Optimizing Culture Conditions For Recombinant Dextransucrase Production In E.coli And Study On Dextran Synthesis

Dextransucrase (EC.2.4.1.5) synthesize high molecular weight glucose polymer dextran by transferring the D-glucosyl moieties to a growing glucan chain using sucrose as substrate. Dextran has several important uses in the pharmaceutical, chemical and food industry. Dextran is produced commercially using Leuconostoc mesenteroides by adding sucrose to the culture medium for enzyme induction and dextran synthesis. Because of the difficulties in investigation of the structure and catalysis of this enzyme, dextransucrase genes have been cloned and expressed successfully in Escherichia coli.In this study,Medium components for cell growth and enzyme expression were optimized using an orthogonal experiment using IPTG and lactose as inducer separately. According to the results, we found the optimal medium for dextransucrase expression,and than the inducing conditions were studied. In the study of dextransucrase induction using IPTG, When the cell density reached 2.0, 0.25 mmol/L IPTG was added and the culture continued for 4 h for enzyme induction.In the study of dextransucrase induction using lactose, when the OD600 reached 3.0, inducer of 1.0% lactiose added and fermentation continued for 6h. Although enzyme expression induced by lactose was fifth of that by IPTG,lactose is suitable for large-scale fermentation due to its low cost and no-toxicity.The dextransucrase gene (dexYG) was expressed in engineered strain after IPTG induction and the crude enzyme was obtained by sonication. We purified the recombinant dextransucrase by using ammonium sulfate precipitation and metal chelate affinity chromatography on a Ni-NTA column. Then we characterized catalytic kinetic parameter of purified enzyme. This purification protocol resulted in a 11.4-fold purification with a yield of 37.5%. The molecular weight of dextransucrase measured by SDS-PAGE was 170 kDa ,which was similar to the enzyme from Leuconostoc mesenteroides. The enzyme had an optimum temperature in the range of 25℃to 30℃and an optimum pH of 5.4. It was relatively stable in the range of pH 5.0 to pH 7.0, but the stability declined rapidly as soon as the temperature rose over 35℃.The enzyme activity remained 59% after stored for 4 days at room temperature (25℃), and lost 50% activity after stored for seven weeks at 4℃. Ca2+ of 0.5 mmol/L could strongly activate the enzyme, Mg2+ of 1 mmol/L had little effect, Cu2+ and SDS could greatly inhibit the enzyme.The recombinant dxtransucrase was used to produced dextran using sucrose as substrate , the temperature,sucrose concentration and pH value effects on synthesized dextran were studied. As to these results, the yield was more than 80% ,the dextran synthesized showed high molecular weight and good dissolubility. Sucrose concentration had the greatest impact on the dextran yield, followed by the temperature, and pH value had minimal impact. Preliminary research on the molecular weight control of dextran synthesis, dextran hydrolysis and fractional precipitation with alcohol may provide an important basis for industrial applications of the recombinant dxtransucrase.