Screening, Characterization And Application Of An Glucosidase From Environmental DNA

The environmental microbiology presents an enormous genetic and biological pool that can be exploited for the discovery of novel genes, entire metabolic pathways and their products. It is reported that more than99%microbe species were un-cultured by the traditional cultivation approach, which as a main limiting factor for the exploitation and application of the microbial resources. Metagenomics as the new fields of studying the gene function and interactions of microbial community, has made some breakthrough progress.In this study, screening of a glycosidase (D141)from deep-sea sludge metagenomic library. The full-length DNA of the clone gene was1395bp, encoding465amino acids and the predicted was52.0kD. A recombinant protein about52.0kD in the Escherichia coli BL21(DE3) was induced and the purpose of protein purification capacity can reach72.7%of the total protein.Characterization of the glycosidase, the optimum reaction temperature for enzyme activity was about of37℃, pH7.5, it has good thermal stability, a long time to maintain the stable at40℃,at45℃able to maintain about50percent of vitality after2h. Glycosidase (GluD142) to maintain the vitality of about80%in the pH range6.0-7.5, the pH5.5and pH8.0to maintain the vitality of about50%, below or above this range, the vitality to decline rapidly. Among the metal ions, Cu2+, Mn2+, Ni2+significantly enhanced the activity, no activation effect addition of Mg2+, Zn2+and Inhibition from K+, Na+, Co2+, Ca2+. The hydrolytic activity of this enzyme in10%methanol, ethyl acetate, acetone and hexane, was ranging from120%-150%increase. Even in40%hexane, the activity can be maintained close to100%, there was a significantly decreased the activity from DMSO.The selection of the immobilization conditions and its application in synthesis of alkyl Glucoside and resveratrol were studied by using MCFs as carrier for the immobilization of glycosidase. The research on synthesis alkyl Glucoside showed that at the substrate molar ratio(Glucose/tyrosol) was4:1, the water phase reached30%, at the pH6.5system when reaction temperature42℃, reaction time was48h. With the fermentation process of polydatin, the optimal reaction system on synthesis resveratrol showed that at substrate molar ratio (solution of glycosidase solution of polydatin buffer) was1:5:5, at the pH6.5, reaction temperature37℃, reaction time was16h.Overall, we isolated a new glycosidase with the metagenomic enzyme technology, through its enzymatic properties characterization, that this enzyme have higher activity in the melting of high expression levels、organic solvents and resistance to ion of stability and substrate specificity. Preliminary studies prove that this enzyme has a higher catalytic efficiency with glycosytransfer, to prove that the enzyme is a new glycosidase molecules has potential applications.