Enzyme-substrate Reaction Research Articles

Comparative Assessment of Lipid Peroxidase in Oral Cancer and Oral Potentially Malignant Disorders.

Introduction Oral potentially malignant disorders (OPMD) are abnormally altered tissues that could potentially develop into oral cancer. From the literature, it is understandable that not all OPMDs develop into oral cancer. Hence, it is essential to identify the high-risk lesions that are more likely to develop into oral cancer. Lipid peroxidase (LPO) is a byproduct of phospholipidmetabolism, and its levels are an oxidative stress marker that can probably help us predict the onset of cancer in OPMDS. This study aimed to assess the levels of LPO in OPMD, oral cancer, and normal patients. Materials and methods The sample size estimated was 15 per group. There were four groups in total. The estimation was done with the Abbkine LPO enzyme-linked immunosorption assay (ELISA) kit (Atlanta, Georgia, USA). An enzyme-substrate reaction was carried out, and the degree of the color change was measured using a microplate reader. The values were tabulated, and statistics were carried out using Statistical Package for Social Sciences(SPSS) version 26.0 (IBM Corp., Armonk, NY, USA). Both descriptive and inferential statistics were carried out. Results LPO levels (nmol/L) in each of the four groups were as follows: Group 1 (oral cancer): 171.86±78.86, Group 2 (controls): 71.66±28.36, Group 3: (oral leukoplakia): 127.50±103.53, and Group 4 (oral submucous fibrosis and oral lichen planus): 100.39±41.06.The results, when compared, were statistically significant (P< 0.05). Discussion From the above results, it is understandable that oral cancer patients experience increased oxidative stress compared to the OPMD group. The current study concluded that the obtained results showed differences in LPO levels, suggesting LPO could be used as a marker and screening tool to assess the rate and severity of cellular damage in patients with oral potentially malignant disorders.

Cry Toxins from &lt;i&gt;Bt&lt;/i&gt; Bollgard II® Cotton Seeds: An Affordable Source for Insect Bioassays

The relative expression of Bacillus thuringiensis (Bt) Cry1Ac and Cry2Ab toxins from seeds of Bt Bollgard II® cotton was quantified using Enzyme Linked Immunosorbent Assays (ELISA) method. The ELISA technique involves binding a sample or calibrator to a solid-phase antibody, forming a "sandwich complex" with an enzyme-labeled antibody. The resulting enzyme substrate reaction produces a product proportional to the antigen concentration. The study reveals that each gram of Bt Bollgard II® seed powder contains 3.25 μg Cry1Ac and 182.32 μg Cry2Ab, with Cry2Ab expression 56 times higher. Variability in Cry protein expression is influenced by factors like temperature, genetic background, and plant maturity. The research emphasizes the importance of Bt plants, particularly Bt Bollgard II® seed powder as a valuable source for studying insect responses to combined Cry1Ac + Cry2Ab expression in bioassay studies.

Influence of the magnetic flux on the dynamics of a self-sustaining system: analytical, numerical and analogical investigations

This paper investigates the nonlinear dynamics of a ferroelectric enzyme-substrate reaction modeled by the birhythmic van der Pol oscillator coupled to the magnetic flux. We derive the equilibrium points and study their stability. We analyze some bifurcation structures and the variation of the Lyapunov exponents. The phenomena of symmetric attractors and the anti-monotonicity are observed. By increasing the magnetic flux, we find that the equilibrium points are stable, tends to control chaotic regimes, and affects regular and quasi-regular ones. As the magnetic flux increases, the amplitude of the oscillations around the equilibrium points decreases and the amplitude of the limit cycles at the Hopf bifurcation tends to disappear. Further increasing the magnetic flux gives rise to chaotic dynamics. The electrical circuit and analogical simulations are derived using the PSpice software. The agreement between analogical and numerical results is acceptable.

Characterization Of Keratinases From Bacillus Spp.

The present study deals with the characterization of keratinases from Bacillus brevis and Bacillus cereus isolated from the poultry wastes. During the enzyme-substrate reaction, the crude keratinases of the two bacteria showed their maximum activity at 35 and 500C and pH 10 and pH 8 with optimum incubation period of 80 and 70 minutes, respectively. The keratinases showed their activity towards casein, keratin, BSA and gelatine. It was found that keratinases belongs to metalloprotease and serine protease. Ca2+, Mg2+ and Triton X-100 increased the activity of both the keratinases, while Cu2+ and Hg2+ decreased the activity. β-Mercaptoethanol and Dimethyl sulfoxide increased the crude keratinase activity of B. brevis and B. cereus respectively, while β-Mercaptoethanol had negative effect on keratinase of B. cereus. The Chittagong Univ. J. B. Sci.,Vol. 7(1 &amp;2):109-119, 2012.

