アノマー反転型糖質加水分解酵素のグライコシンターゼ化

DOI 日本農学文献記事索引 Web Site Web Site 被引用文献2件 参考文献52件

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  • Conversion of an Inverting Glycoside Hydrolase into Glycosynthase

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Reducing end xylose releasing exo-oligoxylanase (REX, EC 3.2.1.156) hydrolyzes the glycosidic bond at reducing end of xylooligosaccharide with anomeric inversion manor. REX catalyzed “Hehre resynthesis-hydrolysis” reaction in the presence of α-xylobiosyl fluoride (α-X2F) and X1. We found that nine catalytic base mutants (D263→G, A, V, T, L, A, C, P or S) catalyzed glycosynthase reaction using α-X2F and X1 as the donor and acceptor substrates. D263C was found to be the best glycosynthase among these mutants. However, the mutant had a little hydrolytic activity, which produced X1 and X2 from glycosynthase reaction product, X3. To obtain mutant with more efficient the glycosynthase activity, we designed new glycosynthase based on the active site structure. We found that the nucleophilic water molecule activated by D263 was supported by Y198 with a hydrogen bond at its phenolic oxygen in the 3D structure of REX. To break the interaction between Y198 and water molecule, the residue was replaced with F residue (Y198F). Y198F mutant expressed a drastic decrease in the hydrolytic activity and a small increase in F- releasing activity from α-X2F in the presence of xylose. To create glycosynthase from inverting GHs, we conclude that an amino acid residue holding the nucleophilic water molecule was better target for mutation than catalytic base residue.

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