Jinsha Huang, Xiaoman Xie, Wanlin Zheng, Li Xu, Jinyong Yan, Ying Wu, Min Yang, Yunjun Yan
{"title":"硅学设计多点突变体,提高兰氏热霉菌脂肪酶的性能,以高效生产生物柴油。","authors":"Jinsha Huang, Xiaoman Xie, Wanlin Zheng, Li Xu, Jinyong Yan, Ying Wu, Min Yang, Yunjun Yan","doi":"10.1186/s13068-024-02478-5","DOIUrl":null,"url":null,"abstract":"<div><h3>Background</h3><p>Biodiesel, an emerging sustainable and renewable clean energy, has garnered considerable attention as an alternative to fossil fuels. Although lipases are promising catalysts for biodiesel production, their efficiency in industrial-scale application still requires improvement.</p><h3>Results</h3><p>In this study, a novel strategy for multi-site mutagenesis in the binding pocket was developed via FuncLib (for mutant enzyme design) and Rosetta Cartesian_ddg (for free energy calculation) to improve the reaction rate and yield of lipase-catalyzed biodiesel production. <i>Thermomyces lanuginosus</i> lipase (TLL) with high activity and thermostability was obtained using the <i>Pichia pastoris</i> expression system. The specific activities of the mutants M11 and M21 (each with 5 and 4 mutations) were 1.50- and 3.10-fold higher, respectively, than those of the wild-type (wt–TLL). Their corresponding melting temperature profiles increased by 10.53 and 6.01 °C, <span>\\(T_{50}^{15}\\)</span> (the temperature at which the activity is reduced to 50% after 15 min incubation) increased from 60.88 to 68.46 °C and 66.30 °C, and the optimum temperatures shifted from 45 to 50 °C. After incubation in 60% methanol for 1 h, the mutants M11 and M21 retained more than 60% activity, and 45% higher activity than that of wt–TLL. Molecular dynamics simulations indicated that the increase in thermostability could be explained by reduced atomic fluctuation, and the improved catalytic properties were attributed to a reduced binding free energy and newly formed hydrophobic interaction. Yields of biodiesel production catalyzed by mutants M11 and M21 for 48 h at an elevated temperature (50 °C) were 94.03% and 98.56%, respectively, markedly higher than that of the wt–TLL (88.56%) at its optimal temperature (45 °C) by transesterification of soybean oil.</p><h3>Conclusions</h3><p>An integrating strategy was first adopted to realize the co-evolution of catalytic efficiency and thermostability of lipase. Two promising mutants M11 and M21 with excellent properties exhibited great potential for practical applications for in biodiesel production.</p></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":null,"pages":null},"PeriodicalIF":6.1000,"publicationDate":"2024-02-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://biotechnologyforbiofuels.biomedcentral.com/counter/pdf/10.1186/s13068-024-02478-5","citationCount":"0","resultStr":"{\"title\":\"In silico design of multipoint mutants for enhanced performance of Thermomyces lanuginosus lipase for efficient biodiesel production\",\"authors\":\"Jinsha Huang, Xiaoman Xie, Wanlin Zheng, Li Xu, Jinyong Yan, Ying Wu, Min Yang, Yunjun Yan\",\"doi\":\"10.1186/s13068-024-02478-5\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><h3>Background</h3><p>Biodiesel, an emerging sustainable and renewable clean energy, has garnered considerable attention as an alternative to fossil fuels. Although lipases are promising catalysts for biodiesel production, their efficiency in industrial-scale application still requires improvement.</p><h3>Results</h3><p>In this study, a novel strategy for multi-site mutagenesis in the binding pocket was developed via FuncLib (for mutant enzyme design) and Rosetta Cartesian_ddg (for free energy calculation) to improve the reaction rate and yield of lipase-catalyzed biodiesel production. <i>Thermomyces lanuginosus</i> lipase (TLL) with high activity and thermostability was obtained using the <i>Pichia pastoris</i> expression system. The specific activities of the mutants M11 and M21 (each with 5 and 4 mutations) were 1.50- and 3.10-fold higher, respectively, than those of the wild-type (wt–TLL). Their corresponding melting temperature profiles increased by 10.53 and 6.01 °C, <span>\\\\(T_{50}^{15}\\\\)</span> (the temperature at which the activity is reduced to 50% after 15 min incubation) increased from 60.88 to 68.46 °C and 66.30 °C, and the optimum temperatures shifted from 45 to 50 °C. After incubation in 60% methanol for 1 h, the mutants M11 and M21 retained more than 60% activity, and 45% higher activity than that of wt–TLL. Molecular dynamics simulations indicated that the increase in thermostability could be explained by reduced atomic fluctuation, and the improved catalytic properties were attributed to a reduced binding free energy and newly formed hydrophobic interaction. Yields of biodiesel production catalyzed by mutants M11 and M21 for 48 h at an elevated temperature (50 °C) were 94.03% and 98.56%, respectively, markedly higher than that of the wt–TLL (88.56%) at its optimal temperature (45 °C) by transesterification of soybean oil.