PURIFICATION AND PROPERTIES OF THE CLOSTRIDIUM-THERMOCELLUM BGLB GENE-PRODUCT EXPRESSED IN ESCHERICHIA-COLI

The Clostridium thermocellum beta-glucosidase B was purified to homoge neity in its recombinant form from Escherichia coli. The purification protocol included ion exchange, hydrophobic interaction and hydroxyapa tite chromatography. The polypeptide was found to have a molecular mas s of 84,000 daltons and a pI of 4.4. There was a differential effect o f temperature on the aryl-beta-glucosidase and cellobiase activities o f the purified protein. The cellobiase activity had an optimum of 45-d egrees-C, and aryl-beta-glucosidase 60-degrees-C. Both activities had an optimum pH of 5.6, although the aryl-beta-glucosidase had a seconda ry peak at 7.0. Both activities were stimulated by divalent cations an d DTT, but inhibited by thiol reagents. The enzyme was found to have a broad substrate specificity. Using cellobiose as substrate and a temp erature of 45-degrees-C, the K(m) and V(max) values were 1.6 mm and 5. 5 U mg-1 respectively. The aryl-beta-glucosidase when assayed against pNP glucopyranoside and a temperature of 60-degrees-C had K(m) and V(m ax) of 2.9 mm and 1.1 U mg-1 respectively. The enzyme was very stable at 45-degrees-C, but rapidly inactivated at 60-degrees-C.