Surface display of MPH on Pseudomonas putida JS444 using ice nucleation protein and its application in detoxification of organophosphates

Methyl parathion hydrolase (MPH) has been displayed on the surface of microorganisms for the first time using only N- and C-terminal domains of the ice nucleation protein (INPNC) from Pseudomonas syringae INA5 as an anchoring motif. A shuttle vector pINCM coding for INPNC-MPH was constructed and used to target MPH onto the surface of a natural p-nitrophenol (PNP) degrader, Pseudomonas putida JS444, overcoming the potential substrate uptake limitation. Over 90% of the MPH activity was located on the cell surface as determined by protease accessibility and cell fractionation experiments. The surface localization of the INPNC-MPH fusion was further verified by Western blot analysis and immunofluorescence microscopy. The engineered P. putida JS444 degraded organophosphates as well as PNP rapidly without growth inhibition. Compared to organophosphorus hydrolase-displaying systems reported, changes in substrate specificity highlight an important potential use of the engineered strain for the clean-up of specific organophosphate nerve agents.     

Metabolic differentiation of Arabidopsis treated with methyl jasmonate using nuclear magnetic resonance spectroscopy

NMR spectroscopy combined with principal component analysis was applied to Arabidopsis thaliana treated with methyl jasmonate in order to obtain macroscopic metabolic changes caused by the treatment. As the first step several chromatographic and NMR spectroscopic techniques were utilized to identify metabolites of Arabidopsis. Sephadex LH-20 showed a high efficiency in the separation of phenolic metabolites in the plant. For identification of minor metabolites two-dimensional J-resolved NMR technique was directly applied to the plant extract and results in a number of elucidation of the metabolites of which signals overlap in ¹H NMR spectra. The chemical structure of the identified metabolites were confirmed by various two-dimensional NMR spectroscopy including correlated spectroscopy, heteronuclear single quantum coherence, and heternuclear multiple bond correlation. As next step, a statistical approach, principal component analysis based on projected J-resolved NMR spectra was performed for metabolic alteration of methyl jasmonate-treated Arabidopsis. The results show that methyl jasmonate caused an increase of flavonoids, fumaric acid, sinapoyl malate, sinigrin, tryptophan, valine, threonine, and alanine and a decrease of malic acid, feruloyl malate, glutamine, and carbohydrates after 24 h treatment.     

Overexpression of methyl parathion hydrolase and its application in detoxification of organophosphates

The coding region of mpd gene corresponding to mature methyl parathion hydrolase (MPH) was heterologously overexpressed in Escherichia coli BL21 (DE3) by using pET expression system. The lactose-induced expression yield of MPH is increased 2-fold compared with IPTG as inducer. Furthermore, it was found that specific activity of MPH increased 48% by reducing the induction temperature to 22°C. The addition of 25 mM lactose at 22°C, the MPH activity of fermentation broth had a specific activity of 1.4 × 10⁴ U/mg protein. Plasmid was no significant decrease in the modified medium. The optimal pH and temperature of MPH were 8.0 and 30°C, respectively. Over a period of 5 months, the dried cells showed no significant decrease in the activity of the detoxifying enzymes. The crude enzymes in 50 mM citrate-phosphate buffer (pH 8.0) were able to degrade about 98% of the organophosphate pesticides sprayed on cabbage. The detoxification efficiency was superior to that of the treatments of water, detergent, and a commercially available enzyme product. Additionally, the products of pesticide hydrolysis generated by treatment with the enzyme extract were determined to be virtually nontoxic.     

