Study On The Fermentation Of 2-keto-D-gluconic Acid

2-keto-D-gluconic acid(2-keto-D-gluconic acid,2KGA)is a synthetic D-sodium erythorbate(EN),and D-iso-ascorbic acid(EA)of the precursor can also be converted to D-ketoses,D-arabinose.2KGA cement itself can be used as plasticizers,detergents and herbicides,with a high industrial value.First,the comparison of several commonly used detection 2KGA ways to build a simple rough iodimetry precise detection technology and intuitive liquid chromatography detection technology.Among them,the liquid chromatography conditions:United States Agilent liquid chromatograph,Bole Aminex HPX-87H column with a mobile phase of 0.005 M H2SO4.The column temperature was 55℃,the flow rate was 0.6 mL/min,detection wavelength was 210 nm,and the sample volume was 20μL.This method is rapid,accurate and reproducible.There was a strain which can produce 2KGA in our laboratory,the 16SrDNA was sequenced and it was compared to database information through the identification of Pseudomonas putida,named P.putida KD-1.Carbon source,nitrogen source,osmotic pressure,pH and other conditions were studied on the yield of 2KGA of P.putida KD-1,the production of 2KGA was 15-22 g/L,the overall yield was low.The strain growed slowly and fermened long period in high osmotic pressure.Development potential was low.Furthermore the deposited strain Gluconobacter suboxydans J12(referred J12)Fermentation experiments conducted with the P.putida KD-1 contrast,J12 could tolerat at higher concentrations of glucose than P.putida KD-1.J12 metabolize glucose fast,fermentating after 72 h,the production of 2KGA can reach 29.5 g/L.However,J12 contains various dehydrogenase,glucose not only can be converted to generate 2KGA,but also can be converted to generate 5KGA,while the yield of production which glucose converts to 2KGA falls and the separation and purification of fermentation products is so difficult.So I hope to knockout the dehydrogenase gene of 5KGA through genetic manipulation techniques to increase the yield of 2KGA.By designing primer,transformation,resistant screening methods,we obtained transformants called J12-1.Molecular validations showed that we achieved a knockout.Fermentation product contains only 2KGA through HPLC.After optimizing fermentation conditions,the production of the mutant strains J12-1 was 39.6 g/L.Further fermentation experiments of the mutant J12-1 was taken by 2 L fermentor,the total amount of glucose is 400 g,after 72 h fermentation,the volume of fermentation broth was 2 164 mL,finally obtained 2KGA was 162.58 g,equivalenting to 75.13 g/L,the quality yield was 40.65%,the rate of molar conversion is 37.99%.Experiments further explored the conversion of glucose to generate 2KGA circumstances under J12-1 resting state,due to the process of glucose dehydrogenase which located on the cell membrane of J12-1 or periplasm of Gluconobacter suboxydans catalyzed to 2KGA.As the living cells still have catalytic activity in the resting state,theoretically glucose can be converted into gluconic acid directly by the cells,then to 2KGA.Through the study we found that collected cells of J12-1 which had been fermentated 36 h,into 60 g/L glucose,aerobicly converting 72 h.The product of 2KGA could achieve a high purity.When the bacteria concentration reaches 20 g/L,can be almost 100%conversion of glucose to 2KGA.