Screening,Cultivation Of Alginate Lyase Producing Strain BY-1 And Enzymatic Properties Of Alginate Lyase

In terms of alginate degradation,enzymatic degradation has its unique advantages over physical and chemical methods.Therefore,alginate lyase is an indispensable tool enzyme for us to make full use of marine resources.The sources and classifications of alginate lyase are diverse.Alginate is degraded into alginate oligosaccharides by?-elimination,which can be used in food,agriculture,medicine and bio-energy.In this study,strains capable of degrading alginate were screened out,and the fermentation conditions were optimized.The enzymatic properties of alginate lyase was studied,which provides a theoretical basis for subsequent applications.The main findings were as follows:(1)Preliminary screening and re-screening of strains.Using rotten sargassum,wakame,carrageenan,oysters and conch viscera as raw materials.Iodine solution was used for transparent circle detection,27 strains were obtained through preliminary screening on flat plates.Strains with transparent circles included MW-1,MW-7,MW-8,ML-1,QD-1,QD-2,QD-4,QD-5 and BY-1.The target strain BY-1 with higher enzymatic activity was obtained by the DNS determination method.(2)Single factor and orthogonal condition optimization.The strain was firstly optimized with single factor conditions,and the optimization results were as follows:carbon source was 1.2%sodium alginate,nitrogen source was 0.4%ammonium sulfate,sodium chloride concentration was 1.5%,the initial p H was 7.0,the temperature was 24?;the rotation speed was 150 rpm,the inoculation amount was3%.The enzyme activity increased by 45.04%.Orthogonal experiments were carried out on the basis of single factor experiments,and the optimal fermentation conditions were obtained as follows:sodium alginate concentration was 1%,ammonium sulfate concentration was 0.5%,temperature was 30?,rotation speed was 130 rpm,and the enzyme activity reaches 94.41 U/m L.Compared with the initial enzyme activity,it increased by 54.67%.(3)Research on the enzymatic properties.The fermentation broth is centrifuged to obtain the crude enzyme solution,which is separated and purified by ammonium sulfate salting-out.And the enzymatic properties of the enzyme solution are studied.The conclusion is that the best enzymatic hydrolysis time was 20 min.The substrate specificity was that it can degrade sodium alginate,Poly M(?-D-mannuronate;M)and Poly G(?-L-guluronate;G)at the same time,but the degradation ability of Poly G was weak.Sodium alginate was mainly degrade,followed by Poly M.The optimal reaction temperature was 30?,enzyme activity was stable at 4-40?for 1h and stable above 80%at 30-40?,enzyme activity was stable when it is higher than 40?;the best reaction p H was 7.0 and enzyme activity was stable when the optimum p H is6.5-9,p H 6.5-8 buffer didn't have much effect on the enzyme activity;the effect of metal ions on the enzyme activity was:Na⁺,Ca²⁺promoted the enzymatic reaction,K⁺,NH₄⁺,Cu²⁺had no obvious effect on the enzymatic reaction Influence,Fe²⁺had a significant inhibitory effect on the enzymatic reaction,EDTA can significantly inhibit its enzymatic effect,but SDS was not obvious.(4)Product analysis:The enzymatic hydrolysis products were preliminary identified by TLC thin layer chromatography.The oligosaccharides degtaded from sodium alginate by the alginate lyase producing by strain BY-1 disaccharides and trisaccharides.The alginate lyase was determined to be an endonuclease.