Separation and quantification of 2-keto-3-deoxy-gluconate (KDG) a major metabolite in pectin and alginate degradation pathways.

A rapid and sensitive High Performance Liquid Chromatography (HPLC) method with photometric and fluorescence detection is developed for routine analysis of 2-Keto-3-deoxy-gluconate (KDG), a catabolite product of pectin and alginate. These polysaccharides are primary-based compounds for biofuel production and for generation of high-value-added products. HPLC is performed, after derivatization of the 2-oxo-acid groups of the metabolite with o-phenylenediamine (oPD), using a linear gradient of trifluoroacetic acid and acetonitrile. Quantification is accomplished with an internal standard method. The gradient is optimized to distinguish KDG from its close structural analogues such as 5-keto-4-deoxyuronate (DKI) and 2,5-diketo-3-deoxygluconate (DKII). The proposed method is simple, highly sensitive and accurate for time course analysis of pectin or alginate degradation.

Combinatorial metabolic engineering of industrial Gluconobacter oxydans DSM2343 for boosting 5-keto-D-gluconic acid accumulation.

BACKGROUND: L-(+)-tartaric acid (L-TA) is an important organic acid, which is produced from the cream of tartar or stereospecific hydrolysis of the cis-epoxysuccinate. The former method is limited by the availability of raw material and the latter is dependent on the petrochemical material. Thus, new processes for the economical preparation of L-TA from carbohydrate or renewable resource would be much more attractive. Production of 5-keto-D-gluconate (5-KGA) from glucose by Gluconobacter oxydans is the first step to produce L-TA. The aim of this work is to enhance 5-KGA accumulation using combinatorial metabolic engineering strategies in G. oxydans. The sldAB gene, encoding sorbitol dehydrogenase, was overexpressed in an industrial strain G. oxydans ZJU2 under a carefully selected promoter, P0169. To enhance the efficiency of the oxidation by sldAB, the coenzyme pyrroloquinoline quinone (PQQ) and respiratory chain were engineered. Besides, the role in sldAB overexpression, coenzyme and respiratory chain engineering and their subsequent effects on 5-KGA production were investigated. RESULTS: An efficient, stable recombinant strain was constructed, whereas the 5-KGA production could be enhanced. By self-overexpressing the sldAB gene in G. oxydans ZJU2 under the constitutive promoter P0169, the resulting strain, G. oxydans ZJU3, produced 122.48 ± 0.41 g/L of 5-KGA. Furthermore, through the coenzyme and respiratory chain engineering, the titer and productivity of 5-KGA reached 144.52 ± 2.94 g/L and 2.26 g/(L · h), respectively, in a 15 L fermenter. It could be further improved the 5-KGA titer by 12.10 % through the fed-batch fermentation under the pH shift and dissolved oxygen tension (DOT) control condition, obtained 162 ± 2.12 g/L with the productivity of 2.53 g/(L · h) within 64 h. CONCLUSIONS: The 5-KGA production could be significantly enhanced with the combinatorial metabolic engineering strategy in Gluconobacter strain, including sldAB overexpression, coenzyme and respiratory chain engineering. Fed-batch fermentation could further enlarge the positive effect and increase the 5-KGA production. All of these demonstrated that the robust recombinant strain can efficiently produce 5-KGA in larger scale to fulfill the industrial production of L-TA from 5-KGA.

A new caffeoylgluconic acid derivative from the nearly ripe fruits of Evodia rutaecarpa.

A new caffeoylgluconic acid derivative, trans-caffeoyl-6-O-D-gluconic acid methyl ester (1), together with two known compounds named trans-caffeoyl-6-O-D-glucono-γ-lactone (2) and trans-caffeoyl-6-O-D-gluconic acid (3), was isolated from the nearly ripe fruits of Evodia rutaecarpa (Juss.) Benth.. These compounds were isolated by various separation methods associated with the UPLC-Q-TOF-MS technique. Their structures were elucidated on the basis of extensive spectroscopic methods.

2-Ketogluconic acid production by Klebsiella pneumoniae CGMCC 1.6366.