Concept of a six-fold multiplex planar bioassay to distinguish endocrine agonist, antagonist, cytotoxic and false-positive responses

To analyze a complex sample for endocrine activity, different tests must be performed to clarify androgen/estrogen agonism, antagonism, cytotoxicity, anti-cytotoxicity, and corresponding false-positive reactions. This means a large amount of work. Therefore, a six-fold planar multiplex bioassay concept was developed to evaluate up to the mentioned six endpoints or mechanisms simultaneously in the same sample analysis. Separation of active constituents from interfering matrix via high-performance thin-layer chromatography and effect differentiation via four vertical stripes (of agonists and end-products of the respective enzyme–substrate reaction) applied along each separated sample track were key to success. First, duplex endocrine bioassay versions were established. For the androgen/anti-androgen bioassay applied via piezoelectric spraying, the mean limit of biological detection of bisphenol A was 14 ng/band and its mean half maximal inhibitory concentration IC50 was 116 ng/band. Applied to trace analysis of six migrate samples from food packaging materials, 19 compound zones with agonistic or antagonistic estrogen/androgen activities were detected, with up to seven active compound zones within one migrate. For the first time, the S9 metabolism of endocrine effective compounds was studied on the same surface and revealed partial deactivation. Coupled to high-resolution mass spectrometry, molecular formulas were tentatively assigned to compounds, known to be present in packaging materials or endocrine active or previously unknown. Finally, the detection of cytotoxicity/anti-cytotoxicity and false-positives was integrated into the duplex androgen/anti-androgen bioassay. The resulting six-fold multiplex planar bioassay was evaluated with positive control standards and successfully applied to one migrate sample. The streamlined stripe concept for multiplex planar bioassays made it possible to assign different mechanisms to individual active compounds in a complex sample. The concept is generic and can be transferred to other assays.

Clinical Efficacy in Skin Hydration and Reducing Wrinkles of Nanoemulsions Containing Macadamia integrifolia Seed Oil.

This study aimed to assess natural oils for their antioxidant and anti-hyaluronidase properties and select the most effective candidate for development into nanoemulsions (NE) for clinical evaluations. The oils were assessed using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS•+) and ferric thiocyanate assays for antioxidant properties and an enzyme-substrate reaction assay for anti-hyaluronidase activity. The most potent oil was formulated into conventional emulsions (CE) and NE, which were characterized and evaluated for their stability, both in accelerated and long-term conditions. The irritation potential was assessed using both the hen's eggs chorioallantoic membrane test and a clinical trial. Skin hydration enhancement and skin wrinkle reduction efficacy were clinically assessed. Macadamia integrifolia oil exhibited significant potency as an ABTS•+ radical scavenger, lipid peroxidation inhibitor, and hyaluronidase inhibitor (p < 0.05). Both the CE and NE, comprising 15% w/w oil, 5% w/w Tween® 80 and Span® 80, and 80% w/w DI water, were found to be optimal. NE with an internal droplet size of 112.4 ± 0.8 nm, polydispersity index of 0.17 ± 0.01, and zeta potential of -31.5 ± 1.0 mV, had good stability and induced no irritation. Both CE and NE enhanced skin hydration and reduced skin wrinkles in human volunteers, while NE was outstanding in skin hydration enhancement.

Setting up of a sensitivity-modulable ELIMC assay for the determination of microcystins/nodularins in water samples

Herein, a sensitivity-modulable ELIMC (Enzyme-Linked Immuno-Magnetic Colorimetric) assay for the determination of microcystins/nodularins in water samples is presented. Firstly, we revisited a commercial ELISA microcystins/nodularins tube kit using Magnetic Beads as a solid phase of the immuno-complex. All immunological reactions occur in a single short-step procedure, followed by a colorimetric enzymatic reaction. The ELIMC assay showed a detection limit, sensitivity, working range and rapidity similar to those of the commercial kit but, thanks to the use of MBs, the analysis number was increased from 40 to 250, minimizing the use of reagents and therefore the waste generation, facilitating environmental sustainability. Satisfactory recovery and precision, obtained by analyzing controls, drinking and freshwater samples, directly without any pre-treatment, proved the suitability of the assay as a rapid screening method for numerous samples. Then, the analytical performance of the ELIMC assay was improved by making simple changes to the protocol. By doubling the enzyme-substrate reaction time and halving the concentration of the primary antibody a lower detection limit (0.03 ng/mL), an increased sensitivity (0.26 ng/mL) and a wider working range (0.05–2.0 ng/mL) were reached. Recovery from 84 to 120% and precision (RSD) from 7.3 to 16.6% demonstrated the reliability of the enhanced ELIMC assay to measure very low concentrations of MCs/NODs in water samples, allowing the adoption of timely corrective actions in the frame of environmental recovery programs.

The basal and major pilins in the Corynebacterium diphtheriae SpaA pilus adopt similar structures that competitively react with the pilin polymerase.

Many species of pathogenic gram-positive bacteria display covalently crosslinked protein polymers (called pili or fimbriae) that mediate microbial adhesion to host tissues. These structures are assembled by pilus-specific sortase enzymes that join the pilin components together via lysine-isopeptide bonds. The archetypal SpaA pilus from Corynebacterium diphtheriae is built by the Cd SrtA pilus-specific sortase, which crosslinks lysine residues within the SpaA and SpaB pilins to build the shaft and base of the pilus, respectively. Here, we show that Cd SrtA crosslinks SpaB to SpaA via a K139(SpaB)-T494(SpaA) lysine-isopeptide bond. Despite sharing only limited sequence homology, an NMR structure of SpaB reveals striking similarities with the N-terminal domain of SpaA (N SpaA) that is also crosslinked by Cd SrtA. In particular, both pilins contain similarly positioned reactive lysine residues and adjacent disordered AB loops that are predicted to be involved in the recently proposed "latch" mechanism of isopeptide bond formation. Competition experiments using an inactive SpaB variant and additional NMR studies suggest that SpaB terminates SpaA polymerization by outcompeting N SpaA for access to a shared thioester enzyme-substrate reaction intermediate.

In silico modelling of the function of disease-related CAZymes.

In silico modelling of proteins comprises a diversity of computational tools aimed to obtain structural, electronic, and/or dynamic information about these biomolecules, capturing mechanistic details that are challenging to experimental approaches, such as elusive enzyme-substrate complexes, short-lived intermediates, and reaction transition states (TS). The present article gives the reader insight on the use of in silico modelling techniques to understand complex catalytic reaction mechanisms of carbohydrate-active enzymes (CAZymes), along with the underlying theory and concepts that are important in this field. We start by introducing the significance of carbohydrates in nature and the enzymes that process them, CAZymes, highlighting the conformational flexibility of their carbohydrate substrates. Three commonly used in silico methods (classical molecular dynamics (MD), hybrid quantum mechanics/molecular mechanics (QM/MM), and enhanced sampling techniques) are described for nonexpert readers. Finally, we provide three examples of the application of these methods to unravel the catalytic mechanisms of three disease-related CAZymes: β-galactocerebrosidase (GALC), responsible for Krabbe disease; α-mannoside β-1,6-N-acetylglucosaminyltransferase V (MGAT5), involved in cancer; and O-fucosyltransferase 1 (POFUT1), involved in several human diseases such as leukemia and the Dowling-Degos disease.

Antioxidant and hypoglycemic potential of phytogenic cerium oxide nanoparticles

Plants provide humans with more than just food and shelter; they are also a major source of medications. The purpose of this research was to investigate the antioxidant and hypoglycemic potential of green synthesized CeONPs using Mentha royleana leaves extract. The morphological and physicochemical features of CeONPs were evaluated by UV–Visible spectrophotometry, Scanning Electron Microscopy, Energy Dispersive X-rays and Fourier-transform infrared spectrometry, Dynamic light scattering, Atomic Force Microscopy, Zeta Potential. The average size range of synthesized CeONPs diameter between 46 and 56 nm, crystalline in shape, with Polydispersity index value of 0.2 and subatomic particles mean diameter was 4.5–9.1 nm. The antioxidant capability of CeONPs was assessed using DPPH, ABTS+, hydrogen peroxide, hydroxyl radical scavenging, and reducing power tests. The hypoglycemic potential of CeONPs was investigated using alpha-amylase, alpha-glucosidase, glucose absorption by yeast cells, and antisucrase. The effective concentrations were 500 and 1000 µg/ml found good in suppressing radical species. To explore the hypoglycemic potential of CeONPs, alpha-amylase, alpha-glucosidase, glucose absorption by yeast cell, and antisucrase assays were performed. Glucose absorb by yeast cells assay was tested for three distinct glucose concentrations: 5 mmol/L, 10 mmol/L, and 25 mmol/L. Green synthesize CeONPs showed a dose-dependent response, higher concentrations of CeONPs imposed a stronger inhibitory impact on the catalytic site of enzymes. This study suggest that CeONPs could possibly binds to the charge carrying species and act as competitive inhibitor which slow down the enzyme substrate reaction and prevents enzymatic degradation. The study’s findings were outstanding, which bodes well for future medicinal applications of CeONPs.

Computationally guided bioengineering of the active site, substrate access pathway, and water channels of thermostable cytochrome P450, CYP175A1, for catalyzing the alkane hydroxylation reaction.

