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Although lipases are promising catalysts for biodiesel production, their efficiency in industrial-scale application still requires improvement.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      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.\n                      <jats:italic>Thermomyces lanuginosus<\/jats:italic>\n                      lipase (TLL) with high activity and thermostability was obtained using the\n                      <jats:italic>Pichia pastoris<\/jats:italic>\n                      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\u2013TLL). Their corresponding melting temperature profiles increased by 10.53 and 6.01\u00a0\u00b0C,\n                      <jats:inline-formula>\n                        <jats:alternatives>\n                          <jats:tex-math>$$T_{50}^{15}$$<\/jats:tex-math>\n                          <mml:math xmlns:mml=\"http:\/\/www.w3.org\/1998\/Math\/MathML\">\n                            <mml:msubsup>\n                              <mml:mi>T<\/mml:mi>\n                              <mml:mrow>\n                                <mml:mn>50<\/mml:mn>\n                              <\/mml:mrow>\n                              <mml:mn>15<\/mml:mn>\n                            <\/mml:msubsup>\n                          <\/mml:math>\n                        <\/jats:alternatives>\n                      <\/jats:inline-formula>\n                      (the temperature at which the activity is reduced to 50% after 15 min incubation) increased from 60.88 to 68.46\u00a0\u00b0C and 66.30\u00a0\u00b0C, and the optimum temperatures shifted from 45 to 50\u00a0\u00b0C. 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\u2013TLL. 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\u00a0h at an elevated temperature (50\u00a0\u00b0C) were 94.03% and 98.56%, respectively, markedly higher than that of the wt\u2013TLL (88.56%) at its optimal temperature (45\u00a0\u00b0C) by transesterification of soybean oil.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats: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.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s13068-024-02478-5","type":"journal-article","created":{"date-parts":[[2024,2,24]],"date-time":"2024-02-24T09:02:02Z","timestamp":1708765322000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":24,"title":["In silico design of multipoint mutants for enhanced performance of Thermomyces lanuginosus lipase for efficient biodiesel production"],"prefix":"10.1186","volume":"17","author":[{"given":"Jinsha","family":"Huang","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaoman","family":"Xie","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wanlin","family":"Zheng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Li","family":"Xu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jinyong","family":"Yan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ying","family":"Wu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Min","family":"Yang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yunjun","family":"Yan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2024,2,24]]},"reference":[{"key":"2478_CR1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jclepro.2022.131588","volume":"355","author":"P Maheshwari","year":"2022","unstructured":"Maheshwari P, Haider MB, Yusuf M, Kleme\u0161 JJ, Bokhari A, Beg M, Al-Othman A, Kumar R, Jaiswal AK. 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