Chemical modification of β-endoglucanase from Trichoderma viridin by methanol and determination of the catalytic functional groups
β-Endoglucanase from Trichoderma viride was modified by methanol to explore the catalytic functional groups of cellulase. Crude cellulase was produced, and the conditions of saturation and pH by salting out with ammonium sulfate were optimized. Under optimal conditions, crude cellulase was isolated and purified. The pure cellulase was modified by excess methanol, and it was found that the carboxyl in the side-chain radical of cellulase proved to be the catalytic functional group by analysis of the infrared spectrum of modification of cellulase. Modification of cellulase with different concentrations of methanol was carried out and results show that the modified side-chain radical lies at the active site of cellulase or on essential groups. Sodium carboxymethyl cellulose (CMC-Na) which protected the cellulase from inactivation by methanol indicated that the carboxyl modified by methanol is not only a structural radical but also lies at the active site. The inactivation degree of cellulase modified by methanol could be decreased by glucose and it showed that the modification occurred in the active site of cellulase. The kinetic analysis according to Keech and Farrant equation demonstrated that one carboxyl was an essential group for cellulase activity.
Keywords: Chemical modification, cellulase, methanol, carboxyl