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...
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2013-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3700896?pdf=render |
| _version_ | 1818529527176888320 |
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| author | Jun-Hui Zhang Yu-Yan Jiang Ying Lin Yu-Fei Sun Sui-Ping Zheng Shuang-Yan Han |
| author_facet | Jun-Hui Zhang Yu-Yan Jiang Ying Lin Yu-Fei Sun Sui-Ping Zheng Shuang-Yan Han |
| author_sort | Jun-Hui Zhang |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-11T17:08:01Z |
| format | Article |
| id | doaj.art-21cea953fc9447909b70c6953e2daea9 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-11T17:08:01Z |
| publishDate | 2013-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-21cea953fc9447909b70c6953e2daea92022-12-22T00:57:37ZengPublic Library of Science (PLoS)PLoS ONE1932-62032013-01-0187e6789210.1371/journal.pone.0067892Structure-guided modification of Rhizomucor miehei lipase for production of structured lipids.Jun-Hui ZhangYu-Yan JiangYing LinYu-Fei SunSui-Ping ZhengShuang-Yan HanTo 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.http://europepmc.org/articles/PMC3700896?pdf=render |
| spellingShingle | Jun-Hui Zhang Yu-Yan Jiang Ying Lin Yu-Fei Sun Sui-Ping Zheng Shuang-Yan Han Structure-guided modification of Rhizomucor miehei lipase for production of structured lipids. PLoS ONE |
| 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 |
| url | http://europepmc.org/articles/PMC3700896?pdf=render |
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