ABSTRACT
Vitamin C is an essential vitamin for human beings. It has a huge market in the fields of food and pharmaceuticals. 2-keto-L-gulonic acid is an important precursor to produce vitamin C by microbial fermentation in industrial. In microbial fermentations, the L-sorbose pathway and the D-gluconate pathway have been the focus of research because of high yield. This article aims at stating recent research progress in dehydrogenases related to biosynthesis of vitamin C in the L-sorbose pathway and the D-gluconate pathway. The properties of dehydrogenase in terms of localization, substrate specificity, cofactors, and electron transport carrier are elaborated. And then, the main problems and strategies are reviewed in the L-sorbose pathway and in the D-gluconate pathway. Finally, future research on the dehydrogenases in the biosynthesis of vitamin C through L-sorbose pathway and D-gluconate pathway is discussed.
Subject(s)
Ascorbic Acid/biosynthesis , Fermentation , Gluconates , Oxidoreductases/metabolism , SorboseABSTRACT
Abstract A new plate method was developed for rapid screening of Ketogulonicigenium vulgare mutants overproducing 2-keto-l-gulonic acid (2-KLG). The screening methodology took the advantage of the acidity caused by 2-KLG, which changes the color of bromothymol blue (pH indicator) from blue to yellow. Using the proposed method, a mutant, K. vulgare 65, was selected from 20,000 colonies produced by a strain subjected to spaceflight mutagenesis. When co-cultured with Bacillus megaterium 2980 in 20-L fermenters, K. vulgare 65 showed a high conversion rate (94.45%) of l-sorbose to 2-KLG. In contrast to the traditional screening method, this one significantly improved the frequency of obtaining positive mutants. The proposed plate screening method is cost-effective and easy to run and is thus useful for the isolation and screening of K. vulgare mutants overproducing 2-KLG.
Subject(s)
Sugar Acids/metabolism , Bacteriological Techniques/methods , Rhodobacteraceae/metabolism , Sorbose/metabolism , Bacteriological Techniques/instrumentation , Rhodobacteraceae/isolation & purification , Rhodobacteraceae/genetics , Fermentation , MutationABSTRACT
The aim of this study was to improve the 2-keto-L-gulonic acid (2-KLG) production efficiency by Ketogulonicigenium vulgare and Bacillus megaterium by using multi-stage pH control strategy. The effect of pH on the cell growths and 2-KLG production showed that the optimum pH for K. vulgare and B. megaterium cell growth were 6.0 and 8.0, respectively, while the optimum pH for 2-KLG production was 7.0. Based on the above results, we developed a three-stage pH control strategy: the pH was kept at 8.0 during the first 8 h, then decreased to 6.0 for the following 12 h, and maintained at 7.0 to the end of fermentation. With this strategy, the titer, productivity of 2-KLG and L-sorbose consumption rate were achieved at 77.3 g/L, 1.38 g/(L x h) and 1.42 g/(L x h), respectively, which were 9.7%, 33.2% and 25.7% higher than the corresponding values of the single pH (pH 7.0) control model.
Subject(s)
Bacillus megaterium , Metabolism , Culture Media , Chemistry , Fermentation , Hydrogen-Ion Concentration , Rhodobacteraceae , Metabolism , Sorbose , Metabolism , Sugar Acids , MetabolismABSTRACT
L-sorbose/L-sorbosone dehydrogenase from Ketogulonigenium vulgare S2 can transform L-sorbose to 2-KLG, which is widely used in production of Vitamin C. In order to obtain the engineering strain producing L-sorbose/L-sorbosone dehydrogenase and simplify the fermentation technology, firstly, this enzyme was purified by the methods of ammonium sulfate precipitation, DEAE Sepharose Fast Flow and Q Sepharose High Performance. Then, the purified L-sorbose/L-sorbosone dehydrogenase was injected to rabbit to obtain antibody. Next, the genomic library of Ketogulonigenium vulgare S2 was constructed by inserting the restriction fragments of chromatosomal DNA digested with Sau3A I into cosmid pKC505 vector digested by Hpa I and Pst I, which were packed with lamda phage package protein and transferred into E. coli DH5alpha in vitro. Finally, the positive strain K719# was selected from more than 12,000 clones via Dot-ELISA. Through the test of SDS-PAGE and thin layer chromatography, the results showed that the engineering strain K719# had the same biological activity as Ketogulonigenium vulgare S2 after adding coenzyme PQQ.
Subject(s)
Carbohydrate Dehydrogenases , Genetics , Metabolism , Cloning, Molecular , Escherichia coli , Genetics , Metabolism , Genomic Library , Gluconobacter oxydans , Genetics , Sorbose , Metabolism , Sugar Acids , Metabolism , Transformation, BacterialABSTRACT
BACKGROUND: Rapid species identification of enterococci is necessary for optimal treatment of infected patients as they are frequently resistant to various antimicrobial agents. Minimal identification scheme is necessary to cut the laboratory cost. In this study, a minimal identification system was modified to expand the identifiable species. METHODS: Performance of MGP test was compared to that of MIO motility test. Colonies on blood agar were used to inoculate primary identification media: SFA, BEAA, mannitol agar, tellurite agar, sorbose agar and MGP agar, which were prepared in biplates. Pigment production was tested when necessary using colonies on a blood agar. Isolates, which were not identifiable by the primary test, were inoculated to secondary test media: ADH, and arabinose-, raffinose- and sucrose-containing CTA. Vitek GPI cards were used to test isolates with a doubtful identification or no identification. RESULTS: MGP test was selected for the modified scheme, as it was more rapid and accurate than motility test. Among the 879 clinical isolates of enterococci, 462 (52.6%) and 3 (0.3%) were identified as E. faecalis and E. casseliflavus, respectively, by the primary test only. With the additional secondary tests, 379 (43.1%) isolates were identified as E. faecium. Vitek test showed the identification of 4 isolates with atypical test results and 5 isolates of rare species by modified scheme were correct. Nine isolates (1.0 %) were not identifiable by the modified scheme. CONCLUSIONS: The modified minimal identification scheme which included MGP test identified most E. faecalis isolates rapidly and accurately. Most of E. faecium isolates were identified with the additional secondary tests. In conclusion, the system is useful for the identification of commonly isolated species of enterococci.
Subject(s)
Humans , Agar , Anti-Infective Agents , Mannitol , SorboseABSTRACT
NAD(P)ase activity was stimulated when 1% sorbose was present in the culture medium of A. nidulans, and this effect was partially reversed by 1% glucose. 2. The level of extracellular NAD(P)ase was more affected by sorbose in the culture medium than the intracellular enzyme and no morphological changes were obtained. 3. The sorbose effect on NAD(P)ase activity appears to be specific sinle two other exoenzymes tested (ß-glucosidase and alkaline protease) show normal secretion patterns. 4. These findings suggest that the sorbose effect on NAD(P)ase production may be the consequence of metabolic disorders not necessarily linked with the morphological changes induced by the ketohexose