Novel GH115 xylan α-1,2-glucuronidases with distinct preferences for xylan-derived oligomers and polymers.

Natural polymers are promising sustainable materials for diverse applications. Xylans, major components of hemicellulose, exhibit origin-dependent substitution patterns that determine their physicochemical properties. Targeted enzymatic modification of these substituents offers a mild and precise approach for tailoring the polymer's characteristics. Glucuronidases (EC 3.2.1.139) and xylan α-1,2-glucuronosidases (EC 3.2.1.131) are carbohydrate active enzymes that specifically remove the common glucuronic acid and 4-O-methyl d-glucuronic acid side branches of xylans/xylooligosaccharides from different hardwood and softwood sources. These enzymes are, dependent on sequence and structure, occurring in three glycoside hydrolase families (GH4, GH67 and GH115). GH115 predominantly includes xylan α-1,2-glucuronosidases, but the number of characterized enzymes is low. In this study, 22 novel GH115 candidates were identified through a combination of automated and manual sequence mining from databases, for evaluation of their production, function and storage possibilities. Twenty enzymes were produced in active form in crude extracts, while only four remained soluble for extended time periods post purification. These four GH115 candidates include first time reported monomeric enzymes, and while all were active on polymeric xylan, their specific activity and activity-ratio on polysaccharides/oligosaccharides of beechwood xylan differed significantly. Structural models created by AlphaFold3, and different length oligosaccharides were docked in the catalytic clefts. The presence of an additional domain, and the position of a mobile loop affected oligomerization and activity on polymeric substrates, respectively, resulting in differences in specific activity and preference for polymeric substrates.