Oxidation Of L-proline Research Articles

Effect of high temperature stress on metabolome and aroma in rice grains

Heat stress poses a significant challenge to global rice production, affecting yield and grain quality. Elevated temperatures during the flowering and grain-filling stages, both day and night, lead to reduced yield and compromised grain quality. This impact is more pronounced during nighttime high-temperature stress, seriously threatening rice productivity. With global temperatures rising, there is a looming threat to rice production. Aromatic rice, prized for superior aroma and grain quality, is particularly vulnerable to heat. Therefore, the present work has been carried out to investigate how high temperature affects the aromatic metabolites in rice grains among the 15 rice genotypes (fourteen aromatic and one non-aromatic rice i.e., Nagina 22). Results from the present study indicated that the inactive (mutated) BADH2 gene expression was down-regulated under high-temperature stress conditions and no 2-acetyl-1-pyrroline (2-AP) accumulation was detected in the selected rice genotypes. However, the increase in levels of L-proline (precursor molecule for 2-AP) was detected, and due to the down-regulation of inactive BADH2, the oxidation of L-proline into 2-AP was affected. Proline amino acid significantly increased under high temperatures, impacting aroma quality. Metabolome studies revealed variations in compound detection among scented rice genotypes. Understanding these metabolites aids in addressing the loss of aroma in fragrant rice genotypes, offering insights into developing stable aromatic rice varieties under elevated temperature conditions. The study aims to identify metabolites causing aroma loss in aromatic rice. Results will aid in understanding aroma depletion mechanisms in scented rice under high-temperature stress, guiding the development of a stable aromatic rice variety in elevated temperatures.

Discovery of New Fe(II)/α-Ketoglutarate-Dependent Dioxygenases for Oxidation of l-Proline

Genome mining for novel Fe(II)/α-ketoglutarate-dependent dioxygenases (αKGDs) to expand the enzymatic repertoire in the oxidation of l-proline is reported. Through clustering of proteins, we predicted regio- and stereoselectivity in the hydroxylation reaction and validated this hypothesis experimentally. Two novel byproducts in the reactions with enzymes from Bacillus cereus and Streptomyces sp. were isolated, and the structures were determined to be a 3,4-epoxide and a 3,4-diol, respectively. The mechanism for the formation of the epoxide was investigated by performing an 18O-labeling experiment. We propose that the mechanism proceeds via initial cis-3-hydroxylation followed by ring closure. A biocatalytic step was run on subgram quantities of starting material without any significant optimization of the conditions. However, the substrate concentration was 40-fold higher than the usual reported titers for recombinant P450-mediated hydroxylations, showing the synthetic potential of αKGDs on a preparative scale.

Kinetic and Mechanism of Mn(II) Catalytic Oxidation of L-Proline by Cerium(IV) in Acidic Medium by Spectrophotometry Method

The rate of reaction of L-proline with Ce(IV) acidic medium has been kinetically studied in presence of Mn(II) as catalyst using a spectrophotometric method. The reactions have been performed at temperatures range from 298 K to 318 K in interval of 10 min. In the case of Ce(IV), this reaction would be a first order reaction. The results exhibits first order each in Ce(IV) and metal ion Mn(II) and positive fractional order with respect to [L-proline]. This would have a first order reaction between the Ce(IV) and L-proline in the presence and absence SDS, CTAB and KCl. The rate constant decreases with increasing the concentration of [HSO4 −] and [H+] ion. The thermodynamical parameter values such as ΔH#, ΔG# and ΔS# increased with increasing temperature so that the rate equation derived for this mechanism could explain all observed results.

