Purification and properties ofClostridium thermocellum endoglucanase 5 produced inEscherichia coli

Endoglucanase 5 (EG5) has been isolated from the strain ofE. coli TGI harboring recombinant plasmid pCU108, which contains thecel5 gene ofC. 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?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?C and pH 6.O. Cellobiose in concentrations up to 200 Μg/mL do not inhibit the hydrolysis of CMC by EG5, but 10–30 Μg/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.