Characterization of a broad-specificity ?-glucanase acting on ?-(1,3)-, ?-(1,4)-, and ?-(1,6)-glucans that defines a new glycoside hydrolase family.

ABSTRACT: Here we report the cloning of the Pa_3_10940 gene from the coprophilic fungus Podospora anserina, which encodes a C-terminal family 1 carbohydrate binding module (CBM1) linked to a domain of unknown function. The function of the gene was investigated by expression of the full-length protein and a truncated derivative without the CBM1 domain in the yeast Pichia pastoris. Using a library of polysaccharides of different origins, we demonstrated that the full-length enzyme displays activity toward a broad range of ?-glucan polysaccharides, including laminarin, curdlan, pachyman, lichenan, pustulan, and cellulosic derivatives. Analysis of the products released from polysaccharides revealed that this ?-glucanase is an exo-acting enzyme on ?-(1,3)- and ?-(1,6)-linked glucan substrates and an endo-acting enzyme on ?-(1,4)-linked glucan substrates. Hydrolysis of short ?-(1,3), ?-(1,4), and ?-(1,3)/?-(1,4) gluco-oligosaccharides confirmed this striking feature and revealed that the enzyme performs in an exo-type mode on the nonreducing end of gluco-oligosaccharides. Excision of the CBM1 domain resulted in an inactive enzyme on all substrates tested. To our knowledge, this is the first report of an enzyme that displays bifunctional exo-?-(1,3)/(1,6) and endo-?-(1,4) activities toward beta-glucans and therefore cannot readily be assigned to existing Enzyme Commission groups. The amino acid sequence has high sequence identity to hypothetical proteins within the fungal taxa and thus defines a new family of glycoside hydrolases, the GH131 family.

SUBMITTER: Lafond M 

PROVIDER: S-EPMC3502917 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature