The wood rot ascomycete Xylaria polymorpha produces a novel GH78 glycoside hydrolase that exhibits α-L-rhamnosidase and feruloyl esterase activities and releases hydroxycinnamic acids from lignocelluloses.

Soft rot (type II) fungi belonging to the family Xylariaceae are known to substantially degrade hardwood by means of their poorly understood lignocellulolytic system, which comprises various hydrolases, including feruloyl esterases and laccase. In the present study, several members of the Xylariaceae were found to exhibit high feruloyl esterase activity during growth on lignocellulosic materials such as wheat straw (up to 1,675 mU g(-1)) or beech wood (up to 80 mU g(-1)). Following the ester-cleaving activity toward methyl ferulate, a hydrolase of Xylaria polymorpha was produced in solid-state culture on wheat straw and purified by different steps of anion-exchange and size-exclusion chromatography to apparent homogeneity (specific activity, 2.2 U mg(-1)). The peptide sequence of the purified protein deduced from the gene sequence and verified by de novo peptide sequencing shows high similarity to putative α-L-rhamnosidase sequences belonging to the glycoside hydrolase family 78 (GH78; classified under EC 3.2.1.40). The purified enzyme (98 kDa by SDS-PAGE, 103 kDa by size-exclusion chromatography; pI 3.7) converted diverse glycosides (e.g., α-L-rhamnopyranoside and α-L-arabinofuranoside) but also natural and synthetic esters (e.g., chlorogenic acid, hydroxycinnamic acid glycoside esters, veratric acid esters, or p-nitrophenyl acetate) and released free hydroxycinnamic acids (ferulic and coumaric acid) from arabinoxylan and milled wheat straw. These catalytic properties strongly suggest that X. polymorpha GH78 is a multifunctional enzyme. It is the first fungal enzyme that combines glycosyl hydrolase with esterase activities and may help this soft rot fungus to degrade lignocelluloses.

Bioconversion of Lignocellulosic Materials with the Contribution of a Multifunctional GH78 Glycoside Hydrolase from Xylaria polymorpha to Release Aromatic Fragments and Carbohydrates.

A bifunctional glycoside hydrolase GH78 from the ascomycete Xylaria polymorpha (XpoGH78) possesses catalytic versatility towards both glycosides and esters, which may be advantageous for the efficient degradation of the plant cell-wall complex that contains both diverse sugar residues and esterified structures. The contribution of XpoGH78 to the conversion of lignocellulosic materials without any chemical pretreatment to release the water-soluble aromatic fragments, carbohydrates, and methanol was studied. The disintegrating effect of enzymatic lignocellulose treatment can be significantly improved by using different kinds of hydrolases and phenoloxidases. The considerable changes in low (3 kDa), medium (30 kDa), and high (> 200 kDa) aromatic fragments were observed after the treatment with XpoGH78 alone or with this potent cocktail. Synergistic conversion of rape straw also resulted in a release of 17.3 mg of total carbohydrates (e.g., arabinose, galactose, glucose, mannose, xylose) per gram of substrate after incubating for 72 h. Moreover, the treatment of rape straw with XpoGH78 led to a marginal methanol release of approximately 17 µg/g and improved to 270 µg/g by cooperation with the above accessory enzymes. In the case of beech wood conversion, the combined catalysis by XpoGH78 and laccase caused an effect comparable with that of fungal strain X. polymorpha in woody cultures concerning the liberation of aromatic lignocellulose fragments.