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

• A 52 kD- endo-β-1, 4-glucanase was purified from Bacillus subtilis DR8806.
• The enzyme revealed optimal pH and temperature of 9.5 and 55 °C.
• Some organic solvents (20% v/v) stimulated glucanase activity.
• Glucanase was a β 1-4 glycosyl hydrolase family 5 with a CBM-3 at its N terminus.
• Three- structure modeling of the enzyme showed two Glu residues at active site.

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 52 kDa. Optimum pH of enzyme was 9.5 and the enzyme was stable at pH range of 8.5–10.5. The optimum temperature of enzyme was found to be 55 °C and it showed a remarkable stability at temperatures between 40 and 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 66.66 μM min−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 great of potential applications in hydrolyzing cellulosic substrates.

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