Novel Function of CtXyn5A from Acetivibrio thermocellus: Dual Arabinoxylanase and Feruloyl Esterase Activity in the Same Active Site**

Abstract

Enzymes' uncharacterised side activities can have significant effects on reaction products and yields. Hence, their identification and characterisation are crucial for the development of successful reaction systems. Here, we report the presence of feruloyl esterase activity in CtXyn5A from Acetivibrio thermocellus, besides its well-known arabinoxylanase activity, for the first time. Activity analysis of enzyme variants mutated in the catalytic nucleophile, Glu279, confirmed removal of all activity for E279A and E279L, and increased esterase activity while removing xylanase activity for E279S, thus allowing the proposal that both reaction types are catalysed in the same active site in two subsequential steps. The ferulic acid substituent is cleaved off first, followed by hydrolysis of the xylan backbone. The esterase activity on complex carbohydrates was found to be higher than that of a designated ferulic acid esterase (E-FAERU). Therefore, we conclude that the enzyme exhibits a dual function rather than an esterase side activity.

Novel thermostable GH5_34 arabinoxylanase with an atypical CBM6, displays activity on oat fibre xylan for prebiotic production

“…The novel GH5_34 arabinoxylanase Hh Xyn5A could be produced at high yields with long storage stability, which makes the enzyme a potential candidate for large scale production, as no domain splitting or aggregation was observed. In contrast, Ct Xyn5A formulation is dependent on imidazole to prevent enzyme aggregation and precipitation ( Schmitz et al. 2022a ).…”