Production of free and substrate-bound cellulases of Cellulomonas flavigena

Abstract

Conditions for the production of extracellular carboxymethyl cellulase ( CMCase) and avicelase activities by a locally isolated Cellulomonas species identified as C. flavigena were optimized. The microbe produced maximal levels of free cellulases after 72 h of fermentation, when cultivated in the presence of 0.2% yeast extract, 0.5% Avicel, and 0.1% Tween 80 at 30°C. Conditions for the elution of substrate-bound cellulases from the residual Avicel were optimized. Higher proportions of the extracellular cellulases were bound to the substrate when higher concentrations of Avicel were used. When 2.0% Avicel was used as a carbon source, maximal levels of extracellular CMCase and avicelase produced were 13.2 and 3 U ml −1 of the culture medium, respectively. Both the cellulases were most active at pH 6.5 and 50°C and were inhibited by β-mercaptoethanol. Calcium chloride activated CMCase but had no effect on avicelase activity. Each of the enzymes lost activity when incubated at 70°C for 30 min. A comparative study of free and substrate-bound CMCase on gradient polyacrylamide gel electrophoresis ( PAGE) showed that free CMCase activity was composed of at least seven active fractions, while bound activity had four major bands of CMCase activity. Since a considerable level of protease was secreted by C. flavigena, the additional fractions in the free CMCase activity could be a result of proteolysis.