Search

This double-blinded, randomized and placebo-controlled, crossover study investigated whether α-ketoglutaric-acid (α-KG) and 5-hydroxymethylfurfural (5-HMF) supplementation improves exercise performance in hypoxia and affects physiological responses during the exercise task. Eight moderately trained male participants (age: 25.3 ± 2.0 y, VO2max: 48.0 ± 8.3 ml/min/kg) performed an incremental exercise test to exhaustion in normoxia and two 2-hour cycle time trial (TT) tests in hypoxia (3,500 m) each separated by 1-week. Prior to the TT, participants supplemented with either α-KG and 5-HMF or placebo (random order). Supplementation did not improve TT performance at altitude and did not affect heart rate, effort perception and oxidative stress levels (p > 0.05). Oxygen saturation (SpO2) was enhanced during the α-KG and 5-HMF supplementation trial (79.5 ± 3.3 vs. 78.2 ± 3.7%, p = 0.026). Even though TT performance was unaffected, the enhanced SpO2– possibly originated from changed O2-affinity – deserves further consideration as the exercise performance decline at altitude is strongly linked to the SpO2decline. The inclusion of moderately fit participants, not specifically cycle trained, might have prevented any visible performance enhancement.

α-Ketoglutaric acid (α-KG) is a multifunctional dicarboxylic acid in the tricarboxylic acid (TCA) cycle, but microbial engineering for α-KG production is not economically efficient, due to the intrinsic inefficiency of its biosynthetic pathway. In this study, pathway engineering was used to improve pathway efficiency for α-KG production in Escherichia coli. First, the TCA cycle was rewired for α-KG production starting from pyruvate, and the engineered strain E. coli W3110Δ4-PCAI produced 15.66 g/L α-KG. Then, the rewired TCA cycle was optimized by designing various strengths of pyruvate carboxylase and isocitrate dehydrogenase expression cassettes, resulting in a large increase in α-KG production (24.66 g/L). Furthermore, acetyl coenzyme A (acetyl-CoA) availability was improved by overexpressing acetyl-CoA synthetase, leading to α-KG production up to 28.54 g/L. Finally, the engineered strain E. coli W3110Δ4-P(H) CAI(H) A was able to produce 32.20 g/L α-KG in a 5-L fed-batch bioreactor. This strategy described here paves the way to the development of an efficient pathway for microbial production of α-KG.

α-Ketoglutaric acid (α-KA) is an important endogenous metabolite in the Krebs cycle and considered a critical marker for various diseases. Several approaches for identifying α-KA have been reported. However, most of them are limited to single-signal transduction modes in one assay and working in the suspension state, which easily cause quantification errors and lead to false-positive detection results. Herein, a multivalue-responsive and semi-solid EuMOF-gel (EuMOG) logic biosensor is first designed and fabricated, which can realize the specific, rapid, and easy-to-differentiate naked-eye detection of the disease-associated marker of α-KA in serum through implementing one-to-two logic operation with three multicolor gates. The implementation of one-to-two logic operation can be rationally achieved by assembling 2,6-naphthalenedicarboxylic acid as both the energy donor and blue-colored output 1, photoactive Eu

Life originated on Earth possibly as a physicochemical process; thus, geological environments and their hypothetical characteristics on early Earth are essential for chemical evolution studies. Also, it is necessary to consider the energy sources that were available in the past and the components that could have contributed to promote chemical reactions. It has been proposed that the components could have been mineral surfaces. The aim of this work is to determine the possible role of mineral surfaces on chemical evolution, and to study of the stability of relevant molecules for metabolism, such as α-ketoglutaric acid (α-keto acid, Krebs cycle participant), using ionizing radiation and thermal energy as energy sources and mineral surfaces to promote chemical reactions. Preliminary results show α-ketoglutaric acid can be relatively stable at the simulated conditions of an impact-generated hydrothermal system; thus, those systems might have been plausible environments for chemical evolution on Earth.

