The gene encoding a Baeyer-Villiger monooxygenase and identified in Pseudomonas putida KT2440 was cloned and functionally expressed in Escherichia coli. The highest yield of soluble protein could be achieved by co-expression of molecular chaperones. In order to determine the substrate specificity, biocatalyses were performed using crude cell extract, growing and resting cells. Examination of aromatic, cyclic and aliphatic ketones revealed a high specificity towards short-chain aliphatic ketones. Interestingly, some open-chain ketones were converted to the alkylacetates, while for others formation of the ester products with oxygen on the other side of the keto group could also be detected yielding the corresponding methyl or ethyl esters.
The Baeyer-Villiger monooxygenase (BVMO) BmoF1 from Pseudomonas fluorescens DSM 50106 was shown before to enantioselectively oxidize different 4-hydroxy-2-ketones to the corresponding hydroxyalkyl acetates, being the first example of a BVMO-catalyzed kinetic resolution of aliphatic acyclic ketones. However, the wild-type enzyme exhibited only moderate E values (E approximately 55). Thus, the enantioselectivity was enhanced by means of directed evolution and optimization of reaction conditions since it was found that higher E values (E approximately 70 for wild-type BmoF1) could already be obtained when performing biotransformations in shake flasks rather than small tubes. In a first step, random mutations were introduced by error-prone polymerase chain reaction, and BmoF1 mutants (>3,500 clones) were screened for improved activity and enantioselectivity using a microtiter-plate-based screening method. Mutations S136L and L252Q were found to increase conversion compared to wild type, while several mutations (H51L, F225Y, S305C, and E308V) were identified enhancing the enantioselectivity to a varying extent (E approximately 75-90). In a second step, beneficial mutations were recombined by consecutive cycles of QuikChange site-directed mutagenesis resulting in a double mutant (H51L/S136L) showing both improved conversion and enantioselectivity (E approximately 86).
A gene encoding a Baeyer-Villiger monooxygenase (BVMO) identified in Pseudomonas fluorescens DSM 50106 was cloned and functionally expressed in Escherichia coli JM109. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis showed an estimated 56 kDa-size protein band corresponding to the recombinant enzyme. Expression in BL21 (DE3) resulted mainly in the formation of inclusion bodies. This could be overcome by coexpression of molecular chaperones, especially the DnaK/DnaJ/GrpE complex, leading to increased production of soluble BVMO enzyme in recombinant E. coli. Examination of the substrate spectra using whole-cell biocatalysis revealed a high specificity of the BVMO for aliphatic open-chain ketones. Thus, octyl acetate, heptyl propionate, and hexyl butyrate were quantitatively formed from the corresponding ketone substrates. Several other esters were obtained in conversion >68%. Selected esters were also produced on preparative scale.
For the investigation of the NADPH-dependent Baeyer-Villiger monooxygenase MekA from Pseudomonas veronii MEK700, the encoding gene mekA with a C-terminal strep-tag was cloned and expressed under the control of a L: -rhamnose inducible promoter from Escherichia coli. The mekA gene was found by analyzing the methylethylketone (MEK) degradation pathway by Onaca et al. J Bacteriol 189:3759-3767, 2007. Sequence analysis of the corresponding protein, which catalyzes the Baeyer-Villiger oxidation of MEK to ethyl acetate, showed two binding sites (Rossman-fold motifs) for cofactors NAD(P)H and FAD. Although expression of mekA resulted in large amounts of inclusion bodies compared to soluble protein, high amounts of purified and active MekA were obtained by affinity chromatography. The substrate spectrum of MekA was investigated with purified enzyme and whole cells using a variety of aliphatic, aromatic, and cyclic ketones including four chiral substrates. The specific activity of MekA with MEK as substrate was determined to be 1.1 U/mg protein. K (M) values were determined for MEK and the cofactors NADPH and NADH to be 6, 11, and 29 microM, respectively.
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