Identification Of Two Novel Esterases From A Marine Metagenomic Library Derived From South China Sea

99%of microbes in the environment cannot be cultured under laboratory conditions, which severely inhibited the full exploration of microbial genetic resources.The development of biotechnology and the criticality of energy crisis make the demand for novel biocatalysts is increasing in the morden world,which greatly stimulates the development of powerful tools to explore the genetic resources in the environment. Metagenomics,a culture independent strategy,provides an access to valuable genetic resources of the uncultured microbes.In this study,we have constructed a high quality metagenomic expression library from surface water microbes of South China Sea,five lipolytic active clones were isolated from this marine microbial metagenomic library by functional screening.Two novel esterase genes desinated as estA and estB,which encoded 277- and 328-amino-acid peptides respectively were discovered by subcloning and sequencing of two clones that displyed superior activity among them. Amino acid sequence comparison and phylogenetic analysis indicated that EstA together with other putative lipolytic enzymes was closely related to Familyâ…¢,and EstB with its relatives formed a subFamily of Familyâ…£.Site-directed mutagenesis showed that EstA contained classical catalytic triad made up of S146-D222-H255, whereas EstB contained an unusual catalytic triad which consisted of S149-E243-H273,an important feature of the subFamily.Both of them were characterized after overexpressed and purified.EstA exhibited habitat-specific characteristics such as its high level of stability in the presence of various divalent cations and at high concentrations of NaCl.EstB was not inhibited by PMSF, suggested which may possesd a lipase-like lid structure.EstB displayed remarkable activity against p-nitrophenyl esters and was highly stable in 30%methanol,ethanol, dimethylformamide,and dimethyl sulfoxide,making EstB a potential candidate for industrial applications.Mining important industrial enzymes from various environments is highly advantageous for industrial development and economic growth by reducing the energy and raw materials consumed in industrial process.In another aspect,it is also important in being a useful reference for exploration of other important functional genes from marine microbes and estabolishing a solid theoretical foundation for sustainable development and full utilization of marine microbial resources.