L-amino acid oxidase (AAO) was reported to be capable of converting L-glutamic acid to α-aketoglutaric acid (α-KG). The sequence of AAO from Kitasatospora cheerisanensis was synthesized based on Pichia pastoris codon-usage preferences. AAO gene was cloned into plasmid pPICZα which was transformed into P. pastoris. Next, multi-copy expression plasmids were constructed by using plasmid pHBM905BDM. High-density fermentation was performed and the recombinant enzyme was characterized. The conversion conditions were optimized. By using Escherichia coli expression system, no soluble or active AAO was obtained from two strains after fermentation and induction. We can’t obtain high-level expression of recombinant strains by using plasmid pPICZα. Therefore, we constructed multi-copy expression plasmids using plasmid pHBM905BDM. By using this plasmid, multi-copy strains were constructed and named as PAAO1, PAAO2, PAAO3, PAAO4, and PAAO5, respectively. The following results showed that expression of AAO in multicopy strains increased as designed and strain PAAO5 was chosen for high-density fermentation and enzyme activity experiments. After high-density fermentation, we achieved an AAO-expression yield of 120.8 U/mL. After temperature and pH optimization, the highest AAO activity was observed at a temperature and pH of 20°C and 6, respectively. After optimization of the conversion conditions, the average production rate of L-glutamic acid to α-KG was 3.46 g/L/h and the highest α-KG titer (103 g/L) was converted from 120 g/L L-glutamic acid. In this study, AAO was abundantly expressed by using P. pastoris expression system. The following experiments indicated that AAO is suitable for use in industrial applications.

In this issue of Cell Metabolism, Asadi Shahmirzadi et al. (2020) demonstrate that late-onset dietary supplementation with calcium alpha-ketoglutarate results in increased survival, compressed morbidity, and reduced frailty in mice. The study provides further evidence for critical links between metabolism, inflammation, and aging.

The inhibition of α-glucosidase is used as a key clinical approach to treat type 2 diabetes mellitus and thus, we assessed the inhibitory effect of α-ketoglutaric acid (AKG) on α-glucosidase with both an enzyme kinetic assay and computational simulations. AKG bound to the active site and interacted with several key residues, including ASP68, PHE157, PHE177, PHE311, ARG312, TYR313, ASN412, ILE434 and ARG439, as detected by protein-ligand docking and molecular dynamics simulations. Subsequently, we confirmed the action of AKG on α-glucosidase as mixed-type inhibition with reversible and rapid binding. The relevant kinetic parameter IC

The article on the basis of an analysis of the literature discusses promising areas for the development of antidote prophylaxis and treatment of acute cyanide poisoning. The chemical compounds mediating anticyanide activity by stimulating the main detoxification mechanisms of endogenous cyanide: sulfangen, cobinamide, α-ketoglutaric acid claimed to be potential cyanide antidotes, effective and safe for intramuscular and intraosseous injections, inhalation and oral administration.

This work demonstrates the ability of the yeast Yarrowia lipolytica cultivated on biodiesel waste to synthesize α-ketoglutaric acid with a minimal content of pyruvic acid as the main byproduct. The key factor promoting the microbial production of α-ketoglutaric acid from the waste is a strong deficiency of thiamine in the cultivation medium. The production of α-ketoglutaric acid by the yeast can be regulated by changing the concentration of nitrogen, iron, zinc, copper, and manganese in the medium, as well as by pH medium and the aeration rate. The optimization of these parameters in flask experiments allowed us to increase the concentration of α-ketoglutaric acid in the medium by 2.6 times and to shift the α-ketoglutaric acid/pyruvic acid ratio from 5:1 to 30:1. During cultivation in a fermentor under optimized conditions, Y. lipolytica produced 80.4 g/L α-ketoglutaric acid with a process selectivity of 96.7% and the product yield (YKGA) equal to 1.01 g/g. • α-Ketoglutaric acid is commercially important biotechnological product. • Biosynthesis of α-ketoglutaric acid from biodiesel waste. • Optimization of cultivation medium and nutrition medium.

The selective and sensitive detection of α-ketoglutaric acid ( KA ) is of great importance not only for biological studies but also for early diagnosis of cancers. In comparison with other assays, fluorescence detection is advantageous. However, the fluorescence turn-on detection of KA remains rare. In this paper, we disclose a new fluorescence detection of KA with the ensemble of silole-A and n -octanohydrazide by utilizing the aggregation induced emission feature of silole compounds and the chemical reaction between KA and n -octanohydrazide. The ensemble displays reasonably good sensitivity and selectivity toward KA , and KA with concentration as low as 1.5 μM can be detected.

