Recombinant Expression Of Alginate Lyase Alg2668 In Bacillus Subtilis

Alginate lyase is the key enzyme for degrading alginate to prepare bioactive oligosaccharides,and has important application value in food,industry,agriculture and medicine,et al.However,due to the low enzyme production capacity of the strains at present,it is difficult to industrialize production,which restricts the wide application.In this paper,the new alginate lyase-producing species HB172198T was used as the research object.The genomic and enzyme-producing gene analysis,the expression of alginate lyase Alg2668 in Bacillus subtilis,the enzymatic properties of recombinase and the optimization of alginate lyase-producing fermentation process were carried out.The specific results of the study are as follows:Based on the new alginate lyase-producing species HB172198T,isolated from the Wenchang sea area of Hainan Province,it was predicted that the strain had the ability to degrade and utilize polysaccharides such as alginate,agar,carrageenan,xylan,cellulose and so on.Four alginate lyases were produced:Alg2660,Alg0773,Alg2668 and Alg3334,which belonged to the PL7,PL 15 and PL38 families.Among them,Alg2668 had a single domain and was secreted extracellular through the SEC pathway.This study focused on Alg2668 for further research.Using the genome of strain HB72198T as template,the primers were designed to amplify the target gene alg2668,connected to the expression vector PBE2-P43-his by seamless cloning,then transformed to Bacillus subtilis WB800n by Spizizen salt chemical conversion method,constructed the recombinant strain Bac-PBE2-P43-alg2668-his,and purified the tag protein by NTA-Ni spharose column to obtain the recombinant protein rAlg2668.The enzymatic properties of recombinant enzyme rAlg2668 were studied.The optimal reaction temperature was 55℃,the optimal reaction pH was 8.0,the enzyme activity below 30℃ for 1h was basically stable,and the enzyme activity for 1h in the pH 5.5-6.5 environment remained above 80%.10mmol/L Mg2+ has a significant promoting effect on enzyme activity.rAlg2668 had enzymatic hydrolysis activity on sodium alginate,polymanuronic acid and polyguronic acid,and preferred to degrade polymanuronic acid.The enzymatic hydrolysate of sodium alginate by rAlg2668 was identified by TLC and ESIMS,and the oligosaccharide polymerization degree was 2 and 3.Using the activity of alginate lyase and biomass as indicators,the fermentation process of recombinant bacteria for enzyme production was optimized.The composition of the optimized medium(g/L)was:10g/L glycerol,20g/L yeast,18g/L soybean peptone,0.23g/L CaCl2,the initial pH of the medium was 7.5,and the enzyme activity reached 807.67 U/mL under the condition of 52℃,200r/min for 38h,which was 1.83 times of that before optimization.This study provides a new resource for the preparation of alginate lyase and lays a foundation for the industrial application of enzyme-producing microorganisms and alginate lyase preparations.