Cellobiohydrolase from Clostridium thermocellum, synthesized by a recombinant E. coli strain]

Clostridium thermocellum cellobiohydrolase was isolated in preparative amounts from the recombinant strain of E. coli K12 C600 carrying plasmid pCU 304 with a C. thermocellum chromosomal DNA insertion. The isolation procedure included chromatography on Ultrogel AcA 44, ion-exchange chromatography on DEAE-Sepharose CL-6B, rechromatography on Ultrogel and FPLC on Mono Q resulting in a 17.6% yield and 1530-fold purification. According to data from sodium dodecylsulfate polyacrylamide gel electrophoresis performed under nondenaturing conditions and analytical gel isoelectrofocusing, the enzyme preparation contains only one active protein band with Mr 56.2 +/- 1.0 kDa and pI 4.15. The enzyme does not reduce the viscosity of the CM-cellulose solution but forms reducing sugars from this soluble substrate. Cellobiose (93-97%) is the major component produced by the enzyme from crystalline and amorphous cellulose (specific activity 2.3 x 10(-3) and 2.8 x 10(-2) U/mg, respectively). The activity optimum of the enzyme is at pH 5.6, 60 degrees C. The half-inactivation time at 60 degrees C and 65 degrees C is 450 and 15.5 min, respectively. The action pattern of the enzyme on the low molecular fluorogenic cellooligosaccharides suggests that the enzyme pertains to typical cellobiohydrolases.