Optimization Of Enzyme Production Conditions By Alginate Degrading Bacteria TS-5

Alginate is a naturally occurring anionic polymer,usually obtained from brown seaweed.The natural decayed brown seaweed and marine organisms eating seaweed were used to screen out the target strains that could degrade alginate,and by optimizing the fermentation conditions for enzyme production,it could efficiently degrade alginate.After alginate is degraded by alginate lyase,it becomes an alginate oligosaccharide with a variety of biological activities,which has a wide range of research and application prospects.In this article,the target strains were screened firstly.The single factor experiment and response surface analysis were applied to optimize fermentation conditions secondly.Some enzymatic properties were studied,which provided a preliminary basis for subsequent practical applications.The main findings were as follows:（1）Preliminary screening and re-screening of strains:The rotten sargassum,asparagus,carrageenas,black fish and conch viscera were used as raw materials.The sodium alginate was the single carbon source for flat plate preliminary screening.The transparent circle detection by iodine solution showed that,the strains with transparent circles included LX-1,LX-2,TS-1,TS-2,TS-5,BY-3,BY-4,BY-6.The target strain TS-5 with higher enzymatic activity was obtained by the DNS determination method.（2）Single factor and response surface analysis method:The strain was optimized by single factor conditions to determine the composition of the most suitable medium:the carbon source was 1%sodium alginate;the nitrogen source was 1.1%peptone;sodium chloride concentration was 1.5%;initial p H was 7;temperature was 30°C;rotation speed was 170 rpm;inoculation amount was 3%.The enzyme activity increased by 50.79%.The response surface analysis was carried out on the basis of single-factor experiments.Temperature,inoculum amount,and carbon source concentration were the three factors that had the most significant impact on the enzyme production capacity.The software predicts the fermentation conditions as follows:temperature 24°C,inoculum amount 3.198%,carbon source concentration 1.161%,and the enzyme activity reached 72.86 U/m L.（3）Enzymatic properties research:the fermentation broth was centrifuged to obtain the crude enzyme solution,and the preliminary separation and purification was carried out by ammonium sulfate fractional salting-out.The enzymatic properties of the enzyme solution were studied.The conclusion showed that,the substrate specificity:at the same time,it degraded sodium alginate,Poly M and Poly G,but the degradation ability of Poly G was weak,mainly degraded Poly M,followed by sodium alginate;the optimal reaction temperature was 40℃,and the enzyme activity was stable at 80%at50℃above.The enzyme activity was stable when the temperature was 10-40℃for1 h,and the enzyme activity gradually decreased when the temperature was higher than50℃;When the optimum p H was 7.0 and the p H was 6.5-8.5,the enzyme activity was stable,and the relative enzyme activity could reach 80%-90%of the highest enzyme activity;The effect of metal ions on the enzyme activity was:Na⁺,K⁺,Mg²⁺,and Ca²⁺all had a promoting effect on the enzyme activity,but Na⁺had a more significant effect on the enzyme activity,although K⁺,Mg²⁺,and Ca²⁺could promote unobviously.Fe²⁺,Cu²⁺,Zn²⁺,Mn²⁺could inhibit enzyme activity.The inhibitor EDTA could obviously inhibit the enzymatic hydrolysis activity,while SDS had no inhibitory effect almostly.