L-gulonic Acid Research Articles

Chirospecific Synthesis of D‐erythro‐ and L‐threo‐Sphinganines from Sugars.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Chirospecific Synthesis of D-erythro- and L-threo-Sphinganines from Sugars

D-erythro-sphinganine 1 and L-threo-sphinganine 2 have been prepared in the enantiomerically pure form by the chirospecific manner. Key intermediates, 2-amino-3-hydroxy-4-pentenoates 8 and 12, were obtained from L-glucono-1,5-lactone and L-gulonic acid g-lactone via a simultaneous dealkoxyhalogenation.

Development of a chiral non-aqueous capillary electrophoretic system using the partial filling technique with UV and mass spectrometric detection

A chiral non-aqueous CE system with UV and mass spectrometric detection has been developed. The enantioseparation was promoted by diastereomeric complex (ion-pair) formation between the amines (e.g. salbutamol, atenolol) and the chiral selector, (−)-2,3:4,6-di- O-isopropylidene-2-keto- l-gulonic acid [(−)-DIKGA]. Different solvent mixtures were studied, as well as different concentrations of (−)-DIKGA and ammonium acetate in the background electrolyte. A partial filling technique was developed with a selector plug composed of (−)-DIKGA and ammonium acetate in a solvent mixture of methanol and 2-propanol. The separated enantiomers of pronethalol were detected by a Q-TOF MS system equipped with a sheath-flow electrospray ionization interface.

Mathematical Modeling of in vitro Enzymatic Production of 2-Keto- L-gulonic Acid Using NAD(H) or NADP(H) as Cofactors

A 2-Keto- L-gulonic acid (2-KLG) production process using stationary Pantoea citrea cells and a Corynebacterium 2,5-diketo- D-gluconic acid (2,5-DKG) reductase enzyme has been developed which may represent an improved method of vitamin C biosynthesis. Experimental data was collected using the F22Y/A272G 2,5-DKG reductase mutant and NADP(H) as a cofactor. An extensive kinetic analysis was performed and a kinetic rate equation model for this process was developed. A recent protein engineering effort has resulted in several 2,5-DKG reductase mutants exhibiting improved activity with NADH as a cofactor. The use of NAD(H) in the bioreactor may be preferable due to its increased stability and lower cost. The kinetic parameters in the rate equation model have been replaced in order to predict 2-KLG production with NAD(H) as a cofactor. The model was also extended to predict 2-KLG production in the presence of a range of combined cofactor concentrations. This analysis suggests that the use of the F22Y/K232G/R238H/A272G 2,5-DKG reductase mutant with NAD(H) combined with a small amount of NADP(H) could provide a significant cost benefit for in vitro enzymatic 2-KLG production.

Efficient synthesis of 3-hydroxyprolines and 3-hydroxyprolinols from sugars

trans-3-Hydroxy-l-proline 1, trans-3-hydroxy-l-prolinol 2, cis-3-hydroxy-d-proline 3, and cis-3-hydroxy-d-prolinol 4 have been prepared in enantiomerically pure form with chirospecific manner. Key intermediates, 2-amino-3-hydroxy-4-pentenoate 9 and 17, were obtained from d-glucono-δ-lactone and l-gulonic acid γ-lactone via a simultaneous dealkoxyhalogenation.

DNA from uncultured organisms as a source of 2,5-diketo-D-gluconic acid reductases.

Total DNA of a population of uncultured organisms was extracted from soil samples, and by using PCR methods, the genes encoding two different 2,5-diketo-D-gluconic acid reductases (DKGRs) were recovered. Degenerate PCR primers based on published sequence information gave internal gene fragments homologous to known DKGRs. Nested primers specific for the internal fragments were combined with random primers to amplify flanking gene fragments from the environmental DNA, and two hypothetical full-length genes were predicted from the combined sequences. Based on these predictions, specific primers were used to amplify the two complete genes in single PCRs. These genes were cloned and expressed in Escherichia coli. The purified gene products catalyzed the reduction of 2,5-diketo-D-gluconic acid to 2-keto-L-gulonic acid. Compared to previously described DKGRs isolated from Corynebacterium spp., these environmental reductases possessed some valuable properties. Both exhibited greater than 20-fold-higher kcat/Km values than those previously determined, primarily as a result of better binding of substrate. The Km values for the two new reductases were 57 and 67 microM, versus 2 and 13 mM for the Corynebacterium enzymes. Both environmental DKGRs accepted NADH as well as NADPH as a cosubstrate; other DKGRs and most related aldo-keto reductases use only NADPH. In addition, one of the new reductases was more thermostable than known DKGRs.

