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S24P/G182A/G196A/H222D/S250E/S254R
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the mutant CbADH-6M exhibits a favorable soluble and highly active expression with an activity of 46.3 U/ml, which is 16times higher than the wild-type (2.9 U/ml) and a more stable protein conformation with an enhanced thermal stability. The activity of CbADH-6M is upgraded to 2401.8 U/ml by high cell density fermentation strategy using recombinant Escherichia coli. Improving CbADH solubility by chaperone buffering. The efficiency for NADPH regeneration of the mutant enzyme is testified in the synthesis of some fine chiral aromatic alcohols coupling with another ADH from Lactobacillus kefir (LkADH). Method optimization, overview. Although the Asp225-His222 salt bridge in the original wild-type CbADH protein disappears due to the amino acid substitution H222D, the replacement of H222D, S250E and S254R leads to the formation of four new salt bridges including Arg254-Glu250, Arg254-Asp225, Arg254-Glu280 and Arg254-Asp222, which constitute a salt bridge network centered on Arg254
D275P
mutation significantly enhances the thermal stability of EhADH1
G37D
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regular PCR with site-specific mutation for both DNA strands, the gene is divided into two separate amplification products, preference for NAD+ rather than NADP+, depends on an a interaction between the adenosine ribose moiety of NAD+ and the inserted aspartate side chain
D194A
mutation in putative metal-coordinating residue, almost complete loss of activity
H198A
mutation in putative metal-coordinating residue, almost complete loss of activity
H267A
mutation in putative metal-coordinating residue, almost complete loss of activity
H281A
mutation in putative metal-coordinating residue, almost complete loss of activity
H363A
107% of wild-type activity
D194A
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mutation in putative metal-coordinating residue, almost complete loss of activity
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H198A
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mutation in putative metal-coordinating residue, almost complete loss of activity
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H267A
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mutation in putative metal-coordinating residue, almost complete loss of activity
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H281A
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mutation in putative metal-coordinating residue, almost complete loss of activity
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H363A
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107% of wild-type activity
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D194A
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mutation in putative metal-coordinating residue, almost complete loss of activity
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H198A
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mutation in putative metal-coordinating residue, almost complete loss of activity
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H267A
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mutation in putative metal-coordinating residue, almost complete loss of activity
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H281A
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mutation in putative metal-coordinating residue, almost complete loss of activity
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H363A
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107% of wild-type activity
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K245R
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mutation leads to improved thermostability
L54Q
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mutant retains 62% of its initial activity after heat treatment at 30°C for 6 h and mutation confers improved enantioselectivity
L54Q/K245R/N271D
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mutant displays improved thermostability and mutation confers improved enantioselectivity
L54Q/R104C
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mutation leads to improved thermostability and enantioselectivity
N271D
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mutation leads to improved thermostability
K245R
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mutation leads to improved thermostability
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L54Q
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mutant retains 62% of its initial activity after heat treatment at 30°C for 6 h and mutation confers improved enantioselectivity
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L54Q/K245R/N271D
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mutant displays improved thermostability and mutation confers improved enantioselectivity
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L54Q/R104C
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mutation leads to improved thermostability and enantioselectivity
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N271D
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mutation leads to improved thermostability
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A18P/K40N/Q165H/D182H/E202G/R219M
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mutant with increased specific activity towards 2,5-hexanedione compared to the wild type enzyme
G211C
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site-directed mutagenesis, cofactor binding and kinetic analysis compared to the wild-type enzyme
G211S
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site-directed mutagenesis, cofactor binding and kinetic analysis compared to the wild-type enzyme
K209T
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mutant with wild type specific activity towards 2,5-hexanedione
L176P
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mutant with the highest activity at 30°C with 2,5-hexanedione, the temperature optimum of the mutant is not changed (90°C), but the activity at lower temperature (60°C and below) is clearly increased when compared to the wild type enzyme
N86D/R213I
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mutant with increased specific activity towards 2,5-hexanedione compared to the wild type enzyme
T153A
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mutant with the highest activity at 30°C with 2,5-hexanedione, the temperature optimum of the mutant is not changed (90°C), but the activity at lower temperature (60°C and below) is clearly increased when compared to the wild type enzyme