</p><h3>Conclusions</h3><p>An integrating strategy was first adopted to realize the co-evolution of catalytic efficiency and thermostability of lipase. Two promising mutants M11 and M21 with excellent properties exhibited great potential for practical applications for in biodiesel production.</p></div>\",\"PeriodicalId\":494,\"journal\":{\"name\":\"Biotechnology for Biofuels\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":6.1000,\"publicationDate\":\"2024-02-24\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://biotechnologyforbiofuels.biomedcentral.com/counter/pdf/10.1186/s13068-024-02478-5\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Biotechnology for Biofuels\",\"FirstCategoryId\":\"5\",\"ListUrlMain\":\"https://link.springer.com/article/10.1186/s13068-024-02478-5\",\"RegionNum\":1,\"RegionCategory\":\"工程技术\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"BIOTECHNOLOGY & APPLIED MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biotechnology for Biofuels","FirstCategoryId":"5","ListUrlMain":"https://link.springer.com/article/10.1186/s13068-024-02478-5","RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
In silico design of multipoint mutants for enhanced performance of Thermomyces lanuginosus lipase for efficient biodiesel production
Background
Biodiesel, an emerging sustainable and renewable clean energy, has garnered considerable attention as an alternative to fossil fuels. Although lipases are promising catalysts for biodiesel production, their efficiency in industrial-scale application still requires improvement.
Results
In this study, a novel strategy for multi-site mutagenesis in the binding pocket was developed via FuncLib (for mutant enzyme design) and Rosetta Cartesian_ddg (for free energy calculation) to improve the reaction rate and yield of lipase-catalyzed biodiesel production. Thermomyces lanuginosus lipase (TLL) with high activity and thermostability was obtained using the Pichia pastoris expression system. The specific activities of the mutants M11 and M21 (each with 5 and 4 mutations) were 1.50- and 3.10-fold higher, respectively, than those of the wild-type (wt–TLL). Their corresponding melting temperature profiles increased by 10.53 and 6.01 °C, \(T_{50}^{15}\) (the temperature at which the activity is reduced to 50% after 15 min incubation) increased from 60.88 to 68.46 °C and 66.30 °C, and the optimum temperatures shifted from 45 to 50 °C. After incubation in 60% methanol for 1 h, the mutants M11 and M21 retained more than 60% activity, and 45% higher activity than that of wt–TLL. Molecular dynamics simulations indicated that the increase in thermostability could be explained by reduced atomic fluctuation, and the improved catalytic properties were attributed to a reduced binding free energy and newly formed hydrophobic interaction. Yields of biodiesel production catalyzed by mutants M11 and M21 for 48 h at an elevated temperature (50 °C) were 94.03% and 98.56%, respectively, markedly higher than that of the wt–TLL (88.56%) at its optimal temperature (45 °C) by transesterification of soybean oil.
Conclusions
An integrating strategy was first adopted to realize the co-evolution of catalytic efficiency and thermostability of lipase. Two promising mutants M11 and M21 with excellent properties exhibited great potential for practical applications for in biodiesel production.
期刊介绍:
Biotechnology for Biofuels is an open access peer-reviewed journal featuring high-quality studies describing technological and operational advances in the production of biofuels, chemicals and other bioproducts. The journal emphasizes understanding and advancing the application of biotechnology and synergistic operations to improve plants and biological conversion systems for the biological production of these products from biomass, intermediates derived from biomass, or CO2, as well as upstream or downstream operations that are integral to biological conversion of biomass.
Biotechnology for Biofuels focuses on the following areas:
• Development of terrestrial plant feedstocks
• Development of algal feedstocks
• Biomass pretreatment, fractionation and extraction for biological conversion
• Enzyme engineering, production and analysis
• Bacterial genetics, physiology and metabolic engineering
• Fungal/yeast genetics, physiology and metabolic engineering
• Fermentation, biocatalytic conversion and reaction dynamics
• Biological production of chemicals and bioproducts from biomass
• Anaerobic digestion, biohydrogen and bioelectricity
• Bioprocess integration, techno-economic analysis, modelling and policy
• Life cycle assessment and environmental impact analysis