Heterologous expression and biochemical studies of a thermostable glucose tolerant β-glucosidase from Methylococcus capsulatus (bath strain)

Glucose inhibition of β-glucosidase (BG) is a bottleneck in biomass hydrolysis. In this study, a glucose resistant GH1 β-glucosidase gene- Mbgl from Methylococcus capsulatus (bath strain) was cloned and overexpressed in E.coli. The Ni-NTA affinity purified Mbgl displayed an optimum temperature of 70 °C and optimum pH was 6.0. The calculated KM of the enzyme was 48.6 mM and 0.12 mM for cellobiose and 4-Nitrophenyl β-d-glucopyranoside (PNPG) respectively. PNPG hydrolysis in presence of various glucose concentrations showed that the enzyme was stimulated by ∼2.2 fold at 50 mM glucose and was not inhibited up to 450-500 mM glucose. Homology modeling and structural comparisons of Mbgl with a glucose tolerant β-glucosidase of Humicola insolens (HiBG) revealed that the Mbgl has a much broader active site unlike to a deep and narrow active site pocket of HiBG. The difference in active site shape reflects on an alternative mechanism of glucose tolerance in Mbgl. Supplementing a commercial cellulase enzyme mixture CTec with Mbgl in the hydrolysis of the pretreated rice straw enhanced the glucose yield by 10-15%. In addition, Mbgl was also stable in organic solvents, detergents and oxidative conditions which would be advantageous for biotechnological applications.