Free radical scavenging potential of L-proline: evidence from in vitro assays

Summary. An assessment of the potential of proline to scavenge free radicals was made in a couple of in vitro assay systems, namely graft co-polymerization and autooxidation of pyrogallol. Both these assays are essentially dependent upon free radical mechanisms. Graft co-polymerization involved a ceric (Ce⁴⁺) ion- or γ-radiation-induced grafting of methyl acrylate (MA) onto a cellulose backbone. The degree of grafting, measured gravimetrically, was taken as a measure of free radical generation. The γ-radiation-dependent grafting was far greater than that due to Ce⁴⁺ ions. Inclusion of proline in the assay, irrespective of the initiator used, led to suppression of grafting in a concentration-dependent manner indicating the ability of proline to scavenge free radicals. The γ-radiation-dependent grafting was also suppressed by hydroquinone and glutathione but not by ascorbate, glycine and spermine. In contrast to graft co-polymerization, proline did not inhibit the autooxidation of pyrogallol, a reaction involving superoxide radical generation. A subset of data constitutes an evidence for the ability of proline to scavenge free radicals in vitro. It is implied by extension that free proline, known to accumulate in plant tissues during abiotic stresses, would contribute to scavenging of surplus free radicals produced under a variety of abiotic stresses. Authors’ address: Shanti S. Sharma, Department of Biosciences, Himachal Pradesh University, Shimla 171 005, India     

Glutathione reductase and catalase as potential biomarkers for synergistic intoxication of pesticides in fish

Abstract

Synergy occurs when chemicals give pronounced effect on combination in contrast to their individual effect. The objective of this study was to investigate the synergistic effect of pesticides carbaryl (C) and methyl parathion (MP) on oxidative stress biomarkers viz catalase (CAT), glutathione reductase (GSSG-R) including different enzymes like lactate dehydrogenase (LDH), succinate dehydrogenase (SDH) and acetyl cholinesterase (AChE) in different tissues of carps Catla catla. Fishes were exposed to 6.25?mg/L of MP and 2.3?mg/L of C in mixture (one-third of LC₅₀ value). CAT and GSSG-R were studied in gills, brain, liver and muscle of carp were found to be elevated significantly (p?<?0.005). LDH activity increased significantly (p?<?0.005) in synergistic group, there was a seven-fold (748%) increase in LDH activity in muscle compared to individual studies with same pesticides. Contrary to LDH, sudden decrease in SDH activity was accounted. Significant (p?<?0.005) decrease in AChE activity after initial 24?h was remarkable addressing to the shift in neurotransmission pathway in organism. Significant increase was observed in activity of CAT and GSSG-R in all tissues compared to control fishes in individual as well as synergistic (MP?+?C) group suggesting that CAT and GSSG-R can be a potential biomarker of oxidative stress when studied in combination.     

A comprehensive analysis of fifteen genes of steviol glycosides biosynthesis pathway in Stevia rebaudiana (Bertoni)

Stevia [Stevia rebuaidana (Bertoni); family: Asteraceae] is known to yield diterpenoid steviol glycosides (SGs), which are about 300 times sweeter than sugar. The present work analyzed the expression of various genes of the SGs biosynthesis pathway in different organs of the plant in relation to the SGs content. Of the various genes of the pathway, SrDXS, SrDXR, SrCPPS, SrKS, SrKO and three glucosyltransferases namely SrUGT85C2, SrUGT74G1 and SrUGT76G1 were reported from stevia. Here, we report cloning of seven additional full-length cDNA sequences namely, SrMCT, SrCMK, SrMDS, SrHDS, SrHDR, SrIDI and SrGGDPS followed by expression analysis of all the fifteen genes vis-à-vis SGs content analysis. SGs content was highest in the leaf at 3rd node position (node position with reference to the apical leaf as the first leaf) as compared to the leaves at other node positions. Except for SrDXR and SrKO, gene expression was maximum in leaf at 1st node and minimum in leaf at 5th node. The expression of SrKO was highest in leaf at 3rd node while in case of SrDXR expression showed an increase up to 3rd leaf and decrease thereafter. SGs accumulated maximum in leaf tissue followed by stem and root, and similar was the pattern of expression of all the fifteen genes. The genes responded to the modulators of the terpenopids biosynthesis. Gibberellin (GA₃) treatment up-regulated the expression of SrMCT, SrCMK, SrMDS and SrUGT74G1, whereas methyl jasmonate and kinetin treatment down-regulated the expression of all the fifteen genes of the pathway.     