This paper presents the synthesis and characterization of three complexes of Mn (II,III), with the a-ketoglutaric acid (H2A) and 1- (o-tolyl) biguanide (TB) as ligands. The complexes corresponding formulas are: [Mn(TB)2(H2A)]Cl2 . 0.5C2H5OH (C1), [Mn2(TB)(A)(H2A)2(H2O)2(NO3)2](NO3)2. C2H5OH (C2) and [Mn2(TB)(A)(H2A)2(H2O)2(CH3COO)2](CH3COO)2 . 3H2O (C3), where A is H2A without 2 protons. C1-C3 compounds were characterized by standard physico-chemical methods: chemical elemental analysis, IR spectroscopy, UV-Vis-NIR spectroscopy, molar conductivity and thermal analysis. For the obtained C1-C3 complexes, but also for the ligands (H2A and TB) was tested the antibacterial activity against Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853 and antitumor activity on HeLa tumour cells. It has been observed a moderate cytotoxic effect of the complexes C1-C3 on growth and metabolic activity of HeLa cells, while the ligands show a very weak effect on them. The obtained complexes inhibits the adhesion on the substrate of bacterial strains Staphylococcus aureus and Pseudomona aeruginosa, which recommends them for possible therapeutic applications.

Probe for α-ketoglutaric acid (α-KA) usually provides low sensitivity, invisible colour change and complex procedure with surfacants, which limits the accurate, sensitive quantification and mornitoring of α-KA in biological samples. Essentially different from the routine sensing mechanism, a special mechanism of photo-induced electron transfer (PET) combining with large absorption red-shift (rather than emission) has been designed to develop a facile yet multifunctional probe for the simultaneous fluorimetric, absorption-ratiometric and colorimetric detections of α-KA in biological samples. Effective inhibition of PET ensured the intense fluorescence turn-on (60 fold) and qutie low detection limit (0.9 μM) for α-KA without need of any surfacants. Dramatical colour change caused by the large absorption red shift (100 nm) from UV–vis region enabled the colormetric analysis of α-KA by naked-eye. Meanwhile, the large absorption red shift also contributed to the ratiometric detection, allowing for the accurate quantification of α-KA in complex biological sample. With this probe, ratiometric detection, naked eye mornitoring and imaging of α-KA in serum, living cells and tissues are realized for the first time.

To improve the production of α-ketoglutaric acid (α-KG) from l-glutamate by whole-cell biocatalysis. A novel and highly active l-glutamate oxidase, SmlGOX, from Streptomyces mobaraensis was overexpressed and purified. The recombinant SmlGOX was approx. 64 kDa by SDS-PAGE. SmlGOX had a maximal activity of 125 ± 2.7 U mg−1 at pH 6.0, 35 oC. The apparent Km and Vmax values of SmlGOX were 9.3 ± 0.5 mM and 159 ± 3 U mg−1, respectively. Subsequently, a co-expression plasmid containing the SmlGOX and KatE genes was constructed to remove H2O2, and the protein levels of SmlGOX were improved by codon optimization. Finally, by optimizing the whole-cell transformation conditions, the production of α-KG reached 77.4 g l−1 with a conversion rate from l-glutamate of 98.5% after 12 h. An efficient method for the production of α-KG was established in the recombinant Escherichia coli, and it has a potential prospect in industrial application.

