Using Metagenomic Approaches To Obtain New Esterase Gene And Study On Enzymatic Properties

Enzyme is a kind of very important multifunctional catalyst, has been widely used in food and biological engineering. The enzyme has been used in the food industry, biological medicine, resolution of chiral drugs and new biological material. Esterase industrial production has become the most widely used enzyme catalyst, is also in the industrial production is focus on the study of enzyme catalyst.Soil microorganism is most can not be cultivated, this limits the development and use of microbial resources. This study using metagenome DNA technology to direct separation of uncultured environmental microorganism genome DNA, construction of soil metagenomic library, through functional screening approach from metagenomic obtained a new esterase gene "EstC28-1". Metagenomic technology, which accounted for99%of the total microbial unculturable microbial resource development become reality. Subsequently, the enzyme sequence analysis, cloning, expression and purification. And the purified enzyme were conventional enzymatic characterization, the optimal reaction temperature determination, determination of thermal stability, substrate specificity analysis.The experimental results show that isolated from the metagenomic library the esterase EstC28-1, the use of recombinant DNA technology will esterase the EstC28-1gene was cloned into recombinant vector electroporation into Pichia pastoris genome by yeast expression vector pPIC9K Colony PCR and tributyrin culture screening of the board, to get positive Pichia yeast genes.30℃,250r/min and cultured for5days, the fermentation broth supernatant were collected by centrifugation, concentrated fermentation broth using PEG20000concentrate with50%saturation of ammonium sulfate salting purification, the purity of greater than90%protein. EstC28-1enzymatic properties:the optimum temperature of45℃when the temperature is25-35℃, the esterase activity retained more than85%, EstC28-1is a thermophilic esterase. On pNPC4showed maximum hydrolysis activity, the most suitable pH value of9.0, EstC28showed remarkable stability in up to20%-50%(v/v) benzene and alkanes (high log P solvents). When incubated for72h in the presence of20%-50% benzene or alkanes, the enzyme maintained its2-3fold elevated activity, good resistance to organic. Mg2+, Ba2+metal ions with the increase of the concentration enhancement of the the esterase EstC28-1activity is also increasingly evident, indicating that these two metal ions on the esterase activation. These results suggest that the the esterase EstC28-1has potential value in use in industrial processes.