Organophosphorus pesticides stress deranged kinetic values of a few midgut carbohydrases of naiad of Trithemis aurora (burm.) (Odonata: Libellulidae)

2016 ◽  
Vol 1 (02) ◽  
pp. 155-160
Author(s):  
Shekhar Chand
Keyword(s):  
Substrate Concentration ◽  
Initial Velocity ◽  
Ph Value ◽  
Organophosphorus Pesticides ◽  
Enzymatic Reaction ◽  
Organophosphorus Pesticide ◽  
Enzymatic Reactions ◽  
Reaction Velocity ◽  
Straight Line ◽  
Maximum Reaction

The 40 Hrs. treatment of last instar naiad of Trithemis aurora (Burm.) in Chlorphyriphos and Quinalphos pesticides concentrations (LC50 = 5.12 ×10-7 ppm and 7.6 × 10-8 ppm) has shown significant variations in the enzyme kinetic parameters and arrested the enzymatic activity in the midgut tissue of last instar naiad of T. aurora causing deleterious effect on various carbohydrases at standard temp. and pH value. The midgut amylase ( μ and β amylase) showed the change in the velocity of enzymatic reaction under LC50 conc. of chlorpyriphos. The data of initial velocity and substrate concentration were processed to achieve their reciprocal values. These values were plotted and a characteristic Lineweaver Burke straight line was observed from the graph and values of maximum reaction velocity (Vmax) and Michaelis Menten constants (km) were assessed. The present organophosphorus pesticide showed an inhibitory impact on midgut amylase reaction velocity. The double reciprocal plot of initial velocity and substrate concentration after exposing the enzyme under LC50 conc. of chlorphyriphos resulted in varied Vmax and Km. values. These carbohydrase on treatment with LC50 conc. of chlorphyriphos showed an inhibitory change in the reaction velocity. The 1/V and 1/S values were plotted to achieve a characteristic Lineweaver – Burke pattern of Vmax and km values obtained as 5.0 × 10-2 [M] and 2.0 under LC50 chlorphyriphos stress for α amylase. The km and Vmax values were obtained from 0.625 × 10-3 [M] to 1.25 × 10-2 [M] for various other midgut carbohydrases with Vmax value obtained from 0.28 to 5.0 under chlorpyriphos stress. The Quinalphos inhibited the enzymatic efficiencies of various carbohydrases severely and changed Km and Vmax values were found under the pesticidal stress and found as potent uncompetitive inhibitor for enzymes as values compared to the controlled enzymatic reactions by deranging the kinetic values. The Km values determined as on 1/V and 1/S basis found deranged from 1.66 × 10-3 [M] to 10 × 10-2 [M]. The Vmax values were found in a range of 0.41 to 3.3 under LC50 Quinalphos stress for midgut hydrolases. The analysis of enzymec kinetic values revealed the great inhibitory and deranged activities of various carbohydrases under both the pesticide constrain. The present toxicants were found to change the enzymatic velocity negatively. The LC50 concentrations of these toxicants were sufficient to inhibit the activity of present hydrolases as α and β amylase, α glucosidase, α galactosidase, β galactosidase, β frictosidase and α trehalase obtaining a meaningful Lineweaver – Burke line of plotted reciprocals of data of reaction velocity and substrate concentration.

scholarly journals On the Velocity of Enzymatic Reactions in Michaelis–Menten-Like Schemes (Ensemble and Single-Molecule Versions)

2020 ◽  
Vol 65 (5) ◽  
pp. 412 ◽  
Author(s):  
L. N. Christophorov
Keyword(s):  
Binding Site ◽  
Single Molecule ◽  
Substrate Inhibition ◽  
Enzymatic Reaction ◽  
Enzymatic Reactions ◽  
Reaction Velocity ◽  
Conformational Regulation ◽  
Regulatory Properties

In searching non-standard ways of conformational regulation, various Michaelis–Menten-like schemes attract relentless attention, resulting in sometimes too sophisticated considerations. With the example of monomeric enzymes possessing an only binding site, we define the minimal schemes capable of bearing peculiar regulatory properties like “cooperativity” or substrate inhibition. The simplest ways of calculating the enzymatic reaction velocity are exemplified, either in the ensemble or single-molecule case.

scholarly journals Factors like Dilution and Mixing Influence Enzymatic Reactions

2020 ◽  
Vol 19 (4) ◽  
Author(s):  
Mahin Basha Syed ◽  
Venkatanagraraju Erumalla
Keyword(s):  
Enzyme Activity ◽  
Substrate Concentration ◽  
Enzymatic Reaction ◽  
Enzyme Reaction ◽  
Enzyme Concentration ◽  
Enzymatic Reactions ◽  
First Report ◽  
Enzyme Substrate ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions

