title: 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.
authors: IFP Energies Nouvelles, Rueil-Malmaison, France.
authors: Laetitia
authors: L
authors: Poidevin
authors: Julia
authors: J
authors: Feliu
authors: Annick
authors: A
authors: Doan
authors: Jean-Guy
authors: JG
authors: Berrin
authors: Mathieu
authors: M
authors: Bey
authors: Pedro M
authors: PM
authors: Coutinho
authors: Bernard
authors: B
authors: Henrissat
authors: Eric
authors: E
authors: Record
authors: Senta
authors: S
authors: Heiss-Blanquet
mesh_terms: D000595:Amino Acid Sequence; D002480:Cellulase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004271:DNA, Fungal / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D048468:Podospora / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment
chemical_list: D018076:DNA, Complementary; D004271:DNA, Fungal; D006026:Glycoside Hydrolases; D002480:Cellulase
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