V66A/L176P/Y229H
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mutant with increased specific activity towards 2,5-hexanedione compared to the wild type enzyme
P275D
mutation reduces the thermostability of the enzyme
W110A
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99.9% conversion of phenylacetone to 84.1% S-product
W110G
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99.9% conversion of phenylacetone to 79% S-product, 95.8% conversion of 1-phenyl-2-butanone to 91.6% S-product, 99.1% conversion of 4-phenyl-2-butanone to 70.5% S-product
W110I
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99.9% conversion of phenylacetone to 99.9% S-product, 99.4% conversion of 1-phenyl-2-butanone to 99.9% S-product, 99.1% conversion of 4-phenyl-2-butanone to 99.9% S-product
W110L
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99.9% conversion of phenylacetone to 99.9% S-product, 98.9% conversion of 1-phenyl-2-butanone to 99.9% S-product, 99.2% conversion of 4-phenyl-2-butanone to 99.9% S-product
W110M
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99.9% conversion of phenylacetone to 99.9% S-product, 97.3% conversion of 1-phenyl-2-butanone to 99.9% S-product, 99.3% conversion of 4-phenyl-2-butanone to 99.9% S-product
W110Q
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99.9% conversion of phenylacetone to 99.9% S-product, 83.5% conversion of 1-phenyl-2-butanone to 99.9% S-product, 99.1% conversion of 4-phenyl-2-butanone to 99.9% S-product
W110V
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99.9% conversion of phenylacetone to 99.9% S-product, 99.2% conversion of 1-phenyl-2-butanone to 99.9% S-product, 99.1% conversion of 4-phenyl-2-butanone to 99.9% S-product
F197W
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site-directed mutagenesis, kinetics compared to the wild-type enzyme
S196A
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site-directed mutagenesis, kinetics compared to the wild-type enzyme
S196A/F197W
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site-directed mutagenesis, kinetics compared to the wild-type enzyme
V84I
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site-directed mutagenesis, kinetics compared to the wild-type enzyme
V84I/Y127M
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site-directed mutagenesis, mutant KpADHV84I/Y127M exhibits a lower KM of 15.1 mM and a higher kcat of 30.1/ min than wild-type KpADH. The ratio of kcat/KM toward 2-hydroxytetrahydrofuran (2-HTHF), in comparison to 1,4-BD, in KpADHV84I/Y127M is dramatically reduced by almost 100fold compared to wild-type KpADH, which is advantageous for NADPH regeneration
V84I/Y12C
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site-directed mutagenesis, kinetics compared to the wild-type enzyme
Y127C
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site-directed mutagenesis, kinetics compared to the wild-type enzyme
Y127C/S196A
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site-directed mutagenesis, kinetics compared to the wild-type enzyme
Y127M
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site-directed mutagenesis, kinetics compared to the wild-type enzyme
R11L/A180V
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mutant with the highest activity at 30°C with 2,5-hexanedione, the temperature optimum of the mutant is not changed (90°C), but the activity at lower temperature (60°C and below) is clearly increased when compared to the wild type enzyme, the maximum specific activity of R11L/A180V with 2,5-hexanedione at 30°C is 10fold higher compared to the activity of the wild type enzyme
R11L/A180V
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the maximum specific activity of the mutant with 2,5-hexanedione at 30°C is 10fold higher compared to the activity of the wild type enzyme
additional information
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computational design of highly stable and soluble alcohol dehydrogenase mutants for NADPH regeneration, several mutants are created and evaluated, overview. A NADP+-specific ADH from Clostridium beijerinckii is engineered for cofactor recycling using an automated algorithm. The mutant is selected for large-scale production and industrial usage
additional information
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a yqhD knockout mutant does not confer resistance to potassium tellurite and other ROS elicitors
additional information
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evaluation of a dehydrogenase-acetone NADP-regeneration system for the enzymatic preparative-scale production of 12-ketochenodeoxycholic acid, overview
additional information
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insertion mutants of Ala, Gly, Ser or Cys between positions 211 and 212 (termed G211InsA, G211InsG, G211InsS, and G211InsC, respectively) are created by site-directed mutagenesis of the wild-type AdhD gene in the pET-20b vector. For some mutants (G211S, G211C, G211InsA and G211InsS), with NAD+ as a cofactor, the KA or KB values are observed to be higher than the maximum concentration of substrate or cofactor utilized. So, for mutants G211S, G211C, G211InsA and G211InsS, kinetic data are also measured using assay reaction mixtures containing 1-450 mM 2,3-butanediol and 1-5500 microM NAD+. In the case of NAD+, the on-rate of cofactor (kss 1 ) is found to decrease in all of the mutants except for the G211S and insertion of Gly (G211InsG). Kinetic analysis, detailed overview
additional information
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gene deletion mutant and combined gene deletion mutant lacking both ADH and ADH7, both are viable and show similar growth curves as wild-type
additional information
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adhA disruption mutant shows no differences in responses to heat shock, salt, or hyperosmotic stress as compared to the wild-type. The adhA mutant strain is unable to grow heterotrophically (added glucose) in darkness, contrary to the wild-type strain
additional information
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evaluation of a dehydrogenase-acetone NADP-regeneration system for the enzymatic preparative-scale production of 12-ketochenodeoxycholic acid, overview
additional information
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engineering an alcohol dehydrogenase for balancing kinetics in NADPH regeneration with 1,4-butanediol as a cosubstrate. Identification of the key residues of KpADH by molecular dynamics simulations