From in vitro to in silico: Modeling and recombinant production of DT-Diaphorase enzyme.

DT-Diaphorase (DTD) belonging to the oxidoreductase family, is among the most important enzymes and is of great significance in present-day biotechnology. Also, it has potential applications in glucose and pyruvate biosensors. Another important role of the DTD enzyme is in the detection of Phenylketonuria disease. According to the above demands, at first, we tried to study molecular cloning and production of recombinant DTD in E. coli BL21 strain. We have successfully cloned, expressed, and purified functionally active diaphorase. The amount of enzyme was increased in 10-h using IPTG induction, and the recombinant protein was purified by Ni-NTA agarose affinity chromatography. After that, the kinetic and thermodynamic parameters of the enzyme, optimum temperature and pH were also investigated to find more in-depth information. In the end, to represent the connections between the structures and function of this enzyme, the molecular dynamics simulations have been considered at two temperatures in which DTD had maximum and minimum activity (310 and 293 K, respectively). The results of MD simulations indicated that the interaction between NADH with phenylalanine 232 residue at 310 K is more severe than other residues. So, to investigate the interaction details of NADH/PHE 232 the DFT calculations were done.

In-cell determination of Lactate Dehydrogenase Activity in a Luminal Breast Cancer Model ⁻ ex vivo Investigation of Excised Xenograft Tumor Slices Using dDNP Hyperpolarized [1-13C]pyruvate.

[1-13C]pyruvate, the most widely used compound in dissolution-dynamic nuclear polarization (dDNP) magnetic resonance (MR), enables the visualization of lactate dehydrogenase (LDH) activity. This activity had been demonstrated in a wide variety of cancer models, ranging from cultured cells, to xenograft models, to human tumors in situ. Here we quantified the LDH activity in precision cut tumor slices (PCTS) of breast cancer xenografts. The Michigan Cancer Foundation-7 (MCF7) cell-line was chosen as a model for the luminal breast cancer type which is hormone responsive and is highly prevalent. The LDH activity, which was manifested as [1-13C]lactate production in the tumor slices, ranged between 3.8 and 6.1 nmole/nmole adenosine tri-phosphate (ATP) in 1 min (average 4.6 ± 1.0) on three different experimental set-ups consisting of arrested vs. continuous perfusion and non-selective and selective RF pulsation schemes and combinations thereof. This rate was converted to an expected LDH activity in a mass ranging between 3.3 and 5.2 µmole/g in 1 min, using the ATP level of these tumors. This indicated the likely utility of this approach in clinical dDNP of the human breast and may be useful as guidance for treatment response assessment in a large number of tumor types and therapies ex vivo.

The Usefulness of Performing Biochemical Tests in the Saliva of Kickboxing Athletes in the Dynamic of Training.

The study aimed to determine the suitability of testing the saliva of kickboxing athletes to show changes in biochemical parameters in dynamic of training. 8 elite male athletes (mean age 17.29± 0.31 years, body mass 66.82± 3.46kg, with 5.62±0.96 years of training experience) participated in the study. Indicators of lipid peroxidation and glycolysis (the concentration of lactic acid and pyruvic acid) were defined before and after a training session. Significant increases in indicators of lipid peroxidation activity indicators and the concentration of lactic acid (4-fold) were observed; analysis of correlation matrices confirms the absence of expressed changes. At the same time, significant decreases in catalase (10-fold from 3.69 µkat/L to 0.39 µkat/L) and pyruvic acid (from 3.92 µl/l to 0.55 µl/l) were observed. Our results confirm the value of using saliva to determine training load in an individual. Moreover, the study provided information on the importance of indexes reflecting a correlation of various biochemical indicators to estimate the sufficiency of training loads. The ease of sampling and informational content of saliva are reasons to use such tests in monitoring athletes' functional state to prevent fatigue.

Separation of α-ketoglutaric acid and pyruvic acid from the culture broth of Yarrowia lipolytica WSH-Z06 by chromatographic methods.

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.

Separation and purification of α-ketoglutarate and pyruvate from the fermentation broth of Yarrowia lipolytica.

