Cloning, biochemical characterization and molecular docking of novel thermostable β-glucosidase BglA9 from Anoxybacillus ayderensis A9 and its application in de-glycosylation of Polydatin

Abstract

This study reports a novel BglA9 gene of 1345 bp encoding β-glucosidase from Anoxybacillus ayderensis A9, which was amplified and expressed in E. coli BL21 (DE3): pLysS cells, purified with Ni-NTA column having molecular weight of 52.6 kDa and was used in the bioconversion of polydatin to resveratrol. The kinetic parameters values using pNPG as substrate were K_m (0.28 mM), V_max (43.8 μmol/min/mg), k_cat (38.43 s^-1) and k_cat/K_m (135.5 s^-1 mM^-1). The BglA9 was active in a broad pH range and had an activity half-life around 24 h at 50 °C. The de-glycosylation efficiency of BglA9 for polydatin was determined by estimating the amount of glucose released after enzymatic reaction by a dinitrosalicylic acid (DNS) assay. The kinetic parameters of BglA9 for polydatin were 5.5 mM, 20.84 μmol/min/mg, 18.28 s^-1and 3.27 s^-1 mM^-1 for K_m, V_max, k_cat, and k_cat/K_m values, respectively. The K_i value for glucose was determined to be 1.7 M. The residues Gln19, His120, Glu355, Glu409, Glu178, Asn222 may play a crucial role in the deglycosylation as revealed by the 3D structure of enzyme docked with polydatin.

Keywords: Anoxybacillus ayderensis A9; Enzymatic de-glycosylation; Polydatin.