Heterologous Expression And Application Of Nitrosospira Multiformis Sucrose Synthase

Sucrose synthase（Sucrose synthase,SuSy,EC 2.4.1.13）is a glycosyltransferase,belonging to the GT-4 glycosyltransferase subfamily and the GT-B glycosyltransferase superfamily.It can use cheap sucrose to generate expensive nucleoside diphosphate sugars in one step with almost no energy consumption.Compared with other methods of synthesizing nucleoside diphosphate sugars,the sucrose synthase pathway is expected to become the most cost-effective synthetic pathway.Nucleoside diphosphate sugars are important sugar donors for nucleotide sugar-dependent glycosyltransferases for glycosylation reactions.These GTs are extremely important sugar groups in glycobiology.Chemical catalysts can synthesize glycosides on a large scale and with high precision.Compared with the complexity of glycoside synthesis by chemical methods,GTs can achieve substrate glycosylation more concisely,accurately and efficiently due to its outstanding regioselectivity and stereoselectivity.However,the high cost of nucleoside diphosphate sugar donors（NDP-sugars）is the main limitation of the industrial application of GTs,which hinders the large-scale synthesis of oligosaccharides and polysaccharides.In this paper,the sucrose synthase（Nm Su Sy）from Nitrospirillum ammonia oxidized by E.coli was expressed and purified,and the enzymatic properties of the recombinase and the characteristics of its catalytic process were explored,and the thermodynamic and kinetic characteristics of this reversible reaction were emphasized.Analysis,and further combined with enzyme immobilization technology to improve the stability of the sucrose synthase in the catalytic process,to achieve high-efficiency synthesis of UDP-glucose.The main conclusions are as follows:（1）The heterologous expression,purification and enzymatic properties analysis of the sucrose synthase gene derived from Nitrosspirillum ammonia oxidase in Escherichia coli were realized.The results showed that the molecular weight of the enzyme was 89 k Da,the specific enzyme activity was 14.4 U·mg^-1,the optimal reaction pH was 6.5,and it had good stability in the range of pH 6-7.5.The optimal reaction temperature is 55℃.When the temperature is lower than 40℃,it has good stability.Mg²⁺,Mn²⁺,Fe²⁺can all increase the enzyme activity of Nm Su Sy.In addition,the optimal fermentation conditions were found by orthogonal experiment:glycerol 5 g·L^-1,Na Cl 5 g·L^-1,complex nitrogen source 25 g·L^-1,Fe Cl₃ 1 mmol·L^-1.Under the optimal conditions,the enzyme yield was 3.4 times that of LB medium.（2）Combining the enzymatic properties of Nm Su Sy and the thermodynamic limitations of the reversible reaction to determine the optimal reaction conditions:pH 6,temperature 40℃,1 mol·L^-1 sucrose,100 mmol·L^-1 UDP.Under the optimal reaction conditions,100 mg·L^-1 of Nm Su Sy can generate 35.8 mmol·L^-1(20.2 g·L^-1)of UDP-glucose for 1 h,and the reversible reaction reaches equilibrium after 6 h,and 41.5 mmol·L^-1 can be generated.(23.5 g·L^-1)UDP-glucose.（3）Using enzyme immobilization technology,using chitosan as a carrier to achieve the IIIimmobilization of Nm Su Sy,using single factor analysis to find the best immobilization conditions:glutaraldehyde activated chitosan concentration is 0.5%,time is 1 h,enzyme The cross-linking time with chitosan is 48 h.Under these conditions,the enzyme activity of 10chitosan beads immobilized was 2.51 U.The calculation showed that the specific enzyme activity was 24.36 U·g^-1,the enzyme activity recovery rate was 5.8%,and the immobilization efficiency was 10.23%.Comparing the enzymatic properties of NmSusy before and after immobilization,it is found that the stability of NmSusy becomes better at pH 5-5.5.Therefore,the immobilized NmSusy is used to synthesize UDP-glucose efficiently at pH 5-5.5,so that the conversion rate of the reversible reaction is changed from free.The 41.5%of the enzyme increased to 75.2%.Moreover,the enzyme activity remained 55.14%after the immobilized NmSusy was reused seven times.