Study On Heterologous Expression Of Bifidobacterium Adolescentis Sucrose Phosphorylase And Its Application And Immobilization

Sucrose phosphorylase?SPase?is a glucosyltransferase that can produce kojibiose from sucrose which reacted as a donor and glucose which reacted as a receptor.Kojibiose can promote the growth of intestinal probiotics,inhibit the activity of?-glucosidase,and delay the digestion and absorption of carbohydrates.It has wide application potential in food and medicine industry,and usually synthesized by SPase.However,the currently reported SPase is only expressed heterologously in E.coli and is not suitable for food industry applications,to the contrast,B.subtilis as the host of food safety is the better choice to express SPase.For the first time,sucrose phosphorylase from Bifidobacterium adolescentis was heterologously expressed in the food-grade safety strain B.subtilis WS11.The reason why SPase was secreted to the extracellular as a natural intracellular enzyme was studied,and its enzymatic properties were also studied.On this basis,the process of preparing the kojibiose by the recombinant enzyme was optimized,and good immobilization effect was obtained.The main research results are as follows:?1?Recombinant strains B.subtilis/pBSMuL3-SPase and B.subtilis/pHY300PLK-SPase were constructed.From which,the SPase activity expressed by recombinant bacteria B.subtilis/pBSMuL3-SPase was higher,and the intracellular enzyme activity was0.56 U·mL^-1.In addition,it was unexpectedly found that its extracellular enzyme activity was as high as 1.58 U·mL^-1,this extracellular expression phenomenon simplifies the extraction process of enzyme.The reason why SPase was efficiently secreted extracellularly was further analyzed,it is speculated that SPase is secreted to extracellular via atypical secretion pathway.?2?The enzyme was purified,and then its enzymatic properties were examined.The results showed that the optimal response of recombinant enzyme pH was 7.5,the optimal temperature was 60°C,its half-life at 60°C and 55°C were respectively 20 h and 80 h,and there was extensive pH tolerance.?3?The synthesis of kojibiose was catalyzed by recombinant SPase.and the L9?3³?orthogonal test was performed on the basis of single factor optimization.Finally,the optimal process conditions were obtained:the enzyme amount was 20 U·g^-1_substrate,the sucrose/glucose ratio was 0.5 M/0.5 M,pH 7.0,the reaction temperature was 50°C and the reaction time was 36 h.Under these conditions,the conversion rate of kojibiose was 40.01%,the yield was 104.45 g·L^-11 and the purity was 44.16%.In order to obtain higher purity kojibiose,the products were treated with Saccharomyces cerevisiae to remove digestible sugar,then the final purity of kojibiose was 95.14%.?4?The recombinant SPase was immobilized by chitosan-polyethylene glycol,LX-1000EA and LX-1000HA,among w hich the immobilized SPase of LX-1000HA has the advantages of convenient operation,good immobilization and application effect,and low cost.The recovery rate of SPase activity immobilized by LX-1000HA reached 101.82%when the amount of enzyme was 9 U·g^-1_carrier and the immobilization time was 16 h.The enzymatic properties indicate that the optimum temperature for immobilized SPase is 55°C,and the optimum pH is 7.0.After continuous reaction with immobilized SPase of LX-1000HA for 8batches,97.03%of residual enzyme activity was retained,and the conversion rate remained at45.24%.The above experimental results show that the immobilized enzyme can effectively promote the synthesis of kojibiose,and has good operational stability,thereby providing a reference value for the industrialization of the solidification enzyme to produce kojibiose.