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Simultaneous improvement of the thermostability and activity of lactic dehydrogenase from Lactobacillus rossiae through rational design
d-Phenyllactic acid, is a versatile organic acid with wide application prospects in the food, pharmaceutical and material industries. Wild-type lactate dehydrogenase LrLDH from Lactobacillus rossiae exhibits a high catalytic performance in the production of d-phenyllactic acid from phenylpyruvic aci...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The Royal Society of Chemistry
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9677063/ https://www.ncbi.nlm.nih.gov/pubmed/36425200 http://dx.doi.org/10.1039/d2ra05599f |
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author | Luo, Xi Wang, Yifeng Zheng, Weilong Sun, Xiaolong Hu, Gaowei Yin, Longfei Zhang, Yingying Yin, Fengwei Fu, Yongqian |
author_facet | Luo, Xi Wang, Yifeng Zheng, Weilong Sun, Xiaolong Hu, Gaowei Yin, Longfei Zhang, Yingying Yin, Fengwei Fu, Yongqian |
author_sort | Luo, Xi |
collection | PubMed |
description | d-Phenyllactic acid, is a versatile organic acid with wide application prospects in the food, pharmaceutical and material industries. Wild-type lactate dehydrogenase LrLDH from Lactobacillus rossiae exhibits a high catalytic performance in the production of d-phenyllactic acid from phenylpyruvic acid or sodium phenylpyruvate, but its industrial application is hampered by poor thermostability. Here, computer aided rational design was applied to improve the thermostability of LrLDH. By using HotSpot Wizard 3.0, five hotspot residues (N218, L237, T247, D249 and S301) were identified, after which site-saturation mutagenesis and combined mutagenesis were performed. The double mutant D249A/T247I was screen out as the best variant, with optimum temperature, t(1/2), and T(10)(50) that were 12 °C, 17.96 min and 19 °C higher than that of wild-type LrLDH, respectively. At the same time, the k(cat)/K(m) of D249A/T247I was 1.47 s(−1) mM(−1), which was 3.4 times higher than that of the wild-type enzyme. Thus rational design was successfully applied to simultaneously improve the thermostability and catalytic activity of LrLDH to a significant extent. The results of molecular dynamics simulations and molecular structure analysis could explain the mechanisms for the improved performance of the double mutant. This study shows that computer-aided rational design can greatly improve the thermostability of d-lactate dehydrogenase, offering a reference for the modification of other enzymes. |
format | Online Article Text |
id | pubmed-9677063 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | The Royal Society of Chemistry |
record_format | MEDLINE/PubMed |
spelling | pubmed-96770632022-11-23 Simultaneous improvement of the thermostability and activity of lactic dehydrogenase from Lactobacillus rossiae through rational design Luo, Xi Wang, Yifeng Zheng, Weilong Sun, Xiaolong Hu, Gaowei Yin, Longfei Zhang, Yingying Yin, Fengwei Fu, Yongqian RSC Adv Chemistry d-Phenyllactic acid, is a versatile organic acid with wide application prospects in the food, pharmaceutical and material industries. Wild-type lactate dehydrogenase LrLDH from Lactobacillus rossiae exhibits a high catalytic performance in the production of d-phenyllactic acid from phenylpyruvic acid or sodium phenylpyruvate, but its industrial application is hampered by poor thermostability. Here, computer aided rational design was applied to improve the thermostability of LrLDH. By using HotSpot Wizard 3.0, five hotspot residues (N218, L237, T247, D249 and S301) were identified, after which site-saturation mutagenesis and combined mutagenesis were performed. The double mutant D249A/T247I was screen out as the best variant, with optimum temperature, t(1/2), and T(10)(50) that were 12 °C, 17.96 min and 19 °C higher than that of wild-type LrLDH, respectively. At the same time, the k(cat)/K(m) of D249A/T247I was 1.47 s(−1) mM(−1), which was 3.4 times higher than that of the wild-type enzyme. Thus rational design was successfully applied to simultaneously improve the thermostability and catalytic activity of LrLDH to a significant extent. The results of molecular dynamics simulations and molecular structure analysis could explain the mechanisms for the improved performance of the double mutant. This study shows that computer-aided rational design can greatly improve the thermostability of d-lactate dehydrogenase, offering a reference for the modification of other enzymes. The Royal Society of Chemistry 2022-11-21 /pmc/articles/PMC9677063/ /pubmed/36425200 http://dx.doi.org/10.1039/d2ra05599f Text en This journal is © The Royal Society of Chemistry https://creativecommons.org/licenses/by-nc/3.0/ |
spellingShingle | Chemistry Luo, Xi Wang, Yifeng Zheng, Weilong Sun, Xiaolong Hu, Gaowei Yin, Longfei Zhang, Yingying Yin, Fengwei Fu, Yongqian Simultaneous improvement of the thermostability and activity of lactic dehydrogenase from Lactobacillus rossiae through rational design |
title | Simultaneous improvement of the thermostability and activity of lactic dehydrogenase from Lactobacillus rossiae through rational design |
title_full | Simultaneous improvement of the thermostability and activity of lactic dehydrogenase from Lactobacillus rossiae through rational design |
title_fullStr | Simultaneous improvement of the thermostability and activity of lactic dehydrogenase from Lactobacillus rossiae through rational design |
title_full_unstemmed | Simultaneous improvement of the thermostability and activity of lactic dehydrogenase from Lactobacillus rossiae through rational design |
title_short | Simultaneous improvement of the thermostability and activity of lactic dehydrogenase from Lactobacillus rossiae through rational design |
title_sort | simultaneous improvement of the thermostability and activity of lactic dehydrogenase from lactobacillus rossiae through rational design |
topic | Chemistry |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9677063/ https://www.ncbi.nlm.nih.gov/pubmed/36425200 http://dx.doi.org/10.1039/d2ra05599f |
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