A strategy to achieve the efficient co-production of α-ketoglutarate (KGA) and pyruvate (PYR) via Yarrowia lipolytica fermentation was established in our previous work. The next big challenge is to achieve an efficient separation of the two keto acids. A strategy for simultaneously separating and purifying KGA and PYR based on their different boiling points was established, leading to the efficient separation and purification of the two keto acids from the fermentation broth of Y. lipolytica. The purity and yield of KGA/PYR reached 99.3/99.5 and 79.8/80.6%, respectively. Application of the separation method on industrial scale could further decrease the cost of the production of the two keto acids by biotechnological routes.

Glycolysis without pyruvate kinase in Clostridium thermocellum.

The metabolism of Clostridium thermocellum is notable in that it assimilates sugar via the EMP pathway but does not possess a pyruvate kinase enzyme. In the wild type organism, there are three proposed pathways for conversion of phosphoenolpyruvate (PEP) to pyruvate, which differ in their cofactor usage. One path uses pyruvate phosphate dikinase (PPDK), another pathway uses the combined activities of PEP carboxykinase (PEPCK) and oxaloacetate decarboxylase (ODC). Yet another pathway, the malate shunt, uses the combined activities of PEPCK, malate dehydrogenase and malic enzyme. First we showed that there is no flux through the ODC pathway by enzyme assay. Flux through the remaining two pathways (PPDK and malate shunt) was determined by dynamic 13C labeling. In the wild-type strain, the malate shunt accounts for about 33±2% of the flux to pyruvate, with the remainder via the PPDK pathway. Deletion of the ppdk gene resulted in a redirection of all pyruvate flux through the malate shunt. This provides the first direct evidence of the in-vivo function of the malate shunt.

Determination of α-ketoglutaric and pyruvic acids in urine as potential biomarkers for diabetic II and liver cancer.

BACKGROUND: A simple and sensitive hollow fiber-liquid phase microextraction with in situ derivatization method was developed for the determination of α-ketoglutaric (α-KG) and pyruvic acids (PA) in small-volume urine samples. 2,4,6-trichloro phenyl hydrazine was used as derivatization agent. RESULTS: Under the optimum extraction conditions, enrichment factors of 742 and 400 for α-KG and PA, respectively, were achieved. Calibration curves were linear over the range 1 to 1000 ng/ml (r(2) ≥ 0.998). Detection and quantitation limits were 0.03 and 0.02, and 0.10 and 0.05 ng/ml for α-KG and PA, respectively. CONCLUSION: The concentrations in diabetic II and liver cancer samples were significantly lower than those from healthy people, showing their potential as biomarkers for these diseases.

In vivo continuous and simultaneous monitoring of brain energy substrates with a multiplex amperometric enzyme-based biosensor device.

Enzyme-based amperometric biosensors are widely used for monitoring key biomarkers. In experimental neuroscience there is a growing interest in in vivo continuous and simultaneous monitoring of metabolism-related biomarkers, like glucose, lactate and pyruvate. The use of multiplex biosensors will provide better understanding of brain energy metabolism and its role in neuropathologies such as diabetes, ischemia, and epilepsy. We have developed and characterized an implantable multiplex microbiosensor device (MBD) for simultaneous and continuous in vivo monitoring of glucose, lactate, and pyruvate. First, we developed and characterized amperometric microbiosensors for monitoring lactate and pyruvate. In vitro evaluation allowed us to choose the most suitable biosensors for incorporation into the MBD, along with glucose and background biosensors. Fully assembled MBDs were characterized in vitro. The calculated performance parameters (LOD, LR, LRS, IMAX and appKM) showed that the multiplex MBD was highly selective and sensitive (LRS≥100 nA/mM) for each analyte and within an adequate range for in vivo application. Finally, MBDs were implanted in the mPFC of anesthetized adult male Wistar rats for in vivo evaluation. Following an equilibration period, baseline brain levels of glucose (1.3±0.2 mM), lactate (1.5±0.4 mM) and pyruvate (0.3±0.1 mM) were established. Subsequently, the MBDs recorded the responses of the animals when submitted to hyperglycemic (40% glucose i.v.) and hypoglycemic (5 U/kg insulin i.v.) challenges. Afterwards, MBDs were recalibrated to convert electrochemical readings into accurate substrate concentrations and to assess biofouling. The presented MBD can monitor simultaneously multiple biomarkers in vivo.

