| D-tagatose is a new type of functional sweetner, which can be used in foods,healthcare products, diabetes drugs and chemical syntheses. Enzymatic method of D-tagatose production has become a research focus during recent years. Using this method, L-arabinose isomerase(AI) is used as the catalyst to convert D-galactose obtained from lactose hydrolysis to D-tagatose. Enzymatic method of D-tagatose production has many advantages, such as mild reaction conditions and very few side products. Therefore, the method is accordant with environmental protection concepts. To improve the conversion rate and yield of enzymatic D-tagatose production, the present study identified a novel AI from Bacillus coagulan NL01 and obtained its mutants through rational design methods based on computational modelling. One of these mutants was finally used in D-tagatose production.This study includes the three following parts of research work:(1) The encoding gene(ara A) of AI from Bacillus coagulan NL01(BCAI) was cloned and expressed in Escherichia coli BL21( DE3)?BCAI was purified by nickel affinity chromatography and the purification fold was near 3.5. Molecular mass of BCAI monomer was about 55 k Da. BCAI showed an optimal temperature and optimal p H of 60 oC and 7.5 respectively. It exhibited perfect stability at 60 oC. It was sensitive to metallic ion s and Mn2 +?Co2+ were its activators. The enzyme possessed a kcat Km-1 of 8.7 min-1 m M-1 toward L-arabinose and a kcat Km-1 of 1.0 min-1 m M-1 toward D-galactose?(2) The structure of BCAI was obtained through performing homology modelling method using crystal structures of Lactobacillus fermentum CGMCC2921 and E. coli AIs as the templates. L-arabinose and D-galactose molecules were then docked to the active site of BCAI by using CDOCKER algorithm. The result showed L-arabinose was better than D-galactose for the enzymatic catalysis in terms of docking energy and hydrogen bond formation. To improve the catalytic capacity of BCAI toward D-galactose, the M185?F279?M349?I370?R186 residues were chosen as the mutation sites. Alanine scanning results showed that mutations of F279 and I370 made remarkble changes on substrate specificity of BCAI. Saturation mutageneses of F279 and I370 and crude enzyme assay showed that D-galactose activity of F279I( 0.63 U mg-1) was higher than that of WT( 0.43 U mg-1), while activity of F279 V was similar to that of WT. However, four I370 mutants almost lost D-galactose activity completely.(3) Purified F279 I was obtained by nickel affinity chromatography. Its optimal temperature and p H for D-galactose catalysis were 50 oC and 8.0 respectively? F279 I possessed kcat Km-1 values of 5.2 m M-1 min- 1 and 1.3 m M-1 min-1 toward L-arabinose and D-galactose respectively under optimal conditions. Its catalytic efficiency and specificity toward D-galactose were apparently higher than those of WT.(4) Results of D-tagatose production catalyzed by whole cells showed tha t E. coli cells containing F279 I possessed optimal reaction temperatures of 50oC-70 oC, while E. coli cells containing WT possessed an optimal reaction temperature of 60 oC. Under 20 g L- 1 D-galactose, conversation rate of the former could reach the maximal value of 53% which was 41% higher than that of the latter one. At D-galactose concentrations of 150 g L-1 and 250 g L-1,E. coli cells containing F279 I achieved convertion rates of 45% and 35.5%, and D-tagatose productions of 67.5 g L-1 and 88.3 g L- 1,both of which were 30%-40% higher than those of E. coli cells containing WT?... |
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