Brevibacillus thermoruber 9x-glc, bacteria isolated from hot compost, producer of a beta-glucosidase resistant to glucose inhibition

The thermotolerant strain Brevibacillus thermoruber 9X-GLC produces large amounts of β-D-glucosidase and cellobiohydrolase when grown on cellobiose and avicel. 9X-GLC was isolated from hot compost of sugarcane bagasse and filter mud and selected for its high enzymatic activity from a Bacilli collection of 77 isolates from soils and compost. Culture supernatants and ultrafiltered concentrates from shaken flask cultures of 9X-GLC were tested for their β-D-glucosidase activity and stability under different conditions of temperature, pH and glucose concentration. The concentrated supernatant from 9X-GLC cultures on cellobiose as sole carbon source had its highest β-D-glucosidase activity at 55°C; pH= 6.0 and retained 60% of its activity at a glucose concentration of up to 200 mM. The effect of different carbon sources on growth and β-D-glucosidase activity of concentrated supernatants was tested in 48 h shaken flask cultures of 9X-GLC. Glucose, cellodextrin and cellobiose supported high cell densities (>2×10⁸ CFU/ml), whereas cellobiose and cellodextrin provided the best substrates for β-D-glucosidase production. The putative gene encoding β-D-glucosidase from 9X-GLC was obtained from PCR amplification and sequenced. The sequence of the putative structural gene of 2085 bp reveals 6 catalytic domains with high (>90%) homology to bacterialglycosidases.