Conformational switching in pyruvic acid isolated in Ar and N2 matrixes: spectroscopic analysis, anharmonic simulation, and tunneling.

Monomers of pyruvic acid (PA) isolated in cryogenic argon and nitrogen matrixes were characterized by mid- and near-infrared spectroscopy. Interpretation of the experiments was aided by fully anharmonic calculations of the fundamental modes, overtones, and combinations up to two quanta, including their infrared intensities. The initially dominating PA conformer (Tc) has a cis CCOH arrangement and is stabilized by a strong intramolecular H-bond. Selective near-infrared excitation of Tc at the first OH overtone (6630 cm(-1) in Ar, 6643 cm(-1) in N2) induced a large scale conformational conversion to the higher-energy conformer (Tt) with trans CCOH arrangement. Tt was then converted back to Tc by selective NIR irradiation at the first Tt OH overtone (6940 cm(-1) in Ar, 6894 cm(-1) in N2). In N2 matrix, the Tt form was stabilized due to interaction between the OH group and the matrix molecules. This stabilization manifested itself in the absence of Tt → Tc relaxation and in a considerable change of the vibrational Tt signature upon going from argon to nitrogen matrix. In argon, the Tt form spontaneously decayed back to Tc in the dark (characteristic lifetime +16 h). In the presence of broad-band near-infrared light, the Tt → Tc relaxation speed considerably increased. The decay mechanisms are discussed.

Breeding of a cyclic imide-assimilating bacterium, Pseudomonas putida s52, for high efficiency production of pyruvate.

A succinimide-assimilating bacterium, Pseudomonas putida s52, was found to be a potent producer of pyruvate from fumarate. Using washed cells from P. putida s52 as catalyst, 400 mM pyruvate was produced from 500 mM fumarate in a 36-h reaction. Bromopyruvate, a malic enzyme inhibitor, was used for the selection of mutants with higher pyruvate productivity. A bromopyruvate-resistant mutant, P. putida 15160, was found to be an effective catalyst for pyruvate production. Moreover, under batch bioreactor conditions, 767 mM of pyruvate was successfully produced from 1,000 mM fumarate in a 72-h reaction with washed cells from P. putida 15160 as catalyst.

Structure characterization of a fucose-containing exopolysaccharide produced by Enterobacter cloacae Z0206.

A novel high molecular weight (1.1 × 10(6)Da) exopolysaccharide (EPS) produced by Enterobacter cloacae Z0206 strain was isolated by column chromatography. Complete hydrolysis of the EPS followed by gas chromatography mass spectrometry (GC-MS) and high performance liquid chromatography (HPLC) analyses showed that the EPS is composed of L-fucose, D-glucose, D-galactose, D-glucuronic acid and pyruvic acid in the approximate molar ratio of 2:1:3:1:1. Partial acid hydrolysis of the purified EPS followed by gel permeation chromatography (GPC) yielded a hexasaccharide. A combination of chemical analysis coupled with mass spectrometry and 1D and 2D NMR spectroscopy applied to the oligosaccharide showed that the EPS comprises a heptasaccharide repeating unit.

Yak lactate dehydgogenase A4: purification, properties, and cDNA cloning.

Lactate dehydrogenase A4 (LDH-A4) was purified for yak skeletal muscle. Michaelis constant (Km) analysis showed that yak LDH-A4 for pyruvate was significantly higher than that of cattle. cDNA cloning of LDH-A revealed two amino acid substitutions between yak and cattle. We suggest that the higher Km of yak LDH-A4 might be a result of molecular adaptation to a hypoxic environment.

New method for reducing the binding power of sweet white wines.

