermostability (at 85°C, 150 min) relative to parental mutant variant 3B9
Q192R/A46P/V159A/C129S/A178D/A88G/N131D/L143Q
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site-directed mutagenesis, analysis of initial activity and thermostability (at 85°C, 150 min) relative to parental mutant variant 1B6
Q192R/A46P/V159A/C129S/A178D/K74E/L143Q
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site-directed mutagenesis, analysis of initial activity and thermostability (at 85°C, 150 min) relative to parental mutant variant 2E4
Q192R/A46P/V159A/C129S/A178D/N131D/L143Q
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site-directed mutagenesis, analysis of initial activity and thermostability (at 85°C, 150 min) relative to parental mutant variant 6F11
Q192R/A46P/V159A/C129S/A77T/N131D
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site-directed mutagenesis, analysis of initial activity and thermostability (at 80°C, 60 min) relative to parental mutant variant 14D4
Q192R/A46P/V159A/C129S/D7H/A178D
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site-directed mutagenesis, analysis of initial activity and thermostability (at 80°C, 60 min) relative to parental mutant variant 13G10
Q192R/A46P/V159A/C129S/E36D/L143Q
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site-directed mutagenesis, analysis of initial activity and thermostability (at 80°C, 60 min) relative to parental mutant variant 1C11
Q192R/A46P/V159A/C129S/I6V/T79R/Y179H
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site-directed mutagenesis, analysis of initial activity and thermostability (at 80°C, 60 min) relative to parental mutant variant 32F5
Q192R/A46P/V159A/C129S/K74E/A88G
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site-directed mutagenesis, analysis of initial activity and thermostability (at 80°C, 60 min) relative to parental mutant variant 23C5
Q192R/A46P/V159A/C129S/L161M/L169P
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site-directed mutagenesis, analysis of initial activity and thermostability (at 80°C, 60 min) relative to parental mutant variant 27E4
Q192R/A46P/V159A/C129S/N14D/L143Q
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site-directed mutagenesis, analysis of initial activity and thermostability (at 80°C, 60 min) relative to parental mutant variant 6F10
Q192R/A46P/V159A/C129S/Y179H
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site-directed mutagenesis, analysis of initial activity and thermostability (at 80°C, 60 min) relative to parental mutant variant 23E4
Q192R/A46P/V159A/Y179H
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site-directed mutagenesis, analysis of initial activity and thermostability (at 60°C, 45 min) relative to parental mutant variant 19E4
Q192R/Y179H
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site-directed mutagenesis, analysis of initial activity and thermostability (at 55°C, 90 min) relative to parental mutant variant 12B8
Y179H
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site-directed mutagenesis, analysis of initial activity and thermostability (at 55°C, 60 min) relative to parental mutant variant K7E3
additional information
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deletion of genes omcB, omcC, and omcE encoding outer membrane proteins of Geobacter sulfurrescens modestly inhibit the decolorization activity by 16%, 42%, and 35%, respectively
additional information
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deletion of genes omcB, omcC, and omcE encoding outer membrane proteins of Geobacter sulfurrescens modestly inhibit the decolorization activity by 16%, 42%, and 35%, respectively
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additional information
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improvement of the kinetic and thermodynamic stability of the azoreductase by directed evolution via rational design approaches, five rounds of mutagenesis/recombination are followed by high-throughput screening. Mutant 1B6 shows a 300fold higher half-life at 50°C compared to the wild-type enzyme. mutant 1B6 has a folded state slightly less stable than the wild-type (with lower melting and optimal temperatures) but in contrast is more resistant to irreversible denaturation. The superior kinetic stability of 1B6 variant is therefore related to an increased resistance of the unfolded monomers to aggregation through the introduction of mutations that disturb hydrophobic patches and increase the surface net charge of the protein. Mutants 2A1 and 2A1-Y179H show increased thermodynamic stability with a 10-20°C higher melting temperature than wild-type, these residues are mostly involved in strengthening the solvent-exposed loops or the inter-dimer interactions of the folded state. Molecular details of mutations that improve stability, overview
additional information
construction of a chimeric artificial bifunctional enzyme chimeric enzyme composed of the two oligomeric enzymes, azoreductase (AZR) from Shewanella oneidensis MR-1 and the alkaline-resistant NAD(P)-dependent glucose 1-dehydrogenase (LsGDH) from Lysinibacillus sphaericus G10. The reaction is extended to a recycling system. The fusion enzyme exhibits a higher stability and suitability for high-temperature application than the parental wild-type enzyme
additional information
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construction of a chimeric artificial bifunctional enzyme chimeric enzyme composed of the two oligomeric enzymes, azoreductase (AZR) from Shewanella oneidensis MR-1 and the alkaline-resistant NAD(P)-dependent glucose 1-dehydrogenase (LsGDH) from Lysinibacillus sphaericus G10. The reaction is extended to a recycling system. The fusion enzyme exhibits a higher stability and suitability for high-temperature application than the parental wild-type enzyme
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