Organic Remobilization of zinc and phosphorus availability to plants by application of mineral solubilizing bacteria Pseudomonas aeruginosa.

Incessant utilization of chemical fertilizers leads to the accumulation of minerals in the soil, rendering them unavailable to plants. Unaware of the mineral reserves present in the soil, farming communities employ chemical fertilizers once during each cultivation, a practice that causes elevated levels of insoluble minerals within the soil. The use of biofertilizers on the other hand, reduces the impact of chemical fertilizers through the action of microorganisms in the product, which dissolves minerals and makes them readily available for plant uptake, helping to create a sustainable environment for continuous agricultural production. In the current investigation, a field trial employing Arachis hypogaea L was conducted to evaluate the ability of Pseudomonas aeruginosa to enhance plant growth and development by solubilizing minerals present in the soil (such as zinc and phosphorus). A Randomized Complete Block Design (RCBD) included five different treatments as T1: Un inoculated Control; T2: Seeds treated with a liquid formulation of P. aeruginosa; T3: Seeds treated with a liquid formulation of P. aeruginosa and the soil amended with organic manure (farmyard); T4: Soil amended with organic manure (farmyard) alone; T5: Seeds treated with lignite (solid) based formulation of P. aeruginosa were used for the study. Efficacy was determined based on the plant's morphological characters and mineral contents (Zn and P) of plants and soil. Survival of P. aeruginosa in the field was validated using Antibiotic Intrinsic patterns (AIP). The results indicated that the combination treatment of P. aeruginosa liquid formulation and organic fertilizer (farmyard) (T3) produced the highest biometric parameters and mineral (Zn and P) content of the groundnut plants and the soil. This outcome is likely attributed to the mineral solubilizing capability of P. aeruginosa. Furthermore, the presence of farmyard manure increased the metabolic activity of P. aeruginosa by inducing its heterotrophic activity, leading to higher mineral content in T3 soil compared to other soil treatments. The AIP data confirmed the presence of the applied liquid inoculant by exhibiting a similar intrinsic pattern between the in vitro isolate and the isolate obtained from the fields. In summary, the Zn and P solubilization ability of P. aeruginosa facilitates the conversion of soil-unavailable mineral form into a form accessible to plants. It further proposes the utilization of the liquid formulation of P. aeruginosa as a viable solution to mitigate the challenges linked to solid-based biofertilizers and the reliance on mineral-based chemical fertilizers.

Impact of Diverse Parameters on the Physicochemical Characteristics of Green-Synthesized Zinc Oxide-Copper Oxide Nanocomposites Derived from an Aqueous Extract of Garcinia mangostana L. Leaf.

Compared to conventional metal oxide nanoparticles, metal oxide nanocomposites have demonstrated significantly enhanced efficiency in various applications. In this study, we aimed to synthesize zinc oxide-copper oxide nanocomposites (ZnO-CuO NCs) using a green synthesis approach. The synthesis involved mixing 4 g of Zn(NO3)2·6H2O with different concentrations of mangosteen (G. mangostana) leaf extract (0.02, 0.03, 0.04 and 0.05 g/mL) and 2 or 4 g of Cu(NO3)2·3H2O, followed by calcination at temperatures of 300, 400 and 500 °C. The synthesized ZnO-CuO NCs were characterized using various techniques, including a UV-Visible spectrometer (UV-Vis), photoluminescence (PL) spectroscopy, Fourier Transform Infrared (FTIR) spectroscopy, X-ray powder diffraction (XRD) analysis and Field Emission Scanning Electron Microscope (FE-SEM) with an Energy Dispersive X-ray (EDX) analyzer. Based on the results of this study, the optical, structural and morphological properties of ZnO-CuO NCs were found to be influenced by the concentration of the mangosteen leaf extract, the calcination temperature and the amount of Cu(NO3)2·3H2O used. Among the tested conditions, ZnO-CuO NCs derived from 0.05 g/mL of mangosteen leaf extract, 4 g of Zn(NO3)2·6H2O and 2 g of Cu(NO3)2·3H2O, calcinated at 500 °C exhibited the following characteristics: the lowest energy bandgap (2.57 eV), well-defined Zn-O and Cu-O bands, the smallest particle size of 39.10 nm with highest surface area-to-volume ratio and crystalline size of 18.17 nm. In conclusion, we successfully synthesized ZnO-CuO NCs using a green synthesis approach with mangosteen leaf extract. The properties of the nanocomposites were significantly influenced by the concentration of the plant extract, the calcination temperature and the amount of precursor used. These findings provide valuable insights for researchers seeking innovative methods for the production and utilization of nanocomposite materials.