Enzyme-catalyzed reactions were influenced by many factors. The enzyme reacts with the substrate and converts it into products. Enzymes are influenced by temperature, pH, enzyme concentration, and substrate concentration. This paper evaluates the hypothesis of factors that may influence enzyme activity. Two more factors that affects enzyme activity are dilution and mixing. In enzyme-substrate reactions, the small amount of dilution and mixing will not affect the enzyme activity. Dilution and mixing do not slowdowns the enzyme reaction but it enhances the enzymatic reaction up to a certain limit. Increase in dilution results in less interaction of enzyme substrate, which causes a decrease in the rate of reactions. To the best of our knowledge, this is the first report to shows that, factors like mixing and dilution also affect enzyme and substrate reactions.

Optimization of sol-gel based fibre-optic cholinesterase biosensor for the determination of organophosphorus pesticides

2000 ◽  
Vol 42 (7-8) ◽  
pp. 283-290 ◽  
Author(s):  
H.-C. Tsai ◽  
R.-A. Doong
Keyword(s):  
Fiber Optic ◽  
Ph Value ◽  
Rapid Determination ◽  
Organophosphorus Pesticides ◽  
Sol Gel ◽  
Neutral Red ◽  
Fluorescent Indicators ◽  
Low Leakage ◽  
Fiber Optic System

A sol-gel based fiber-optic biosensor with acetylcholinesterase as the biorecognition element has been developed for the rapid determination of organophosphorus pesticides. Nine fluorescent indicators, acridine, acridine orange, neutral red, DAPI, rhodamine B, fluorescein, umbelliferone, FITC on celite and FITC-dextran, have been examined to optimize the fiber-optic system. Results showed that acridine and FITCs were sensitive to the change of pH value caused by the enzyme-substrate catalysis reaction. However, the sensitivity of acridine was 260 times lower than that of FITCs. Higher toxicity of acridine to acetylcholinesterase than FITC was also observed. Moreover, the high-molecular-weight FITC-dextran showed low leakage rate when immobilizing using sol-gel technology, showing that the FITC-dextran was a suitable pH sensitive fluorescent indicator for the OPPs biosensor. The response of the fiber-optic biosensor to the substrate, acetylcholine, was highly reproducible (RSD=3.5%). A good linearity of acetylcholine in the range from 0.5 to 20 mM was also obtained (R2=0.98). Furthermore, a 30% inhibition can be achieved in 30min when 152 ppb paraoxon was added into the system. The results show the possibility for real-time determination of organophosphorus pesticides by using the biosensor developed in this study.

scholarly journals Evaluation of Enzyme Inhibitory Activity of Flavonoids by Polydopamine-Modified Hollow Fiber-Immobilized Xanthine Oxidase

Molecules ◽  
2021 ◽  
Vol 26 (13) ◽  
pp. 3931
Author(s):  
Cong-Peng Zhao ◽  
Guo-Ying Chen ◽  
Yuan Wang ◽  
Hua Chen ◽  
Jia-Wen Yu ◽  
...  
Keyword(s):  
Xanthine Oxidase ◽  
Hollow Fiber ◽  
Inhibitory Activity ◽  
Ph Value ◽  
Enzymatic Reaction ◽  
Amino Acid Residues ◽  
Reaction Conditions ◽  
Docking Analysis ◽  
Dopamine Concentration ◽  
Immobilization Time

In this study, a polydopamine (PDA)-modified hollow fiber-immobilized xanthine oxidase (XOD) was prepared for screening potential XOD inhibitors from flavonoids. Several parameters for the preparation of PDA-modified hollow fiber-immobilized XOD, including the dopamine concentration, modification time, XOD concentration and immobilization time, were optimized. The results show that the optimal conditions for immobilized XOD activity were a dopamine concentration of 2.0 mg/mL in 10.0 mM Tris-HCl buffer (pH 8.5), a modification time of 3.0 h, an XOD concentration of 1000 μg/mL in 10.0 mM phosphate buffer (pH 7.5) and an immobilization time of 3.0 h. Subsequently, the enzymatic reaction conditions such as the pH value and temperature were investigated, and the enzyme kinetics and inhibition parameters were determined. The results indicate that the optimal pH value (7.5) and temperature (37 °C) of the PDA-modified hollow fiber-immobilized XOD were consistent with the free enzyme. Moreover, the PDA-modified hollow fiber-immobilized XOD could still maintain above 50% of its initial immobilized enzyme activity after seven consecutive cycles. The Michaelis–Menten constant (Km) and the half-maximal inhibitory concentration (IC50) of allopurinol on the immobilized XOD were determined as 0.25 mM and 23.2 μM, respectively. Furthermore, the PDA-modified hollow fiber-immobilized XOD was successfully applied to evaluate the inhibitory activity of eight flavonoids. Quercetin, apigenin, puerarin and epigallocatechin showed a good inhibition effect, and their percentages of inhibition were (79.86 ± 3.50)%, (80.98 ± 0.64)%, (61.15 ± 6.26)% and (54.92 ± 0.41)%, respectively. Finally, molecular docking analysis further verified that these four active compounds could bind to the amino acid residues in the XOD active site. In summary, the PDA-modified hollow fiber-immobilized XOD is an efficient method for the primary screening of XOD inhibitors from natural products.

