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 ␤-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.

Cello-Oligosaccharide Oxidation Reveals Differences between Two Lytic Polysaccharide Monooxygenases (Family GH61) from Podospora anserina

“…Analysis of the genome sequence of P. anserina demonstrated an unexpected enzymatic arsenal (32,37,39). This coprophilic fungus displays a large array of GMC oxidoreductases, including two CDHs, a pyranose oxidase, a galactose oxidase, a copper radical oxidase, and several laccases, that could be relevant for in vivo synergy with LPMOs upon lignocellulose oxidative reactions.…”

A Novel Glycoside Hydrolase Family 5 β-1,3-1,6-Endoglucanase from Saccharophagus degradans 2-40 T and Its Transglycosylase Activity

“…However, ␤-1,3-glucosidases or ␤-1,3-exoglucanases (EC 3.2. 1.58) in the GH5, GH7, GH12, GH16, and GH17 families act on the nonreducing end of ␤-1,3-glucans to release glucose (29). GH5 is one of the largest GH families, previously known as cellulase family A (30).…”

The Quaternary Structure of a Glycoside Hydrolase Dictates Specificity toward β-Glucans

“…All assays were carried out in triplicate. The specificity constants were calculated using the Matsui equation for oligosaccharides (27,28). Activities toward pNP-substrates (1 mM) were determined by measuring the release of 4-nitrophenol in McIlvaine's buffer, pH 7.0, 30°C in 100-l reaction volume.…”