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Directed Evolution of Pseudomonas fluorescens Lipase Variants With Improved Thermostability Using Error-Prone PCR
Lipases catalyze the hydrolysis of fats and oils, and have been widely used in various industrial fields. However, bacterial lipases have a lower thermostability in industrial processes, which was a limiting factor in their industrial application. In this study, we obtained an improve variant of Pse...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7492553/ https://www.ncbi.nlm.nih.gov/pubmed/32984290 http://dx.doi.org/10.3389/fbioe.2020.01034 |
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author | Guan, Lijun Gao, Yang Li, Jialei Wang, Kunlun Zhang, Zhihong Yan, Song Ji, Nina Zhou, Ye Lu, Shuwen |
author_facet | Guan, Lijun Gao, Yang Li, Jialei Wang, Kunlun Zhang, Zhihong Yan, Song Ji, Nina Zhou, Ye Lu, Shuwen |
author_sort | Guan, Lijun |
collection | PubMed |
description | Lipases catalyze the hydrolysis of fats and oils, and have been widely used in various industrial fields. However, bacterial lipases have a lower thermostability in industrial processes, which was a limiting factor in their industrial application. In this study, we obtained an improve variant of Pseudomonas fluorescens lipase (PFL) with enhanced thermostability using classical error-prone PCR. Wild-type PFL showed an optimal temperature and pH of 50°C and pH 7.5, respectively. Due to the low thermostability of PFL, a library containing over 3000 individual mutants as constructed using error-prone PCR. Screening for thermotolerance yielded the mutants L218P and P184C/M243C with T(m) values of 62.5 and 66.0°C, which was 2.5 and 6°C higher than that of the WT, respectively. The combination of the two mutants (P184C/M243C/L218P) resulted in an approximately additive effect with a T(m) value of 68.0°C. Although the increase of T(m) was not substantial, the mutant also had dramatically increased methanol tolerance. Structural analysis revealed that the introduction of a disulfide bond between P184C and M243C and the substitution of Pro to reduce the flexibility of a loop increased the thermostability of PFL, which provides a theoretical foundation for improving the thermostability and methanol tolerance of lipase family I.1 to resist the harsh conditions of industrial processes. |
format | Online Article Text |
id | pubmed-7492553 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-74925532020-09-25 Directed Evolution of Pseudomonas fluorescens Lipase Variants With Improved Thermostability Using Error-Prone PCR Guan, Lijun Gao, Yang Li, Jialei Wang, Kunlun Zhang, Zhihong Yan, Song Ji, Nina Zhou, Ye Lu, Shuwen Front Bioeng Biotechnol Bioengineering and Biotechnology Lipases catalyze the hydrolysis of fats and oils, and have been widely used in various industrial fields. However, bacterial lipases have a lower thermostability in industrial processes, which was a limiting factor in their industrial application. In this study, we obtained an improve variant of Pseudomonas fluorescens lipase (PFL) with enhanced thermostability using classical error-prone PCR. Wild-type PFL showed an optimal temperature and pH of 50°C and pH 7.5, respectively. Due to the low thermostability of PFL, a library containing over 3000 individual mutants as constructed using error-prone PCR. Screening for thermotolerance yielded the mutants L218P and P184C/M243C with T(m) values of 62.5 and 66.0°C, which was 2.5 and 6°C higher than that of the WT, respectively. The combination of the two mutants (P184C/M243C/L218P) resulted in an approximately additive effect with a T(m) value of 68.0°C. Although the increase of T(m) was not substantial, the mutant also had dramatically increased methanol tolerance. Structural analysis revealed that the introduction of a disulfide bond between P184C and M243C and the substitution of Pro to reduce the flexibility of a loop increased the thermostability of PFL, which provides a theoretical foundation for improving the thermostability and methanol tolerance of lipase family I.1 to resist the harsh conditions of industrial processes. Frontiers Media S.A. 2020-09-02 /pmc/articles/PMC7492553/ /pubmed/32984290 http://dx.doi.org/10.3389/fbioe.2020.01034 Text en Copyright © 2020 Guan, Gao, Li, Wang, Zhang, Yan, Ji, Zhou and Lu. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Bioengineering and Biotechnology Guan, Lijun Gao, Yang Li, Jialei Wang, Kunlun Zhang, Zhihong Yan, Song Ji, Nina Zhou, Ye Lu, Shuwen Directed Evolution of Pseudomonas fluorescens Lipase Variants With Improved Thermostability Using Error-Prone PCR |
title | Directed Evolution of Pseudomonas fluorescens Lipase Variants With Improved Thermostability Using Error-Prone PCR |
title_full | Directed Evolution of Pseudomonas fluorescens Lipase Variants With Improved Thermostability Using Error-Prone PCR |
title_fullStr | Directed Evolution of Pseudomonas fluorescens Lipase Variants With Improved Thermostability Using Error-Prone PCR |
title_full_unstemmed | Directed Evolution of Pseudomonas fluorescens Lipase Variants With Improved Thermostability Using Error-Prone PCR |
title_short | Directed Evolution of Pseudomonas fluorescens Lipase Variants With Improved Thermostability Using Error-Prone PCR |
title_sort | directed evolution of pseudomonas fluorescens lipase variants with improved thermostability using error-prone pcr |
topic | Bioengineering and Biotechnology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7492553/ https://www.ncbi.nlm.nih.gov/pubmed/32984290 http://dx.doi.org/10.3389/fbioe.2020.01034 |
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