scholarly journals A Novel Sucrose Isomerase Producing Isomaltulose from Raoultella terrigena

2021 ◽  
Vol 11 (12) ◽  
pp. 5521
Author(s):  
Li Liu ◽  
Shuhuai Yu ◽  
Wei Zhao
Keyword(s):  
Ph Value ◽  
Wild Type ◽  
Michaelis Constant ◽  
Wild Type Enzyme ◽  
Maximum Reaction Rate ◽  
E Coli ◽  
Maximum Reaction ◽  
Conversion Rates ◽  
Raoultella Terrigena ◽  
Sucrose Isomerase

Isomaltulose is widely used in the food industry as a substitute for sucrose owing to its good processing characteristics and physicochemical properties, which is usually synthesized by sucrose isomerase (SIase) with sucrose as substrate. In this study, a gene pal-2 from Raoultella terrigena was predicted to produce SIase, which was subcloned into pET-28a (+) and transformed to the E. coli system. The purified recombinant SIase Pal-2 was characterized in detail. The enzyme is a monomeric protein with a molecular weight of approximately 70 kDa, showing an optimal temperature of 40 °C and optimal pH value of 5.5. The Michaelis constant (Km) and maximum reaction rate (Vmax) are 62.9 mmol/L and 286.4 U/mg, respectively. The conversion rate of isomaltulose reached the maximum of 81.7% after 6 h with 400 g/L sucrose as the substrate and 25 U/mg sucrose of SIase. Moreover, eight site-directed variants were designed and generated. Compared with the wild-type enzyme, the enzyme activities of two mutants N498P and Q275R were increased by 89.2% and 42.2%, respectively, and the isomaltulose conversion rates of three mutants (Y246L, H287R, and H481P) were improved to 89.1%, 90.7%, and 92.4%, respectively. The work identified a novel SIase from the Raoultella genus and its mutants showed a potential to be used for the production of isomaltulose in the industry.

scholarly journals Identification and characterization of a phospholipase C activity in resident mouse peritoneal macrophages. Inhibition of the enzyme by phenothiazines

1981 ◽  
Vol 197 (2) ◽  
pp. 523-526 ◽  
Author(s):  
Paul D. Wightman ◽  
Mary Ellen Dahlgren ◽  
James C. Hall ◽  
Philip Davies ◽  
Robert J. Bonney
Keyword(s):  
Phospholipase C ◽  
High Activity ◽  
Peritoneal Macrophages ◽  
Ph Optimum ◽  
Reaction Velocity ◽  
Neutral Ph ◽  
Maximum Reaction ◽  
Mouse Peritoneal Macrophages ◽  
Identification And Characterization

Resident mouse peritoneal macrophages contain a phospholipase C of high activity that is specific for phosphatidylinositol. The activity has a neutral pH optimum, is Ca2+-dependent and has a maximum reaction velocity of 525nmol/h per mg of protein. Certain phenothiazines are potent inhibitors of this activity.

scholarly journals Pre-fermentative treatment of a model wine with aim to serve as a functional food with decreased alcohol content

2017 ◽  
Vol 63 (01) ◽  
pp. 47-53
Author(s):  
Irina Mladenoska ◽  
Verica Petkova ◽  
Tatjana Kadifkova Panovska
Keyword(s):  
Enzyme Activity ◽  
Gluconic Acid ◽  
Functional Food ◽  
Ph Value ◽  
Enzymatic Reaction ◽  
Alginate Beads ◽  
High Concentration ◽  
Ph Optimum ◽  
Enzyme Preparations ◽  
Initial Ph

