title: Characterization of a broad-specificity β-glucanase acting on β-(1,3)-, β-(1,4)-, and β-(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 β-glucan polysaccharides, including laminarin, curdlan, pachyman, lichenan, pustulan, and cellulosic derivatives. Analysis of the products released from polysaccharides revealed that this β-glucanase is an exo-acting enzyme on β-(1,3)- and β-(1,6)-linked glucan substrates and an endo-acting enzyme on β-(1,4)-linked glucan substrates. Hydrolysis of short β-(1,3), β-(1,4), and β-(1,3)/β-(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-β-(1,3)/(1,6) and endo-β-(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.
authors.affiliation: INRA, Aix Marseille Université, UMR1163 BCF, Polytech Marseille, Marseille, France.
authors.forename: Mickael
authors.initials: M
authors.lastname: Lafond
authors.forename: David
authors.initials: D
authors.lastname: Navarro
authors.forename: Mireille
authors.initials: M
authors.lastname: Haon
authors.forename: Marie
authors.initials: M
authors.lastname: Couturier
authors.forename: Jean-Guy
authors.initials: JG
authors.lastname: Berrin
mesh_terms: D003001:Cloning, Molecular; D015870:Gene Expression; D005936:Glucans / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics; D048468:Podospora / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D005936:Glucans; D006026:Glycoside Hydrolases