Biochemical characterization and gene cloning of a novel alkaline endo-1-4-glucanase from Bacillus subtilis DR8806

Publication date: Available online 5 July 2016 Source:Journal of Molecular Catalysis B: Enzymatic Author(s): Somayeh Ramezani, Ahmad Asoodeh In the present study, an endo-1-4-glucanase was isolated from Bacillus subtilis DR8806. The enzyme was purified to homogeneity via salt precipitation and ion-exchange chromatography. SDS-PAGE analysis revealed a molecular mass of 52kDa. Optimum pH of enzyme was 9.5 and the enzyme was stable at pH range of 8.5 to 10.5. The optimum temperature of enzyme was found to be 55°C and it showed a remarkable stability at temperatures between 40–60°C. The enzyme activity was stimulated by Co2+, K+, Mg2+, Ca2+ and Na+ ions while Hg2+, Mn2+, Pb2+ and Zn2+ ions were found to inhibit the enzyme activity. The enzyme activity was decreased by increasing in concentration of β-mercaptoethanol, EDTA, SDS, PMSF and Triton X-100. Organic solvents such as hexane, 2-propanol, acetone and ethanol (20% v/v) stimulated enzyme activity by 110%, 114%, 119% and 128%, respectively. Imidazolium-based ionic liquids (ILs) had inhibitory effects on endo-1-4-glucanase activity. Using carboxymethyl cellulose as substrate, kinetic parameters of Km apparent and Vmax were calculated to be 1.49% (W/V) and 66.66μMmin−1 mg−1, respectively. Endo-1-4-glucanase coding gene of B. subtilis DR8806 was identified by T/A cloning. The computational modeling of endo-1-4-glucanase showed that two Glu residues are at active site. Our results suggest that the enzyme has grea...

Source: Journal of Molecular Catalysis B: Enzymatic - July 5, 2016 Category: Biochemistry Source Type: research