Strain Screening Of α-amylase And Proteomic Analysis Of Streptomyces Sp.D227Synthesized Amylase

Since21st century, the energy crisis has been a hot spot. Renewable energy will play an important role as substitutes in the future energy utilization while disposable energy is exhausting. Among them, fuel ethanol is the typical representative of renewable energy. Fuel ethanol is mainly obtained through polysaccharide ways such as the lignin, starch, α-amylase can efficiently open the a-1,4glycoside bond inside starch molecules, thus producing dextrin, maltose, oligosaccharides and glucose, which is the first step of starch-ethanol transformation. At present, the amylase is mainly obtained from bacteria, fungi and other microorganisms. Compared to other microorganisms, Streptomycin is more common, which can be found either high mountains or deep oceans. All these extreme environmental conditions bring incomparable advantages of streptomycin-original amylase to other origins. These advantages make the streptomycin-original amylase a permanent resource for industrialization, and the application of the streptomycin-original amylase could be operated widely.In this study, we isolated Streptomyces.sp.D227from soil, which has high starch hydrolysis ability. By comparative proteomics techniques, the differentially expressed intracellular proteins in starch culture medium and the key regulatory factors in amylase biosynthesis were studied.After plate culture and purification, there were a total of35strains of actinomycetes isolated. We then detected hydrolysis circle by refrigeration at4℃overnight. After measuring the radius of transparent zone and colony, we found the ratio was4.51±0.71. After enlarged culture mycelium was inoculated to different carbon media, the selected mycelium in the stage of starch content decreased sharply, and the extracted intracellular proteins were collected for comparative proteomics analysis. After electrophoresis analysis, we got2-D gel maps, in which about1700highly repeatable protein spots were found. By the use of ImageMaster software, we found that84protein spots were of significant difference (P<0.05), and43protein spots only detected in one gel. The selected protein spots were identified by MALDI-TOF-MS. Finally, we had successfully identified12protein spots, and6of them were specifically expressed in the treatment of starch, and the rest spots were of significant difference (P<0.05) between the treatments. By Blast2GO analysis, we found that lpxtg-motif cell wall anchor domain protein, aspartyl-trna synthetase, sensor histidine kinase, bi-functional protein, n-6DNA methylase, carboxyl-terminal protease were specifically expressed in the starch treatment. Compared to glucose treatment, the50S ribosomal protein, adenosine deaminase, dihydrolipoyl dehydrogenase, acylphosphatase were down-regulated, while aldolase was significantly up-regulated.In this study, we selected Streptomyces sp.D227as the research object, which have strong hydrolysis ability of starch. Using comparative proteomics technology, we investigated the differentially expressed proteins in amylase synthesis and secretion biological process which might be key regulatory factors in in amylase synthesis. The results would provide useful references for further study of amylase synthesis and secretion biological process in Streptomyces.