Non-aqueous capillary electrophoretic separation of enantiomeric amines with (−)-2,3:4,6-di- O-isopropylidene-2-keto- l-gulonic acid as chiral counter ion

(−)-2,3:4,6-Di- O-isopropylidene-2-keto- l-gulonic acid [(−)-DIKGA] has been introduced as a chiral counter ion in non-aqueous capillary electrophoresis. High enantioresolutions ( R S ≥3) were obtained for amines, e.g., pronethalol, labetalol and bambuterol. Methanol containing NaOH and (−)-DIKGA was used as the background electrolyte. The counter ion concentration and the nature of the injection medium were found to affect the chiral separation. Covalent coating of the fused-silica capillary reduced the electro-osmotic flow resulting in improved enantioresolutions.

Purification and molecular characterization of a quinoprotein alcohol dehydrogenase from Pseudogluconobacter saccharoketogenes IFO 14464

We have cloned and verified a gene for a novel quinoprotein alcohol dehydrogenase (ADH) from Pseudogluconobacter saccharoketogenes IFO 14464 that has the ability to oxidize l-sorbose to 2-keto- l-gulonic acid (2-KLGA). The enzyme was purified from the soluble fraction of the bacterium and was estimated to be a monomeric protein with a molecular weight of 65 kDa from the analyses of SDS-PAGE and gel-filtration chromatography. An open reading frame of 1824 bp for 608 amino acid residues was estimated as the gene for ADH because of the consistency of the calculated molecular mass and the elucidated partial amino acid sequences of the native enzyme. Homology search revealed that the enzyme showed close similarity to quinoprotein alcohol dehydrogenases isolated from Methylobacterium extorquens and Acetobacter aceti, particularly in the tryptophan docking motifs in the α-subunits of those dehydrogenases. The ability to convert l-sorbose to 2-KLGA was found when the lysate of recombinant Escherichia coli DH10B transformed with the gene for ADH was mixed with CaCl 2 and pyrroloquinoline quinone (PQQ). These data indicate that the cloned DNA is the desired gene for the ADH in which CaCl 2 and PQQ are essential for enzymatic activity.

Farmacologia e toxicologia do ácido ascórbico: uma revisão

L-Ascorbic acid (AA) or vitamin C is a six carbon cetolactone, structurally related to glucose and other hexoses. The major sources of AA are citrus fruits, strawberry, melon, green pepper, potato, tomato and leafy green vegetables. AA interferes with a broad spectrum of oxidation-reduction reactions, acting in at least 10 enzymatic systems. In this way, vitamin C influences the synthesis of collagen, carnitine, and neurotransmitters; the transformation of cholesterol into bile acids; biotransformation of xenobiotics substances; absorption of iron; and formation and scavenging of oxygen free radicals. AA is used as food addictive because of its antioxidant properties.Therapeutically, it is used as nutritional supplement during scurvy. Human beings and other primates, as well as guinea pigs and some species of bats are mammals that are unable to synthesize AA; thus, they need AA in the diet to prevent scurvy. Rats are able to synthesize AA using glucose, through intermediary formation of D-glucuronic acid, L-gulonic acid and gulonolactone.Homo sapiens lack the hepatic enzyme gulonolactone oxidase, which catalyses the last reaction of the biosynthesis pathway (L-gulonolactone conversion to ascorbic acid). The functions of central nervous, immune, and cardiovascular systems, and the periodontal tissue, as well as the detoxification function of the liver, are negatively influenced by vitamin C deficiency. In this way, it has been described several benefits of vitamin supplement ingestion, as decreasing of LDL cholesterol, including mega doses that can reach as much as 18g daily. Although there have been raised many literatures about vitamin C use in a wide variety of diseases, there is a lack of clinical efficiency of mega doses; besides, some side effects can come up, as diarrhea and oxalate stones in the kidneys. However, the ideal daily intake of vitamin C is still unknown. This happen because the recommended daily intake is based in a single role of AA, the scurvy prevention. Daily ingestion of AA should be the same quantity excreted or destroyed by oxidation, taking into consideration AA actions on the enzymatic systems. Actually, vitamin C is necessary for health in little quantities and is harmful in large doses. It happens because the cells are always walking a balance between oxidation and reduction processes, and AA in great quantities assume oxidative characteristics, interfering in this balance. Although the existence of several evidences indicating AA toxicity in large doses, there are some authors who believe that the ingestion of large doses is safe, but they admit that the disposable data are very contradictory.