Effect of Calcination Temperature on Structural, Morphological and Optical Properties of Copper Oxide Nanostructures Derived from Garcinia mangostana L. Leaf Extract.

Synthesis of copper oxide (CuO) nanostructures via biological approach has gained attention to reduce the harmful effects of chemical synthesis. The CuO nanostructures were synthesized through a green approach using the Garcinia mangostana L. leaf extract and copper (II) nitrate trihydrate as a precursor at varying calcination temperatures (200-600 °C). The effect of calcination temperatures on the structural, morphological and optical properties of CuO nanostructures was studied. The red shifting of the green-synthesized CuO nanoparticles' absorption peak was observed in UV-visible spectrum, and the optical energy bandgap was found to decrease from 3.41 eV to 3.19 eV as the calcination temperatures increased. The PL analysis shown that synthesized CuO NPs calcinated at 500 °C has the maximum charge carriers separation. A peak located at 504-536 cm-1 was shown in FTIR spectrum that indicated the presence of a copper-oxygen vibration band and become sharper and more intense when increasing the calcination temperature. The XRD studies revealed that the CuO nanoparticles' crystalline size was found to increase from 12.78 nm to 28.17 nm, and dislocation density decreased from 61.26 × 1014 cm-1 to 12.60 × 1014 cm-1, while micro strain decreased from 3.40 × 10-4 to 1.26 × 10-4. From the XPS measurement, only CuO single phase without impurities was detected for the green-mediated NPs calcinated at 500 °C. The morphologies of CuO nanostructures were examined using FESEM and became more spherical in shape at elevated calcination temperature. More or less spherical nanostructure of green-mediated CuO calcinated at 500 °C were also observed using TEM. The purity of the green-synthesized CuO nanoparticles was evaluated by EDX analysis, and results showed that increasing calcination temperature increases the purity of CuO nanoparticles.

Muntingia calabura Leaves Mediated Green Synthesis of CuO Nanorods: Exploiting Phytochemicals for Unique Morphology.

In this study, phytochemical assisted nanoparticle synthesis was performed using Muntingia calabura leaf extracts to produce copper oxide nanoparticles (CuO NPs) with interesting morphology. Scanning electron microscope (SEM) and transmission electron microscope (TEM) analysis of the biosynthesized CuO NPs reveal formation of distinct, homogeneous, and uniform sized CuO nanorods structure with thickness and length of around 23 nm and 79 nm, respectively. Based on Fourier-transform infrared (FTIR) analysis, the unique combinations of secondary metabolites such as flavonoid and polyphenols in the plant extract are deduced to be effective capping agents to produce nanoparticles with unique morphologies similar to conventional chemical synthesis. X-ray diffraction (XRD) analysis verified the monoclinical, crystalline structure of the CuO NPs. The phase purity and chemical identity of the product was consolidated via X-Ray photoelectron spectroscopy (XPS) and Raman spectroscopic data which indicate the formation of a single phase CuO without the presence of other impurities. The direct and indirect optical band gap energies of the CuO nanorods were recorded to be 3.65 eV and 1.42 eV.

The role of large-scale motions in catalysis by dihydrofolate reductase.

