IQUIR   05412
INSTITUTO DE QUIMICA ROSARIO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
BIOCATALYTIC CHARACTERIZATION OF THREE BACTERIAL BAEYER-VILLIGER MONOOXYGENASES
Autor/es:
BIANCHI DARIO A.; RIAL, DANIELA V.; CECCOLI, ROMINA D.
Reunión:
Congreso; Reunión conjunta SAIB-SAMIGE 2020 (on line) (LVI SAIB Meeting - XV SAMIGE Meeting; 2020
Institución organizadora:
Sociedad Argentina de Investigación en Bioquímica y Biología Molecular - Asociación Civil de Microbiología
Resumen:
Baeyer-Villiger monooxygenases (BVMOs) are flavoenzymes that catalyze the insertion of one atom of oxygen from molecular oxygen into the substrate while the other one is reduced to water. Ketones are the typical BVMO substrates, therefore their oxidation produces esters or lactones in an environmentally friendly reaction. We detected seven putative type I BVMOs in Bradyrhizobium diazoefficiens USDA 110 by genome mining. We cloned and functionally expressed in Escherichia coli three of these flavoenzymes, which we named BVMO2, BVMO4 and BVMO5. Each of these sequences belongs to a different group of an inferred phylogenetic tree of type I BVMOs. The aim of this work was to characterize new BVMOs in order to expand the set of this type of biocatalysts available for synthetic applications. First, we assessed their biocatalytic potential in whole cell systems by challenging them with several ketones as candidate substrates. We found out that these enzymes oxidize linear, aromatic, cyclic and bicyclic ketones but with different preferences. Then, we purified the recombinant BVMO2, BVMO4 and BVMO5 to homogeneity and characterized them in vitro. We determined the molar absorption coefficient of each of these enzymes, and investigated the dependence of their activities with the cofactor, temperature (20 to 45 °C) and pH (6.0 to 9.0) as well as their pH and temperature stability. We determined the steady-state kinetic parameters of the three recombinant flavoenzymes from B. diazoefficiens for phenylacetone and heptan-3-one by following the consumption of NADPH. We observed that the catalytic efficiency of BVMO2 was similar for both substrates, BVMO4 performed very well on phenylacetone under the established assay conditions, and both ketones were very good substrates for BVMO5. Thus, according to the substrate scope and selectivities obtained in vivo and the performance of each enzyme in vitro, we propose a complementary behavior among the three BVMOs. While BVMO2 oxidized ketones with variable structure, BVMO4 and BVMO5 showed a narrow substrate profile with preference for linear ketones and with particular regioselectivities for a bicyclic ketone tested. Hence, these enzymes expand our toolbox toward regioisomeric esters by the oxidation of certain linear ketones, providing eco-friendly alternatives for synthetic purposes.