Gene Cloning,Expression And Application Of Levansucrase From Bacillus Amyloliquefaciens H47

Levan is one of fructans. It is often found in many plants and microorganisms. There are broad application prospects in the fields of chemical industry, food and medicine of levan. The content of levan in plants is low and the cost of extraction is high. As well as, the yield of levan from microbial fermentation is usually low and purification is more complex. But levan is easily obtained by the method of levansucrase which can hydrolyze sucrose into glucose and fructose and form levan. The purification method is relatively simple, too. So there is much certain theoretical significance and potential application value on the study of levan synthesising from levansucrase. In this study, Bacillus amyloliquefaciens which was isolated from honey sources in our laboratory was used as materials. The gene of levansucrase was cloned and heterologously expressed in Escherichia coli BL21（DE3）. After purification, the enzymatic properties were analyzed, and then the research on the synthesis of levan by recombinant levansucrase was conducted. Main research results are as follows:1) Through the results of whole genome sequence of B. amyloliquefaciens H47 and sequence alignment analysis of NCBI, the levansucrase gene in B. amyloliquefaciens H47 is at a length of 1422 bp, encoding 473 amino acid. The target gene was cloned by PCR and connected with the expression vector p ET28a（+）, and then constructed into E. coli BL21（DE3）. After inducing expression, the molecular weight of levansucrase was predicted to be 52 k Da. The induced expression conditions of the recombinant enzyme were optimized, and the optimal induction conditions were determined as follows: the initial induction bacteria concentration OD600 was 0.6, IPTG concentration was 0.4 mmol·L^-1, temperature was 30°C,and induction time was 12 h.2) The recombinant enzyme was purified by the affinity column. After purification, the enzymatic characterization of levansucrase was determined and analyzed. The optimum temperature of hydrolytic activity was 45°C and the maximal transfructosylation activity was measured at 40°C. The optimum p H for hydrolytic and transfructosylation activities were 6.5 and 6.0, respectively. Fe3+ and Mg²⁺ stimulated the transfructosylation activity and hydrolytic activity slightly. Besides, the Km and Vmax values for levansucrase were calculated to be 150.7 mmol·L^-1 sucrose and 153.6 U·mg^-1, respectively.3) The structure of polysaccharides synthesized by levansucrase were evaluated by sugars analysis and fourier transform-infrared analysis. The results showed that the polysaccharide synthesized by levansucrase was levan. In addition, sucrose concentrations was a important factor affected the molecular weight of levan. The molecular weight of levan was 8.0 and 2.8 k Da when the sucrose concentration was at 100 and 600 g·L^-1, respectively. But the effect of temperature and enzyme dosage on the molecular weight were few.4) The optimal conditions of the conversion rate for levan were studied. The optimal sucrose concentration, enzyme dosage, p H, temperature and time of levan biosynthesis were 300 g·L^-1, 10 U·m L^-1, 6.0, 35°C and 16 h, respectively. In the conditions, the conversion rate of levan was the highest, which are reached 38.8%.