Dihydrofolate reductase has long been used as a model system to study the coupling of protein motions to enzymatic hydride transfer. By studying environmental effects on hydride transfer in dihydrofolate reductase (DHFR) from the cold-adapted bacterium Moritella profunda (MpDHFR) and comparing the flexibility of this enzyme to that of DHFR from Escherichia coli (EcDHFR), we demonstrate that factors that affect large-scale (i.e., long-range, but not necessarily large amplitude) protein motions have no effect on the kinetic isotope effect on hydride transfer or its temperature dependence, although the rates of the catalyzed reaction are affected. Hydrogen/deuterium exchange studies by NMR-spectroscopy show that MpDHFR is a more flexible enzyme than EcDHFR. NMR experiments with EcDHFR in the presence of cosolvents suggest differences in the conformational ensemble of the enzyme. The fact that enzymes from different environmental niches and with different flexibilities display the same behavior of the kinetic isotope effect on hydride transfer strongly suggests that, while protein motions are important to generate the reaction ready conformation, an optimal conformation with the correct electrostatics and geometry for the reaction to occur, they do not influence the nature of the chemical step itself; large-scale motions do not couple directly to hydride transfer proper in DHFR.

Catalysis by dihydrofolate reductase from the psychropiezophile Moritella profunda.

The influence of temperature and pH on the stability and catalytic activity of dihydrofolate reductase (MpDHFR) from the cold-adapted deep-sea bacterium Moritella profunda was studied. The thermal melting temperature was found to be ∼38 °C and was not affected by pH, while activity measurements demonstrated that its stability was maximal at pH 7 and was reduced dramatically below pH 6 or above pH 8. The steady-state rate constant (k(cat)) was maximal at neutral pH and higher temperatures, while the Michaelis constants (K(M)) for both substrate and cofactor were optimal at lower temperatures and at elevated or reduced pH. For both temperature and pH, any change in k(cat) was therefore offset by a similar change in K(M). Both the activation enthalpy and entropy of the MpDHFR-catalysed reaction were lower than those of DHFR from E. coli leading overall to a very small difference in activation free energy and therefore similar steady-state rate constants at the same temperature. The chemical step of the reaction is not rate limiting at pH 7, but becomes progressively more rate limiting as the pH increases. These results demonstrate adaptation of MpDHFR to its environment and show compromises between enthalpic and entropic contributions to the reaction, and between k(cat) and K(M).

Highly site-selective stability increases by glycosylation of dihydrofolate reductase.

Post-translational glycosylation is one of the most abundant forms of covalent protein modification in eukaryotic cells. It plays an important role in determining the properties of proteins, and stabilizes many proteins against thermal denaturation. Protein glycosylation may establish a surface microenvironment that resembles that of unglycosylated proteins in concentrated solutions of sugars and other polyols. We have used site-directed mutagenesis to introduce a series of unique cysteine residues into a cysteine-free double mutant (DM, C85A/C152S) of dihydrofolate reductase from Escherichia coli (EcDHFR). The resulting triple mutants, DM-N18C, DM-R52C, DM-D87C and DM-D132C EcDHFR, were alkylated with glucose, N-acetylglucosamine, lactose and maltotriose iodoacetamides. We found little effect on catalysis or stability in three cases. However, when DM-D87C EcDHFR is glycosylated, stability is increased by between 1.5 and 2.6 kcal.mol(-1) in a sugar-dependent manner. D87 is found in a hinge region of EcDHFR that loses structure early in the thermal denaturation process, whereas the other glycosylation sites are found in regions involved in the later stages of temperature-induced unfolding. Glycosylation at this site may improve the stability of EcDHFR by protecting a region of the enzyme that is particularly prone to denaturation.

Solvent effects on catalysis by Escherichia coli dihydrofolate reductase.

