Cloning of Clostridiumcellulovoransendo-1,4-β-glucanase genes

Abstract A Clostridium cellulovorans lambda gt1l gene bank was screened for endo-1,4-β-glucanase [EC 3.2.1.4, EGase, Carboxy Methyl Cellulase (CMCase)] genes using a chromogenic substrate. Three genes ( engA , engB , and engC ) were isolated. The engB expressed the most active CMCase. The engA encoded a bifunctional enzyme that displayed endo-1,4-β-glucanase and β-glucosidase activities. The three recombinant glucanases, when expressed in Escherichia coli , were partially degraded into multiform active enzymes as evidenced by their SDS-PAGE-CMC zymograms. None of the clones could degrade crystalline cellulose, thus supporting the hypothesis that the integrity of the C . cellulovorans cellulase complex was essential for its ‘true cellulase’ activity.