Alteration of the formate dehydrogenase isoelectric point by rational design

In order to extend the pH stability optimum for NAD⁺-dependent formate dehydrogenase (FDH, EC 1.2.1.2) from the bacterium Pseudomonas sp. 101 (PseFDH), four mutant enzymes with Lys112Pro, Lys231Ala, Lys231Ser, and Lys317Asn substitutions were obtained by site-directed mutagenesis. The choice of the mutation sites and the types of substituting amino acids were based on the alignment of amino acid sequences of FDHs from various sources and the analysis of the three-dimensional structure of PseFDH. The kinetic properties and temperature stability were studied for all obtained mutant forms. It is shown that the substitutions in positions 112 and 231 slightly improved the kinetic properties; meanwhile, the Lys317Asn mutant possessed a decreased affinity for the coenzyme. A thermal stability assay for the obtained mutants revealed that the substitutions in positions 112 and 231 result in just a slight destabilization of the enzyme, while Lys317Asn substitution causes a significant decrease in thermal stability. The isoelectric point was decreased by 0.1 points for all obtained mutant forms.     

Mutation of Tyr-218 to Phe in <Emphasis Type="Italic">Thermoanaerobacter ethanolicus</Emphasis> Secondary Alcohol Dehydrogenase: Effects on Bioelectronic Interface Performance

Bioelectronic interfaces that facilitate electron transfer between the electrode and a dehydrogenase enzyme have potential applications in biosensors, biocatalytic reactors, and biological fuel cells. The secondary alcohol dehydrogenase (2° ADH) from Thermoanaerobacter ethanolicus is especially well suited for the development of such bioelectronic interfaces because of its thermostability and facile production and purification. However, the natural cofactor for the enzyme, β-nicotinamide adenine dinucleotide phosphate (NADP⁺), is more expensive and less stable than β-nicotinamide adenine dinucleotide (NAD⁺). PCR-based, site-directed mutagenesis was performed on 2° ADH in an attempt to adjust the cofactor specificity toward NAD⁺ by mutating Tyr²¹⁸ to Phe (Y218F 2° ADH). This mutation increased the K _m(app) for NADP⁺ 200-fold while decreasing the K _m(app) for NAD⁺ 2.5-fold. The mutant enzyme was incorporated into a bioelectronic interface that established electrical communication between the enzyme, the NAD⁺, the electron mediator toluidine blue O (TBO), and a gold electrode. Cyclic voltammetry, impedance spectroscopy, gas chromatography, mass spectrometry, constant potential amperometry, and chronoamperometry were used to characterize the mutant and wild-type enzyme incorporated in the bioelectronic interface. The Y218F 2° ADH exhibited a fourfold increase in the turnover ratio compared to the wild type in the presence of NAD⁺. The electrochemical and kinetic measurements support the prediction that the Rossmann fold of the enzyme binds to the phosphate moiety of the cofactor. During the 45 min of continuous operation, NAD⁺ was electrically recycled 6.7 × 10⁴ times, suggesting that the Y218F 2° ADH-modified bioelectronic interface is stable.     

Attempting to remove the substrate inhibition ofL-lactate dehydrogenase fromBacillus stearothermophilus by site-directed mutagenesis

L-lactate dehydrogenase (LDH) catalyzes the interconversion of an oxoacid (pyruvate) and hydroxy-acid (lactate) using the NADH/NAD⁺ pair as a redox cofactor. The enzyme has a commercial significance, as it can be used to produce chiral building blocks for the synthesis of key pharmaceuticals and agrochemicals. However, the substrate inhibition which is due to an abortive NAD⁺-pyruvate complex reducing the steady state concentration of functional LDH limits its use in industry. This substrate inhibition can be overcome by weaking the binding of NAD⁺. The conserved aspartic acid residue at position 38 was replaced by the longer basic arginine side chain (D38R) using PCR based overlap extension mutagenesis technique in the hope of weakening NAD⁺-binding. The mutant gene was overexpressed in theEscherichia coli high-expression vector pKK223-3 in JM105 cells; then, the mutant protein was produced. Comparing the effect of substrate inhibition in the arginine-38 mutant with wild-type, substrate inhibition is decreased threefold.     