Summary: Inhibition of oxidative phosphorylation (OXPHOS) by 1-cyclopropyl-4-(4-[(5-methyl-3-(3-[4-(trifluoromethoxy)phenyl]-1,2,4-oxadiazol-5-yl)-1H-pyrazol-1-yl)methyl]pyridin-2-yl)piperazine (BAY87-2243, abbreviated as B87), a complex I inhibitor, fails to kill human cancer cells in vitro. Driven by this consideration, we attempted to identify agents that engage in synthetically lethal interactions with B87. Here, we report that dimethyl α-ketoglutarate (DMKG), a cell-permeable precursor of α-ketoglutarate that lacks toxicity on its own, kills cancer cells when combined with B87 or other inhibitors of OXPHOS. DMKG improved the antineoplastic effect of B87, both in vitro and in vivo. This combination caused MDM2-dependent, tumor suppressor protein p53 (TP53)-independent transcriptional reprogramming and alternative exon usage affecting multiple glycolytic enzymes, completely blocking glycolysis. Simultaneous inhibition of OXPHOS and glycolysis provoked a bioenergetic catastrophe culminating in the activation of a cell death program that involved disruption of the mitochondrial network and activation of PARP1, AIFM1, and APEX1. These results unveil a metabolic liability of human cancer cells that may be harnessed for the development of therapeutic regimens. : Sica et al. show that respiratory chain inhibition by 1-cyclopropyl-4-(4-[(5-methyl-3-(3-[4-(trifluoromethoxy)phenyl]-1,2,4-oxadiazol-5-yl)-1H-pyrazol-1-yl)methyl]pyridin-2-yl)piperazine (BAY87-2243, abbreviated as B87) becomes lethal for cancer cells when glycolysis is simultaneously suppressed. When combined with B87, dimethyl α-ketoglutarate acquires the capacity to suppress glycolysis, thus lethally poisoning bioenergetics metabolism. This therapeutic combination effect relies on transcriptional reprogramming that can be reverted by pharmacological inhibition of MDM2. Keywords: MDM2, Krebs cycle, glycolysis, mitochondrial fragmentation, regulated cell death, parthanatos, cancer metabolism

The metabolite α-ketoglutarate is membrane-impermeable, meaning that it is usually added to cells in the form of esters such as dimethyl α-ketoglutarate (DMKG), trifluoromethylbenzyl α-ketoglutarate (TFMKG) and octyl α-ketoglutarate (O-KG). Once these compounds cross the plasma membrane, they are hydrolyzed by esterases to generate α-ketoglutarate, which remains trapped within cells. Here, we systematically compared DMKG, TFMKG and O-KG for their metabolic and functional effects. All three compounds similarly increased the intracellular levels of α-ketoglutarate, yet each of them had multiple effects on other metabolites that were not shared among the three agents, as determined by mass spectrometric metabolomics. While all three compounds reduced autophagy induced by culture in nutrient-free conditions, TFMKG and O-KG (but not DMKG) caused an increase in baseline autophagy in cells cultured in complete medium. O-KG (but neither DMKG nor TFMK) inhibited oxidative phosphorylation and exhibited cellular toxicity. Altogether, these results support the idea that intracellular α-ketoglutarate inhibits starvation-induced autophagy and that it has no direct respiration-inhibitory effect.

Keto acids are essential organic acids that are widely applied in pharmaceuticals, cosmetics, food, beverages, and feed additives as well as chemical synthesis. Currently, most keto acids on the market are prepared via chemical synthesis. The biochemical synthesis of keto acids has been discovered with the development of metabolic engineering and applied toward the production of specific keto acids from renewable carbohydrates using different metabolic engineering strategies in microbes. In this review, we provide a systematic summary of the types and applications of keto acids, and then summarize and compare the chemical and biochemical synthesis routes used for the production of typical keto acids, including pyruvic acid, oxaloacetic acid, α-oxobutanoic acid, acetoacetic acid, ketoglutaric acid, levulinic acid, 5-aminolevulinic acid, α-ketoisovaleric acid, α-keto-γ-methylthiobutyric acid, α-ketoisocaproic acid, 2-keto-L-gulonic acid, 2-keto-D-gluconic acid, 5-keto-D-gluconic acid, and phenylpyruvic acid. We also describe the current challenges for the industrial-scale production of keto acids and further strategies used to accelerate the green production of keto acids via biochemical routes.

In this contribution, we demonstrate that it is possible to obtain the lactol tautomer of alpha-ketoglutaric acid (AKGA) in the solid state by cocrystallizing it with Leviteracetam, a chiral nootropic drug used to treat epilepsy. In addition, we show that a cocrystal can be isolated with the racemic equivalent of Levetiracetam, Etiracetam (Eti), in which AKGA stays in the keto-form. We also report the existence of a cocrystal conglomerate in the Etiracetam-AKGA system, which is more stable than the racemic cocrystal at room temperature. The existence of a stable conglomerate is put in relation to the enantiospecificity of the Levetiracetam cocrystals, which is likely related to the ability of the Etiracetam enantiomers to stabilize one lactol tautomer at a time in solution or to promote its formation by hydrogen bonding. By comparing the peculiarities of the system in hand to the general behavior of cocrystallizing chiral systems with and without zwitterionic coformers, we suggest that for a pseudoquaternary cocrystal (made up of two racemate compounds) to exist the pseudoternary combinations (made up of one racemate and an enantiomer of the second compound) should exist and the enantiomers of the two compounds should form a diastereomeric pair at the binary level, rather than behave enantiospecifically.

