Improving the catalytic activity of lipase LipK107 from Proteus sp. by site-directed mutagenesis in the lid domain based on computer simulation |
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| Affiliation: | 1. State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, PR China;2. College of Life Science, Hubei University, Wuhan 430062, PR China;1. Division of Bacterial Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 15000, PR China;2. School of Medicine, Tsinghua University, Beijing 100084, PR China;3. Fujian Agriculture and Forestry University, Fuzhou 350002, PR China;1. University of Sfax, Laboratory of Biochemistry and Enzymatic Engineering of Lipases, ENIS, BP 3038 Sfax, Tunisia;2. CNRS, Enzymologie Interfaciale et Physiologie de la Lipolyse, Aix-Marseille University, UMR7282, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France;3. University Tunis-ElManar, Institute of Pasteur, Laboratory of Venoms and Therapeutiques Biomolecules LR11IPT08, Tunis 1002, Tunisia;4. Univ Lyon, Université Lyon 1, Institut de Chimie et de Biochimie Moléculaires et Supramoléculaires, UMR 5246, Métabolisme, Enzymes et Mécanismes Moléculaires (MEM2), 43, Bd du 11 novembre 1918, F-69622 Villeurbanne Cedex, France;5. University of Sfax, Laboratory of Molecular and Cellular Screening Processes, Center of Biotechnology of Sfax, BP 1117, Route Sidi Mansour Km 6, Sfax, Tunisia;6. Laboratoire de biogenèse Membranaire, CNRS, UMR5200, INRA Bordeaux Aquitaine, BP81, 71 Edouard Bourlaux, 33883 Villenave d’Ornon cedex, Bordeaux, France;1. Department of Electrical and Electronic Information Engineering, Toyohashi University of Technology, 1-1 Hibarigaoka, Tempaku, Toyohashi, Aichi 441-8580, Japan;2. Fuji Research Laboratory, Tokai Carbon Co., Ltd., 394-1 Subashiri, Oyama, Sunto, Shizuoka 410-1431, Japan;3. Shonan Plastic Mfg. Co., Ltd., 31-27 Daikan, Hiratsuka, Kanagawa 254-0807, Japan;1. Department of Applied Molecular Bioscience, Yamaguchi University, Tokiwadai 2-16-1, Ube, 755-8611, Japan;2. Department of Applied Chemistry, Yamaguchi University, Tokiwadai 2-16-1, Ube, 755-8611, Japan;3. Division of Chemical Engineering, Graduate School of Engineering Science, Osaka University, Machikaneyama 1-3, Toyonaka 560-8531, Japan |
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| Abstract: | The capacity of lipase LipK107 from Proteus sp. catalyzing the kinetic resolution of racemates was investigated. The resolution of racemic 1-phenylethanol in organic medium was selected as model reaction. The conversion was dramatically dependent on the water content and the LipK107 showed high activity in a wide range of water content without appreciable loss of enzyme enantiodiscrimination. Besides, the chain length of acyl donor also had a significant effect on the conversion, and the highest enantioselectivity was achieved when methyl palmitate was used. Based on the analysis of computer model structure of LipK107, different mutations were introduced into the lid region. Each derivative of LipK107 was expressed, purified, and assessed of the activity. According to the prediction, using mutants E130L + K131I and T138V as catalyst, respectively, the conversions of 1-phenylethanol improved greatly with a slight increase of enantiodiscrimination. In addition, the effects of hydrophobicity and electrostatic of the lid on lipase activity were determined. This work indicated that the modification of the lid might considerably enhance the activity and improve the yield of catalytic reactions, which could apply to other lipases. The computer simulations would make the process of identifying amino acids for substitution efficiently. |
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