Determination of the concentration of active sites and the catalytic rate constant of recombinant formate dehydrogenase from <Emphasis Type="Italic">Glycine max</Emphasis>

The analysis of a formate dehydrogenase (FDH) structure in the apo form and in a complex with nicotinamide (NAD⁺) and azide ion has shown a high probability of efficient fluorescence quenching during the formation of such a triple complex. The excitation and fluorescence spectra indicated that the enzyme fluorescence is determined by tryptophan residues. The dependence of FDH fluorescence quenching on the NAD⁺ and azide concentrations was studied. The obtained data were used to determine the concentration of active sites and the catalytic rate constant of recombinant FDH from Glycine max.     

Characterization of the CYP3A4 Enzyme Inhibition Potential of Selected Flavonoids

Acacetin, apigenin, chrysin, and pinocembrin are flavonoid aglycones found in foods such as parsley, honey, celery, and chamomile tea. Flavonoids can act as substrates and inhibitors of the CYP3A4 enzyme, a heme containing enzyme responsible for the metabolism of one third of drugs on the market. The aim of this study was to investigate the inhibitory effect of selected flavonoids on the CYP3A4 enzyme, the kinetics of inhibition, the possible covalent binding of the inhibitor to the enzyme, and whether flavonoids can act as pseudo-irreversible inhibitors. For the determination of inhibition kinetics, nifedipine oxidation was used as a marker reaction. A hemochromopyridine test was used to assess the possible covalent binding to the heme, and incubation with dialysis was used in order to assess the reversibility of the inhibition. All the tested flavonoids inhibited the CYP3A4 enzyme activity. Chrysin was the most potent inhibitor: IC₅₀ = 2.5 ± 0.6 µM, K_i = 2.4 ± 1.0 µM, k_inact = 0.07 ± 0.01 min⁻¹, k_inact/K_i = 0.03 min⁻¹ µM⁻¹. Chrysin caused the highest reduction of heme (94.5 ± 0.5% residual concentration). None of the tested flavonoids showed pseudo-irreversible inhibition. Although the inactivation of the CYP3A4 enzyme is caused by interaction with heme, inhibitor-heme adducts could not be trapped. These results indicate that flavonoids have the potential to inhibit the CYP3A4 enzyme and interact with other drugs and medications. However, possible food–drug interactions have to be assessed clinically.     

NADP+-Specific Isocitrate Dehydrogenase from Oleaginous Yeast Yarrowia lipolytica CLIB122: Biochemical Characterization and Coenzyme Sites Evaluation

NADP⁺-dependent isocitrate dehydrogenase from Yarrowia lipolytica CLIB122 (YlIDP) was overexpressed and purified. The molecular mass of YlIDP was estimated to be about 81.3 kDa, suggesting its homodimeric structure in solution. YlIDP was divalent cation dependent and Mg²⁺ was found to be the most favorable cofactor. The purified recombinant YlIDP displayed maximal activity at 55 °C and its optimal pH for catalysis was found to be around 8.5. Heat inactivation studies revealed that the recombinant YlIDP was stable below 45 °C, but its activity dropped quickly above this temperature. YlIDP was absolutely dependent on NADP⁺ and no NAD-dependent activity could be detected. The K _m values displayed for NADP⁺ and isocitrate were 59 and 31 μM (Mg²⁺), 120 μM and 58 μM (Mn²⁺), respectively. Mutant enzymes were constructed to tentatively alter the coenzyme specificity of YlIDP. The K _m values for NADP⁺ of R322D mutant was 2,410 μM, being about 41-fold higher than that of wild type enzyme. NAD⁺-dependent activity was detected for R322D mutant and the K _m and k _cat values for NAD⁺ were 47,000 μM and 0.38 s^?1, respectively. Although the R322D mutant showed low activity with NAD⁺, it revealed the feasibility of engineering an eukaryotic IDP to a NAD⁺-dependent one.     

