Purification And Properties Of The Clostridium-Thermocellum BGLB Gene-Product Expressed In Escherichia-Coli

Romaniec, Marek P.M. and Huskisson, N. and Barker, Patrick J. and Demain, Arnold L. (1993) Purification And Properties Of The Clostridium-Thermocellum BGLB Gene-Product Expressed In Escherichia-Coli. Enzyme and Microbial Technology, 15 (5). pp. 393-400. ISSN 0141-0229. (The full text of this publication is not currently available from this repository. You may be able to access a copy if URLs are provided)

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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 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(max) 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.

Item Type: Article
Uncontrolled keywords: Beta-Glucosidase; Clostridium-Thermocellum; BGLB Gene; Cellulase; Anaerobic Bacteria; Thermophiles
Subjects: Q Science
Divisions: Faculties > Science Technology and Medical Studies > School of Biosciences
Depositing User: E.C. Henry-Duru
Date Deposited: 04 Oct 2009 18:15
Last Modified: 26 Jun 2014 10:47
Resource URI: https://kar.kent.ac.uk/id/eprint/20630 (The current URI for this page, for reference purposes)
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