Method for producing dextransucrase through fermentation of leuconostoc mesenteroides and application of dextransucrase
A technology of dextran sucrase and Leuconostoc enterocolitica, which is applied in the field of producing glucan sucrase by fermentation of Leuconostoc enterococcus, achieves the effects of reduced fermentation cost, mild conditions and stable enzyme activity
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Embodiment 1
[0023] Example 1: Dextran sucrase fermentation optimization
[0024] 1) Preparation of seed solution
[0025] Leuconostoc enterococcus was inoculated in the basic MRS liquid medium (glucose 20g / L, tryptone 10g / L, beef extract powder 10g / L, yeast extract powder 5g / L, K 2 HPO 4 2 g / L, ammonium citrate 2g / L, anhydrous sodium acetate 5g / L, MgSO 4 ·7H 2 O0.58g / L, MnSO 4 4H 2 O 0.25g / L, Tween 801mL / L, pH 6), static culture at 30°C, so that the initial cell concentration in the medium was 1.0×10 8 unit / mL is the seed solution.
[0026] 2) Single factor optimization
[0027] On the basis of the MRS basal medium, the seed solution was inoculated in the enzyme-producing medium, and the concentration of sucrose, tryptone, beef extract powder, yeast extract powder, and K 2 HPO 4 concentration, ammonium citrate concentration, anhydrous sodium acetate concentration, CaCl 2 Effects of addition amount, inoculum amount, initial pH value, and fermentation time on the production of dex...
Embodiment 2
[0031] Embodiment 2: Dextran sucrase optimum reaction temperature and optimum reaction pH value
[0032] 1) Take 1.0mL buffer solution with pH of 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0 and mix with 1.0mL crude dextran sucrase enzyme solution respectively, place it statically for 30min, measure the enzyme activity retention rate under different pH value conditions, determine Optimal pH of dextran sucrase.
[0033] 2) Mix 1.0mL 0.1M pH 5.5 sodium acetate buffer with 1.0mL crude dextran sucrase enzyme solution, incubate at 20, 30, 40, 50 and 60°C for 30min, then store at 4°C to dissolve The crude enzyme activity in 0.1M pH 5.5 sodium acetate is the original enzyme activity, and the enzyme activity retention rate under different temperature conditions is determined to determine the optimum temperature of dextran sucrase.
[0034] like figure 2 As shown, the optimum reaction pH value of the dextran sucrase obtained in this example is 5.0, and the optimum reaction temperature is 30°C. ...
Embodiment 3
[0035] Example 3: In vitro synthesis of exopolysaccharide by dextran sucrase
[0036]Add 2mL of dextran sucrase to 100mL of sucrose substrate, and let stand at 30°C for 30h. After the reaction was completed, the reaction product was incubated in a water bath at 80°C for 30 min to inactivate unreacted enzymes. An equal volume of 10% (w / v) trichloroacetic acid solution was added to the reaction solution, and after continuous stirring at 4°C for 4 hours, centrifuged at 10,000×g for 60 minutes at 4°C to remove protein precipitates and retain the supernatant. Three volumes of pre-cooled 95% (w / v) ethanol was added to the supernatant, and after standing overnight at 4°C, centrifuged at 10,000×g for 60 min at 4°C to obtain exopolysaccharide precipitates. The exopolysaccharide precipitate was dissolved with 100 mL deionized water, and the catalytic synthesis efficiency of exopolysaccharide was determined by the phenol-sulfuric acid method.
[0037] In this example, dextran sucrase c...
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