Invitro Toxicity of Binary Mixtures of Glyphosate and 2, 2 Dichlorovinyl Dimethyl Phosphate on Bacterial Isolates

The in vitro toxicity of glyphosate (Gly) and 2, 2 Dichlorovinyl dimethyl phosphate (DDVP) single compound and binary mixtures was assessed against Pseudomonas sp. and Bacillus sp. isolated from Otamiri River, Imo state, Nigeria was investigated. The toxicity response was assessed using the inhibitory effect of the single and binary mixtures on isolates dehydrogenase activity; and 2,3,5 triphenyltetrazolium chloride (TTC) was used as the artificial electron acceptor. The binary mixtures were composed using fixed ratios of glyphosate and 2, 2 Dichlorovinyl dimethyl phosphate in ratios of 20% Gly:80% DDVP, 40% Gly: 60% DDVP, 50% Gly: 50% DDVP, 60% Gly: 40% DDVP and 80% Gly: 20% DDVP. Results obtained showed that the isolates exhibited different degrees of logistic and sigmoidal toxicity trends with areas of hormesis at low concentrations of the toxicants. Furthermore, isobolographic analysis on the toxic interaction of the mixtures presented both synergism and antagonism, based on the relative ratio of the component mixtures. Increasing concentration of glyphosate in the binary mixture caused a shift in the interaction effect from antagonism to synergism. Our findings showed that isolates exhibited tolerance to glyphosate and 2,2 dichlorovinyl dimethyl phosphate and their binary mixtures exposure at concentration range of 0-1000mg/L; above which has deleterious effects on the aquatic organisms. It is evident that there are considerable differences in pesticide sensitivity among the bacterial species and that the presence of glyphosate and 2, 2-dichlorovinyl dimethyl phosphate in the aquatic environment may present toxicological risk to microbial diversity.

Appropriate Activity Assays Are Crucial for the Specific Determination of Proline Dehydrogenase and Pyrroline-5-Carboxylate Reductase Activities

Accumulation of proline is a widespread plant response to a broad range of environmental stress conditions including salt and osmotic stress. Proline accumulation is achieved mainly by upregulation of proline biosynthesis in the cytosol and by inhibition of proline degradation in mitochondria. Changes in gene expression or activity levels of the two enzymes catalyzing the first reactions in these two pathways, namely pyrroline-5-carboxylate (P5C) synthetase and proline dehydrogenase (ProDH), are often used to assess the stress response of plants. The difficulty to isolate ProDH in active form has led several researchers to erroneously report proline-dependent NAD+ reduction at pH 10 as ProDH activity. We demonstrate that this activity is due to P5C reductase (P5CR), the second and last enzyme in proline biosynthesis, which works in the reverse direction at unphysiologically high pH. ProDH does not use NAD+ as electron acceptor but can be assayed with the artificial electron acceptor 2,6-dichlorophenolindophenol (DCPIP) after detergent-mediated solubilization or enrichment of mitochondria. Seemingly counter-intuitive results from previous publications can be explained in this way and our data highlight the importance of appropriate and specific assays for the detection of ProDH and P5CR activities in crude plant extracts.

Cloning, expression and characterization of thermostable YdaP from Bacillus licheniformis 9A

The Bacillus licheniformis ydaP gene encodes for a pyru­vate oxidase that catalyses the oxidative decarboxyla­tion of pyruvate to acetate and CO2. The YdaP form of this enzyme was purified about 48.6-folds to homoge­neity in three steps. The enzyme was recovered in a soluble form and demonstrated significant activity on pyruvate using 2, 6-dichlorophenolindophenol (DCPIP) as an artificial electron acceptor. HPLC analysis of the YdaP-enzyme catalysed conversion of pyruvate showed acetate as the sole product, confirming the putative identity of pyruvate oxidase. Analysis of the substrate specificity showed that the YdaP enzyme demonstrated preference for short chain oxo acids; however, it was activated by 1% Triton X-100. The YdaP substrate-bind­ing pocket from the YdaP protein differed substantially from the equivalent site in all of the so far character­ized pyruvate oxidases, suggesting that the B. licheni­formis YdaP might accept different substrates. This could allow more accessibility of large substrates into the active site of this enzyme. The thermostability and pH activity of the YdaP enzyme were determined, with optimums at 50ºC and pH 5.8, respectively. The amino acid residues forming the catalytic cavity were identi­fied as Gln460 to Ala480.

Membrane-Associated Glucose-Methanol-Choline Oxidoreductase Family Enzymes PhcC and PhcD Are Essential for Enantioselective Catabolism of Dehydrodiconiferyl Alcohol

ABSTRACTSphingobiumsp. strain SYK-6 is able to degrade various lignin-derived biaryls, including a phenylcoumaran-type compound, dehydrodiconiferyl alcohol (DCA). In SYK-6 cells, the alcohol group of the B-ring side chain of DCA is initially oxidized to the carboxyl group to generate 3-(2-(4-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-7-methoxy-2,3-dihydrobenzofuran-5-yl) acrylic acid (DCA-C). Next, the alcohol group of the A-ring side chain of DCA-C is oxidized to the carboxyl group, and then the resulting metabolite is catabolized through vanillin and 5-formylferulate. In this study, the genes involved in the conversion of DCA-C were identified and characterized. The DCA-C oxidation activities in SYK-6 were enhanced in the presence of flavin adenine dinucleotide and an artificial electron acceptor and were induced ca. 1.6-fold when the cells were grown with DCA. Based on these observations, SLG_09480 (phcC) and SLG_09500 (phcD), encoding glucose-methanol-choline oxidoreductase family proteins, were presumed to encode DCA-C oxidases. Analyses ofphcCandphcDmutants indicated that PhcC and PhcD are essential for the conversion of (+)-DCA-C and (−)-DCA-C, respectively. WhenphcCandphcDwere expressed in SYK-6 andEscherichia coli, the gene products were mainly observed in their membrane fractions. The membrane fractions ofE. colithat expressedphcCandphcDcatalyzed the specific conversion of DCA-C into the corresponding carboxyl derivatives. In the oxidation of DCA-C, PhcC and PhcD effectively utilized ubiquinone derivatives as electron acceptors. Furthermore, the transcription of a putative cytochromecgene was significantly induced in SYK-6 grown with DCA. The DCA-C oxidation catalyzed by membrane-associated PhcC and PhcD appears to be coupled to the respiratory chain.

