Purification and characterization of endo-β-1,4-glucanase encoded by clostridium thermocellum gene in escherichia coli

Abstract

The cellulase produced by E. coli harbouring C. thermocellum endo-$\beta$-1,4-glucanase gene was purified and characterized. Streptomycin sulfate fractionation, heat treatment, ammonium sulfate fractionation, DEAE-Sephadex A-50, and Hydroxyapatite column chromatography were employed for the purification. The purification procedures yielded a 40-fold purification of the enzyme with yield of 29\%. The molecular weight of the purified enzyme was estimated to be 40,000 dalton by SDS-polyacrylamide gel electrophoresis and gel filtration. The enzyme showed optimum activity at pH 5.0 and at $65\,^\circ\!C$. Viscometric assay showed that the enzyme behaved as an endoglucanase. The enzyme was insensitive against end product inhibition by glucose and cellobiose and resistant to organic solvents (ethanol, acetate, and lactate). The enzyme was sensitive to Mn$^{++}$, CO$^{++}$, EDTA, and Hg$^{++}$, but not to Ca$^{++}$, Mg$^{++}$, and Zn$^{++}$. Enzyme activity was stimulated by low concentrations of Cu$^{++}$, but inhibited by high concentrations of Cu$^{++}$. The values of Km and Vm of this enzyme for CMC were 0.39 \% and 268 units per mg protein, respectively. The Km an Vm for pNPC hydrolysis were 0.75 mM and 1.32 units per mg protein, respectively.