Electrochemical detection of nicotinamide adenine dinucleotide based on molecular beacon-like DNA and E. coli DNA ligase

An electrochemical method for nicotinamide adenine dinucleotide (NAD⁺) detection with high sensitivity and selectivity has been developed by using molecular beacon (MB)-like DNA and Escherichia coli DNA ligase. In this method, MB-like DNA labeled with 5′-SH and 3′-biotin was self-assembled onto a gold electrode in its duplex form by means of facile gold-thiol chemistry, which resulted in blockage of electronic transmission. It was eT OFF state. In the presence of NAD⁺, E. coli DNA ligase was activated, and the two nucleotide fragments which were complementary to the loop of the MB-like DNA could be ligated by the NAD⁺-dependent E. coli DNA ligase. Hybridization of the ligated DNA with the MB-like DNA induced a large conformational change in this surface-confined DNA structure, which in turn pushed the biotin away from the electrode surface and made the electrons exchange freely with the electrode. Then the generated electrochemical signals can be measured by differential pulse voltammetry (DPV). Under optimized conditions, a linear response to logarithmic concentration of NAD⁺ range from 3 nM to 5 μM and a detection limit of 1.8 nM were obtained. Furthermore, the proposed strategy had sufficient selectivity to discriminate NAD⁺ from its analogues.     

l-myo-Inositol-1-Phosphate Synthase Expressed in Developing Organ: Isolation and Characterisation of the Enzyme from Human Fetal Liver

l-myo-inositol-1-phosphate synthase (MIPS; EC: 5.5.1.4) activity has been detected and partially purified for the first time from human fetal liver. Crude homogenate from the fetal liver was subjected to streptomycin sulphate precipitation and 0?C60?% ammonium sulphate fractionation followed by successive chromatography through DEAE cellulose and BioGel A 0.5-m columns. After the final chromatography, the enzyme was purified 51-fold and 3.46?% of MIPS could be recovered. The human fetal liver MIPS specifically utilised d-glucose-6-phosphte and NAD⁺ as its substrate and coenzyme, respectively. It shows pH optima between 7.0 and 7.5 while the temperature maximum was at 40?°C. The enzyme activity was remarkably stimulated by NH ₄ ⁺ , slightly stimulated by K⁺ and Ca²⁺ and highly inhibited by Zn²⁺, Cu²⁺ and Hg²⁺. The K _m values of MIPS for d-glucose-6-phosphate and NAD⁺ were found to be as 1.15 and 0.12?mM respectively while the V _max values were 280?nM and 252?nM for d-glucose-6-phosphate and NAD⁺ correspondingly. The apparent molecular weight of the native enzyme was determined to be 170?kDa.     

MiodesinTM Positively Modulates the Immune Response in Endometrial and Vaginal Cells

Endometriosis presents high prevalence and its physiopathology involves hyperactivation of endometrial and vaginal cells, especially by bacteria. The disease has no cure and therapies aiming to inhibit its development are highly desirable. Therefore, this study investigated whether Miodesin^TM (10 µg/mL = IC₈₀; 200 µg/mL = IC₅₀), a natural compound constituted by Uncaria tomentosa, Endopleura uchi, and astaxanthin, could exert anti-inflammatory and anti-proliferative effects against Lipopolysaccharides (LPS) stimulation in endometrial and Candida albicans vaginal cell lines. VK2 E6/E7 (vaginal) and KLE (epithelial) cell lines were stimulated with Candida albicans (1 × 10⁷ to 5 × 10⁷/mL) and LPS (1 μg/mL), respectively. Miodesin^TM inhibited mRNA expression for Nuclear factor kappa B (NF-κB), ciclo-oxigenase 1 (COX-1), and phospholipase A2 (PLA2), beyond the C–C motif chemokine ligand 2 (CCL2), CCL3, and CCL5 in VK2 E6/E7 cells (p < 0.05). In addition, the inhibitory effects of both doses of Miodesin^TM (10 µg/mL and 200 µg/mL) resulted in reduced secretion of interleukin-1β (IL-1β), IL-6, IL-8, tumor necrosis factor α (TNF-α) (24 h, 48 h, and 72 h) and CCL2, CCL3, and CLL5 (p < 0.05) by VK2 E6/E7 cells. In the same way, COX-1 Miodesin^TM inhibited LPS-induced hyperactivation of KLE cells, as demonstrated by reduced secretion of IL-1β, IL-6, IL-8, TNF-α (24 h, 48 h, and 72 h) and CCL2, CCL3, and CLL5 (p < 0.05). Furthermore, Miodesin^TM also inhibited mRNA expression and secretion of matrix metalloproteinase-2 (MMP-2), MMP-9, and vascular endothelial growth factor (VEGF), which are key regulators of invasion of endometrial cells. Thus, the study concludes that Miodesin^TM presents beneficial effects in the context of endometriosis, positively affecting the inflammatory and proliferative response.     