Sulfur dioxide is now considered to be a toxic chemical by most world health authorities. However, it remains an irreplaceable additive in enology for wine conservation, combining antioxidant and antibacterial properties. Sweet white wines from botrytized grapes retain particularly high SO 2 levels due to their high sulfur dioxide binding power. This paper presents a new method for reducing this binding power by removing some of the carbonyl compounds responsible, which are naturally present in these wines. The main carbonyl compounds responsible for the SO 2 binding power of sweet wines were removed, that is, acetaldehyde, pyruvic acid, 2-oxoglutaric acid, and 5-oxofructose. The method retained was selective liquid-solid removal, using phenylsulfonylhydrazine as a scavenging agent. The scavenging function was grafted on different classes of porous polymer supports, and its efficiency was evaluated on sweet white wines under conditions intended to conserve their organoleptic qualities. The results obtained showed that the method was efficient for removing carbonyl compounds and significantly reduced the binding power of the wines. Sensory analysis revealed that this process did not deteriorate their organoleptic qualities.

New methodology for removing carbonyl compounds from sweet wines.

Sweet white wines from botrytized grapes present high SO2 levels because of their high sulfur dioxide binding power. The objective of this work was to develop a new method for reducing this binding power by partially eliminating the carbonyl compounds naturally present in these wines that are responsible for this phenomenon. A selective liquid-solid removal technique was developed. Phenylsulfonylhydrazine was selected as the best candidate for removing carbonyl compounds. Its reactivity in the presence or absence of sulfur dioxide was verified in model media containing acetaldehyde, pyruvic acid, and 2-oxoglutaric acid, some of the main carbonyl compounds responsible for the SO2 binding power of sweet wines. The scavenging function was grafted on porous polymer supports, and its efficiency was evaluated in model wines. Dependent upon the supports used, different quantities of carbonyl compounds (over 90% in some cases) were removed in a few days. The presence of sulfur dioxide delayed removal without changing its quality. The results obtained showed that the method removed carbonyl compounds efficiently and was applicable to wines at any stage in winemaking.

Recovery of pyruvic acid from biotransformation solutions.

The aim of this investigation was to separate pyruvic acid of biotransformation solutions from lactic acid through complex extraction. For this purpose, complex extraction was investigated from model solutions. Tri-n-octanylamine (TOA) was used as the extractant. The effects of various diluents, the stoichiometry of pyruvic acid to TOA, and the initial pH of the aqueous phase on the extraction process were investigated in this study. The effects of sodium hydroxide (NaOH) and trimethylamine (TMA) on the back extraction process were also studied, respectively. The optimal conditions attained from the model solutions proved efficient on the biotransformation solutions of different concentrations. A total recovery of 71-82% of pyruvic acid was obtained, whereas 89-92% of lactic acid was removed. The purity of pyruvic acid reached 97% after the removal of TMA by a simple distillation.

The chemometric resolution and quantification of overlapped peaks form comprehensive two-dimensional liquid chromatography.

The chemometric resolution and quantification of overlapped peaks from comprehensive two-dimensional (2D) liquid chromatography (LCxLC) data are demonstrated. The LCxLC data is produced from an in-house LCxLC analyzer that couples an anion-exchange column via a multi-port valve with a reversed-phase column connected to a UV absorbance detector. Three test mixtures, each containing a target analyte, are subjected to partial LCxLC separations to simulate likely cases of signal overlap. The resulting unresolved target-analyte signals are then analyzed by the standard-addition method and two chemometric methods. The LCxLC analyses of a test mixture and its corresponding standard-addition mixture results in two data matrices, one for each mixture. The stacking of these two data matrices produces a data structure that can then be analyzed by trilinear chemometric methods. One method, the generalized rank annihilation method (GRAM), uses a non-iterative eigenvalue-based approach to mathematically resolve overlapped trilinear signals. The other method, parallel factor analysis (PARAFAC), uses an iterative approach to resolve trilinear signals by the optimization of initial estimates using alternating least squares and signal constraints. In this paper, GRAM followed by PARAFAC analysis is shown to produce better qualitative and quantitative results than using each method separately. For instance, for all three test mixtures, the GRAM-PARAFAC approach improved quantitative accuracy by at least a factor of 4 and quantitative precision by more than 2 when compared to GRAM alone. This paper also introduces a new means of correcting run-to-run retention time shifts in comprehensive 2D chromatographic data.

