Expression of the lasB gene encoding an organic solvent-stable elastase in Pichia pastoris and potential applications of the recombinant enzymes in peptide synthesis |
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| Affiliation: | 1. Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, No. 1800 Lihu Avenue, Wuxi 214122, China;2. Jiangsu Key Laboratory for Biomass-based Energy and Enzyme Technology, Jiangsu Key Laboratory for Eco-Agricultural Biotechnology around Hongze Lake, Huaiyin Normal University, No. 111 Changjiang West Road, Huaian 223300, China;1. Metabolic Biophysics, Professional Toxicology and Applied Environmental Laboratory, Department of Biophysics, Medicine Faculty of Sousse, Sousse University, Sousse 4002, Tunisia;2. Laboratory of Natural Substances, National Institute of Research and Physical–Chemical Analysis (INRAP), Technopole Sidi Thabet 2020, Tunisia;3. Neurology Department of Central Hospital University (CHU), Sousse University, Sousse 4002, Tunisia;1. Key Lab of Organic Optoelectronics and Molecular Engineering of Ministry of Education, Department of Chemistry, Tsinghua University, Beijing 100084, China;2. Key Lab of Cigarette Smoke of State Tobacco Monopoly Administration, Technical Center of Shanghai Tobacco Corporation, Shanghai 200082, China;1. Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, C.P. 476, 88040-900, Florianópolis, SC, Brazil;2. Laboratório de Entomologia e Fitopatologia, Centro de Ciências e Tecnologia Agropecuárias, Universidade Estadual do Norte Fluminense Darcy Ribeiro, 28013-602, Campos dos Goytacazes, RJ, Brazil;3. Departamento de Tecnologia de Alimentos e Saúde Pública, Universidade Federal de Mato Grosso do Sul, C.P. 549, 79070-900, Campo Grande, MS, Brazil;1. MOE Key Laboratory of Modern Acoustics, Nanjing University, Nanjing 210093, China;2. Department of Nephrology, Nanjing Children''s Hospital, Nanjing Medical University, Nanjing 210008, China;3. Department of Radiology, University of Michigan School of Medicine, Ann Arbor, MI 48109, USA;4. Department of Automation, Nanjing College of Chemical Technology, Nanjing 210048, China;1. Instituto de Química Orgánica General (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain;2. Instituto de Química-Física “Rocasolano” (CSIC), Serrano 119, 28006 Madrid, Spain |
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| Abstract: | The lasB gene encoding a solvent-stable elastase from Pseudomonas aeruginosa (PAE) was isolated and heterologously expressed in Pichia pastoris, resulting in production of three heterogeneously glycosylated recombinant elastases (rPAEs). rPAEs showed higher solvent-stability and thermostability than native PAE, but these recombinant and native enzymes achieved similar values of specific activity (2393 U/mg and 2427 U/mg for rPAEs and the native one, respectively), apparent Km (2.55 and 2.48 g/l for rPAEs and the native one, respectively) and kcat (0.0489 and 0.0496/s for rPAEs and the native one, respectively) for casein hydrolysis. While rPAEs and their native counterpart displayed similar substrate specificity in bipeptide synthesis reactions in water-miscible organic solvents, the former gave higher synthesis rates and yields than the latter. The yields and rates of rPAEs-catalyzed bipeptide synthesis reactions substantially varied with the type of solvent, and dimethylsulfoxide (DMSO) was found to be more suitable for these reactions than methanol, ethanol, isopropanol, and n-butanol. The optimal reaction conditions for rPAEs-catalyzed Cbz-Ala-Phe-NH2 synthesis were the presence of 50% (v/v) DMSO, and at pH 8.0 and temperature 20–30 °C. |
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| Keywords: | Pseudolysin Organic solvent-stability Peptide synthesis Gene expression |
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