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Enhancing the Thermostability of Serratia plymuthica Sucrose Isomerase Using B-Factor-Directed Mutagenesis
The sucrose isomerase of Serratia plymuthica AS9 (AS9 PalI) was expressed in Escherichia coli BL21(DE3) and characterized. The half-life of AS9 PalI was 20 min at 45°C, indicating that it was unstable. In order to improve its thermostability, six amino acid residues with higher B-factors were select...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4757035/ https://www.ncbi.nlm.nih.gov/pubmed/26886729 http://dx.doi.org/10.1371/journal.pone.0149208 |
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author | Duan, Xuguo Cheng, Sheng Ai, Yixin Wu, Jing |
author_facet | Duan, Xuguo Cheng, Sheng Ai, Yixin Wu, Jing |
author_sort | Duan, Xuguo |
collection | PubMed |
description | The sucrose isomerase of Serratia plymuthica AS9 (AS9 PalI) was expressed in Escherichia coli BL21(DE3) and characterized. The half-life of AS9 PalI was 20 min at 45°C, indicating that it was unstable. In order to improve its thermostability, six amino acid residues with higher B-factors were selected as targets for site-directed mutagenesis, and six mutants (E175N, K576D, K174D, G176D, S575D and N577K) were designed using the RosettaDesign server. The E175N and K576D mutants exhibited improved thermostability in preliminary experiments, so the double mutant E175N/K576D was constructed. These three mutants (E175N, K576D, E175N/K576D) were characterized in detail. The results indicate that the three mutants exhibit a slightly increased optimal temperature (35°C), compared with that of the wild-type enzyme (30°C). The mutants also share an identical pH optimum of 6.0, which is similar to that of the wild-type enzyme. The half-lives of the E175N, K576D and E175N/K576D mutants were 2.30, 1.78 and 7.65 times greater than that of the wild-type enzyme at 45°C, respectively. Kinetic studies showed that the K(m) values for the E175N, K576D and E175N/K576D mutants decreased by 6.6%, 2.0% and 11.0%, respectively, and their k(cat)/K(m) values increased by 38.2%, 4.2% and 19.4%, respectively, compared with those of the wild-type enzyme. After optimizing the conditions for isomaltulose production at 45°C, we found that the E175N, K576D and E175N/K576D mutants displayed slightly improved isomaltulose yields, compared with the wild-type enzyme. Therefore, the mutants produced in this study would be more suitable for industrial biosynthesis of isomaltulose. |
format | Online Article Text |
id | pubmed-4757035 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-47570352016-02-26 Enhancing the Thermostability of Serratia plymuthica Sucrose Isomerase Using B-Factor-Directed Mutagenesis Duan, Xuguo Cheng, Sheng Ai, Yixin Wu, Jing PLoS One Research Article The sucrose isomerase of Serratia plymuthica AS9 (AS9 PalI) was expressed in Escherichia coli BL21(DE3) and characterized. The half-life of AS9 PalI was 20 min at 45°C, indicating that it was unstable. In order to improve its thermostability, six amino acid residues with higher B-factors were selected as targets for site-directed mutagenesis, and six mutants (E175N, K576D, K174D, G176D, S575D and N577K) were designed using the RosettaDesign server. The E175N and K576D mutants exhibited improved thermostability in preliminary experiments, so the double mutant E175N/K576D was constructed. These three mutants (E175N, K576D, E175N/K576D) were characterized in detail. The results indicate that the three mutants exhibit a slightly increased optimal temperature (35°C), compared with that of the wild-type enzyme (30°C). The mutants also share an identical pH optimum of 6.0, which is similar to that of the wild-type enzyme. The half-lives of the E175N, K576D and E175N/K576D mutants were 2.30, 1.78 and 7.65 times greater than that of the wild-type enzyme at 45°C, respectively. Kinetic studies showed that the K(m) values for the E175N, K576D and E175N/K576D mutants decreased by 6.6%, 2.0% and 11.0%, respectively, and their k(cat)/K(m) values increased by 38.2%, 4.2% and 19.4%, respectively, compared with those of the wild-type enzyme. After optimizing the conditions for isomaltulose production at 45°C, we found that the E175N, K576D and E175N/K576D mutants displayed slightly improved isomaltulose yields, compared with the wild-type enzyme. Therefore, the mutants produced in this study would be more suitable for industrial biosynthesis of isomaltulose. Public Library of Science 2016-02-17 /pmc/articles/PMC4757035/ /pubmed/26886729 http://dx.doi.org/10.1371/journal.pone.0149208 Text en © 2016 Duan et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Duan, Xuguo Cheng, Sheng Ai, Yixin Wu, Jing Enhancing the Thermostability of Serratia plymuthica Sucrose Isomerase Using B-Factor-Directed Mutagenesis |
title | Enhancing the Thermostability of Serratia plymuthica Sucrose Isomerase Using B-Factor-Directed Mutagenesis |
title_full | Enhancing the Thermostability of Serratia plymuthica Sucrose Isomerase Using B-Factor-Directed Mutagenesis |
title_fullStr | Enhancing the Thermostability of Serratia plymuthica Sucrose Isomerase Using B-Factor-Directed Mutagenesis |
title_full_unstemmed | Enhancing the Thermostability of Serratia plymuthica Sucrose Isomerase Using B-Factor-Directed Mutagenesis |
title_short | Enhancing the Thermostability of Serratia plymuthica Sucrose Isomerase Using B-Factor-Directed Mutagenesis |
title_sort | enhancing the thermostability of serratia plymuthica sucrose isomerase using b-factor-directed mutagenesis |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4757035/ https://www.ncbi.nlm.nih.gov/pubmed/26886729 http://dx.doi.org/10.1371/journal.pone.0149208 |
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