PURIFICATION AND PROPERTIES OF CLOSTRIDIUM-THERMOCELLUM ENDOGLUCANASE-5 PRODUCED IN ESCHERICHIA-COLI

Endoglucanase 5 (EG5) has been isolated from the strain of E. coli TG1 harboring recombinant plasmid pCU108, which contains the cel5 gene of C. thermocellum. The enzyme has been produced with 98-fold purification and a final yield of 27% by using subsequent twofold high performance ion-exchange chromatography on Mono Q and high performance chromatofocusing on Mono P. The protein has a mol mass of 35 kDa and includes 3 multiple forms with pI 4.4-4.8 as evidenced by analytical gel isoelectrofocusing. EG5 cleaves CMC (K(m) = 0.097 g/L, V(max) = 8.2 mg/min.mg of protein), amorphous cellulose, xylan, lichenan as a substrate with an optimum temperature of 80-degrees-C and pH 6.0 and Avicel (K(m) = 18.2 g/L, V(max) = 0.035 mg/min.mg of protein) with an optimum temperature of 60-degrees-C and pH 6.0. Cellobiose in concentrations up to 200 mug/mL do not inhibit the hydrolysis of CMC by EG5, but 10-30 mug/mL of glucose significantly decrease the activity of this enzyme. The stimulating role of calcium chloride and concentration of protein in the system has been demonstrated for Avicel hydrolysis by EG5.