Characterization of xylitol dehydrogenase from debaryomyces hansenii

The xylitol dehydrogenase (EC 1.1.1.9) from xylose-grown cells ofDebaryomyces hansenii was partially purified in two Chromatographic steps, and characterization studies were carried out in order to inves tigate the role of the xylitol dehydrogenase-catalyzed step in the regu lation of D-xylose metabolism. The enzyme was most active at pH 9.0–9.5, and exhibited a broad polyol specificity. The Michaelis con stants for xylitol and NAD⁺ were 16.5 and 0.55 mM, respectively. Ca²⁺, Mg²⁺, and Mn²⁺ did not affect the enzyme activity. Conversely, Zn²⁺, Cd²⁺, and Co²⁺ strongly inhibited the enzyme activity. It was concluded that NAD⁺-xylitol dehydrogenase from D.hansenii has similarities with other xylose-fermenting yeasts in respect to optimal pH, substrate specificity, and K_m value for xylitol, and therefore should be named L-iditol:NAD⁺-5-oxidoreductase (EC 1.1.1.14). The reason D.hansenii is a good xylitol producer is not because of its value of K_m for xylitol, which is low enough to assure its fast oxidation by NAD⁺ xylitol dehydrogenase. However, a higher K_m value of xylitol dehydro genase for NAD⁺ compared to theK _m values of other xylose-ferment ing yeasts may be responsible for the higher xylitol yields.     

In Silico and Ex Vivo Studies on the Spasmolytic Activities of Fenchone Using Isolated Guinea Pig Trachea

Fenchone is a bicyclic monoterpene found in a variety of aromatic plants, including Foeniculum vulgare and Peumus boldus, and is used in the management of airways disorders. This study aimed to explore the bronchodilator effect of fenchone using guinea pig tracheal muscles as an ex vivo model and in silico studies. A concentration-mediated tracheal relaxant effect of fenchone was evaluated using isolated guinea pig trachea mounted in an organ bath provided with physiological conditions. Sustained contractions were achieved using low K⁺ (25 mM), high K⁺ (80 mM), and carbamylcholine (CCh; 1 µM), and fenchone inhibitory concentration–response curves (CRCs) were obtained against these contractions. Fenchone selectively inhibited with higher potency contractions evoked by low K⁺ compared to high K⁺ with resultant EC₅₀ values of 0.62 mg/mL (0.58–0.72; n = 5) and 6.44 mg/mL (5.86–7.32; n = 5), respectively. Verapamil (VRP) inhibited both low and high K⁺ contractions at similar concentrations. Pre-incubation of the tracheal tissues with K⁺ channel blockers such as glibenclamide (Gb), 4-aminopyridine (4-AP), and tetraethylammonium (TEA) significantly shifted the inhibitory CRCs of fenchone to the right towards higher doses. Fenchone also inhibited CCh-mediated contractions at comparable potency to its effect against high K⁺ [6.28 mg/mL (5.88–6.42, n = 4); CCh] and [6.44 mg/mL (5.86–7.32; n = 5); high K⁺]. A similar pattern was obtained with papaverine (PPV), a phosphodiesterase (PDE), and Ca²⁺ inhibitor which inhibited both CCh and high K⁺ at similar concentrations [10.46 µM (9.82–11.22, n = 4); CCh] and [10.28 µM (9.18–11.36; n = 5); high K⁺]. However, verapamil, a standard Ca²⁺ channel blocker, showed selectively higher potency against high K⁺ compared to CCh-mediated contractions with respective EC₅₀ values of 0.84 mg/mL (0.82–0.96; n = 5) 14.46 mg/mL (12.24–16.38, n = 4). The PDE-inhibitory action of fenchone was further confirmed when its pre-incubation at 3 and 5 mg/mL potentiated and shifted the isoprenaline inhibitory CRCs towards the left, similar to papaverine, whereas the Ca²⁺ inhibitory-like action of fenchone pretreated tracheal tissues were authenticated by the rightward shift of Ca²⁺ CRCs with suppression of maximum response, similar to verapamil, a standard Ca²⁺ channel blocker. Fenchone showed a spasmolytic effect in isolated trachea mediated predominantly by K⁺ channel activation followed by dual inhibition of PDE and Ca²⁺ channels. Further in silico molecular docking studies provided the insight for binding of fenchone with Ca²⁺ channel (−5.3 kcal/mol) and K⁺ channel (−5.7), which also endorsed the idea of dual inhibition.     

