Homologous expression and characterization of Coniochaeta ligniaria glycoside hydrolase family 115 α-glucuronidase

Abstract

Generation of fermentable sugars from biomass is necessary for microbial production of bioproducts. Due to its inherent complexity, xylan hydrolysis to monosaccharides requires several different enzymatic activities. To completely depolymerize glucuronoarabinoxylan from biomass, α-glucuronidase activity is necessary. The ascomycete fungus Coniochaeta ligniaria has the ability to degrade xylan and is an unutilized source of biomass degrading enzymes. Therefore, a gene encoding a putative Coniochaeta ligniaria GH115 α-glucuronidase was cloned and expressed homologously in Coniochaeta ligniaria as a native secreted protein. Culture supernatants were concentrated and purified by a combination of ultrafiltration, anion-exchange, and size-exclusion chromatography. The purified protein behaved as dominantly dimeric complexes as determined by size-exclusion chromatography. The expressed protein liberated: 4-O-methyl glucuronic acid from beech xylan, birch xylan, and beech-derived glucuronoxylooligosaccharides as the sole product of hydrolysis; and 4-O-methyl glucuronic acid and glucuronic acid from oat spelt xylan. The expressed α-1,2-glucuronidase had greater activity on glucuronoxylooligosaccharides than on full length beech xylan. The expressed α-1,2-glucuronidase, herein designated ClAgu115, had K_m values of: 1.3 mM; 1.2 mM; and 1.0 mM for beech xylan, GH10-hydrolyzed glucuronooligosaccharides, and GH11-hydrolyzed glucuronooligosaccharides, respectively. The measured kinetic constants show that the enzyme prefers an oligosaccharide substrate with the 4-O-methyl glucuronic acid on the non-reducing end. The enzyme had activity between pH 3.0–6.0 and temperatures 10°C-60°C, with optima at pH 4.3 and 40°C. The expression and characterization of ClAgu115 expands the repertoire of fungal GH115 enzymes for use in biomass conversion.