Cloning,Expression,and Application Of New Marine-deriyed Esterases

Enzymes from marine bacteria are expected to have unusual habitat-related properties such as salt tolerance, hyperthermostability, barophilicity, cold adaptivity, and often excellent region-and stereospecificity, which make them attractive for industrial application. Optically pure methyl (R)-3-(4-fluorophenyl)glutarate ((R)-3-MFG) is a pharmaceutically important precursor in the synthesis of the widely used antidepressant-(-)-paroxetine hydrochloride. However the existing methods for the synthesis of (R)-3-MFG were not suitable for industrial application due to various reasons.In the present study, a series of new esterases were cloned from the marine bacterium Pelagibacterium halotolerans B2T, heterologously expressed in E. coli in soluble forms and biochemically characterized. These enzymes were then applied in the synthesis of (R)-3-MFG through enantioselective hydrolysis of dimethyl3-(4-fluorophenyl)glutarate (3-DFG) in an aqueous-organic biphase system.Firstly, eleven new esterases from the marine bacterium Pelagibacterium halotolerans B2T were successsfully cloned. Among these new enzymes, pe2, pe8, pe9, pe10,pe11ere expressed in E. coli Rosseta(DE3) in soluble forms while pe6, pe7were expressed as inclusion bodies. Then, pe6and pe7were successfully expressed in E.coli BL21(DE3) in soluble forms by coexpression of chaperone plasmid pGro7.Secondly, these seven new esterses were screened for the activity and enantioselectivity in the synthesis of (R)-3-MFG from3-DFG. It turned out that one of the esterase PE8exhibited relatively high activity and enantioselectivity towards3-DFG. Then the effect of organic co-solvents, buffer pH, ionic strength, reaction temperature, reaction time and protein purification were investigated.(R)-3-MFG was obtained in71.6%ee and73.2%yield after36h reaction under optimized conditions (0.6M phosphate buffer (pH8.0) containing17.5%1,4-dioxane under30℃). To our knowledge, PE8is the first marine-derived esterase reported as a potential biocatalyst for the production of (-)-paroxetine.Finally, phylogenetic and enzyme properties analysis were conducted. The results showed that PE8(660bp,219aa) has a theoretical molecular weight of23.19kDa and theoretica pI of4.73. BLASTP of the translated protein sequence showed maximum identity (46%) with the esterase from Parvibaculum lavamentivorans DS-1. Phylogenetic analysis of the protein showed it was a new member of family VI lipolytic enzymes. Biochemical characterization analysis showed that PE8was an esterase which exhibited maximum activity towards p-nitrophenyl acetate. PE8was stable in the presence of many metal ions, and it could retain more than50%of activity with most of the ions except for Ni2+, Zn2+and Cu2+. PE8was an alkaline esterase with an optimal pH of9.5and an optimal temperature of45℃toward p-nitrophenyl acetate. Furthermore, the enzyme still exhibited activity under0℃,65℃and pH11.0.Given its highly soluble expression, alkalitolerance, halotolerance and enantioselectivity, PE8could 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.