Process strategies to enhance pyruvate production with recombinant Escherichia coli: from repetitive fed-batch to in situ product recovery with fully integrated electrodialysis.

Using the pyruvate production strain Escherichia coli YYC202 ldhA::Kan different process alternatives are studied with the aim of preventing potential product inhibition by appropriate product separation. This strain is completely blocked in its ability to convert pyruvate into acetyl-CoA or acetate, resulting in acetate auxotrophy during growth in glucose minimal medium. Continuous experiments with cell retention, repetitive fed-batch, and an in situ product recovery (ISPR) process with fully integrated electrodialysis were tested. Although the continuous approach achieved a high volumetric productivity (QP) of 110 g L(-1) d(-1), this approach was not pursued because of long-term production strain instabilities. The highest pyruvate/glucose molar yield of up to 1.78 mol mol(-1) together with high QP 145 g L(-1) d(-1) and high pyruvate titers was achieved by the repetitive fed-batch approach. To separate pyruvate from fermentation broth a fully integrated continuous process was developed. In this process electrodialysis was used as a separation unit. Under optimum conditions a (calculated) final pyruvate titer of >900 mmol L(-1) (79 g L(-1)) was achieved.

Ascosonchine, the enol tautomer of 4-pyridylpyruvic acid with herbicidal activity produced by Ascochyta sonchi.

A new phytotoxic enol tautomer of 4-pyridylpyruvic acid, named ascosonchine, was isolated from the culture filtrate of Ascochyta sonchi. Such a leaf pathogen is a potential biocontrol agent of Sonchus arvensis, a perennial herbaceous weed occurring throughout the temperate regions of the world. Ascosonchine, characterised as (Z)-2-hydroxy-3-(4-pyridyl)-2-propenoic acid by spectroscopic methods, showed selective herbicidal properties, that are not associated with antibacterial, antifungal or zootoxic activities.

Highly efficient conversion of lactate to pyruvate using whole cells of Acinetobacter sp.

On an industrial scale, the production of pyruvate at a high concentration from the cheaper lactate substrate is a valuable process. To produce pyruvate from lactate by whole cells, various lactate-utilizing microorganisms were isolated from soil samples. Among them, strain WLIS, identified as Acinetobacter sp., was screened as a pyruvate producer. For the pyruvate preparation from lactate, the preparative conditions were optimized with whole cells of the strain. The cells cultivated in the medium containing 100 mM of l-lactate showed the highest biotransformation efficiency from lactate to pyruvate. The optimized dry-cell concentration, pH, and temperature of reaction were 6 g/L, pH 7.0-7.5, and 30 degrees C, respectively. The influences of ethylenediaminetetraacetic acid (EDTA) and aeration on a biotransformation reaction were carried out under the test conditions. Under the optimized reaction conditions, l-lactate at concentrations of 200 and 500 mM were almost totally stoichiometrically converted into pyruvate in 8 and 12 h, respectively. About 60% of 800 mM of l-lactate was transformed into pyruvate in 24 h. This reduced conversion rate is probably due to the high substrate inhibition in biotransformation.

Kinetic modeling of pyruvic acid ozonation in aqueous solutions catalyzed by Mn(II) and Mn(IV) ions.

The ozonation of pyruvic acid (2-ketopropionic acid) in aqueous solutions, catalyzed by Mn(II) and Mn(IV) ions, has been studied at three different pH values (pH = 1.1, 2.0 and 3.0). A mathematical model of the unsteady operation of the experimental reactor has been developed, which takes into account the reactions occurring in the liquid phase and the ozone mass transfer from the gas bubbles. Those reactions have been described with two alternative kinetic models, both made out of four elementary steps. The two kinetic models correlate the experimental data with a fair accuracy, respectively at the lowest and at the highest pH examined. In particular, at pH = 3.0, the ozonation results are inhibited by the acetate ions produced by the reaction itself. This effect has been correctly described in the terms of a complex formed with the low oxidation-state manganese, which successively reacts with the dissolved ozone.