Cloning,expression and characterization of a new enantioselective esterase from a marine bacterium Pelagibacterium halotolerans B2T |
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| Institution: | 1. Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China;2. State Key Laboratory for Diagnosis and Treatment of Infectious Disease, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China;3. College of Life Sciences, Zhejiang University, Room 209, 866 Yuhangtang Road, Hangzhou 310058, China;3. Department of Biochemistry and Biophysics, Stockholm University SE-10691 Stockholm, Sweden;4. Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, United Kingdom;5. Science for Life Laboratories, Stockholm University, SE-171 21 Solna, Sweden;6. Department of Experimental Medical Science, Lund University, SE-221 84 Lund, Sweden;1. Wetsus, European Centre of Excellence for Sustainable Water Technology, PO Box 1113, 8900 CC Leeuwarden, The Netherlands;2. CEB - Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-57 Braga, Portugal;1. School of Chemical Engineering, Universiti Sains Malaysia, Engineering Campus, 14300 Nibong Tebal, Pulau Pinang, Malaysia;2. Department of Chemical Engineering, Universitas Negeri Semarang, Kampus Sekaran, Gunungpati, 50229 Semarang, Indonesia;1. Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Nijenborgh 4 Groningen, 9747 AG, The Netherlands |
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| Abstract: | An esterase, designated as PE8 (219 aa, 23.19 kDa), was cloned from a marine bacterium Pelagibacterium halotolerans B2T and overexpressed in Escherichia coli Rosetta, resulting an active, soluble protein which constituted 23.1% of the total cell protein content. Phylogenetic analysis of the protein showed it was a new member of family VI lipolytic enzymes. Biochemical characterization analysis showed that PE8 preferred short chain p-nitrophenyl esters (C2–C6), exhibited maximum activity toward p-nitrophenyl acetate, and was not a metalloenzyme. PE8 was an alkaline esterase with an optimal pH of 9.5 and an optimal temperature of 45 °C toward p-nitrophenyl acetate. Furthermore, it was found that PE8 exhibited activity and enantioselectivity in the synthesis of methyl (R)-3-(4-fluorophenyl)glutarate ((R)-3-MFG) from the prochiral dimethyl 3-(4-fluorophenyl)glutarate (3-DFG). (R)-3-MFG was obtained in 71.6% ee and 73.2% yield after 36 h reaction under optimized conditions (0.6 M phosphate buffer (pH 8.0) containing 17.5% 1,4-dioxane under 30 °C). In addition, PE8 was tolerant to extremely strong basic and high ionic strength solutions as it exhibited high activity even at pH 11.0 in 1 M phosphate buffer. Given its highly soluble expression, alkalitolerance, halotolerance and enantioselectivity, PE8 could be a promising candidate for the production of (R)-3-MFG in industry. The results also demonstrate the potential of the marine environment as a source of useful biocatalysts. |
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| Keywords: | Esterase Marine Enzyme biocatalysis |
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