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 (PaCel6A), PaCel6B, and PaCel6C, 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. PaCel6A had a catalytic efficiency on cellotetraose comparable to that of Trichoderma reesei Cel6A (TrCel6A), but PaCel6B and PaCel6C were clearly less efficient. PaCel6A was the enzyme with the highest stability at 45°C, while PaCel6C 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 TrCel6A, 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 PaCel6A being the enzyme most similar to TrCel6A. PaCel6B 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 PaCel6B is an endoglucanase.

Nanoscale Engineering of Designer Cellulosomes

“…For example, even appended to a CBM, it is not clear how an endoglucanase would be capable of pulling a single polysaccharide chain out of its crystalline environment and forcing the chain productively into its active site cleft. So far, crystal structures [131] and sequence alignments [132] suggest that the shape and charge balance in the pocket induces a local distortion of a few units of a cellulose chain and it is postulated that this distortion may be essential for the catalysis to take place. Single-molecule techniques have permitted the study and manipulation of individual biomolecules, providing unprecedented insights into their function and dynamics.…”

Exo‐exo synergy between Cel6A and Cel7A from Hypocrea jecorina: Role of carbohydrate binding module and the endo‐lytic character of the enzymes

“…Relative activity Cel7A 0.0133 AE 0.0001 4.23 Â 10 À4 Cel6A 0.0058 AE 0.0003 1.84 Â 10 À4 Cel7B 31.300 AE 0.2498 1 (Boisset et al, 2000;Poidevin et al, 2013), and this understanding has been mainly based on structural evidence (see Introduction section and Payne et al, 2015 for a review). However, this explanation was not supported by the biochemical data presented here.…”

“…One interpretation is based on a potential endo-lytic activity of CBHs; particularly Cel6A (Boisset et al, 2000(Boisset et al, , 2001Divne et al, 1994;Medve et al, 1994;Poidevin et al, 2013;Ståhlberg, 1993). One interpretation is based on a potential endo-lytic activity of CBHs; particularly Cel6A (Boisset et al, 2000(Boisset et al, , 2001Divne et al, 1994;Medve et al, 1994;Poidevin et al, 2013;Ståhlberg, 1993).…”

Characterization of a Glycoside Hydrolase Family 31 α-Glucosidase Involved in Starch Utilization inPodospora anserina

“…[2][3][4] In this study, P. anserina was cultivated with soluble starch as carbon source to investigate its amylolytic enzymes. A survey of the sugar composition of the culture supernatant indicated that soluble starch converted into glucose andglucobioses, in which mainly nigerose and maltose were found, but the supernatant contained no maltooligosaccharides longer than disaccharides (Fig.…”

“…1) Several studies of P. anserina's carbohydrate active enzymes focusing on cellulose metabolism have been reported. [2][3][4] In contrast, amylolytic enzymes of P. anserina are less well understood. Among the filamentous fungi, amylolysis by Aspergillus oryzae, Aspergillus niger, and Aspergillus nidulans has been well studied.…”