L-Amino acid deaminases (LAADs; EC 1.4.3.2) belong to a family of amino acid dehydrogenases that catalyze the formation of α-keto acids from L-amino acids. In a previous study, a whole cell biocatalyst with the L-amino acid deaminase (pm1) from Proteus mirabilis was developed for the one-step production of α-ketoglutarate (α-KG) from L-glutamic acid, and the α-KG titer reached 12.79 g L−1 in a 3 L batch bioreactor. However, the product α-KG strongly inhibited pm1 activity, and the titer of α-KG was comparatively lower than expected. Therefore, in this study, multiple rounds of error-prone polymerase chain reaction (PCR) and gene shuffling were integrated for the molecular engineering of pm1 to further improve the catalytic performance and α-KG titer. A variant (pm1338g4), which contained mutations in 34 amino acid residues, was found to have enhanced catalytic efficiency. In a batch system, the α-KG titer reached 53.74 g L−1 when 100 g of monosodium glutamate was used as a substrate. Additionally, in a fed-batch biotransformation system, the maximum α-KG titer reached 89.11 g L−1 when monosodium glutamate was continuously fed at a constant rate of 6 g L−1 h−1 (from 4 to 23 h) with an initial concentration of 50 g L−1. Analysis of the kinetics of the mutant variant showed that these improvements were achieved due to enhancement of the reaction velocity (from 56.7 μM min−1 to 241.8 μM min−1) and substrate affinity (the Km for glutamate decreased from 23.58 to 6.56 mM). A possible mechanism for the enhanced substrate affinity was also evaluated by structural modeling of the mutant. Our findings showed that the integration of error-prone PCR and gene shuffling was an effective method for improvement of the catalytic performance of industrial enzymes.

Glutaric acid is a C5 dicarboxylic acid that can be used as a building block for bioplastics. Although high concentrations of glutaric acid can be produced by fermentation or bioconversion, a large amount of α-ketoglutaric acid (α-KG) is necessary to accept the amine group from 5-aminovaleric acid. To decrease the demand for α-KG, we introduced l-glutamate oxidase (GOX) from Streptomyces mobaraensis in our previous system for cofactor regeneration in combination with a glutaric acid production system from 5-aminovaleric acid. To enhance glutaric acid production, critical factors were optimized such as the expression vector, pH, temperature, and cell ratio. As a result, the demand for α-KG was decreased by more than 6-fold under optimized conditions. Additionally, the effect of catalase was also demonstrated by blocking the degradation of α-KG to succinic acid because of the hydrogen peroxide. Finally, 468.5 mM glutaric acid was produced from 800 mM 5-aminovaleric acid using only 120 mM α-KG. Moreover, this system containing davBA, gabTD-nox, and gox can be applied to produce glutaric acid from L-lysine by reusing α-KG with GOX. This improved cofactor regeneration system has a potential to apply much larger production of glutaric acid.

A novel kinetic method was developed for the quantitation of α-ketoglutaric acid (AKG) in cardioplegic solution and athletic supplements. The assay relied on an enzymatic transamination of AKG and d -4-hydroxyphenylglycine to form 4-hydroxybenzoylformic acid and l -glutamic acid using d -phenylglycine aminotransferase. Since 4-hydroxybenzoylformic acid absorbed UV strongly at 334 nm, the initial rate of the reaction was determined by the increasing absorbance at this wavelength without the need for colorimetric probes or coupling reactions, and this information was used for the construction of a standard curve against AKG concentration. The method showed good linearity (r2 = 0.9994) over an AKG concentration range of 20–160 μM. The limits of detection and quantitation were 4.09 and 13.62 μM respectively. It was simple, inexpensive, accurate and precise, as well as repeatable, and was not interfered with by excipients in the samples. Regarding the environmental friendliness, the method was free from the use of organic solvents or hazardous reagents and required no chemical pre-treatment of samples. The proposed method gave assay results tested in real samples in agreement with the HPLC method and commercial assay kits, therefore being suitable for routine analysis of AKG in quality control laboratories.

