Improving the Catalytic Property of the Glycoside Hydrolase LXYL-P1-2 by Directed Evolution.
Improving the Catalytic Property of the Glycoside Hydrolase LXYL-P1-2 by Directed Evolution.
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通过定向进化提高糖苷水解酶 LXYL-P1-2 的催化性能。
DOI：
10.3390/molecules22122133
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发表时间：
2017-12-04
期刊：
影响因子：
--
通讯作者：
Zhu P
中科院分区：
文献类型：
--
作者：
Chen JJ;Liang X;Li HX;Chen TJ;Zhu P
香菇糖苷水解酶LXYL-P1-2能特异性水解7-β-木糖基紫杉烷，生成7-β-羟基紫杉烷，用于紫杉醇的半合成。为了提高酶的催化性能，我们采用易错PCR技术构建了LXYL-P1-2的随机突变体库，并采用甲醇诱导平板法筛选了催化性能提高的突变体。从文库中获得两个突变体LXYL-P1-2-EP 1（EP 1，S91 D突变）和LXYL-P1-2-EP 2（EP 2，T368 E突变），它们对7-β-木糖基-10-脱乙酰紫杉醇的催化效率分别提高了17%和47%。与LXYL-P1-2相比，EP 1和EP 2在pH ≥ 6的条件下稳定性更好。在pH 12条件下处理48 h后，EP 1和EP 2分别保留了77%和63%的活性，而野生型仅保留了33%的活性。分子对接结果表明，S91 D突变导致R链与底物之间的距离缩短，而T368 E突变增加了酶表面的负电荷，并可能引入活性口袋附近环的改变，这两者都可能导致酶的稳定性和催化活性的提高。该研究为探索催化改进的糖苷水解酶提供了一种实用的定向进化方法。
The 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.
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