Production of endoglucanase A of Clostridium thermocellum in Bacillus subtilis

Abstract

Endoglucanase A (CelA) is a component of thermostable complex - cellulosome which is produced by anaerobic bacterium Clostridium thermocellum. In this complex, CelA has the highest catalytic activity and it can cleave randomly β-1,4 glucosidic linkage of cellulose to smaller oligosaccharides. Bacillus subtilis a Gram positive and aerobic fast growth bacterium, is commonly used to produce industrial enzymes. This bacterium can be used to replace the anaerobic slow growth bacterium, C. thermocellum, to produce CelA. To create B. subtilis strains which can secret recombinant CelA enzyme, celA gene was amplified using C. thermocellum DNA genome by PCR and inserted in the C-terminus of signal peptide of amyQ (samyQ) in plasmid pHT43. The plasmid pHT43-celA was then transformed into B. subtilis 1012 and WB800N, an extracellular protease deficiency strain. Next, CelA production was induced by IPTG at different concentrations. Cultural supernatant was collected from 2 to 24 hours after addition of IPTG. The expression levels were evaluated by SDS-PAGE and endo-β-1,4- glucanase activity. This report demonstrates that CelA can be expressed in B. subtilis and its potentials for development of bacterial strains which can produce CelA to hydrolyze cellulose in the future.