Immobilization of Proline Dehydrogenase on Functionalized Silica Mesoporous Nanomaterial Towards Preparation of a Novel Thermostable Enzyme Biosensor

In this report, a thermostable electrochemical biosensor for sensitive determination of L-proline in whole blood was prepared by immobilization of proline dehydrogenase within n-Propylamine functionalized mobile crystalline material-41 doped with iron oxide nanoparticles. By combining of magnetic mesoporous silica with proline dehydrogenase and using voltammetry analysis, a proline biosensor was fabricated on glassy carbon electrode pre-modified by poly cysteine supported beta cyclodextrin. The magnetic mesoporous silica networks having a high surface area (362 m2 g−1) exhibited excellent properties for entrapment of proline dehydrogenase. The prepared sensor was used as an electrochemical detector for selective detection of L-proline at physiological pH. The results show that the oxidation of L-proline is ir-reversible and controlled by diffusion. Using differential pulse voltammetry, the oxidation peak current was proportional to L-proline concentration in the range of 1–100 mM and 1 μM–1 nM, while the low limit of quantification was 1 nM under physiological pH. For proof-of-technology, the biosensor was used for the detection of L-proline in biological samples such as whole blood and human blood serum of neonates. It is demonstrated that engineered biosensor provides a rapid and sensitive method for determination of L-proline in neonatal blood serum samples. The current response of electrocatalytic oxidation of L-Pro was maintained in the analytical solution temperature up to 310 °K. The applied approach led to better resistance to temperature and pH inactivation in comparison to the free enzyme. The method is a potential tool for diagnostic of neonatal hyperprolinemia. Taking a good facet of the biosensor into account, it was implemented for the detection of the L-Pro in normal cell (L929) and various cancer cell lines such as gastric cancer cell-CAT 3, colon cancer cell-HCT, colon cancer cell-SW, and breast cancer cell-MCF7. Finally, this sensor was used to determine of L-Pro in unprocessed blood samples.

Proline dehydrogenase-entrapped mesoporous magnetic silica nanomaterial for electrochemical biosensing of L-proline in biological fluids.

In this work, physical adsorption was used for immobilization of proline dehydrogenase onto a magnetic mesoporous silica nanomaterial. The immobilization and electrocatalytical activity of proline dehydrogenase entrapped in a magnetic mesoporous silica nanomaterial was studied using cyclic voltammetry, differential pulse voltammetry, and square wave voltammetry. The magnetic mesoporous silica networks having a high surface area (362m2g-1) exhibited excellent properties for entrapment of proline dehydrogenase. The applied approach led to better resistance to temperature and pH inactivation in comparison to the free enzyme. The electrocatalytic current response of proline dehydrogenase entrapped in a magnetic mesoporous silica nanomaterial toward oxidation of L-proline was maintained in the analytical solution temperature up to 70°C. The entrapped proline dehydrogenase was casted onto a polycysteine-modified glassy carbon electrode. The electrode was evaluated as an electrochemical biosensor for electrooxidation and determination of L-proline in phosphate buffer solution. A cyclic voltammetry study indicated that the oxidation process of proline is irreversible and is diffusion controlled. The electrochemical behavior was further exploited as a sensitive detection scheme for L-proline determination by differential-pulse voltammetry. Under optimized conditions, the concentration range and detection limit were 0.01-0.15μM and 0.006μM, respectively. The method was applied to the assay of L-proline in whole blood and normal and malignant cell line lysates (normal cell (L929); gastric cancer cell-CAT 3, colon cancer cell-HCT, colon cancer cell-SW, and breast cancer cell-MCF7).

L-Proline sensor based on layer-by-layer immobilization of thermostable dye-linked L-proline dehydrogenase and polymerized mediator

L-Proline sensor has been fabricated through multilayer assembly of recombinant dye-linked L-proline dehydrogenase (L-proDH) originally from hyperthermophilic archaeon (Therococcus profundus) and polymerized mediator, poly(allylamine) ferrocene (PAA-Fc), on a gold electrode by electrostatic layer-by-layer alternate adsorption method. The characteristic redox peaks of ferrocene groups were obvious exhibited in cyclic voltammograms, and both anodic and cathodic peaks increased with PAA-Fc/L-proDH bilayer number which confirmed the formation of a multilayer structure. Further experiments showed that an electrode fabricated in this way catalyzed the oxidation of L-proline. It suggested that polymerized mediator could transfer electrons between electrode surface and immobilized L-proDH. Ampeormetric experiments showed that the current response to L-proline increased with PAA-Fc/L-proDH bilayer number. The stability of the sensor was measured, and it kept a 40% relative response after 30 days storage in buffer under 4 °C.

