0100491634

open access

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Synthesis of a natural core substrate with lignin-xylan cross-linkage for unveiling the productive kinetic parameters of glucuronoyl esterase

English (英語)

Biochemical and Biophysical Research Communications

734

150642

Elsevier

2024-09-23

2024-09-24

Lignin-carbohydrate complexes (LCCs) present a considerable hurdle to the economic utilization of lignocellulosic biomass. Glucuronoyl esterase (GE) is an LCC-degrading enzyme that catalyzes the cleavage of the cross-linkages between lignin and xylan in LCCs. Benzyl-d-glucuronate (Bn-GlcA), a commercially available substrate, is widely used to evaluate GE activity assays. However, since Bn-GlcA lacks the structural backbone of naturally occurring LCCs, the mechanisms underlying the activity of GEs and their diversity in the structure-activity relationship are not fully understood. Herein, we provided a synthesis scheme for designing 1,2³-α-d-(6-benzyl-4-O-methyl-glucuronyl)-1,4-β-d-xylotriose (Bn-MeGlcA³Xyl₃) as a natural core substrate bearing cross-linkage between lignin and glucuronoxylan. A well-defined and yet more realistic synthetic substrate was successfully synthesized via a key step of the benzyl esterification of 4-O-methyl-glucuronyl-1,4-β-d-xylotriose (MeGlcA³Xyl₃), a minimized fragment of glucuronoxylan enzymatically digested by β-1,4-xylanase. To the best of our knowledge, this is the first report of the productive GE kinetic analysis using this substrate. Kinetic parameters of the GE from the fungal Pestalotiopsis sp. AN-7 (PesGE), i.e., the Km, Vmax, and kcat of Bn-MeGlcA³Xyl₃, were 0.43 mM, 55.5 μmol min⁻¹·mg⁻¹, and 35.8 s⁻¹, respectively. On the other hand, as reported to date, the productive kinetic parameters for Bn-GlcA were not obtained because of its excessively high Km value (>16 mM). The substantial variance in the enzymatic activity of PesGE regarding substrate-binding affinity between Bn-MeGlcA³Xyl₃ and Bn-GlcA was also demonstrated using in silico docking simulation. These results suggested that the extended xylan fragment is a key structural determinant affecting PesGE's substrate recognition. Furthermore, the presence of a natural xylan backbone allows for evaluating the enzyme activity of xylan-degrading enzymes. Accordingly, the synthesized substrate with the natural core structure of LCC allowed us to unveil the productive kinetic parameters of GEs, serving as a versatile substrate for further elucidating the cascade reaction of GE and xylan-degrading enzymes.

Delignification

学術雑誌論文

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