| Alginate lyases, also known as alginases or alginate depolymerases, were important polysaccharide lyases. They have been widely used to analyze the fine structure of alginate, extract protoplast for the food and regeneration of a variety of algal species, and prepare of special product of alginate. The iyases have been isolated from a wide range of organisms, including algae, marine mollusca, and marine and terrestrial microorganisms.In this paper, the alginate lyase was extracted and purified from Haloitis diversicolor aquatilis. The purification, and enzymatic dynamics, and the impact factors on enzymatic activity, and the specificity of lyase, and the changes of molecular weight parameter on degradation were investigated. The magnetic chitosan microspheres (M-CS) were preparated by reverse-phase suspension polymerization in the presence of magnetic fluid. The lyase was immobilized on the carrier of M-CS by absorption and cross-linkage. The adsorptive properties of M-CS and influences of the immobilization on enzymatic activities were also studied.In the means of (NH4)2SO4 fractionated salting-out, the 50-80% was the optimum saturation degree for the lyase, multiple of purification was 2.37, and recovery rate was 22.5%. In the DEAE-52 ion exchange chromatography , the fraction with 0.3-0.6mol/ml NaCl as eluant held a maximal enzymatic activity, its multiple of purification was 8.46, and recovery rate was 6.6%. The result of SDS-PAGE (10% gel) of purified lyase showed only one band, and the molecular weight of the lyase was 63.4kDa.The enzymatic activity of the lyase was affected by many factors, such as temperature, pH, buffer, concentration of substract or lyase or product, metal ions. The temperature and the pH strongly affected the enzyme activity; and the optimal temperature and pH of the lyase were 45 C and 7.0, respectively. Compared with phosphate buffer, the lyase showed the higher activity in Tris-HCl buffer with Km, Vmax corresponding to 0.0189mg/mL and 0.258 UA/min. The progress curves indicated that an apparent endpoint level of maximal conversion wasreached within 2h. This level not only affected by the initial substrate concentration, but also dependent on the enzyme dose. When the substrate concentration reached to 0.4%, enzymatic activity raised slowly, the result indicated that the combination between enzyme and substrate reached saturation. When the lyase concentration reached to 0.250 UA/mL, quantity of product didn't nearly increase. Addition of more enzyme or substrate during incubation revealed that the apparent inhibition was not of an irreversible nature. Adding the enzymatic breakdown products from a commercial alginate, the enzyme activity was clearly affected by the product inhibition. The effect of the ions indicated that the activity of lyase was activated by Co2+, Mg2+ and significantly inhibited by Cu2+, Age and Zn2+. Storing temperature distinctly affected the enzymatic activity, the activity reduce a fat lot. Storing time was also influenced the enzymatic activity especially during the first two months. Intrinsic Viscosity of enzymatic breakdown product is affected by the enzymatic property and the reacting time, the result indicated that the viscosity was mainly changed in the first hour because of the strong reversible product inhibition. 1H NMR monitoring of the degradation of a commercial alginate sample found that GGM, MGM and GOG was degradated by acid; different to the acid degradation, M block was attacked during the lyase degradation with the fraction of MM dimmers decreased while the fraction of MG dimmers, GGM and MGM trimmers increased after epimerization. It indicated that the alginate lyase from Haliotis diversicoloe aquatilis was a kind of mannuronate (EC 4.2.2.3).The magnetic liquid was prepared using NH4Fe(SO4)2 12H2O and (NH4)2Fe(SO4)2 '6H2O. The preparation condition and characteristic was discussed. R(=8NaOH/3 ?Fe) 2:6.67, PH 11 is necessary for the preparation. When the Fe2+/Fe3+ was between 1:2 and 2:3, the ideal magnetic liquid can be gained with...
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