Bioprospecting of novel thermostable β-glucosidase from Bacillus subtilis RA10 and its application in biomass hydrolysis.

Background: Saccharification is the most crucial and cost-intensive process in second generation biofuel production. The deficiency of β-glucosidase in commercial enzyme leads to incomplete biomass hydrolysis. The decomposition of biomass at high temperature environments leads us to isolate thermotolerant microbes with β-glucosidase production potential.

Results: A total of 11 isolates were obtained from compost and cow dung samples that were able to grow at 50 °C. On the basis of qualitative and quantitative estimation of β-glucosidase enzyme production, Bacillus subtilis RA10 was selected for further studies. The medium components and growth conditions were optimized and β-glucosidase enzyme production was enhanced up to 19.8-fold. The β-glucosidase from B. subtilis RA10 retained 78% of activity at 80 °C temperature and 68.32% of enzyme activity was stable even at 50 °C after 48 h of incubation. The supplementation of β-glucosidase from B. subtilis RA10 into commercial cellulase enzyme resulted in 1.34-fold higher glucose release. Furthermore, β-glucosidase was also functionally elucidated by cloning and overexpression of full length GH1 family β-glucosidase gene from B. subtilis RA10. The purified protein was characterized as thermostable β-glucosidase enzyme.

Conclusions: The thermostable β-glucosidase enzyme from B. subtilis RA10 would facilitate efficient saccharification of cellulosic biomass into fermentable sugar. Consequently, after saccharification, thermostable β-glucosidase enzyme would be recovered and reused to reduce the cost of overall bioethanol production process.