Study On Screening Of High-yielding Amylosucrase Strain And Fermentation Technology

Amylosucrase is a glucosyltransferase, which synthesises an insoluble α-1,4-linked glucan polymer from sucrose, and releases fructose. Unlike most amylopolysaccharide synthases, this enzyme does not require any α-D-glucosylnucleotide-diphosphate, such as ADP-or UDP-glucose.Indeed, amylosucrase only uses the energy produced by the splitting of the osidic linkage of sucrose to synthesise other osidic linkages.so amylosucrase can be regarded as an efficient glucosylation tool, very useful for producing novel amylopolysaccharides. This paper takes strains Neisseria polysaccharea (ATCC43768) as starting strain, physics mutagenesis was used to screen high production strain of Amylosucrase, and optimizing the conditions of resistant mutant strain fermentation by response surface method, to obtain high yield Amylosucrase strain. the main results are as follows:1.Using the UV mutagenesis methods to enhance Amylosucrase yield. The UV mutagenesis results show that:The best dilution degrees used for UV mutation was10-3, the best ultraviolet mutation time is10seconds. After Three-circled UV mutagenesis for strain screening, obtain high-yield mutant strain UV3-6, which Amylosucrase yield for6.825IU/mL, increasing4.95IU/mL to the former bacteria, revealing better genetic stability in five generations.2.Bsed on the mono-factor optimization experiment,using the response surface method establish regression model for optimal medium and the optimal fermentation conditions.The result shows the best compounding of nutrition condition was2.8%star,0.5%Bacto-peptone,0.5%yeast extract,0.3%sodium citrate,the optimal combination of fermentation process was pH6.0,fermentation temperature was33℃, inoculation was5%and the time of fermentation was3d,under these optimized conditions the yield of was19.84U/ml.3.Salting precipitation crystallization of isolated and purified amylosucrase,and then study the enzymatic properties of the pure enzyme. the results showed that the optimum pH that strain produced the amylosucrase was6.5,the optimum temperature was30℃, when pH was at the range of5.5to6.5,the Amylosucrase activity was high.above40℃thermal stabilitythe was poor,resist of temperature was was bad.