title: Structure-guided modification of Rhizomucor miehei lipase for production of structured lipids.
abstract: 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.
authors.affiliation: Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, PR China.
authors.forename: Jun-Hui
authors.initials: JH
authors.lastname: Zhang
authors.forename: Yu-Yan
authors.initials: YY
authors.lastname: Jiang
authors.forename: Ying
authors.initials: Y
authors.lastname: Lin
authors.forename: Yu-Fei
authors.initials: YF
authors.lastname: Sun
authors.forename: Sui-Ping
authors.initials: SP
authors.lastname: Zheng
authors.forename: Shuang-Yan
authors.initials: SY
authors.lastname: Han
mesh_terms: D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry; D002384:Catalysis; D004789:Enzyme Activation; D005227:Fatty Acids / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008055:Lipids / Q000096:biosynthesis*; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering; D020103:Rhizomucor / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D005227:Fatty Acids; D008055:Lipids; D008049:Lipase