Both α-ketoglutaric acid (KGA) and pyruvic acid (PYR) are important keto acids. Efficient co-production of KGA and PYR has been achieved in our previous work, and could significantly decrease the cost of fermentation production. KGA and PYR have similar physical and chemical properties. Hence, finding a way to separate the two keto acids efficiently has become a key challenge. In this study, different chromatographic methods have been investigated, including ion-exchange chromatography, aluminum oxide chromatography and silica gel chromatography. The results show that the two keto acids can be well separated with silica gel chromatography, whereas ion-exchange chromatography and aluminum oxide chromatography could not separate them. Using the pretreated fermentation broth of Yarrowia lipolytica WSH-Z06, the purity and yield of KGA/PYR reached 98.7%/99.1% and 86.7%/70.9%, respectively, with an optimized silica gel chromatography-based procedure. This study provides an efficient method for separating KGA and PYR from fermentation broth, which might be applied on an industrial scale and significantly decrease the cost of biotechnological production of keto acids. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1370-1379, 2018.

Kinetics of the MenD-catalyzed 1, 4-addition of α-ketoglutaric acid to acrylonitrile were investigated in detail and based on the results a mathematical model of the process was developed and experimentally validated. The reaction kinetics were described by the double substrate Michaelis–Menten kinetics where the reaction rate linearly depends on acrylonitrile concentration. Non-competitive enzyme inhibition by p-methoxyphenol, i.e. acrylonitrile stabilizer, was also included in the model. Spontaneous reactivity of acrylonitrile towards polymerization was noted. Based on the experimental results, as well as model simulations, a fed-batch reactor was selected as the best reactor mode to carry out the reaction due to shortened reaction time compared to the repetitive batch reactor mode, and the ability to obtain maximum yield on the product, i.e. 6-cyano-4-oxohexanoic acid. Additionally, the fed-batch reactor method enables to set up the feeding strategy of acrylonitrile necessary to keep its concentrations below the level which significantly affects enzyme operational stability. The mathematical model was used for process optimization and as an assessment tool for the economic evaluation. Results have shown that in the investigated variable range all the requirements for the industrial implementation can be achieved ; i.e. product concentration of 70 g L–1, volume productivity of 58 g L–1 d–1, product yield of 96%, and biocatalyst yield of 28.9 gP gMenD–1. Based on the optimized production procedure, economic analysis was performed to determine the breakeven point of the process. In addition, influence of uncertainties in estimation of input variables on overall economic performance was assessed using sensitivity analysis. Project CARBAZYMES- This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 635595.

The growth behavior of anodic oxide films formed via anodizing in glutaric and its derivative acid solutions was investigated based on the acid dissociation constants of electrolytes. High-purity aluminum foils were anodized in glutaric, ketoglutaric, and acetonedicarboxylic acid solutions under various electrochemical conditions. A thin barrier anodic oxide film grew uniformly on the aluminum substrate by glutaric acid anodizing, and further anodizing caused the film to breakdown due to a high electric field. In contrast, an anodic porous alumina film with a submicrometer-scale cell diameter was successfully formed by ketoglutaric acid anodizing at 293 K. However, the increase and decrease in the temperature of the ketoglutaric acid resulted in non-uniform oxide growth and localized pitting corrosion of the aluminum substrate. An anodic porous alumina film could also be fabricated by acetonedicarboxylic acid anodizing due to the relatively low dissociation constants associated with the acid. Acid dissociation constants are an important factor for the fabrication of anodic porous alumina films.

We previously developed a novel one-step biotransformation process for the production of α-ketoglutarate (α-KG) from L-glutamic acid by a Bacillus subtilis whole-cell biocatalyst expressing an L-amino acid deaminase (pm1) of Proteus mirabilis. However, the biotransformation efficiency of this process was low owing to low substrate specificity and high α-KG degradation. In this study, we further improved α-KG production by protein engineering P. mirabilis pm1 and deleting the B. subtilis α-KG degradation pathway. We first performed three rounds of error-prone polymerase chain reaction and identified mutations at six sites (F110, A255, E349, R228, T249, and I352) that influence catalytic efficiency. We then performed site-saturation mutagenesis at these sites, and the mutant F110I/A255T/E349D/R228C/T249S/I352A increased the biotransformation ratio of L-glutamic acid from 31% to 83.25% and the α-KG titer from 4.65 g/L to 10.08 g/L. Next, the reaction kinetics and biochemical properties of the mutant were analyzed. The Michaelis constant for L-glutamic acid decreased from 49.21 mM to 23.58 mM, and the maximum rate of α-KG production increased from 22.82 μM min(-1) to 56.7 μM min(-1). Finally, the sucA gene, encoding α-ketodehydrogenase, was deleted to reduce α-KG degradation, increasing the α-KG titer from 10.08 g/L to 12.21 g/L. Protein engineering of P. mirabilis pm1 and deletion of the α-KG degradation pathway in B. subtilis improved α-KG production over that of previously developed processes.