Immunochemical Detection of Microquantities of Bacterial Formate Dehydrogenase

Abstract

Two immunochemical methods were developed for detection of NAD⁺?dependent formate dehydrogenase (EC 1.2.1.2, FDH) isolated from the methylotrophic bacteria Pseudomonas sp. 101:1) the dot-blot analysis using rabbit polyclonal antibodies; and 2) the indirect competitive ELISA using poly- or monoclonal mouse antibodies. The first method was used for screening the bacterial gene bank, the sensitivity is 5 and 1 pg enzyme per sample using the anti-rabbit antibodies - horse radish peroxidase conjugate or the biotinylated anti-rabbit antibodies and avidin - peroxidase conjugate, respectively. The second method was applied for precise determination of FDH concentration in cell-free extracts of selected recombinant clones. Mouse polyclonal antibodies to bacterial FDH have exibited a rather high affinity binding also to FDH from the methylotrophic yeast Candida methylica. In the indirect competitive ELISA the sensitivity of bacterial FDH determination is 1 ng per sample.     

Exploring the Dual Characteristics of CH3OH Adsorption to Metal Atomic Structures on Si (111)-7 × 7 Surface

Metal atoms were deposited on an Si (111)-7 × 7 surface, and they were adsorbed with alcohol gases (CH₃OH/C₂H₅OH/C₃H₇OH). Initially, C_nH_2n+1OH adsorption was simply used as an intermediate layer to prevent the chemical reaction between metal and Si atoms. Through scanning tunneling microscopy (STM) and a mass spectrometer, the C_nH_2n+1OH dissociation process is further derived as the construction of a surface quasi-potential with horizontal and vertical directions. With the help of three typical metal depositions, the surface characteristics of CH₃OH adsorption are more clearly presented in this paper. Adjusting the preheating temperature, the difference of thermal stability between CH₃O^– and H⁺ could be obviously derived in Au deposition. After a large amount of H⁺ was separated, the isolation characteristic of CH₃O^– was discussed in the case of Fe deposition. In the process of building a new metal-CH₃O^–-H⁺ model, the dual characteristics of CH₃OH were synthetically verified in Sn deposition. CH₃O^– adsorption is prone to influencing the interaction between the metal deposition and substrate surface in the vertical direction, while H⁺ adsorption determines the horizontal behavior of metal atoms. These investigations lead one to believe that, to a certain extent, the formation of regular metal atomic structures on the Si (111)-7 × 7-CH₃OH surface is promoted, especially according to the dual characteristics and adsorption models we explored.     

