Charaterization Of Alginate Lyase From Chlorostoma Rustica And The Study Of Bio-chemical Activity Of Alginate Oligosaccharides

The advances and prospects on alginate lyase and the biochemical activity of alginate oligosaccharides were reviewed in this paper. And the importance of the study on alginate lyase and alginate oligosaccharides for the development of national economy was illuminated. The alginate lyase activity from Chlorostoma rustica was detected. Separation and purification of alginate lyase and preparation techniques were discussed. Physical and chemical characteristic and catalysis kinetics were studied. The objectives of this paper were to obtain valuable performance parameters of alginate lyase. This work establishes theoretical base for the study, reconstruct, production and application of the alginate lyase. And further more the bio-chemical activities of alginate oligosaccharides were studied.The alginate lyase from Chlorostoma rustica was purified by using ammonium sulfate precipitations, and successive fractionation on DEAE Sephadex II5, Sephadex G-100 and Sephacryl S-300 HR column chromatography. The enzyme was purified to an electrophoretically homogenous state. Its molecular weight was determined to be about 28KD on SDS-PAGE. The purified enzyme showed maximum activity at pH 7.0 and 40癈, and This alginate lyase activity is not stable in high temperature; The lyase activity can be affected by several ions. It was activated by Mg2+, Zn2+, Na+and K+, and inhibited strongly by Mn2+, Co2+ and Cd2+; The substrate specificity of the lyase was studied by discriminating between the types of gellation (i.e. halo or ring formation) caused by the interaction between calcium ions and depolymerized alginates. The result indicated that the lyase was highly specific for poly G. Membrane technique and gel filter chromatography were applied to prepare oligomers. And TLC and HPLC were used for checking isolation and purification effect, and there are goodresults.The free radical scavenging activity of alginate oligosaccharides I , II and III was studied by the method for determination of Luminesecence. The protective effects of alginate oligosaccharides on the killing of Escherichia coli. by hypochlorous acid(HOCl) were examed. And the studies on inhibition of peroxidation of polyunsaturated fatty acid from yolk lipoprotein induced by iron were curried out. The results showed that alginate oligosaccharides I , II and III had strong scavenging effects on three free-radical in a concentration-dependent manner. Alginate oligosaccharides I had stronger scavenging than Vc on scavenging O2-, and its IC50 was 8ng/mL. The effect of alginate oligosaccharides I and II was better than III on scavenging OH , and their IC50 were 0.01mg/mL,0.03mg/mL and 0.23mg/mL. The survival rate of E.coli. suggested that alginate oligosaccharides could significantly scavenge HOC1 and their IC50 were 0.8mg/ mL, 1.3mg/ mL and 1.6mg/ mL. The inhibition of the peroxidization of polyunsaturated fatty acid was very prominent. The results showed molecule weight had great influence on the scavenging ability.