Improving the Catalytic Property of the Glycoside Hydrolase LXYL-P1–2 by Directed Evolution
AbstractThe glycoside hydrolase LXYL-P1–2 from Lentinula edodes can specifically hydrolyze 7-β-xylosyltaxanes to form 7-β-hydroxyltaxanes for the semi-synthesis of paclitaxel. In order to improve the catalytic properties of the enzyme, we performed error-prone PCR to construct the random mutant library of LXYL-P1–2 and used the methanol-induced plate method to screen the mutants with improved catalytic properties. Two variants, LXYL-P1–2-EP1 (EP1, S91D mutation) and LXYL-P1–2-EP2 (EP2, T368E mutation), were obtained from the library and exhibited 17% and 47% increases in their catalytic efficiencies on 7-β-xylosyl-10-deacetyltaxol. Meanwhile, compared with LXYL-P1–2, EP1 and EP2 showed elevated stabilities in the range of pH ≥ 6 conditions. After treatment at pH 12 for 48 h, EP1 and EP2 retained 77% and 63% activities, respectively, while the wild-type only retained 33% activity under the same condition. Molecular docking results revealed that the S91D mutation led to a shorter distance between the R-chain and the substrate, while the T368E mutation increased negative charge at the surface of the enzyme, and may introduce alterations of the loop near the active pocket, both of which may result in improved stabilities and catalytic activities of enzymes. This study provides a practical directed evolution method for exploring catalytically improved glycoside hydrolase. View Full-Text
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Chen, J.-J.; Liang, X.; Li, H.-X.; Chen, T.-J.; Zhu, P. Improving the Catalytic Property of the Glycoside Hydrolase LXYL-P1–2 by Directed Evolution. Molecules 2017, 22, 2133.
Chen J-J, Liang X, Li H-X, Chen T-J, Zhu P. Improving the Catalytic Property of the Glycoside Hydrolase LXYL-P1–2 by Directed Evolution. Molecules. 2017; 22(12):2133.Chicago/Turabian Style
Chen, Jing-Jing; Liang, Xiao; Li, Hui-Xian; Chen, Tian-Jiao; Zhu, Ping. 2017. "Improving the Catalytic Property of the Glycoside Hydrolase LXYL-P1–2 by Directed Evolution." Molecules 22, no. 12: 2133.
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