Cloning, expression and characterization of a new enantioselective esterase from a marine bacterium Pelagibacterium halotolerans B2T

Abstract

• An esterase was cloned from a marine bacterium and overexpressed in Escherichia coli . • The enzyme was alkalitolerance and halotolerance. • The enzyme preferred short chain p -nitrophenyl esters, and was not a metalloenzyme. • The enzyme was useful in the synthesis of methyl ( R )-3-(4-fluorophenyl)glutarate. • ( R )-3-MFG was obtained in 71.6% ee and 73.2% yield after 36 h reaction. An esterase, designated as PE8 (219 aa, 23.19 kDa), was cloned from a marine bacterium Pelagibacterium halotolerans B2 T 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.