Gene and primary structures of dye-linked L-proline dehydrogenase from the hyperthermophilic archaeon Thermococcus profundus show the presence of a novel heterotetrameric amino acid dehydrogenase complex.

Dye-linked l-proline dehydrogenase catalyzes the oxidation of l-proline in the presence of artificial electron acceptors such as 2, 6-dichloroindophenol and ferricyanide. The enzyme from the hyperthermophilic archaeon Thermococcus profundus was purified and characterized for the first time in archaea by Sakuraba et al. in 2001. In this study, cloning and sequencing analyses of the gene encoding the enzyme and functional analysis of the subunits were performed. The gene formed an operon that consisted of four genes, pdhA, pdhB, pdhF, and pdhX, which are tandemly arranged in the order of pdhA-F-X-B. SDS-PAGE analysis of the purified recombinant enzyme showed four different bands corresponding to alpha (54 kDa), beta (43 kDa), gamma (19 kDa), and delta (8 kDa) subunits encoded by pdhA, pdhB, pdhF, and pdhX, respectively, and the molecular ratio of these subunits was determined to be equal. This indicates that the enzyme consists of a heterotetrameric alphabetagammadelta structure. Functional analysis of each subunit revealed that the beta subunit catalyzed the dye-linked l-proline dehydrogenase reaction by itself and that, unexpectedly, the alpha subunit exhibited dye-linked NADH dehydrogenase activity. This is the first example showing the existence of a bifunctional dye-linked l-proline/NADH dehydrogenase complex. On the basis of genome analysis, similar gene clusters were observed in the genomes of Pyrococcus horikoshii, Pyrococcus abyssi, Pyrococcus furiosus, and Archaeoglobus fulgidus. These results indicate that the dye-linked l-proline dehydrogenase is a novel type of heterotetrameric amino acid dehydrogenase that might be widely distributed in the hyperthermophilic archaeal strain.

Identification of a trans-dominant mutation affecting proline dehydrogenase in Escherichia coli.

L-Proline dehydrogenase catalyzes the oxidation of L-proline to delta 1-pyrroline-5-carboxylate, a reaction that is an important step in the utilization of proline as a carbon or nitrogen source by bacteria. A mutant of Escherichia coli K-12 lacking L-leucyl-tRNA:protein transferase had been found previously to contain about five times as much proline dehydrogenase activity as its parent strain. This difference has now been shown to be due to the presence in the parent strain of a previously unrecognized mutation. This mutation, which has been designated put-4977, specifically affects proline dehydrogenase rather than proline uptake. Although proline dehydrogenase remains inducible by L-proline in strains carrying the mutation, there is a premature cessation of differential synthesis during induction that results in a lower specific activity. The mutation shows about 50% P1-mediated cotransduction with pyrC and is therefore located at about 22 min on the E. coli chromosome. Merodiploids containing a normal F' factor still exhibit decreased enzyme activity, indicating that the put-4977 mutation is trans-dominant. The mutation cannot be detected in present stocks of the transferase-deficient mutant, suggesting that this mutant is a revertant for put-4977.

Kinetic study on the oxidation of L-proline by D-amino-acid oxidase.

1. Oxidation of L-proline by D-amino-acid oxidase [EC 1.4.3.3], at high concentrations, was confirmed by polarographic and spectroscopic studies under aerobic and anaerobic conditions, respectively. 2. Under anaerobic conditions, the formation of the enzyme-substrate complex was demonstrated spectroscopically. The absorption spectrum indicated that the enzyme in the complex exists in its oxidized state. 3. The rate constant of the formation of the enzyme-substrate complex is definitely larger than that of its breakdown. 4. L-Proline behaves as an inhibitor of this oxidase in competition with D-alanine.

OXIDATION OF L-PROLINE AND L-3,4-DEHYDROPROLINE BY D-AMINO ACID OXIDASE.

OXIDATION OF L-PROLINE AND L-3,4-DEHYDROPROLINE BY D-AMINO ACID OXIDASE.