1.Studies On Cloning And Expression Of Thermophilic α-glucosidase In E.Coli, And Its Purification And Characterization, 2.The Stability Of Drosomycin.

Background and Objective a-glucosidase ( EC 3.2.1.20) was a monomeric enzyme of 62kDa which functions as glycoside Hydrolase and transferase. The essential glucoprotein can cut down the alpha-1,4-glycosidic bond in the structure of maltose and malto-oligosaccharide, then transfer the dissociated glucose residue to another glucose, maltose, isomaltotriose maltotetraose and panose. Research indicates that, the enzyme on the intestinal brush border has connection with utilization and absorption of carbohydrate, as starch, dextrin and sucrose, which would be hydrolyzed by this enzyme after having been hydrolyzed by diastase.a-glucosidase was widely utilized industrially in isomaltose production and beer saccharification, but the high temperature and high pressure has great influence in activity during the fermentation. Hence, recently the characteristics and industry application of a-glucosidase with thermal stability from thermophilic microbes was under the spotlight. Because of the low production and extreme cultivation environment that the thermophile needs, we focused on constructing recombinant with the a-glucosidase gene.In this research, we multiplied a-glucosidase gene (1587bp) by PCR, with the Thermus thermophilus genome as the template, ligated with plasmid pET28a(+), transferred into the E.coli (DH5a), sequenced and expressed in Escherichia coli(528a(+)a). According to its thermal stability, we chose the optimal method of purification and activity measurement, and calculate about its parameter of kinetics equation of enzyme-catalyzed reaction.Method PCR for ampliation of a-glucosidase gene and sequencing was carried out. Prokaryotic expression system was constructed by traditional molecular cloning-TA cloning. The protein was purified by heat incubation, afterwards sephadex G-200 chromatography was performed to extract the expressed target recombinant enzymes, then verified by SDS-PAGE. BCA method was used to measure the concentration of protein. The relative activity was measured in different grades of pH value and temperature. positive-cross method for the optimal condition was carried out. The dynamics parameter Km and Kcat was calculated at pH6.8 at 37℃and 90℃referring to kinetics equation of enzyme- catalyzed reaction.Result The similarity of nucleotide sequence of the cloned a-glucosidase gene was 100% compared to the reported a-glucosidase sequence from Thermus thermophilus (TT_C0107). After two steps of purification, the purity of a-glucosidase was above 95%. According to the positive-cross method, the optimal temperature is around 90℃while the optimal pH value is approximately 6.8. The dynamics parameter Km and Kcat was calculated at pH6.8, which is 0.74 mmol/L and 34.64 s^-1 at 37℃respectively, 0.57 mmol/L and 226.8 s^-1 at 90℃respectively.Conclusion a-glucosidase gene recombinant and prokaryotic expression system was constructed successfully in this research. As had been expected, the enzyme was proved to high stability at high temperature, and much more active under neutral pH value and higher temperature. Antimicrobial peptides(AMPs), produced by insects, plants and vertebrate, are elements of innate immune system of the creatures above. It's particularly difficult to classified these peptides by organism or structure because of the diversity and continuous discovery. Drosomycin(DRS), a strictly antifungal defensin secreted by immunity system of the fruit-fly Drosophila melanogaster, was the first inducible antifungal peptide as we know. This AMP is a 44-residue peptide including eight cysteine residues engaged in the formation of four internal disulfide bridges: Cys1±Cys8, Cys2±Cys5, Cys3±Cys6 and Cys4±Cys7 , the C-terminal residue conferring a highly compact structure to the molecule. This peptide displays antifungal activity while it is inactive against yeasts and bacteria. It can influence the level of serum lysosome and detroy the cell membrane of tumor cell. The stability of drosomycin was researched and the practical prospect in agriculture and medicine was proved in this paper.Objective research the accurate affect of temperature, pH value and denaturants on drosomycin.Method in thermal stability aspect, gradient temperature from 20-90℃was applied to bathe the drosomycin for 30mins, then the fluorescence measurement and CD were carried out to observe the shift of structure. The protein was bathed at 37℃for 30mins with Gradient pH value buffer from 2.0-10.0 to assess the stability. The denaturants urea and GdmC1 with the maximum concentration of 8M and 6M respectively are prepared to incubate with drosomycin at 37℃for 30mins, the same measurement as thermal stability was carried out.Conclusion Drosomycin could keep stable at 20-50℃, in the pH4.0-8.0 buffer, and while the concentration of urea and GdmC1 were below 5M and 3.5M respectively.