Study On Key Enzymes And Its Confactor For Converting L-Sorbose To 2-Keto-Gulonic Acid In The Two-step Fermentation Of Vitamin C

In the two-step fermentation of vitamin C, the first step is the conversion of D-sorbitol into L-sorbose by Acetobacter melanogenum, and the second step is the conversion of L-sorbose into 2-Keto-L-gulonic acid (KGA) by ketogulonigenum vulgare. The so-called two-step fermentation of vitamin C is virtually the bio-oxidation of D-sorbitol and L-sorbose. The elucidation of the biochemistry process is important to the production of vitamin C and design of the engineered bacteria, in which the related genes from Acetobacter melanogenum and ketogulonigenum vulgare were introduced, by using the recombinant DNA technology.In the former work, we have purified an enzyme from ketogulonigenum vulgare, which is able to transform L-sorbose directly to KGA. We have also constructed the genomic library of the ketogulonigenum vulgare, from which the genes incuding the sdh gene were isolated.On the base of the work above, the SDH were highly expressed and purified, its optimal temperature and pH, the stability of the enzyme and some other important properties have been studied. The dynamic studies demonstrate that it is a typital Michslis-Menten enzyme .We also study the activator and inhibitor of SDH.We study the effect of Acetobacter melanogenum to SDH. Constructing the SDH-HyuC difussion expression vector, the hyuC gene as a report gene. In E.coli, we can detect the SDH and HyuC activity at the same time. But in Acetobacter melanogenum, we can only detect the HyuC activity. The plasmid is stability in the Acetobacter melanogenum. Because the hyuC gene on the backward position of the SDH gene, the transcription and translation order of the diffusion gene is from sdh to hyuC, the expression of hyuC gene explain that the diffusion protein can be transcripted and translated in the Acetobacter melanogenum, the SDH may be degradated in the host bacterium.The vitro experiment prove this fact too. We speculate that the C-terminal degradation of SDH , through the analyzing of SDH open reading frame ,we find that on the amino acid sequence ,the Met to Gln there are 23 amino acid, these amino acid are most hydrophobe, may be a signal peptide ,which orientate the SDH.PQQ is the third cofactor of redox-enzyme of Gram-negative bacteria. It is considered a new vitamin B and widely used in pharmaceutical industry and agricultural production. In recent years, some studies about the biological function of PQQ have been reported. A screening method that allows available, efficient, and reliable detection and selection of high lever pqq prdouctivity micro-organisms was devised. We finaly get two strains from soil samples which PQQ can reach 60μg/ml.