Dehydrogenase activity of forest soils depends on the assay used

Dehydrogenases are exclusively intracellular enzymes, which play an important role in the initial stages of oxidation of soil organic matter. One of the most frequently used methods to estimate dehydrogenase activity in soil is based on the use of triphenyltetrazolium chloride as an artificial electron acceptor. The purpose of this study was to compare the activity of dehydrogenases of forest soils with varied physicochemical properties using different triphenyltetrazolium chloride assays. The determination was carried out using the original procedure by Casida et al., a modification of the procedure which involves the use of Ca(OH)2 instead of CaCO3, the Thalmann method, and the assay by Casida et al. without addition of buffer or any salt. Soil dehydrogenase activity depended on the assay used. Dehydrogenase determined by the Casida et al. method without addition of buffer or any salt correlated with the pH values of soils. The autoclaved strongly acidic samples of control soils showed high concentrations of triphenylformazan, probably due to chemical reduction of triphenyltetrazolium chloride. There is, therefore, a need for a sterilization method other than autoclaving, ie a process that results in significant changes in soil properties, thus helping to increase the chemical reduction of triphenyltetrazolium chloride.

Pyruvate:Quinone Oxidoreductase from Corynebacterium glutamicum: Purification and Biochemical Characterization

ABSTRACT Pyruvate:quinone oxidoreductase catalyzes the oxidative decarboxylation of pyruvate to acetate and CO2 with a quinone as the physiological electron acceptor. So far, this enzyme activity has been found only in Escherichia coli. Using 2,6-dichloroindophenol as an artificial electron acceptor, we detected pyruvate:quinone oxidoreductase activity in cell extracts of the amino acid producer Corynebacterium glutamicum. The activity was highest (0.055 ± 0.005 U/mg of protein) in cells grown on complex medium and about threefold lower when the cells were grown on medium containing glucose, pyruvate, or acetate as the carbon source. From wild-type C. glutamicum, the pyruvate:quinone oxidoreductase was purified about 180-fold to homogeneity in four steps and subjected to biochemical analysis. The enzyme is a flavoprotein, has a molecular mass of about 232 kDa, and consists of four identical subunits of about 62 kDa. It was activated by Triton X-100, phosphatidylglycerol, and dipalmitoyl-phosphatidylglycerol, and the substrates were pyruvate (k cat = 37.8 ± 3 s−1; Km = 30 ± 3 mM) and 2-oxobutyrate (k cat = 33.2 ± 3 s−1; Km = 90 ± 8 mM). Thiamine pyrophosphate (Km = 1 μM) and certain divalent metal ions such as Mg2+ (Km = 29 μM), Mn2+ (Km = 2 μM), and Co2+ (Km = 11 μM) served as cofactors. In addition to several dyes (2,6-dichloroindophenol, p-iodonitrotetrazolium violet, and nitroblue tetrazolium), menadione (Km = 106 μM) was efficiently reduced by the purified pyruvate:quinone oxidoreductase, indicating that a naphthoquinone may be the physiological electron acceptor of this enzyme in C. glutamicum.

Kinetics and thermodynamics of activation of quinoprotein glucose dehydrogenase apoenzyme in vivo and catalytic activity of the activated enzyme in Escherichia coli cells

Apo-glucose dehydrogenase existing in Escherichia coli is converted to the holoenzyme with exogenous pyrroloquinoline quinone (PQQ) and Mg2+. Catalytic behaviour of the E. coli cells with the holoenzyme is characterized by a Michaelis–Menten-type equation with a catalytic constant of the cell and apparent Michaelis constants for d-glucose and an artificial electron acceptor added to the E. coli suspension. The catalytic constant is expressed as the product of the number of molecules of the enzyme contained in an E. coli cell (z) and the catalytic constant of the enzyme (kcat), which were determined to be 2.2×103 and 6.8±0.8×103s-1 (phenazine methosulphate as an electron acceptor) respectively. Kinetics of the in vivo holoenzyme formation can be followed by an enzyme-electrochemical method developed by us. The rate constants for the reactions of apoenzyme with PQQ (kf,PQQ) and with Mg2+ (kf,Mg) were determined to be 3.8±0.4×104 M-1·s-1 and 4.1±0.9M-1·s-1 respectively. Equilibrium constants for the binding of apoenzyme to PQQ and Mg2+ were determined as the dissociation constants Kd,PQQ(Mg) and Kd,Mg to be 1.0±0.1nM and 0.14±0.01mM respectively. The dissociation constants for Ca2+ were also determined. The holoenzyme, once formed in E. coli, returns gradually to the apoenzyme in the absence of PQQ and/or Mg2+ in solution. EDTA was effective to remove Mg2+ from the enzyme in the cells to deactivate the enzyme completely, while PQQ remained in the E. coli cells.