The effect of substrate concentration on the enzyme activity in the reaction of glucose conversion into gluconic acid was investigated by using three different enzyme preparations in media with two different glucose concentrations. The media were simulating the conditions in the must, thus named as minimal model must, and were composed form combination of several organic acids and glucose. Those media were having initial pH of 3.5 that is a very unfavorable for glucose oxidase activity having a pH optimum at the pH value of 5.5. Among the three preparations used, the bakery additive, Alphamalt Gloxy 5080, was the most active in the medium with glucose concentration of 10 g/L, showing conversion of more than 70% for the period of 24 h, while the same enzyme preparation in the medium with 100 g/L glucose converted only about 7% of glucose. The pH value of the medium at the beginning and at the end of the enzymatic reaction was a good indicator of the enzyme activity. It seems that for the conversion of glucose in higher concentration, enzymatic preparation in high concentration should also be used. The preliminary attempt of immobilization of two preparations of glucose oxidases in alginate beads was also performed and a successful immobilization procedure for utilization in food industry was preliminarily developed. Keywords: glucose oxidases, enzymatic pretreatment, glucose, gluconic acid, model wine, functional food

Ultrasound promotes enzymatic reactions by acting on different targets: Enzymes, substrates and enzymatic reaction systems

2018 ◽  
Vol 119 ◽  
pp. 453-461 ◽  
Author(s):  
Danli Wang ◽  
Lufeng Yan ◽  
Xiaobin Ma ◽  
Wenjun Wang ◽  
Mingming Zou ◽  
...  
Keyword(s):  
Enzymatic Reaction ◽  
Enzymatic Reactions ◽  
Reaction Systems

scholarly journals Establishing MALDI-TOF as Versatile Drug Discovery Readout to Dissect the PTP1B Enzymatic Reaction

2018 ◽  
Vol 23 (6) ◽  
pp. 561-573 ◽  
Author(s):  
Martin Winter ◽  
Tom Bretschneider ◽  
Carola Kleiner ◽  
Robert Ries ◽  
Jörg P. Hehn ◽  
...  
Keyword(s):  
Drug Discovery ◽  
High Throughput Screening ◽  
Tyrosine Phosphatase ◽  
Enzymatic Reaction ◽  
Enzymatic Reactions ◽  
Label Free ◽  
Ionization Time ◽  
Maldi Tof ◽  
Ptp1b Inhibitors ◽  
Promising Perspective

Label-free, mass spectrometric (MS) detection is an emerging technology in the field of drug discovery. Unbiased deciphering of enzymatic reactions is a proficient advantage over conventional label-based readouts suffering from compound interference and intricate generation of tailored signal mediators. Significant evolvements of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, as well as associated liquid handling instrumentation, triggered extensive efforts in the drug discovery community to integrate the comprehensive MS readout into the high-throughput screening (HTS) portfolio. Providing speed, sensitivity, and accuracy comparable to those of conventional, label-based readouts, combined with merits of MS-based technologies, such as label-free parallelized measurement of multiple physiological components, emphasizes the advantages of MALDI-TOF for HTS approaches. Here we describe the assay development for the identification of protein tyrosine phosphatase 1B (PTP1B) inhibitors. In the context of this precious drug target, MALDI-TOF was integrated into the HTS environment and cross-compared with the well-established AlphaScreen technology. We demonstrate robust and accurate IC50 determination with high accordance to data generated by AlphaScreen. Additionally, a tailored MALDI-TOF assay was developed to monitor compound-dependent, irreversible modification of the active cysteine of PTP1B. Overall, the presented data proves the promising perspective for the integration of MALDI-TOF into drug discovery campaigns.

scholarly journals Extraction and Cleanup of Organochlorine and Organophosphorus Pesticide Residues in Fats by Supercritical Fluid Techniques

1997 ◽  
Vol 80 (3) ◽  
pp. 639-646 ◽  
Author(s):  
Marvin L Hopper
Keyword(s):  
Supercritical Fluid ◽  
Pesticide Residues ◽  
Corn Oil ◽  
Organophosphorus Pesticides ◽  
Organophosphorus Pesticide ◽  
Cleanup Procedure ◽  
Diet Study ◽  
Study Methodology ◽  
Integrated Extraction ◽  
Organochlorine And Organophosphorus Pesticides

Abstract A supercritical fluid extraction and cleanup procedure was developed for separating organochlorine and organophosphorus pesticides from fats. Supercritical carbon dioxide modified with 3% (v/v) acetonitrile was used to extract the pesticides at 6O°C and separate the pesticides from the fats at 4000 psi and 95°C on an in-line C? silica-based column. The extraction and cleanup procedure gave good recoveries for 43 of 62 nonpolar to moderately polar organochlorine and organophosphorus pesticides from fats, whereas 49 were recovered through conventional Florisil column cleanup before quantitation. This procedure can extract and clean up pesticide residues from 0.65 g animalbased fat and 1.0 g oils. Coeluted residues in the pesticide fraction ranged from 2.5 mg for butterfat to 0.8 mg for corn oil. Results for samples analyzed with this integrated extraction cleanup procedure were reproducible and comparable with results obtained with the current Total Diet Study methodology.

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