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

Authors: Bowman, Michael; Nichols, Nancy

Submitted to: Enzyme and Microbial Technology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/8/2025
Publication Date: N/A
Citation: N/A

Interpretive Summary: It has been estimated that the United States can produce up to 1.5 billion tons of biomass annually. Due to its abundance and high concentration of fermentable sugars, biomass is seen as a promising feedstock to produce a variety of biochemical products. Engineered microorganisms are available to convert these sugars to higher value fuels and chemicals but biomass sugars exist in long interconnected chains, making the sugars inaccessible to microorganisms. One of these chains called xylan is a major type of fiber in biomass, making up 20 to 30% of cell wall material in energy crops and agricultural residues. For microbes to effectively convert these sugar components to fuels and chemicals, the chain needs to be broken down to individual sugars. One of the problems associated with using biomass as a feedstock is that breakdown of the sugar chain is obstructed due to the presence of additional side structures. This study identified an enzyme capable of removing one of these side structures. When using biomass sources such as soybean hulls, sorghum, and corn fiber, which are difficult to break down due to higher levels of these side structures, up to 6% more sugar from xylan can be obtained from the same amount of starting material. Thus, using a modified process including this enzyme would reduce the cost of production for any biochemical made from biomass derived sugars and generate more revenue for the manufacturers.

Technical 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, alpha 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 alpha-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 alpha 1,2 glucuronidase had greater activity on glucuronoxylooligosaccharides than on full length beech xylan. The expressed alpha 1,2-glucuronidase, herein designated ClAgu115, had Km values of: 1.3 mM; 1.2 mM; and 1.0 mM for beech xylan, GH10 hydrolyzed glucuronooligosaccharides, and GH11 hydrolyzed glucuronooligosaccharides, respectively. The enzyme had activity between pH 3-6 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 GH115s for use in biomass conversion.