Characterization of endoglucanase-encoding gene of indigenous Bacillus subtilis isolated from rice bran by in silico analysis

Abstract

Cellulase enzyme is a multi-enzyme system consists of endo-β-1,4-glucanase (CMCase; EG; EC 3.2.1.4), exo- β-1,4-glucanase (cellobiohydrolase; CBH; EC 3.2.1.91), and β-1,4-glucosidase (GH; EC 3.2.1.21). The enzyme system works synergistically to degrade cellulosic substrates to glucose. The endo-β-1,4-glucanase enzyme has an important role in the initiation of cellulose hydrolysis. The exploration of endoglucanase is still continuing in order to achieve the best activity and efficiency. The aim of this study was to characterize the endoglucanase-encoding gene of indigenous Bacillus subtilis, a cellulolytic bacterium isolated from rice bran, by in silico analysis. The endoglucanase gene was isolated using forward (5'-TTCGCAGCAAGTGTAACGGA-3′) and reverse (5′-CTCGCCGTACATCGCATAGT-3′) primer, and amplified through PCR in this following condition; pre-denaturation at 94°C for 4 min, denaturation at 94°C for 30 sec, annealing at 54°C for 30 sec and extension at 72°C for 1 min, the final extension was performed at 72°C for 2 min, all repeated to 35 cycles. The endoglucanase gene of indigenous Bacillus subtilis from rice bran comprises of 862 bp (246 amino acid). In silico analysis showed that endoglucanase gene of Bacillus subtilis has a catalytic cellulase domain constituted of BglC gene and CBM (Cellulose Binding Module). The binding region of the enzyme is located at residues 228 - 246 and the constituent amino acids of the active site are Asp44, Glu84 and Glu172. The enzyme belongs to Glycosyl Hydrolase/GH1 and GH5 family.