Klebsiella pneumoniae CGMCC 1.6366 is a bacterium isolated for 1,3-propanediol or 2,3-butanediol production previously. K. pneumoniae ΔbudA, a 2,3-butanediol synthesis pathway truncated mutant with the gene deletion of budA which encodes alpha-acetolactate decarboxylase, was found to execrate an unknown chemical at a high titer when grown in the broth using glucose as carbon source. Later this chemical was identified to be 2-ketogluconic acid, which was formed through the glucose oxidation pathway in K. pneumoniae. It was found that 2-ketogluconic can also be produced by the wild strain. The fermentation studies showed that the production of this metabolite is strictly pH dependent, when the fermenting broth was maintained at pH 6-7, the main metabolite produced by K. pneumoniae CGMCC 1.6366 was 2,3-butanediol, or some organic acids in the budA mutated strain. However, if the cells were fermented at pH 4.7, 2-ketogluconic acid was formed, and the secretion of all other organic acids or 2,3-butanediol were limited. In the 5L bioreactors, a final level of 38.2 and 30.2 g/L 2-ketogluconic acid were accumulated by the wild type and the budA mutant K. pneumoniae, respectively, in 26 and 56 h; and the conversion ratios of glucose to 2-ketogluconic acid reached 0.86 and 0.91 mol/mol for the wild and the budA mutant, respectively.

In vitro antioxidant and in vivo prophylactic effects of two gamma-lactones isolated from Grewia tiliaefolia against hepatotoxicity in carbon tetrachloride intoxicated rats.

Grewia tiliaefolia is widely used in traditional Indian medicines to cure jaundice, biliousness, dysentery and the diseases of blood. Bioassay-guided fractionation of methanolic extract of the G. tiliaefolia bark has resulted in the isolation of D-erythro-2-hexenoic acid gamma-lactone (EHGL) and gulonic acid gamma-lactone (GAGL). Hepatoprotective activity of the methanolic extract and the isolated constituents were evaluated against CCl(4)-induced hepatotoxicity in rats. The treatment with methanolic extract, EHGL and GAGL at oral doses of 100, 150 and 60 mg/kg respectively with concomitant CCl(4) intraperitoneal injection (1 ml/kg) significantly reduced the elevated plasma levels of aminotransferases, alkaline phosphatase and the incidence of liver necrosis compared with the CCl(4)-injected group without affecting the concentrations of serum bilirubin and hepatic markers. EHGL and GAGL significantly inhibited the elevated levels of thiobarbituric acid reactive substances and glutathione in liver homogenates. Histology of the liver tissues of the extract and isolated constituents treated groups showed the presence of normal hepatic cords, absence of necrosis and fatty infiltration as similar to the normal control. The results revealed that the hepatoprotective activity of EHGL is significant as similar to the standard drug silymarin. To clarify the influence of the extract and isolated constituents on the protection of oxidative-hepatic damage, we examined in vitro antioxidant properties of the test compounds. The extract and the constituents showed significant free radical scavenging activity. These results suggest that the extract as well as the constituents could protect the hepatocytes from CCl(4)-induced liver damage perhaps, by their anti-oxidative effect on hepatocytes, hence eliminating the deleterious effects of toxic metabolites from CCl(4).

Preparation of enzymes required for enzymatic quantification of 5-keto-D-gluconate and 2-keto-D-gluconate.

For easy measurement of 5-keto D-gluconate (5KGA) and 2-keto D-gluconate (2KGA), two enzymes, 5KGA reductase (5KGR) and 2KGA reductase (2KGR) are useful. The gene for 5KGR has been reported, and a corresponding gene was found in the genome of Gluconobacter oxydans 621H and was identified as GOX2187. On the other hand, the gene for 2KGR was identified in this study as GOX0417 from the N-terminal amino acid sequence of the partially purified enzyme. Several plasmids were constructed to express GOX2187 and GOX0417, and the final constructed plasmids showed good expression of 5KGR and 2KGR in Escherichia coli. From the two E. coli transformants, large amounts of each enzyme were easily prepared after one column chromatography, and the preparation was ready to use for quantification of 5KGA or 2KGA.

