| As a low-calorie sweetener and substitute for sucrose, D-tagatose has been used in food industry such as fruit juice, beverages and gum. D-tagatose is also shown to be a potential antidiabetic drug to treat type 2 diabetes and a prebiotic to increase beneficial bacteria in colon, lower cholesterol and prevent colon cancer. L-arabinose isomerase could catalyze the reversible conversion between L-arabinose and L-ribulose in vivo, and could also take D-galactose as substrate to synthesis D-tagatose in vitro. Since the complex purification process, the by-product formation and the high cost of waste treatment in the chemical manufacturing method, the bioconversion of D-galactose to D-tagatose by L-arabinose isomerase has been the focus of researches.Bacillus subtilis is a non-pathogenic host strain in genetic engineering for heterologous protein expression. Spores, produced from B. subtilis cells when starved of nutrients, are able to survive under extreme environments. Displaying catalytic heterologous protein on spore coat broadens application of enzyme preparations to industry. This study concentrates on the display of L-AI from lactic acid bacteria and attempt to obtain recombinant spore with high stability and activity. Then analysis and compare the enzymatic activity with L-AI from recombinant Escherichia Coli when using D- galactose as substrate.The main research contents and results of this study are as follows:Firstly, gram-positive lactic acid bacteria with the ability to produce acids were isolated from fresh pickles. Then 18 strains using D- galactose as substrate to produce D-tagatose were selected depending on the Cysteine-carbazole method, of those 4 strains that significantly express L-AI to produce D-tagatose were further screened using the method of TLC. Through analysis of morphological characteristics, physiological and biochemical identification and 16 S rDNA sequencing, 2 target strains were obtained and named Lactobacillus plantarum PC5 and Lactobacillus brevis PC14. And the latter was finally chosen as template for further study.Design primers according to known gene sequence encoding for L-AI, and amplify araA with 1425 bp from genome in screened L. brevis PC14. Construct recombinant plasmid pSE-araA using vector pSE380 and recombinant E.coli strain JM109/pSE-araA. Under the induction of IPTG, the target protein L-AI was successfully expressed with a molecular weight of 55 kDa. The measure results of enzymatic activity of L-AI demonstrated optimum condition for catalytic reaction of temperature 65 ℃ and pH value 6.8 when using D-galactose as substrate. In this condition, the apparent Km and Vmax values for D-galactose were 77.3 mmol/L and 6.0 μmol/(L·min), respectively and the conversion rate from D-galactose to D-tagatose was 53%.The spore coat protein CotG encoding gene cotG was amplified using genome of Bacillus subtilis 168 as template, and inserted into multiple cloning site of plasmid pHS with gene araA together to construct fusion plasmid pHS/cotG-araA and recombinant B. subtilis DB403 strain B.s/cotG-araA. SDS-PAGE and Western Blot analyses of recombinant spore suspension revealed that fusion protein CotG-L-AI was expressed with a molecular weight of 75 kDa and L-AI successfully displayed on spore coat under the anchor of CotG. In accordance with L-AI from recombinant E.coli, spore surface-displayed L-AI showed the same optimal temperature and pH value but higher thermal stability and wider pH range. In this condition, the apparent Km and Vmax values for D-galactose were respectively 48.0 mmol/L and 9.7 μmol/(L·min) and the bioconversion rate was 62%. The assay of recycling of recombinant spore indicated that the enzymatic activity of L-AI had no significant decrease after 4 recycles. |
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