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Literature summary for 1.14.13.22 extracted from

  • van Beek, H.L.; Romero, E.; Fraaije, M.W.
    Engineering cyclohexanone monooxygenase for the production of methyl propanoate (2017), ACS Chem. Biol., 12, 291-299 .
    View publication on PubMed

Application

Application Comment Organism
synthesis the enzyme catalyzes the Baeyer-Villiger oxidation of 2-butanone, yielding ethyl acetate and methyl propanoate as products. Methyl propanoate is of industrial interest as a precursor of acrylic plastic. Various residues near the substrate and NADP+ binding sites are subjected to saturation mutagenesis to enhance both the activity on 2-butanone and the regioselectivity toward methyl propanoate Acinetobacter calcoaceticus

Cloned(Commentary)

Cloned (Comment) Organism
expression in Escherichia coli Acinetobacter calcoaceticus

Protein Variants

Protein Variants Comment Organism
F281H cultures expressing the F281H variant produce 30 to 40% methyl propanoate, while the wild-type enzyme produce 26% methyl propanoate Acinetobacter calcoaceticus
I491A cultures expressing the I491A variant produce 30 to 40% methyl propanoate, while the wild-type enzyme produce 26% methyl propanoate Acinetobacter calcoaceticus
I491A mutant exhibits a significant improvement over the wild type enzyme in the desired regioselectivity using 2-butanone as a substrate (40% vs 26% methyl propanoate, respectively) Acinetobacter calcoaceticus
L244R cultures expressing the L244R variant produce 30 to 40% methyl propanoate, while the wild-type enzyme produce 26% methyl propanoate Acinetobacter calcoaceticus
L435N cultures expressing the L435N variant produce 30 to 40% methyl propanoate, while the wild-type enzyme produce 26% methyl propanoate Acinetobacter calcoaceticus
T56S mutant, which exhibits a higher conversion yield (92%) and kcat (0.5/s) than wild type AcCHMO (52% and 0.3/s, respectively). The uncoupling rate for the T56S mutant is also significantly lower than that for the wild type enzyme Acinetobacter calcoaceticus
T56S/I491A mutant with higher conversion and improved regioselectivity Acinetobacter calcoaceticus
T56S/L435N/I491A mutant produces less total product than wild-type enzyme Acinetobacter calcoaceticus

Organism

Organism UniProt Comment Textmining
Acinetobacter calcoaceticus
-
-
-

Purification (Commentary)

Purification (Comment) Organism
-
Acinetobacter calcoaceticus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2-butanone + NADPH + H+ + O2
-
Acinetobacter calcoaceticus methyl propanoate + NADP+ + H2O
-
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Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.16
-
2-butanone pH 7.0, 25°C, mutant enzyme L435N Acinetobacter calcoaceticus
0.19
-
2-butanone pH 7.0, 25°C, mutant enzyme I491A Acinetobacter calcoaceticus
0.2
-
2-butanone pH 7.0, 25°C, mutant enzyme F281H Acinetobacter calcoaceticus
0.24
-
2-butanone pH 7.0, 25°C, mutant enzyme T56S/L244R/I491 Acinetobacter calcoaceticus
0.26
-
2-butanone pH 7.0, 25°C, mutant enzyme T56S/I491A Acinetobacter calcoaceticus
0.28
-
2-butanone pH 7.0, 25°C, wild-type enzyme Acinetobacter calcoaceticus
0.31
-
2-butanone pH 7.0, 25°C, mutant enzyme L244R Acinetobacter calcoaceticus
0.33
-
2-butanone pH 7.0, 25°C, mutant enzyme T56S/L435N/I491 Acinetobacter calcoaceticus
0.41
-
2-butanone pH 7.0, 25°C, mutant enzyme T56S/L244R Acinetobacter calcoaceticus
0.47
-
2-butanone pH 7.0, 25°C, mutant enzyme T56S Acinetobacter calcoaceticus