Biotechnological production of gluconic acid: future implications.

Gluconic acid (GA) is a multifunctional carbonic acid regarded as a bulk chemical in the food, feed, beverage, textile, pharmaceutical, and construction industries. The favored production process is submerged fermentation by Aspergillus niger utilizing glucose as a major carbohydrate source, which accompanied product yield of 98%. However, use of GA and its derivatives is currently restricted because of high prices: about US$ 1.20-8.50/kg. Advancements in biotechnology such as screening of microorganisms, immobilization techniques, and modifications in fermentation process for continuous fermentation, including genetic engineering programmes, could lead to cost-effective production of GA. Among alternative carbohydrate sources, sugarcane molasses, grape must show highest GA yield of 95.8%, and banana must may assist reducing the overall cost of GA production. These methodologies would open new markets and increase applications of GA.

Zymomonas mobilis as catalyst for the biotechnological production of sorbitol and gluconic acid.

The conversion of glucose and fructose into gluconic acid (GA) and sorbitol (SOR) was conducted in a batch reactor with free (CTAB-treated or not) or immobilized cells of Zymomonas mobilis. High yields (more than 90%) of gluconic acid and sorbitol were attained at initial substrate concentration of 600 g/L (glucose plus fructose at 1:1 ratio), using cells with glucose-fructose-oxidoreductase activity of 75 U/L. The concentration of the products varied hyperbolically with time according to the equations (GA) = t (GA)max /(W(GA) + t), (SOR) = t (SOR)max/(W(SOR) + t), V(GA) = [W(GA) (GA)max]/(W(GA) + t)2 and V(SOR) = [W(SOR) (SOR)max]/(W(SOR) + t)2. Taking the test carried out with free CTAB-treated cells as an example, the constant parameters were (GA)max = 541 g/L, (SOR)max = 552 g/L, WGA = 4.8 h, W(SOR) = 4.9 h, v(GA) = 112.7 g/L x h and v(SOR) = 112.7 g/L x h.

Gluconic acid: an antifungal agent produced by Pseudomonas species in biological control of take-all.

Pseudomonas strain AN5 (Ps. str. AN5), a non-fluorescent Australian bacterial isolate, is an effective biological control (biocontrol) agent of the take-all disease of wheat caused by the fungus Gaeumannomyces graminis var. tritici (Ggt). Ps. str. AN5 controls Ggt by producing an antifungal compound which was purified by thin layer and column chromatography, and identified by NMR and mass spectroscopic analysis to be d-gluconic acid. Commercially bought pure gluconic acid strongly inhibited Ggt. Two different transposon mutants of Ps. str. AN5 which had lost take-all biocontrol did not produce d-gluconic acid. Gluconic acid production was restored, along with take-all biocontrol, when one of these transposon mutants was complemented with the corresponding open reading frame from wild-type genomic DNA. Gluconic acid was detected in the rhizosphere of wheat roots treated with the wild-type Ps. str. AN5, but not in untreated wheat or wheat treated with a transposon mutant strain which had lost biocontrol. The antifungal compounds phenazine-1-carboxylic acid and 2,4-diacetylphloroglucinol, produced by other Pseudomonads and previously shown to be effective in suppressing the take-all disease, were not detected in Ps. str. AN5 extracts. These results suggest that d-gluconic acid is the most significant antifungal agent produced by Ps. str. AN5 in biocontrol of take-all on wheat roots.

Zymomonas mobilis as catalyst for the biotechnological production of sorbitol and gluconic acid.

The conversion of glucose and fructose into gluconic acid (GA) and sorbitol (SOR) was conducted in a batch reactor with free (CTAB-treated or not) or immobilized cells of Zymomonas mobilis. High yields (more than 90%) of gluconic acid and sorbitol were attained at initial substrate concentration of 600 g/L (glucose plus fructose at 1:1 ratio), using cells with glucose-fructose-oxidoreductase activity of 75 U/L. The concentration of the products varied hyperbolically with time according to the equations (GA)=t(GA)(max)/(W(GA) +t), (SOR)=t (SOR)(max)/(W(Sor)+t), v(GA)=[W(GA) (GA)(max)]/(W(GA)+t)(2) and V(SOR)=[W(SOR) (SOR)(max)]/(W(SOR)+t)(2). Taking the test carried out with free CTAB-treated cells as an example, the constant parameters were (GA)(max)= 541 g/L, (SOR)(max)=552 g/L, W(GA)=4.8h, W(SOR)=4.9h, upsilon(GA)=112.7 g/L. and upsilon(SOR)=112.7 g/L.

