EXPLORING THE SUBSTRATE PREFERENCE OF A NOVEL BAEYER-VILLIGER MONOOXYGENASE

ROMINA D. CECCOLI; BIANCHI, DARIO A.; RIAL, DANIELA V.

Rosario

Congreso; IX Congreso de Microbiología General; 2013

Sociedad Argentina de Microbiología General (SAMIGE)

Biocatalysis is a modern and green strategy to produce chemical compounds based on the use of biological systems as catalysts. Enzymes (both in their pure forms and as protein extracts), whole-cell systems (either native or recombinant microorganisms) or plant organs are the most widely accepted sources of biocatalysts. A great variety of compounds can be obtained chemo-enzymatically in convenient processes due to the high selectivity and efficiency of the enzymes and the mild conditions required for enzymatic reactions. Within oxidative biocatalysts, Baeyer-Villiger monooxygenases (BVMOs) bécame invaluable tools for the production of lactones using cyclic ketones as substrates. These enzymes comprise FAD- or FMN-dependent oxidoreductases that catalyze the insertion of an oxygen atom from molecular oxygen next to a carbonyl group at the expense of NAD(P)H and the other atom is reduced to water. Lactones are very versatile precursors for the synthesis of natural products, analogs and bioactive compounds. Moreover, some BVMOs can catalyze the oxidization of linear ketones as well as selenium- and boron-containing compounds, sulfoxidations, epoxidations and N-oxidations. The aim of our study is to expand the number of biocatalysts available for chemical applications. In this work we explored the predicted proteome of Leptospira biflexa (Paris) using protein sequences of known BVMOs as queries for protein blast searches and, as a result, one putative BVMO sequence was retrieved. To examine the relationships between the identified sequence and previously characterized BVMOs, they were aligned and phylogenetic trees were inferred. The putative BVMO from L. biflexa is related to BVMOs with variable substrate preferences. This novel protein exhibits the characteristic consensus sequence and dinucleotide-binding motifs of BVMOs. In order to evaluate its substrate preference, the identified BVMO-encoding gene was cloned and functionally expressed in Escherichia coli BL21(DE3). Whole-cell biotransformations were carried out and linear aliphatic, monocyclic, bicyclic and aromatic ketones were tested as substrates. We observed that the BVMO from L. biflexa was able to oxidize some cyclic compounds and linear short-chain ketones.