Enhancing the Activity of a Dietzia sp. D5 Baeyer-Villiger Monooxygenase towards Cyclohexanone by Saturation Mutagenesis

A recombinant Baeyer-Villiger monooxygenase, BVMO4 from Dietzia sp. D5 has been previously reported. The enzyme exhibited good thermostability and was active with a wide range of cyclic ketone substrates but catalysed poorly the conversion of cyclohexanone to caprolactone. The present work focuses on protein engineering of BVMO4 to improve the conversion of cyclohexanone. Thus, a structural model of BVMO4 was generated and used in combination with literature information on substrate specificity of BVMOs to identify ‘hotspots’ whose mutation would influence substrate specificity. Site saturation mutagenesis was performed on 12 selected sites and 528 mutants were screened with expected coverage of about 98 %. About one-fourth of the mutants screened exhibited more than 50 % increase in cyclohexanone oxidation activity. Compared to the wild type BVMO, the best mutants, Y499I, Y499F and Y499 L have shown about 12-fold increase for caprolactone production.