Cloning, expression and characterization of a novel esterase from marine sediment microbial metagenomic library

Abstract:

Abstract: [Objective] To clone, express and characterize a novel esterase from marine sediment microbial metagenomic library. [Methods] Using esterase segregation agar containing tributyrin, we obtained esterase positive fosmid clone FL10 from marine sediment microbial metagenomic library. This fosmid was partially digested with Sau3A I to construct the sublibrary, from which the esterase positive subclone pFLS10 was obtained. The full length of the esterase gene was amplified and cloned into the expressing vector pET28a, and the recombinant plasmid was transformed into E. coli BL21 cells. We analyse the enzyme activity and study the characterization of the esterase after its expression and purification. [Results] An ORF (Open Reading Frame) of 924 bp was identified from the subclone pFLS10. Sequence analysis indicated that it showed 71% amino acid identity to esterase (ADA70030) from a marine sediment metagenomic library. The esterase is a novel low-temperature-active esterase and had highest lipolytic activity to the substrate of 4-nitrophenyl butyrate (C4).The optimum temperature of the esterase was 20 ℃, the optimum pH was 7.5. The esterase in this study had good thermostability at 20 ℃ and good pH stability at pH8－10. Significant increase in lipolytic activity was observed with addition of K+ and Mg2+, while decrease with Mn2+ etc. [Conclusion] We obtained the novel esterase gene fls10 from the marine sediment microbial metagenomic library. The esterase had good thermostability and high lipolytic activity at low temperature and under basic conditions, which laid a basis for industrial application.

Key words:Keywords: marine sediment; metagenomic library; low-temperature-active esterase; characterization of esterase