title: Purification and properties of the Clostridium thermocellum bglB gene product expressed in Escherichia coli.
abstract: The Clostridium thermocellum beta-glucosidase B was purified to homogeneity in its recombinant form from Escherichia coli. The purification protocol included ion exchange, hydrophobic interaction and hydroxyapatite chromatography. The polypeptide was found to have a molecular mass of 84,000 daltons and a pI of 4.4. There was a differential effect of temperature on the aryl-beta-glucosidase and cellobiase activities of the purified protein. The cellobiase activity had an optimum of 45 degrees C, and aryl-beta-glucosidase 60 degrees C. Both activities had an optimum pH of 5.6, although the aryl-beta-glucosidase had a secondary peak at 7.0. Both activities were stimulated by divalent cations and DTT, but inhibited by thiol reagents. The enzyme was found to have a broad substrate specificity. Using cellobiose as substrate and a temperature of 45 degrees C, the Km and Vmax 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 Km and Vmax 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.
authors.affiliation: Biological Laboratory, University of Kent, Canterbury, UK.
authors.forename: M P
authors.initials: MP
authors.lastname: Romaniec
authors.forename: N
authors.initials: N
authors.lastname: Huskisson
authors.forename: P
authors.initials: P
authors.lastname: Barker
authors.forename: A L
authors.initials: AL
authors.lastname: Demain
mesh_terms: D002240:Carbohydrate Sequence; D002475:Cellobiose / Q000032:analysis; D002480:Cellulase / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005947:Glucose / Q000032:analysis; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000302:isolation & purification; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D011994:Recombinant Proteins; D002475:Cellobiose; D002482:Cellulose; D001617:beta-Glucosidase; D002480:Cellulase; D005947:Glucose