Metabolic engineering study on the direct fermentation of 2-keto- l-gulonic acid, a key intermediate of l-ascorbic acid in Pseudomonas putida IFO3738

We have achieved production of 2-keto- l-gulonic acid (2-KLGA) in recombinant Pseudomonas putida IFO3738. Firstly, the genes for sorbose dehydrogenase (SDH)/sorbosone dehydrogenase (SNDH) were introduced into P. putida. The recombinant P. putida/pBBR-SDH produced 0.7 mg/ml of 2-KLGA in a culture broth containing 5% l-sorbose. Replacement of the native SNDH promoter by the Escherichia coli tufB promoter (pBBR-SDH-tufB) improved the productivity of 2-KLGA up to 11.4 mg/ml. Secondly, the sorbitol dehydrogenase (SLDH) gene was also introduced into P. putida. The recombinant P. putida/pUCP19-3DH carrying the genes for SDH, SNDH and SLDH had the ability to produce 2-KLGA (7.5 mg/ml) in a 5% d-sorbitol broth. The productivity of 2-KLGA was improved up to 9.8 mg/ml by changing to an expression system with two plasmids, pBBR-SDH-tufB (for SDH/SNDH) and pUCP19-SLDH (for SLDH), respectively. Moreover, the replacement of the native SLDH promoter by the E. coli tufB promoter (pUCP19-SLDH-tufB) improved the 2-KLGA productivity up to 11.6 mg/ml. Optimization of cultivation conditions increased the conversion yield of 2-KLGA to 32% and that of l-idonate, a metabolite of 2-KLGA, to 40%. These results indicate P. putida IFO3738 is one of the candidate strains for direct fermentation of 2-KLGA.

Regio- and stereo-selectivity in homogeneous catalytic hydrogenation of [formula omitted] acid

2,5- Diketo- d-threo- hexonic acid (2,5-diketo- d-gluconic acid, 1), a crucial intermediate in the microbial production of l-threo- hex-2-enono-1,4-lactone ( l-ascorbic acid, vitamin C), was isolated from the fermentation broth of bacterium Erwinia citreus ATCC 31623, and its regio- and stereo-selective hydrogenation, catalyzed by the water-soluble Ru(II) complex of tris( m-sulfophenyl)phosphine (TPPTS), was performed. The effect of hydrogen pressure, temperature, pH, and catalyst-to-substrate ratio on regio- and stereo-selectivity of the process was studied. At low pH, over 90% regioselectivity in favor of the reduction of the 5-keto group in 1 was achieved, affording l-xylo-2- hexulosonic acid (2-keto- l-gulonic acid, 2) as the main product. Maximal diastereoselectivity, i.e. ratio between 2 and 2-keto- d-gulonic acid (3) expressed as diastereomeric excess (d.e.%), amounted to ca. 50% and was not influenced by any of the above reaction parameters.

Oxidation of l-sorbose with molecular oxygen on platinum modified by metals, amines and phosphines

The oxidative dehydrogenation of l-sorbose to 2-keto- l-gulonic acid in aqueous medium is reviewed with special emphasis on the use of promoted platinum catalysts. The activity of alumina- or carbon-supported platinum can be increased by selective deposition of bismuth or lead onto the supported platinum particles, and by preadsorbed strongly basic tertiary and quaternary amines. Both methods have a negative influence on the selectivity, but the effect of supported N-bases is less detrimental. Aromatic and cycloaliphatic amines, some phosphines and aminophosphines improve the selectivity to 2-keto- l-gulonic acid. The amine and phosphine modifiers are used in very small amounts, less than 600 ppm related to sorbose. The best modifier is hexamethylenetetramine, which provides 99-95% selectivity up to 30% conversion. The efficiency of metal, amine and phosphine type promoters is compared.

