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Structure-Guided Modification of Rhizomucor miehei Lipase for Production of Structured Lipids
To improve the performance of yeast surface-displayed Rhizomucor miehei lipase (RML) in the production of human milk fat substitute (HMFS), we mutated amino acids in the lipase substrate-binding pocket based on protein hydrophobicity, to improve esterification activity. Five mutants: Asn87Ile, Asn87...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3700896/ https://www.ncbi.nlm.nih.gov/pubmed/23844120 http://dx.doi.org/10.1371/journal.pone.0067892 |
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author | Zhang, Jun-Hui Jiang, Yu-Yan Lin, Ying Sun, Yu-Fei Zheng, Sui-Ping Han, Shuang-Yan |
author_facet | Zhang, Jun-Hui Jiang, Yu-Yan Lin, Ying Sun, Yu-Fei Zheng, Sui-Ping Han, Shuang-Yan |
author_sort | Zhang, Jun-Hui |
collection | PubMed |
description | To improve the performance of yeast surface-displayed Rhizomucor miehei lipase (RML) in the production of human milk fat substitute (HMFS), we mutated amino acids in the lipase substrate-binding pocket based on protein hydrophobicity, to improve esterification activity. Five mutants: Asn87Ile, Asn87Ile/Asp91Val, His108Leu/Lys109Ile, Asp256Ile/His257Leu, and His108Leu/Lys109Ile/Asp256Ile/His257Leu were obtained and their hydrolytic and esterification activities were assayed. Using Discovery Studio 3.1 to build models and calculate the binding energy between lipase and substrates, compared to wild-type, the mutant Asp256Ile/His257Leu was found to have significantly lower energy when oleic acid (3.97 KJ/mol decrease) and tripalmitin (7.55 KJ/mol decrease) were substrates. This result was in accordance with the esterification activity of Asp256Ile/His257Leu (2.37-fold of wild-type). The four mutants were also evaluated for the production of HMFS in organic solvent and in a solvent-free system. Asp256Ile/His257Leu had an oleic acid incorporation of 28.27% for catalyzing tripalmitin and oleic acid, and 53.18% for the reaction of palm oil with oleic acid. The efficiency of Asp256Ile/His257Leu was 1.82-fold and 1.65-fold that of the wild-type enzyme for the two reactions. The oleic acid incorporation of Asp256Ile/His257Leu was similar to commercial Lipozyme RM IM for palm oil acidolysis with oleic acid. Yeast surface-displayed RML mutant Asp256Ile/His257Leu is a potential, economically feasible catalyst for the production of structured lipids. |
format | Online Article Text |
id | pubmed-3700896 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-37008962013-07-10 Structure-Guided Modification of Rhizomucor miehei Lipase for Production of Structured Lipids Zhang, Jun-Hui Jiang, Yu-Yan Lin, Ying Sun, Yu-Fei Zheng, Sui-Ping Han, Shuang-Yan PLoS One Research Article To improve the performance of yeast surface-displayed Rhizomucor miehei lipase (RML) in the production of human milk fat substitute (HMFS), we mutated amino acids in the lipase substrate-binding pocket based on protein hydrophobicity, to improve esterification activity. Five mutants: Asn87Ile, Asn87Ile/Asp91Val, His108Leu/Lys109Ile, Asp256Ile/His257Leu, and His108Leu/Lys109Ile/Asp256Ile/His257Leu were obtained and their hydrolytic and esterification activities were assayed. Using Discovery Studio 3.1 to build models and calculate the binding energy between lipase and substrates, compared to wild-type, the mutant Asp256Ile/His257Leu was found to have significantly lower energy when oleic acid (3.97 KJ/mol decrease) and tripalmitin (7.55 KJ/mol decrease) were substrates. This result was in accordance with the esterification activity of Asp256Ile/His257Leu (2.37-fold of wild-type). The four mutants were also evaluated for the production of HMFS in organic solvent and in a solvent-free system. Asp256Ile/His257Leu had an oleic acid incorporation of 28.27% for catalyzing tripalmitin and oleic acid, and 53.18% for the reaction of palm oil with oleic acid. The efficiency of Asp256Ile/His257Leu was 1.82-fold and 1.65-fold that of the wild-type enzyme for the two reactions. The oleic acid incorporation of Asp256Ile/His257Leu was similar to commercial Lipozyme RM IM for palm oil acidolysis with oleic acid. Yeast surface-displayed RML mutant Asp256Ile/His257Leu is a potential, economically feasible catalyst for the production of structured lipids. Public Library of Science 2013-07-03 /pmc/articles/PMC3700896/ /pubmed/23844120 http://dx.doi.org/10.1371/journal.pone.0067892 Text en © 2013 Zhang 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, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Zhang, Jun-Hui Jiang, Yu-Yan Lin, Ying Sun, Yu-Fei Zheng, Sui-Ping Han, Shuang-Yan Structure-Guided Modification of Rhizomucor miehei Lipase for Production of Structured Lipids |
title | Structure-Guided Modification of Rhizomucor miehei Lipase for Production of Structured Lipids |
title_full | Structure-Guided Modification of Rhizomucor miehei Lipase for Production of Structured Lipids |
title_fullStr | Structure-Guided Modification of Rhizomucor miehei Lipase for Production of Structured Lipids |
title_full_unstemmed | Structure-Guided Modification of Rhizomucor miehei Lipase for Production of Structured Lipids |
title_short | Structure-Guided Modification of Rhizomucor miehei Lipase for Production of Structured Lipids |
title_sort | structure-guided modification of rhizomucor miehei lipase for production of structured lipids |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3700896/ https://www.ncbi.nlm.nih.gov/pubmed/23844120 http://dx.doi.org/10.1371/journal.pone.0067892 |
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