Study on the kinetics of enzymatic interesterification of triglycerides for biodiesel production with methyl acetate as the acyl acceptor

A new route for biodiesel production using methyl acetate instead of methanol as the acyl acceptor was proposed in our previous research, and it has been found that this novel route could enhance the stability of the immobilized lipase greatly. In this paper, the kinetics of lipase-catalyzed interesterification of triglycerides for biodiesel production with methyl acetate as the acyl acceptor was further studied. First, a simplified model based on Ping Pong Bi Bi with substrate competitive inhibition mechanism was proposed to describe the reaction kinetics of the interesterification. During our further study, it was observed that three consecutive and reversible reactions occurred in the interesterification of triglycerides and methyl acetate. So, a kinetic model based on mass balance of three second-order reversible reactions was developed and the reaction rate constant, k, was determined by solving the differential rate equations of the reaction system. The results showed that k_DG–MG (0.1124) and k_MG–TA (0.1129) were much higher than k_TG–DG (0.0311), which indicated that the first step reaction was the limit step for the overall interesterification.     

A combined nuclear magnetic resonance and molecular dynamics study of the two structural motifs for mixed-linkage β-glucans: methyl β-cellobioside and methyl β-laminarabioside

The conformational and hydration properties of the two disaccharides methyl β-cellobioside and methyl β-laminarabioside were investigated by NMR spectroscopy and explicit solvation molecular dynamics simulations using the carbohydrate solution force field (CSFF). Adiabatic maps produced with this force field displayed 4 minima A: (Φ = 300°, Ψ = 280°), B: (Φ = 280°, Ψ = 210°), C: (Φ = 260°, Ψ = 60°), and D: (Φ = 60°, Ψ = 260°) for methyl β-cellobioside and 3 minima A: (Φ = 290°, Ψ = 130°), B: (Φ = 270°, Ψ = 290°), and C: (Φ = 60°, Ψ = 120°) for methyl β-laminarabioside. Molecular dynamics simulations were initiated from all minima. For each disaccharide, the simulation started from the A minimum was conducted for 50 ns, while the other minima were explored for 10 ns. The simulations revealed two stable minima for both compounds. For methyl β-cellobioside, the simulation minima in aqueous solution were shifted from their adiabatic map counterparts, while the simulation minima for methyl β-laminarabioside coincided with the corresponding adiabatic map minima. To validate the simulation results, NMR-derived NOEs and coupling constants across the glycoside linkage, ³J_HC and ³J_CH, were compared with values calculated from the MD trajectories. For each disaccharide, the best agreement was obtained for the simulations started at the A minimum. For both compounds, inter-ring water bridges in combination with the direct hydrogen bonds between the same groups were found to be determining factors for the overall solution structure of the disaccharides which differed from solid-state structures. Comparison with helical parameters showed that the preferred glycosidic dihedral configurations in the methyl β-cellobioside simulation were not highly compatible with the structure of cellulose, but that curdlan helix structures agreed relatively well with the methyl β-laminarabioside simulation. Polymers generated using glycosidic dihedral angles from the simulations revealed secondary structure motifs that that may help to elucidate polymer associations and small-molecule binding.     

Lipase-catalyzed enantioselective hydrolysis of methyl 2-fluoro-2-arylpropionates in water-saturated isooctane

Lipases from Candida rugosa, Candida antartica B and Carica papaya are employed as the biocatalyst for the hydrolytic resolution of methyl 2-fluoro-2-arylpropionates in water-saturated isooctane, in which excellent to good enantioselectivity without the formation of byproducts is obtained for the papaya lipase when using (R,S)-2-fluoronaproxen methyl ester (1) and methyl (R,S)-2-fluoro-2-(4-methoxyphenyl)propionate (2), but not methyl (R,S)-2-fluoro-2-(naphth-1-yl)propionate (3) as the substrates. The thermodynamic analysis indicates that the enantiomer discrimination for the papaya lipase is driven by the difference in activation enthalpy for compound 1, 2 or (R,S)-naproxen methyl ester (4). The kinetic analysis also demonstrates that in comparison with (S)-4, the insertion of the 2-fluorine moiety in (R)-1 has increased k₂, but not K_m, and consequently the lipase activity.