Glucuronoyl esterases (CE15 family) enable targeted cleavage of ester linkages in lignin-carbohydrate complexes (LCCs), particularly those linking lignin and glucuronoyl residues in xylan. A substantial challenge in characterization and kinetic analysis of CE15 enzymes has been the lack of proper substrates. Here, we present an assay using an insoluble LCC-rich lignin fraction from birch; lignin-rich pellet (LRP). The assay employs quantification of enzyme reaction products by LC-MS. The kinetics of four fungal CE15 enzymes, PsGE, CuGE, TtGE, and AfuGE originating from lignocellulose-degrading fungi Punctularia strigosozonata, Cerrena unicolor, Thielavia terrestris, and Armillaria fuscipes respectively were characterized and compared using this new assay. All four enzymes had activity on LRP and showed a clear preference for the insoluble substrate compared with smaller soluble LCC mimicking esters. End-product profiles were near identical for the four enzymes but differences in kinetic parameters were observed. TtGE possesses an alternative active site compared with the three other enzymes as it has the position of the catalytic glutamic acid occupied by a serine. TtGE performed poorly compared with the other enzymes. We speculate that glucuronoyl LCCs are not the preferred substrate of TtGE. Removal of an N-terminal CBM on CuGE affected the catalytic efficiently of the enzyme by reducing K cat by more than 30%. Reaction products were detected from all four CE15s on a similar substrate from spruce indicating a more generic GE activity not limited to the hardwood. The assay with natural substrate represents a novel tool to study the natural function and kinetics of CE15s.
- Aldouronic acids
- Glucuronoyl esterases