Insights into Exo- and Endoglucanase Activities of Family 6 Glycoside Hydrolases from Podospora anserina

Abstract

The ascomycete Podospora anserina is a coprophilous fungus that grows at late stages on droppings of herbivores. Its genome encodes a large diversity of carbohydrate-active enzymes. Among them, four genes encode glycoside hydrolases from family 6 (GH6), the members of which comprise putative endoglucanases and exoglucanases, some of them exerting important functions for biomass degradation in fungi. Therefore, this family was selected for functional analysis. Three of the enzymes, P. anserina Cel6A ( Pa Cel6A), Pa Cel6B, and Pa Cel6C, were functionally expressed in the yeast Pichia pastoris . All three GH6 enzymes hydrolyzed crystalline and amorphous cellulose but were inactive on hydroxyethyl cellulose, mannan, galactomannan, xyloglucan, arabinoxylan, arabinan, xylan, and pectin. Pa Cel6A had a catalytic efficiency on cellotetraose comparable to that of Trichoderma reesei Cel6A ( Tr Cel6A), but Pa Cel6B and Pa Cel6C were clearly less efficient. Pa Cel6A was the enzyme with the highest stability at 45�C, while Pa Cel6C was the least stable enzyme, losing more than 50% of its activity after incubation at temperatures above 30�C for 24 h. In contrast to Tr Cel6A, all three studied P. anserina GH6 cellulases were stable over a wide range of pHs and conserved high activity at pH values of up to 9. Each enzyme displayed a distinct substrate and product profile, highlighting different modes of action, with Pa Cel6A being the enzyme most similar to Tr Cel6A. Pa Cel6B was the only enzyme with higher specific activity on carboxymethylcellulose (CMC) than on Avicel and showed lower processivity than the others. Structural modeling predicts an open catalytic cleft, suggesting that Pa Cel6B is an endoglucanase.