[Direct UV detection of glucose and its derivatives in capillary zone electrophoresis].

UV labeling detection has been commonly used to determine the association constants between lectins and saccharides, but the interaction is always between the labeled carbohydrates, rather than the truly underivatized carbohydrates, and lectins. In order to directly detect saccharides during the study on the interaction of glucose and its derivatives with lectins (e.g., concanavalin A), a capillary zone electrophoretic method with detection at a wavelength of 195 nm has been developed. The influences of various separation conditions including buffer concentration, pH and voltage were investigated. By using an uncoated silica capillary (50 microns i.d., 375 microns o.d., 48.5 cm of total length, and 44.0 cm to the detector) and 50 mmol/L Na2HPO(4)-50 mmol/L NaH2PO4 solution (near to the physiological pH of 7.4) as buffer, the underivatized sugars, including glucosamine, N-acetylglucosamine, glucose, and sodium gluconate, were sufficiently separated within 11 min at an applied voltage of 10 kV. On-column UV monitoring allowed the detection of these compounds at less than 4 mmol/L level, and quantification by the peak area method allowed reproducible determination of them at least at their respective concentration ranges. The method is characterized by its simplicity, rapidity, and reproducibility, and should be useful for the analysis of the interaction of glucose and its derivatives with lectins.

Production of gluconic acid using Micrococcus sp.: optimisation of carbon and nitrogen sources.

A process for production of gluconic acid from glucose by a Micrococcus sp. is described. More than 400 bacterial cultures isolated from local soil were tested for gluconic acid production. Three isolates, were selected on basis of their ability to produce gluconic acid and high titrable acidity. These were identified as Micrococcus sp. and were named M 27, M 54 and M 81. Nutritional and other parameters for maximum production of gluconic acid by the selected isolates were optimised. It was found that Micrococcus sp. isolate M 27 gave highest yield of 8.19 g gluconic acid from 9 g glucose utilised giving 91% conversion effeciency.

Microbial synthesis of 3-deoxy-D-erythro-hex-2-ulosonic acid 6-phosphate.

A microbial route was explored for the synthesis of 3-deoxy-D-erythro-hex-2-ulosonic acid 6-phosphate (2-keto-3-deoxy-6-phosphogluconate, KDPG). Two strains of bacteria, Alcaligenes eutrophus H16 F34 (DSM 529) and Escherichia coli DF 71 (CGSC 4880), lacking in KDPG-aldolase activity were tested for excretion of KDPG. Using pyruvate and gluconate as carbon sources, Alcaligenes eutrophus H16 F34 accumulated and excreted 3-deoxy-D-erythro-hexulosonic acid 6-phosphate into the culture broth, while the E. coli strain, using pyruvate and glucuronate, failed. KDPG was isolated from the culture supernatant of Alcaligenes eutrophus H16 F34 in 78% yield and 5 g scale with respect to the consumed gluconate.

The metabolism of carbohydrates by extremely halophilic bacteria: the identification of lactobionic acid as a product of lactose metabolism by Halobacterium saccharovorum.

Nongrowing cells of Halobacterium saccharovorum oxidized lactose to a product identified as lactobionic acid by thin-layer, paper, and column chromatography, and by identification of the galactose and gluconic acid produced from it after acid hydrolysis. Growing cells oxidized lactose to a product that was identical with lactobionate except that it did not serve as a substrate for galactose oxidase. While the identity of this compound has not been established, it is suggested that the product is lactobionic acid in which the galactose moeity is in the furanose form. Neither lactobionate nor the product produced by growing cells was further metabolized, suggesting that lactose oxidation is not coupled to growth.