Currently, α-ketoglutaric acid (α-KG) is industrially produced by multi-step chemical synthesis, which can cause heavy environmental pollution. Here we reported a simple one-step approach for the production of α-KG by transforming l-glutamic acid with an engineered l-amino acid deaminase (l-AAD) from Proteus mirabilis. First, to facilitate the purification of membrane-bound l-AAD, one N-terminal transmembrane region (from 21 to 87th nucleotide) was removed from l-AAD to block the binding of l-AAD with membrane, and the relatively low-usage codons were replaced by high-usage codons in Escherichia coli to improve the expression level. However, inclusion bodies formed when expressing the ΔN-LAAD in E. coli BL 21, and then the soluble and active ΔN-LAAD was obtained by the solubilization and renaturation of ΔN-LAAD. Furthermore, the biochemical properties of the refolded ΔN-LAAD were characterized and compared with those of full-length l-AAD. Finally, the ΔN-LAAD was used to synthesize α-KG and the maximal formation rate of α-KG reached 12.6% (w/w) at 6h under the following conditions: 12g/L l-glutamic acid, 0.1g/L ΔN-LAAD, 5mM MgCl2, temperature 45°C and pH 8.0. Compared with the multi-step chemical synthesis, the transformation approach has less environmental pollution and has a great potential for α-KG production.

Oxidative cleavage of cycloalkene-1-carboxylates, made from the corresponding carboxylic acids, and subsequent oxidation of the resulting ketoaldehyde afforded the important 1-monoesters of 2-ketoalkanedioic acids. Thus ozonolysis of octyl cyclobutene-1-carboxylate followed by sodium chlorite oxidation afforded the 1-monooctyl 2-ketoglutarate. This is a cell-permeable prodrug form of α-ketoglutarate, an important intermediate in the tricarboxylic acid (TCA, Krebs) cycle and a promising therapeutic agent in its own right.

A practical synthetic procedure for the synthesis of dihydro-2H-pyran-3(4H)-one is reported. The method commenced from the readily available -ketoglutaric acid and allowed preparation of the title compound in four steps in 31% overall yield.

The yeast Yarrowia lipolytica VKM Y-2412 was selected as a prospective producer of α-ketoglutaric acid (KGA) from ethanol. The following peculiarities were found: (1) the intensive KGA production occurred only under the limitation of cell growth by thiamine and the excess of ethanol and nitrogen, (2) the production of KGA from ethanol required increased amount of zinc and iron ions, and (3) KGA production increased significantly with a high aeration at pH medium equal to 3.5. Under optimal conditions, the Y. lipolytica VKM Y-2412 produced up to 172 g l(-1) of KGA with the mass yield coefficient of 0.70 g g(-1).

Effects of different inclusion levels of α-ketoglutaric acid (AKG; 0, 4, 8, and 12 mM) on gas production, ruminal microorganism fermentation and bacterial diversity were evaluated after 72 h in vitro incubations with buffered rumen fluid and wheat straw. Our results showed that the asymptote increased in a linear trend, while the lag time decreased linearly with higher AKG concentrations (P

Alpha-ketoglutaric acid (alpha-KG) and 5-hydroxymethylfurfural (5-HMF) are currently under investigation as promising cancer cell damaging agents. A method for the simultaneous quantitative determination of alpha-KG and 5-HMF in human plasma was established for screening these compounds in human plasma. Plasma samples were directly treated with O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine hydrochloride to form the corresponding oximes, thus facilitating subsequent liquid-liquid extraction. After formation of the trimethylsilyl ethers, samples were analyzed by gas chromatography with electron ionization mass spectrometry. Stable isotope labeled standards were used, the preparation of (13)C(6)-5-HMF is described. Limits of quantitation were set to 0.938 microg/mL for alpha-KG and 0.156 microg/mL for 5-HMF. Inter-day accuracy wasor = 93.7% (alpha-KG) andor = 92.8% (5-HMF). Inter-day precision wasor = 6.0% (alpha-KG) andor = 4.6% (5-HMF). The method has been successfully applied to pharmacokinetic profiling of the compounds after intravenous application.

