Isolation and characterisation of a major cellobiohydrolase (S-8) and a major endoglucanase (S-11) subunit from the cellulosome of Clostridium thermocellum

The cellulosome of Clostridium thermocellum was dissociated under mild cond itions, and mg quantities of pure cellobiohydrolose (CBH)(dagger) (S-g) and endoglucanase (S-11) were isolated in active form. The CBH (79kDa) and end oglucanase (61 kDa) were optimally active between pH 4.5-6.0 at temperature s of 60 and 70 degreesC, respectively. Between pH 6.0-8.4, the CBH was stab le at 60 degreesC for 5 h, whereas the endoglucanase was stable at 70 degre esC for 48 h. Both enzymes were active on natural and derivatised Glc(n), H 3PO4-swollen cellulose, Avicel, laminarin, lichenan and barley glucan, whil e only endoglucanase was active on carboxymethyl (CM)-cellulose and CM-pach yman. Cellobiose inhibited the CBH competitively with a Ki of 0.28 mM. CBH cleaved preferentially either the second (between putative sub-sites -2 and -1 or +1 and +2) or the fourth glycosidic bond (between putative sub-sites -1 and +1) of MeUmbGlc(n) from the non-reducing end, while the endoglucana se required a MeUmbGlC(n) with at least three glycosidic bonds and was spec ific for internal linkages (between putative sub-sites -1 and +1). (C) 2001 Academic Press.