Hydride transfer catalyzed by dihydrofolate reductase (DHFR) has been described previously within an environmentally coupled model of hydrogen tunneling, where protein motions control binding of substrate and cofactor to generate a tunneling ready conformation and modulate the width of the activation barrier and hence the reaction rate. Changes to the composition of the reaction medium are known to perturb protein motions. We have measured kinetic parameters of the reaction catalyzed by DHFR from Escherichia coli in the presence of various cosolvents and cosolutes and show that the dielectric constant, but not the viscosity, of the reaction medium affects the rate of reaction. Neither the primary kinetic isotope effect on the reaction nor its temperature dependence were affected by changes to the bulk solvent properties. These results are in agreement with our previous report on the effect of solvent composition on catalysis by DHFR from the hyperthermophile Thermotoga maritima. However, the effect of solvent on the temperature dependence of the kinetic isotope effect on hydride transfer catalyzed by E. coli DHFR is difficult to explain within a model, in which long-range motions couple to the chemical step of the reaction, but may indicate the existence of a short-range promoting vibration or the presence of multiple nearly isoenergetic conformational substates of enzymes with similar but distinct catalytic properties.

Protein motions during catalysis by dihydrofolate reductases.

Dihydrofolate reductase (DHFR) maintains the intracellular pool of tetrahydrofolate through catalysis of hydrogen transfer from reduced nicotinamide adenine dinucleotide to 7,8-dihydrofolate. We report results for pre-steady-state kinetic studies of the temperature dependence of the rates and the hydrogen/deuterium-kinetic isotope effects for the reactions catalysed by the enzymes from the mesophilic Escherichia coli and the hyperthermophilic Thermatoga maritima. We propose an evolutionary pattern in which catalysis progressed from a relatively rigid active site structure in the ancient thermophilic DHFR to a more flexible and kinetically more efficient structure in E. coli that actively promotes hydrogen transfer at physiological pH by modulating the tunnelling distance. The E. coli enzyme appeared relatively robust, in that kinetically severely compromised mutants still actively propagated the reaction. The reduced hydrogen transfer rates of the extensively studied Gly121Val mutant of DHFR from E. coli were most likely due to sterically unfavourable long-range effects from the introduction of the bulky isopropyl group.

Coupling of protein motions and hydrogen transfer during catalysis by Escherichia coli dihydrofolate reductase.

The enzyme DHFR (dihydrofolate reductase) catalyses hydride transfer from NADPH to, and protonation of, dihydrofolate. The physical basis of the hydride transfer step catalysed by DHFR from Escherichia coli has been studied through the measurement of the temperature dependence of the reaction rates and the kinetic isotope effects. Single turnover experiments at pH 7.0 revealed a strong dependence of the reaction rates on temperature. The observed relatively large difference in the activation energies for hydrogen and deuterium transfer led to a temperature dependence of the primary kinetic isotope effects from 3.0+/-0.2 at 5 degrees C to 2.2+/-0.2 at 40 degrees C and an inverse ratio of the pre-exponential factors of 0.108+/-0.04. These results are consistent with theoretical models for hydrogen transfer that include contributions from quantum mechanical tunnelling coupled with protein motions that actively modulate the tunnelling distance. Previous work had suggested a coupling of a remote residue,Gly121, with the kinetic events at the active site. However, pre-steady-state experiments at pH 7.0 with the mutant G121V-DHFR, in which Gly121 was replaced with valine, revealed that the chemical mechanism of DHFR catalysis was robust to this replacement. The reduced catalytic efficiency of G121V-DHFR was mainly a consequence of the significantly reduced pre-exponential factors, indicating the requirement for significant molecular reorganization during G121V-DHFR catalysis. In contrast, steady-state measurements at pH 9.5, where hydride transfer is rate limiting, revealed temperature-independent kinetic isotope effects between 15 and 35 degrees C and a ratio of the pre-exponential factors above the semi-classical limit, suggesting a rigid active site configuration from which hydrogen tunnelling occurs. The mechanism by which hydrogen tunnelling in DHFR is coupled with the environment appears therefore to be sensitive to pH.