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Improving the Catalytic Property of the Glycoside Hydrolase LXYL-P1–2 by Directed Evolution
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 L...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6149855/ https://www.ncbi.nlm.nih.gov/pubmed/29207529 http://dx.doi.org/10.3390/molecules22122133 |
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author | Chen, Jing-Jing Liang, Xiao Li, Hui-Xian Chen, Tian-Jiao Zhu, Ping |
author_facet | Chen, Jing-Jing Liang, Xiao Li, Hui-Xian Chen, Tian-Jiao Zhu, Ping |
author_sort | Chen, Jing-Jing |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-6149855 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-61498552018-11-13 Improving the Catalytic Property of the Glycoside Hydrolase LXYL-P1–2 by Directed Evolution Chen, Jing-Jing Liang, Xiao Li, Hui-Xian Chen, Tian-Jiao Zhu, Ping Molecules Article 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. MDPI 2017-12-04 /pmc/articles/PMC6149855/ /pubmed/29207529 http://dx.doi.org/10.3390/molecules22122133 Text en © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Chen, Jing-Jing Liang, Xiao Li, Hui-Xian Chen, Tian-Jiao Zhu, Ping Improving the Catalytic Property of the Glycoside Hydrolase LXYL-P1–2 by Directed Evolution |
title | Improving the Catalytic Property of the Glycoside Hydrolase LXYL-P1–2 by Directed Evolution |
title_full | Improving the Catalytic Property of the Glycoside Hydrolase LXYL-P1–2 by Directed Evolution |
title_fullStr | Improving the Catalytic Property of the Glycoside Hydrolase LXYL-P1–2 by Directed Evolution |
title_full_unstemmed | Improving the Catalytic Property of the Glycoside Hydrolase LXYL-P1–2 by Directed Evolution |
title_short | Improving the Catalytic Property of the Glycoside Hydrolase LXYL-P1–2 by Directed Evolution |
title_sort | improving the catalytic property of the glycoside hydrolase lxyl-p1–2 by directed evolution |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6149855/ https://www.ncbi.nlm.nih.gov/pubmed/29207529 http://dx.doi.org/10.3390/molecules22122133 |
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