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Literature summary for 1.14.13.92 extracted from

  • Parra, L.P.; Acevedo, J.P.; Reetz, M.T.
    Directed evolution of phenylacetone monooxygenase as an active catalyst for the Baeyer-Villiger conversion of cyclohexanone to caprolactone (2015), Biotechnol. Bioeng., 112, 1354-1364 .
    View publication on PubMed

Application

Application Comment Organism
synthesis phenylacetone monooxygenase (PAMO) is an exceptionally robust Baeyer-Villiger monooxygenase, which makes it ideal for potential industrial applications, usage as an active catalyst for the Baeyer-Villiger conversion of cyclohexanone to caprolactone, which is important as monomer in polymer science Thermobifida fusca

Protein Variants

Protein Variants Comment Organism
A442P random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 81% conversion rate Thermobifida fusca
A442P/ L443I/S444Q random and site-directed mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 43% conversion rate Thermobifida fusca
A442P/ L443V/S444Q random and site-directed mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 45% conversion rate Thermobifida fusca
A442P/L443I random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 45% conversion rate Thermobifida fusca
A442P/L443L/S444Q random and site-directed mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 41% conversion rate Thermobifida fusca
A442P/L443T/S444Q random and site-directed mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 56% conversion rate Thermobifida fusca
A442P/L443V random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 90% conversion rate Thermobifida fusca
A442P/L443W random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 74% conversion rate Thermobifida fusca
A442P/L443W/ S444Q random and site-directed mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 33% conversion rate Thermobifida fusca
L443V random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 53% conversion rate Thermobifida fusca
L443V/S444M random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 53% conversion rate Thermobifida fusca
L443V/S444Q random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 40-45% conversion rate Thermobifida fusca
L443V/S444Q random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 59% conversion rate Thermobifida fusca
L443V/S444T random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 57% conversion rate Thermobifida fusca
additional information directed evolution of phenylacetone monooxygenase as an active catalyst for the Baeyer-Villiger conversion of cyclohexanone to caprolactone using iterative saturation mutagenesis, mutant screening, overview. Molecular dynamics simulations and induced fit docking of wild-type and mutant enzymes with cyclohexanone. The mutants are used in the whole cell system of Escherichia coli cells Thermobifida fusca
Q93N/P94D/P440F random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at low rate Thermobifida fusca
S441D/A442E random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 73% conversion rate Thermobifida fusca
S441G/A442P/L443T/S444Q site-directed mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at about 90% conversion rate Thermobifida fusca
S441G/A442T random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 48% conversion rate Thermobifida fusca
S441H random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 34% conversion rate Thermobifida fusca
S441H/A442P random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 78% conversion rate Thermobifida fusca

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.266
-
cyclohexanone pH 8.0, 25┬░C, recombinant mutant S441G/A442P/L443T/S444Q Thermobifida fusca
0.698
-
cyclohexanone pH 8.0, 25┬░C, recombinant mutant A442P/L443V Thermobifida fusca

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
phenylacetone + NADPH + H+ + O2 Thermobifida fusca
-
benzyl acetate + NADP+ + H2O
-
?

Organism

Organism UniProt Comment Textmining
Thermobifida fusca Q47PU3
-
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
cyclohexanone + NADPH + H+ + O2 substrate of enzyme mutants, not of wild-type, overview Thermobifida fusca epsilon-caprolactone + NADP+ + H2O
-
?
phenylacetone + NADPH + H+ + O2
-
Thermobifida fusca benzyl acetate + NADP+ + H2O
-
?

Synonyms

Synonyms Comment Organism
PAMO
-
Thermobifida fusca

Temperature Optimum [┬░C]

Temperature Optimum [┬░C] Temperature Optimum Maximum [┬░C] Comment Organism
25
-
in vitro assay at Thermobifida fusca
30 37 in vivo assay at Thermobifida fusca

Temperature Stability [┬░C]

Temperature Stability Minimum [┬░C] Temperature Stability Maximum [┬░C] Comment Organism
57.5
-
Tm value for enzyme PAMO mutant A442P/L443V Thermobifida fusca
58.5
-
Tm value for enzyme PAMO mutant S441G/A442P/L443T/S444Q Thermobifida fusca
60.5
-
Tm value for wild-type enzyme PAMO Thermobifida fusca

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.156
-
cyclohexanone pH 8.0, 25┬░C, recombinant mutant S441G/A442P/L443T/S444Q Thermobifida fusca
0.304
-
cyclohexanone pH 8.0, 25┬░C, recombinant mutant A442P/L443V Thermobifida fusca

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
in vitro assay at Thermobifida fusca

Cofactor

Cofactor Comment Organism Structure
NADPH
-
Thermobifida fusca

General Information

General Information Comment Organism
additional information molecular dynamics simulations and induced fit docking of wild-type and mutant enzymes with cyclohexanone Thermobifida fusca

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.436
-
cyclohexanone pH 8.0, 25┬░C, recombinant mutant A442P/L443V Thermobifida fusca
0.577
-
cyclohexanone pH 8.0, 25┬░C, recombinant mutant S441G/A442P/L443T/S444Q Thermobifida fusca