Modification of supported Pt catalysts by preadsorbed phosphines: enhanced selectivity in the oxidation of l-sorbose

The influence of phosphine and aminophosphine modifiers on the performance of 5 wt.-% Pt/C and Pt/alumina catalysts was studied in the partial oxidation of l-sorbose with molecular oxygen. The molar ratio of modifier to surface Pt atoms (Pt s) was varied between 0.1 and 1.0. In general, higher modifier/Pt s ratios resulted in higher selectivity to 2-keto- l-gulonic acid and lower reaction rate. The activity and selectivity of Pt strongly depended on the chemical structure of phosphine. Tributhylphosphine in trace amounts (modifier: Pt s = 0.1 mol/mol ; modifier: sorbose = 600 ppm) proved to be an outstanding modifier, which improved the selectivity of Pt/C by 28–37% in the whole investigated conversion range. Monitoring the catalyst potential during the reaction revealed that the oxidation state of Pt decreased by promotion, which is an indication that some side reactions leading to catalyst poisoning are suppressed.

Determination of 2-keto- l-gulonic, 2-keto- d-gluconic and 2,5-diketo- d-gluconic acids by capillary zone electrophoresis

During the biosynthetic processing of 2-keto- l-gulonic acid (2-KLG) from 2,5-diketo- d-gluconic acid (2,5-DKG) by Corynebacterium sp., 2-keto- d-glutonic acid (2-KDG) is produced as a byproduct. These organic acids have been analyzed by high-performance liquid chromatography (HPLC) on an Aminex HPX-87H column, but the resolution was not good enough for quantitative analysis. We investigated the quantitation of 2-KLG, 2-KDG and 2,5-DKG using capillary electrophoresis (CE) and the results were compared with those of HPLC. With CE, in contrast to HPLC, good resolution, efficiency and rapid analysis were demonstrated as well as low consumption of solvent and samples. The CE system was applied at 15 kV with UV detection at 195 nm using 100 m M sodium borate (pH 8.4) as an electrolyte. The results were shown within 5 min with efficiency approaching 100 000 theoretical plates. The relative standard deviations of migration time and peak area were less than 0.9% and 1.6%, respectively. The detection limits for quantitative determination were 0.5–1.3 μM level. The above compounds, in fermentation broth, were analyzed under the optimum conditions. Considering the results of our study, the CE method should be highly suitable for the separation of 2-KLG, 2-KDG and 2,5-DKG in the fermentation broth.

Flocculation of cell, cell debris and soluble protein with methacryloyloxyethyl trimethylammonium chloride—acrylonitrile copolymer

The flocculation of cell, cell debris and soluble proteins from 2-keto- L-gulonic acid process media with six different charge densities of methacryloyloxyethyl trimethylammonium chloride—acrylonitrile (DMMC-AN) copolymers as flocculant was studied to investigate the influence of charge density on flocculation. It was found that the effectiveness of flocculation and optimal flocculation dosages depend on the cationic density of the flocculant. With optimal cationic density, DMMC-AN copolymer can remove 97% of the cell debris and 95% soluble proteins by 30 min sedimentation under gravity. The optimal flocculation dosage varies between 500 and 700 ppm. The study also showed that DMMC-AN copolymer has no selectivity on removal of soluble proteins in the spent medium, since the soluble proteins with different sizes can be removed efficiently by the test of Sephadex G-150 column chromatography, which contributed to the purification of the non-protein biological products solution.

Synthesis of sugar-like amino-carboxylic acids from D-mannitol

6-Amino-2,5-anhydro-6-deoxy- D-gluconic and L-gulonic acid derivatives, conformationally restricted sugar-like amino-carboxylic acids which mimic dipeptides, have been synthesized by a silica gel assisted azidolysis of enantiomerically pure bis-epoxides.

Development of a stable shuttle vector and a conjugative transfer system for Gluconobacter oxydans

A shuttle vector pGE1 (11.9 kb) which can replicate both in Gluconobacter oxydans and Escherichia coli was constructed from the cryptic Gluconobacter plasmid pGO3293S (9.9 kb, relaxed type) and E. coli plasmid pSUP301 (5 kb, Km r, Ap r, relaxed type). The plasmid pGO3293S is one of the endogenous plasmids of G. oxydans IFO 3293 which converts l-sorbose to 2-keto- l-gulonic acid (2KGA), an intermediate of vitamin C synthesis. The other plasmid, pSUP301, is a conjugative plasmid which contains pACYC177 and the mob region from plasmid RP4. The plasmid pGE1 could be transferred into G. oxydans IFO 3293 with a high frequency (10 −1 transconjugants/recipient) by a conjugal transfer system, and maintained very stably without antibiotic selection. pGE1 can be introduced and maintained in other acetic acid bacteria including Gluconobacter and Acetobacter. The presence of pGE1 did not inhibit the growth or 2KGA productivity of 2KGA-producing strains derived from G. oxydans IFO 3293. The usefulness of pGE1 as a vector was confirmed by subcloning the membrane-bound l-sorbosone dehydrogenase gene of A. liquefaciens IFO 12258 in G. oxydans IFO 3293 derivatives; in this subcloning, pGE1 could be further shortened to the 9.8 kb plasmid, pGE2.

