| Esterase is a group of hydrolases that catalyze the cleavage and formation of ester bonds. Esterase is usually from animals, plants and microorganisms. Marine environment is the new and huge resource for esterase. Marine environment is extremely diverse, cold seep, hydrothermal vents, abyssal plain, oceanic trench, seamounts, oceanic volcano and deep-sea whale fall with high-pressure, salinity, low or high temperature, complex nutrients and special light. Therefore, the extreme marine environment represents a vast pool of novel genes and biocatalysts, such as esterase.In this study, we extracted the metagenomic DNA from South China Sea sediment and constructed the fosmid library. The esterase gene est424was screened and cloned from the fosmid library, which was921bp and encoded307amino acids. It was homologous with the existed esterase lower than85%at amino acid level. Est424gene was cloned into the pET28a vector and achieved recombinant expression in Escherichia coli BL-21(DE3). However the recombinant esterase was expressed as no bioactive inclusion body. Chaperone Plasmid Sets were used for co-transformation with the est424expression vector. The soluble recombinant esterase,35kDa, was obtained by using co-expression with the chaperone plasmid dnaK-dnaJ-grpE.The recombinant esterase est424was purified by the Ni-nitrilotriacetic affinity chromatography system and characterized using the p-nitrophenol butyrate method. The est424had the highest lipolytic activity at35℃and pH8.0, which revealed that it was meso-thermophilic alkaline esterase. Est424was thermostable at20℃. The lipolytic activity of est424was strongly increased by Fe2+, Mn2+and1%Tween80or Tween20.The recombinant enzyme from marine environment is easy to form inclusion body in E. coli expression system. In this study, we solved the problem by using the chaperone, which provides the theory and methodology for the development and the utilization of the esterase from the marine environment. |
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