Recombination of KrD and XeD ions with electrons

Recombination rate coefficients of protonated and deuterated ions KrH⁺, KrD⁺, XeH⁺ and XeD⁺ were measured using Flowing Afterglow with Langmuir Probe (FALP). Helium at 1600 Pa and at temperature 250 K was used as a buffer gas in the experiments. Kr, Xe, H₂ and D₂ were introduced to a flow tube to form the desired ions. Because of small differences in proton affinities of Kr, D₂ and H₂ mixtures of ions, KrD⁺/D₃⁺ and KrH⁺/H₃⁺ are formed in the afterglow plasma, influencing the plasma decay. To obtain a recombination rate coefficient for a particular ion, the dependencies on partial pressures of gases used in the ion formation were measured. The obtained rate coefficients, α_KrD+(250 K) = (0.9 ± 0.3) × 10⁻⁸ cm³ s⁻¹ and α_XeD+(250 K) = (8 ± 2) × 10⁻⁸ cm³ s⁻¹ are compared with α_KrH+(250 K) = (2.0 ± 0.6) × 10⁻⁸ cm³ s⁻¹ and α_XeH+(250 K) = (8 ± 2) × 10⁻⁸ cm³ s⁻¹.     

The rhodium complex of a tris(bipyridine) ligand—Its electrochemical behaviour and function as mediator for the regeneration of NADH from NAD+

The tris-bipyridine ligand3a and its stoichiometric Rh³⁺ complex have been prepared. Cyclovoltammograms of the complex at pH 7.4 using a glassy carbon disk electrode reveal a strong reduction peak at –620 mV and two weak reduction peaks at more negative voltage. The reduction potential of the new complex is shifted by 300 mV to more positive values as compared to [Rh(bipy)₃]³⁺. There is no reversible reoxidation peak of the Rh(I) complex formed due to the decomplexation of one of the three bipyridine units in the course of the transition Rh(III)Rh(I). The Rh(III) complex of3a was also studied with respect to its function as a possible redox mediator for the electrochemical regeneration of NADH from NAD⁺. The preparative electrolysis of the Rh³⁺ complex of3a in the presence of NAD⁺ yields a selective formation of NADH, whereas NAD dimers were not detected. On the other hand, a significant acceleration of this reaction compared to [Rh(bipy)₃]³⁺ was not observed.     

L-Glutaminase Synthesis by Marine Halomonas meridiana Isolated from the Red Sea and Its Efficiency against Colorectal Cancer Cell Lines

L-glutaminase is an important anticancer agent that is used extensively worldwide by depriving cancer cells of L-glutamine. The marine bacterium, Halomonas meridian was isolated from the Red Sea and selected as the more active L-glutaminase-producing bacteria. L-glutaminase fermentation was optimized at 36 h, pH 8.0, 37 °C, and 3.0% NaCl, using glucose at 1.5% and soybean meal at 2%. The purified enzyme showed a specific activity of 36.08 U/mg, and the molecular weight was found to be 57 kDa by the SDS-PAGE analysis. The enzyme was highly active at pH 8.0 and 37 °C. The kinetics’ parameters of Km and Vmax were 12.2 × 10⁻⁶ M and 121.95 μmol/mL/min, respectively, which reflects a higher affinity for its substrate. The anticancer efficiency of the enzyme showed significant toxic activity toward colorectal adenocarcinoma cells; LS 174 T (IC50 7.0 μg/mL) and HCT 116 (IC50 13.2 μg/mL). A higher incidence of cell death was observed with early apoptosis in HCT 116 than in LS 174 T, whereas late apoptosis was observed in LS 174 T more than in HCT 116. Also, the L-glutaminase induction nuclear fragmentation in HCT 116 was more than that in the LS 174T cells. This is the first report on Halomonas meridiana as an L-glutaminase producer that is used as an anti-colorectal cancer agent.     

