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Literature summary for 1.14.13.22 extracted from

  • Schmidt, S.; Genz, M.; Balke, K.; Bornscheuer, U.T.
    The effect of disulfide bond introduction and related Cys/Ser mutations on the stability of a cyclohexanone monooxygenase (2015), J. Biotechnol., 214, 199-211 .
    View publication on PubMed
Search for more literature for 1.14.13.22

Application

Application Comment Organism
synthesis cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871 is a prototype Baeyer-Villiger monooxygenases (BVMO). The enzyme shows an impressive substrate scope with a high chemo-, regio- and/or enantioselectivity. BVMO reactions are often difficult, if not impossible to achieve by chemical approaches and this makes these enzymes highly desired candidates for industrial applications. The industrial use is hampered by several factors related to the lack of stability of these biocatalysts. An easy computational method is used for the prediction of stabilizing disulfide bonds in the cyclohexanone monooxygenase-scaffold. The most promising predicted disulfide pairs are created and biochemically characterized. The T415C single point variant is the most stable variant with a 30fold increased long-term stability (33% residual activity after 24 h incubation at 25°C) Acinetobacter johnsonii

Cloned(Commentary)

Cloned (Comment) Organism
expression in Escherichia coli Acinetobacter johnsonii

Protein Variants

Protein Variants Comment Organism
T415C most stable variant with a 30fold increased long-term stability (33% residual activity after 24 h incubation at 25°C), the melting temperature of the variant is increased by 6°C Acinetobacter johnsonii
T415C/A463C the melting temperature of the variant is increased by 5°C with simultaneous improved long-term stability Acinetobacter johnsonii
Y411C/A463C the most oxidatively stable variant Y411C-A463C retains nearly 60% activity after incubation with 25 mM H2O2 whereas the wild type retains only 16% Acinetobacter johnsonii

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.003
-
 cyclohexanone 25°C, pH 8.0, mutant enzyme P254C/D290C Acinetobacter johnsonii
0.0046
-
 cyclohexanone 25°C, pH 8.0, wild-type enzyme Acinetobacter johnsonii
0.0109
-
 cyclohexanone 25°C, pH 8.0, mutant enzyme T415C/A463C Acinetobacter johnsonii
0.0138
-
 cyclohexanone 25°C, pH 8.0, mutant enzyme Y411C/A463C Acinetobacter johnsonii
0.0209
-
 cyclohexanone 25°C, pH 8.0, mutant enzyme T415C Acinetobacter johnsonii

Organism

Organism UniProt Comment Textmining
Acinetobacter johnsonii Q9R2F5
-
-

Purification (Commentary)

Purification (Comment) Organism
-
 Acinetobacter johnsonii

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4-methylcyclohexanone + NADPH + H+ + O2
-
 Acinetobacter johnsonii ?
-
 ?
cyclohexanone + NADPH + H+ + O2
-
 Acinetobacter johnsonii hexano-6-lactone + NADP+ + H2O
-
 ?

Synonyms

Synonyms Comment Organism
CHMO prototype Baeyer-Villiger monooxygenases (BVMO) Acinetobacter johnsonii

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
25
-
 mutant T415C is the most stable variant with a 30fold increased long-term stability, 33% residual activity after 24 h incubation at 25°C Acinetobacter johnsonii
31.6
-
 Tm-value of wilde-type enzyme Acinetobacter johnsonii
36.4
-
 Tm-value of mutant enzyme T415C/A463C Acinetobacter johnsonii
37.5
-
 Tm-value of mutant enzyme T415C Acinetobacter johnsonii

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
6.1
-
 cyclohexanone 25°C, pH 8.0, mutant enzyme P254C/D290C Acinetobacter johnsonii
6.15
-
 cyclohexanone 25°C, pH 8.0, mutant enzyme Y411C/A463C Acinetobacter johnsonii
38.5
-
 cyclohexanone 25°C, pH 8.0, wild-type enzyme Acinetobacter johnsonii
44.9
-
 cyclohexanone 25°C, pH 8.0, mutant enzyme T415C/A463C Acinetobacter johnsonii
57.4
-
 cyclohexanone 25°C, pH 8.0, mutant enzyme T415C Acinetobacter johnsonii