Cloning,Expression And Enzymatic Characterization Of Esterases Identified By The Genomic And Metagenomic Approaches

Microorganisms are widely distributed and possess kinds of different characteristics. Extremophiles are the microorganisms that can survive in extreme environments where other normal microorganisms cannot live. So extremophiles are getting big interests. In this paper, we focused on the uncultured and cultured microorganisms from deep-sea and salt lake, and both of them are extreme environments. We applied metagenomic and genomic approaches in the study of the functional genes and genomes.A fosmid metagenomic library was constructed using the sample of deep-sea sediment from seamount region with a water depth of5,886m. The library contains200,000fosmid clones with average insert length of36kb. Then the whole metagenomic library contained7,200Mb genomic DNAs of the microorganisms from the deep-sea sediment. So it was a huge pool of bacterial genes.An extremely halophilic archaeon Halobiforma lacisalsi AJ5T, which was isolated from Ayakekum Lake, Xinjiang, China, was subjected to genome sequencing and analysis. The genome contains a main chromosome and two plasmids. H. lacisalsi AJ5T habors many genes with potential industrial use such as rhodopsins, lipases, a-amylases and polyhydroxyalkanoate synthase, etc. H. lacisalsi AJ5T contains sets of genes encoding proteins for various DNA repair systems to resist the UV radiation in its habitat. The isoelectric point distribution for the predict proteins was observed with an acidic peak, and this phenomenon reflected the adaption of H. lacisalsi AJ5T to the halophilic environment.Using the activity based approach,24esterase-postive fosmid clones were obtained from the deep-sea sediment metagenomic library and10esterase genes were indentified by subcloning or fosmid sequencing. Using the genomic approach,13putative esterase genes were identified from a marine bacterium Pelagibacterium halotolerans B2T. These novel esterase genes can be used in the further study. Two of the esterases named Est6and PE10, which were identified by the metagenomic and genomic approaches respectively, were successfully cloned, heterologous expressed and purified. The characterization of Est6revealed that it was a cold-active esterase and exhibited the highest activity toward p-nitrophenyl butyrate at20℃and pH7.5. Est6was stable in the presence of Mn2+, and we suggest that Est6is a potential candidate for use in industry. The characterization showed PE10exhibited maximum activity toward p-nitrophenyl acetate and its optimal temperature and pH were45℃and pH7.5, respectively. In addition, PE10was a halotolerant esterase as it was still active under4M NaCl. PE10was also stable under some organic solvents such as DMSO and methanol. These properties made PE10a candidate for industrial use. Three dimensional modeling of PE10suggested that the high negative electrostatic potential on the surface may relevant to its tolerance to high salt environment.