Bioinspired Photocatalytic NADH Regeneration by Covalently Metalated Carbon Nitride for Enhanced CO2 Reduction

Natural photosynthesis is a highly unified biocatalytic system, which coupled cofactor (NAD(P)H) regeneration and enzymatic CO₂ reduction efficiently for solar energy conversion. Mimicking nature, a novel system with Rh complex covalently grafted onto NH₂-functionalized polymeric carbon nitride (NH₂-PCN) was constructed. The integrated connection of the light-harvesting and electron mediation modules as Rh_m3-N-PCN could promote the efficient NAD⁺ reduction to NADH. As a result, the integrated system exhibited a conversion of ∼66 % within 20 minutes. By further coupling in situ generated NADH with formate dehydrogenase (FDH), a photoenzymatic production of formic acid (HCOOH) from CO₂ was accomplished. Moreover, by immobilizing FDH onto a hydrophobic membrane, an enhanced HCOOH production of ∼5.0 mM can be obtained due to the concentrated CO₂ on the gas-liquid-solid three-phase interface. Our work herein provides an integrated strategy for coupling the anchored electron mediator with immobilized enzyme for enhanced artificial photosynthesis.     

Enzymatic Characterization of a Type II Isocitrate Dehydrogenase from Pathogenic Leptospira interrogans serovar Lai Strain 56601

Leptospira interrogans, a Gram-negative pathogen, could cause infections in a wide variety of mammalian hosts, but due to their fastidious cultivation requirements and the lack of genetic systems, the pathogenic factor is still not clear. Isocitrate dehydrogenase (IDH) is a key enzyme in the tricarboxylation (TCA) cycle, which could have an important impact on the growth and pathogenesis of the bacteria. In the present study, we first report the cloning, heterologous expression, and detailed characterization of the IDH gene from L. interrogans serovar Lai strain 56601(LiIDH). The molecular weight of LiIDH was determined to be 87 kDa by filtration chromatography, suggesting LiIDH is a typical homodimer. The optimum activity of LiIDH was found at 60 °C, and its optimum pH was 7.0 (Mn²⁺) and 8.0 (Mg²⁺). Heat inactivation studies showed that heat treatment for 20 min at 50 °C caused a 50 % loss of enzyme activity. LiIDH was completely divalent cation dependent as other typical dimeric IDHs and Mg²⁺ was its best activator. The recombinant LiIDH specificities (k _cat/K _m values for NADP⁺ and NAD⁺) in the presence of Mg²⁺ and Mn²⁺ were 6,269-fold and 1,000-fold greater for NADP⁺ than NAD⁺, respectively. This current work is expected to shed light on the functions of metabolic enzymes in L. interrogans and provide useful information for LiIDH to be considered as a possible candidate for serological diagnostics and detection of L. interrogans infection.     

Ion-exchange chromatography using vanadyl and vandate salts as mobile phases with indirect detection

Summary Vanadyl sulfate, VOSO₄, was characterized as the mobile phase for the ion exchange separation of Li⁺, Na⁺, NH ₄ ⁺ , and K⁺ using indirect photometric detection at 254 nm. Detection limits ranged from 0.2 ppm for Li⁺ to 1 ppm for K⁺. Indirect electrochemical detection of these separated cations by reduction of VO (II) to V³⁺ was compared to spectrophotometric detection. The potential of the vanadate species, HVO ₄ ^2– , for the separation of F^–, Cl^–, and SO ₄ ^2– , with indirect photometric detection was also demonstrated.     

Recombination of HCO and DCO ions with electrons

Recombination of HCO⁺ and DCO⁺ ions with electrons was studied in afterglow plasma. The flowing afterglow with Langmuir probe (FALP) apparatus was used to measure the recombination rate coefficients and their temperature dependencies in the range 150–270 K. To obtain a recombination rate coefficient for a particular ion, the dependencies on partial pressures of gases used in the ion formation were measured. The variations of α_HCO⁺(T) and α_DCO⁺(T) seem to obey the power law: α_HCO⁺(T) = (2.0 ± 0.6) × 10⁻⁷ (T/300)^−1.3 cm³ s⁻¹ and α_DCO⁺(T) = (1.7 ± 0.5) × 10⁻⁷ (T/300)^−1.1 cm³ s⁻¹ over the studied temperature range.