Optimization Of Expression And Property Analysis Of Recombinant Sucrose Phosphorylase In Escherichia Coli BL21

Sucrose phosphorylase?EC2.4.1.7,SPase?belongs to family 13 of the glycosyl hydrolases.It is a specific enzyme that catalyzes the transfer of glucoside bonds,and it can catalyze the reversible phosphorylation of sucrose to glucose-1-phosphate and D-fructose.The enzyme has a wide range of substrate specificity,which has an important application value in the industrial production of food and cosmetics and so on.However,the yield of sucrose phosphorylase from wild strain by fermentation is relatively low,and it is difficult to meet large-scale industrial production.In this paper,the expression and enzymatic properties of recombinant sucrose phosphorylase were studied to improve the expression level and yield of sucrose phosphorylase,and to improve the stability.The sucrose phosphorylase gene from the Bifidobacterium longum JCM 1217 is connected with pET30 to construct the recombinant expression plasmid.The recombinant plasmid pET30-SPase was transformed into Escherichia coli BL21 to construct the recombinant strain E.coli BL21/pET30-SPase.Subsequently,recombinant strain was induced by IPTG to express sucrose phosphorylase in Escherichia coli.Then the expression conditions of recombinant E.coli BL21/pET30-SPase were optimized by single factor test,including the initial cell density,inducer concentration,induction temperature,induction time,shaking speed of the culture.As well as the optimization of medium components,including the different carbon and nitrogen sources,protective agents and metal ions.Then the orthogonal experiment was carried out to determine the best medium formula.In addition,Ni-NTA column was used to purify the crude the,and the property of recombinant sucrose phosphorylase was analyzed,including optimum temperature and thermal stability,optimum pH and pH stability,and the influence of chemical reagents on the stability of the the.The results of experiment are as follows:1.The optimum initial bacteria density of the recombinant strain E.coli BL21/pET30-SPase is OD₆₀₀=0.5,the final concentration of IPTG is 0.05 mmol/L,the optimum induction temperature is 30?,the optimum induction time is 15 h,The optimum shaking speed of the culture is 180 rpm;the optimum medium is based on the LB culture medium,and adding 0.5%soluble starch,0.1%K⁺and 0.05%Mg²⁺.2.After optimization,the total enzyme activity of the crude enzyme was 1207.83 U?50mL?,which was raised by 3.64-fold more than that before optimization,and specific enzyme of sucrose phosphorylase was 122.1 U/mg after purification,which was raised by 1.85-fold.The condition for the purification of recombinant sucrose phosphorylase by Ni-NTA column is,that the target protein is eluted with elution buffer containing 60 mmol/L imidazole.The purification multiplier is 8.4 and the recovery rate is 86%,which reached a good effect of purification.3.The optimum reaction temperature of recombinant sucrose phosphorylase is 45?.After incubating at 20-50?for 1 h,the enzyme activity is almost not affected,and at60-90?for 1 h,the remaining relative enzyme activity is more than 70%,and the enzyme has the better thermal stability.The optimum pH is 6.5,and the enzyme has the better stability in buffer with pH 6.0-6.8.Compared with the untreated enzyme,0.1%Tween 80,1%BSA,10mmol/L Fe²⁺and Mg²⁺can improve the sucrose phosphorylase activity and stability by16.69%,18.31%,43.09%and 26.36%of the enzyme activity.The yield of recombinant sucrose phosphorylase has been significantly increased after optimizing the induced expression conditions and medium component of recombinant E.coli BL21/pET30-SPase.And methods and conditions for improving the stability and activity of the enzyme were found,which provided technical support and guidance for industrial production of recombinant sucrose phosphorylase.