Intergeneric protoplast fusion between Gluconobacter oxydans and Corynebacterium species

Intergeneric protoplast fusion between 2,5-diketo-gluconic acid producing Gluconobacter oxydans (ATCC 9937) and a mutant strain of Corynebacterium species (ATCC 31090), capable of reducing 2,5-diketo-gluconic acid to 2-keto- l-gulonic acid, a penultimate step in vitamin C production) resulted in viable recombinants. Some of the fusion products exhibited the capacity to convert d-glucose to 2-keto- l-gulonic acid, but the conversion rate is low.

アスコルビン酸およびその関連物質のホームベーカリーの使用による製パン効果の検討

L-Ascorbic acid (AsA) and its related compounds were tested for the improver of Breadmaking by Homebakery. AsA and dehydro-L-ascorbic acid (DHA) showed almost similar effects on the improvement of specific volume of the bread by adding around 8 ppm to wheat flour. But, 2, 3-diketo- L-gulonic acid (DKG) had no appreciable effect on the specific volume of the bread with the concentration up to 50 ppm. When the mixing time of the breadmaking was changed from 25 min to 85 min by 10-min intervals, the specific volume of the bread baked by 4-hr course was quite different from that of 25-min mixing : AsA gave no distinct decrease in the specific volume ; however, in the case of DKG and the control, the specific volume decreased with the increase of the mixing time. When the breadmaking time was extended from 4 hr to 8 hr, the specific volumes of the bread baked by the addition of AsA were distinctly decreased with the increase of the amount of AsAadded, but DHA showed no distinct effect on the increase of the loaf volume of bread baked. The SH concentrations in dough increased 4-fold after the addition of dry yeast, compared with the dough just before the addition of the yeast. This may suggest that the SH compounds in the yeast affect the cross-linking by SS linkages in the dough. For the breadmaking with an automatic breadmaking apparatus, some interactions between AsA, its related compounds and SH groups in the dough and yeast must be important for the breadmaking.

Inhibition of aldehyde reductase by aldose reductase inhibitors

A broad group of structurally diverse aldose reductase inhibitors including flavonoids, carboxylic acids and hydantoins, have been examined for their ability to inhibit rat kidney aldehyde reductase (EC 1.1.1.19, EC 1.1.1.20) versus rat lens aldose reductase (EC 1.1.1.21). All aldose reductase. inhibitors examined inhibited aldehyde reductase to some extent both in the reductive reaction as determined with glyceraldehyde as substrate and NADPH as coenzyme, and in the oxidative reaction where l-gulonic acid was oxidized to d-glucuronic acid in the presence of NADP + Of the inhibitors examined, 2,7-dinuorospirofluorene-9,5′-imidazolidine-2′,4′-dione (A11576) was the most potent inhibitor requiring only concentrations in the 10 −8 M range to inhibit 50% of the in vitro activity of rat kidney aldehyde reductase ( ic 50 value), whereas 3-dioxo-1- H-benz[de]isoquinoline-2(3H)-acetic acid (alrestatin) was the least potent inhibitor requiring concentrations in the 10 −5 M range. Both the reductive and oxidative steps appeared equally inhibited by these aldose reductases inhibitors. Moreover, all compounds appeared to inhibit either crude or highly purified rat kidney aldehyde reductase to essentially the same extent. Marked differences in the selectivity of these inhibitors, expressed as the ratio of ic 50 values for rat kidney aldehyde reductase versus rat lens aldose reductase with glyceraldehyde as substrate, were observed with selectivity for aldose reductase ranging from ca. 2-fold for A11576 to 119-fold for 3-(4-bromo-2-nuorobenzyl-4-oxo-3-phthalazine-1-ylacetic acid (Ponalrestat). Kinetic and competition studies suggest that these inhibitors interact with aldehyde reductase at a common site that is not identical to either the substrate or nucleotide binding site. These results suggest that the inhibitor binding sites of rat kidney aldehyde reductase and aldose reductase contain several common characteristics.