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Chitin, also known as chitin, on the planet in addition to cellulose outside the largest number of natural macromolecular polysaccharide, is also on the planet in addition to the protein outside the largest number of nitrogen-containing natural organic compounds, widely present in shrimp, crabs, insects, etc. crustaceans and cell walls of fungi and plants in each of the biosynthesis of 100 million tons. Chitin is an important raw material in food, medicine, chemical, but because it does not dissolve in acids, alkalis and organic solvents, severely limits its application. Chitin enzyme can be specifically cut chitin oligosaccharide chain of beta-1, 4 glycosidic bond, its hydrolysis crustaceans oligosaccharide and N-acetyl glucosamine. Enzymatic degradation of chitin mild reaction conditions, product uniformity, high yield, no pollution, very good application prospects. Chitin enzyme has a variety of potential uses, such as: anti-fungal, prevention and treatment of diseases and pests, preparation functional Chitooligosaccharides, processing chitin waste, preparation of fungal protoplasts and so on. Chitin enzyme enzyme activity is low, the production cycle is long, and the enzyme is easily inactivated, there is no large-scale application, therefore, how to get the high activity of chitin enzyme has important research significance. In this study, the chitin as sole carbon source from the seaside soil screened strains with high enzyme activity of a chitin production, number HD002 and initially the strain, strain identification, optimization of fermentation conditions Purification enzymatic properties, enzymatic substrate. First, the partial sequence of the 16S rDNA of strain HD002 landing NCBI website with the existing sequence comparison and combination of physiological and biochemical characteristics of the strains based on the \Through the optimization of fermentation conditions, strains the HD002 producing chitin enzyme optimum medium component (w / v): (NH4) 2SO4, 0.50%, K2HPO4 0.07%, KH2PO4 0.03%, MgSO4 · 7H2O 0.10% colloidal chitin 1.0%; Optimum Fermentation culture conditions: medium initial pH value of 6.0, incubation time 192h. In the the the strain fermentation process, the initial pH value, carbon, nitrogen Fermentation strains. Optimized fermentation conditions, the the fermentation broth enzyme activity reached 1.314U/mL. 4 ° C conditions, the fermentation broth of strain HD002 refrigerated centrifugation, the supernatant was fermented by 70% saturation of ammonium sulfate salting out, dialysis, freeze-dried to give a crude enzyme, a crude enzyme by DEAE-Sepharose Fast Flow anion exchange column chromatography, dialysis and freeze-dried enzyme powder Ⅰ, the the enzyme powder Ⅰ via Sephadex G-75 gel filtration column chromatography, freeze-dried enzyme powder II, through the above three steps of the chitin enzyme purification of the enzyme powder obtained by SDS-PAGE to show a band proved to electrophoretic homogeneity has been reached. The final purification factor of 7.3, the yield was 31.1%. The calculated molecular weight of the enzyme according to the results of SDS-PAGE electrophoresis as 60.2 kDa; analysis by mass spectrometry to obtain the molecular weight of 59.0 kDa enzyme, the difference is small, the initial molecular weight of the chitin enzyme for the 60.2 kDa. Enzymatic properties of the purified chitin enzyme studies to determine the optimum reaction temperature of 55 ° C. Temperature is below 45 ℃, the thermal stability of the enzyme, water bath for 3h enzyme activity almost no loss, 65 ° C insulation for 30 min, the enzyme activity has been basically lost. The the enzyme optimum reaction pH value of 5.0, but no obvious differences in enzyme activity between pH4.6-7.2. Optimal substrate for colloidal chitin. Colloidal chitin concentration in a range of 0.1% -4.0%, the greater the higher the concentration of enzyme activity. Metal ions on the enzyme activity impact, 1 mmol / L Cu2 and Fe3 significantly inhibited the enzyme activity, 1 mmol / L of Ca2 and Na role in promoting the enzyme activity is the most obvious. Silica gel plates by thin layer chromatography results, the chitin enzyme degradation products of colloidal chitin oligosaccharide. Thus the enzyme chitin outer cutting endonuclease enzymes or chitin is not a beta-N-acetyl glucosamine enzyme.
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