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 (Km = 0.097 g/L, Vmax = 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 (Km = 18.2 g/L, Vmax = 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 micrograms/mL do not inhibit the hydrolysis of CMC by EG5, but 10-30 micrograms/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.
Mosolova
T P
TP
Department of Chemical Enzymology, Chemistry Faculty, M. V. Lomonosov Moscow University, Russia.
Kalyuzhnyi
S V
SV
Varfolomeyev
S D
SD
Velikodvorskaya
G A
GA
D002475:Cellobiose / Q000494:pharmacology; D002480:Cellulase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005947:Glucose / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008970:Molecular Weight; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D013379:Substrate Specificity; D013696:Temperature
D011994:Recombinant Proteins; D002475:Cellobiose; D002482:Cellulose; D002480:Cellulase; D005947:Glucose