Reaction of methyl esters of acylpyruvic acids with substituted o-aminophenols led to 6(7)-substituted 3-acylmethylene-1,4-benzoxazin-2-ones. Reaction of substituted o-aminophenols with α-ketoglutaric acid led to 6-substituted 3-carbonyl-1,4-benzoxazin-2-ones. The structures of the synthesized compounds were confirmed by IR and NMR spectroscopy and mass spectrometry. The antibacterial activity of the compounds was studied.

This paper reports a novel method for identification of two aliphatic position isomers between α-ketoglutaric acid (α-KA) and β-ketoglutaric acid (β-KA) by their different perturbation effects on a Briggs-Rauscher oscillating system, in which tetraaza-macrocyclic complex [NiL](ClO4)2 is used as the catalyst. The ligand L in the complex is 5,7,7,12,14,14-hexamethyl-1,4,8,11-tetraazacyclotetradeca-4,11-diene. The experimental results have shown that addition of α-KA into the system does not affect the oscillating patterns, while the presence of β-KA in a dynamic system influences the oscillatory amplitude. A more interesting feature is that, in the presence of a higher concentration of β-KA, there are damped oscillations after the initial spike, followed by quenching (more exactly: very small oscillations) of the oscillations before the subsequent regeneration of oscillations. A qualitative approach was thus established by employing a Briggs-Rauscher system for identification of these two isomers. The concentrations of these two isomers that can be distinguished lie over the range between 5.0 × 10(-6) and 2.5 × 10(-3) mol/L. A reaction mechanism based on the FCA model has been proposed. An explanation is that β-KA reacts with HOO(•) radicals to form acetone, whereas the α-KA does not.

The nutrition conditions needed to redirect the carbon flux in Torulopsis glabrata, a pyruvate hyper-production yeast, from pyruvate to alpha-ketoglutaric acid (KG) were investigated in a stirred fermentor. A minor amount of KG (1.3 gl(-1)) was produced when NaOH was used to control the pH, while 12 g KG l(-1) was produced when CaCO(3) was used instead. When thiamine and biotin were included in the medium, 13 g KG l(-1) and 68 g pyruvate l(-1) were produced after 48 h when glucose was nearly consumed (approximately 5 gl(-1)). With fermentation continuing for a further 16 h, the concentration of pyruvate decreased to 31 gl(-1), and KG increased to 30 gl(-1). KG thus accumulated at the expense of pyruvate consumption.

Alpha-oxoglutaric acid was attached to trypsin via reductive alkylation with NaBH4 thereby introducing metal-chelating groups at the protein surface. The thermostability of the modified enzyme was increased by 6.5-13 degrees C and its resistance to autolytic degradation was improved 2- to 4-fold in 5 mM ZnCl2, MnCl2 or MgCl2.

It has recently been reported that aluminum plays a very important role in reducing the activity of Krebs-cycle enzymes and glutamate dehydrogenase in rat brain homogenate. Therefore, it is necessary to identify the aluminum binding ability with the pivotal substrate alpha-ketoglutarate in biological systems. The interactions of aluminum with alpha-ketoglutarate were studied with pH-potentiometry, cyclic voltammetry, UV-vis, 1H, 27Al-NMR and Raman spectra multi-analytical techniques in acidic aqueous solution to measure the stoichiometries and stability constants of the complexes and its keto-enol tautomerism. The alpha-ketoglutarate was found to bind Al in a bidentate manner at the carboxylate and carbonyl moieties. The mononuclear 1:1 (AlLH(-1), AIL+, AlHL2+) and 2:1 (AlL2-, AlL2H(-2)3-) species, and dinuclear 2:1 (Al2L4+) species were found in acidic aqueous solution. Meanwhile, Al can promote alpha-KG tautomerize to its enolic-structure compounds in solutions. These findings may help to further understand the influence of Al on GDH enzyme reactions in biological systems.