Cloning, expression, and characterization of a Baeyer-Villiger monooxygenase from Pseudomonas fluorescens DSM 50106 in E. coli.
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.
Kirschner
Anett
A
Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Greifswald University, Friedrich-Ludwig-Jahn-Str. 18c, 17487, Greifswald, Germany.
Altenbuchner
Josef
J
Bornscheuer
Uwe T
UT
D000085:Acetates / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001711:Biotransformation; D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000032:analysis / Q000302:isolation & purification; D015870:Gene Expression; D002479:Inclusion Bodies; D007659:Ketones / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018832:Molecular Chaperones / Q000096:biosynthesis / Q000235:genetics; D008970:Molecular Weight; D020543:Proteome / Q000032:analysis / Q000302:isolation & purification; D011551:Pseudomonas fluorescens / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D000085:Acetates; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D007659:Ketones; D018832:Molecular Chaperones; D020543:Proteome; D011994:Recombinant Proteins; D006899:Mixed Function Oxygenases; C059676:octyl acetate