Understanding structure-function relationships in proteins is pivotal in their development as industrial biocatalysts. In this regard, rational engineering of protein active site access pathways and various tunnels and channels plays a central role in designing competent enzymes with high stability and enhanced efficiency. Here, we report the rational evolution of a thermostable cytochrome P450, CYP175A1, to catalyze the C-H activation reaction of longer-chain alkanes. A strategy combining computational tools with experiments has shown that the substrate scope and enzymatic activity can be enhanced by rational engineering of certain important channels such as the substrate entry and water channels along with the active site of the enzyme. The evolved enzymes showed an improved catalytic rate for hexadecane hydroxylation with high regioselectivity. The Q67L/Y68F mutation showed binding of the substrate in the active site, water channel mutation L80F/V220T showed improved catalytic activity through the peroxide shunt pathway and substrate entry channel mutation W269F/I270A showed better substrate accessibility to the active pocket. All-atom MD simulations provided the rationale for the inactivity of the wild-type CYP175A1 for hexadecane hydroxylation and predicted the above hot-spot residues to enhance the activity. The reaction mechanism was studied by QM/MM calculations for enzyme-substrate complexes and reaction intermediates. Detailed thermal and thermodynamic stability of all the mutants were analyzed and the results showed that the evolved enzymes were thermally stable. The present strategy showed promising results, and insights gained from this work can be applied to the general enzymatic system to expand substrate scope and improve catalytic activity.

Ultrasound-assisted green extraction methods: An approach for cosmeceutical compounds isolation from Macadamia integrifolia pericarp.

This study aimed was to examine the potential of several green extraction methods to extract cosmetic/cosmeceutical components from Macadamia integrifolia pericarps, which were a by-product of the macadamia nut industry. M. integrifolia pericarps were extracted by conventional solvent extraction process using 95% v/v ethanol and various green extraction methods, including infusion, ultrasound, micellar, microwave, and pulsed electric field extraction using water as a clean and green solvent. The extracts were evaluated for total phenolic content using Folin-Ciocalteu method.The antioxidant activities were evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferric reducing/antioxidant power, and ferric-thiocyanate method. The anti-skin ageing activities were investigated by means of collagenase, elastase, and hyaluronidase inhibition using enzyme-substrate reaction assay. The irritation profile of the extracts was evaluated by the hen's egg test-chorioallantoic membrane (HET-CAM) test. The results noted that ultrasound-assisted extraction yielded the significantly highest extract amount with the significantly highest total phenolic content (p<0.05), especially when the extraction time was 10min. The aqueous extract from ultrasound-assisted extraction possessed the most potent antioxidant and anti-skin ageing activities (p<0.05). Its antioxidant activities were comparable to ascorbic acid and Trolox, whereas the anti-skin ageing activities were equivalent to epigallocatechin-3-gallate and oleanolic acid. Besides, the extract was safe since it induced no irritation in the HET-CAM test. Therefore, ultrasound-assisted extraction was suggested as an environmentally friendly extraction method for M. integrifolia pericarp extraction and further application in the cosmetic/cosmeceutical industries.

Development of Carbon Cloth/PMG/PAMAM/MWCNTs/ADH Bioanode for Ethanol Biofuel Cell

: More recently, enzyme-based biofuel cells have attracted special attention in different areas. The alcohol dehydrogenase (ADH) due to its cheap and easily available substrate (ethanol) is one of the dehydrogenase enzymes that has been used on researches extensively. This paper explains procurement and performance of bioanode based ADH. In present paper, we used multi-walled carbon nanotubes (MWCNTs) and polyamidoamine dendrimer (PAMAM) on poly methylene green (PMG) modified carbon cloth to fabricate bioanode for an ethanol biofuel cell. The cyclic voltammetry experiments have used to study the performance of the immobilized ADH in presence of β-nicotinamide adenine dinucleotide (NAD+) and ethanol as cofactor and substrate respectively. Also, the kinetic parameter values like kcat, KMS, and k were calculated using analytical equations based on results obtained from cyclic voltammetry. The maximum anodic current of 153.37 µA obtained from the modified bioanode at a concentration of 300 mM ethanol. The kinetic parameter values kcat, KM, and k (rate constant) for enzyme-substrate reaction calculated 1.6 ± 0.1 S-1, 4.4 mM and 1.9 × 105 M-1S-1 respectively. The results showed that the composition of PAMAM and MWCNTs provides a good electronic communication for activity of ADH based on PMG/PAMAM/MWCNTs.

Drought monitoring in tobacco plants by in-vivo electrochemical biosensor

Real-time monitoring along with early detection of drought levels was studied in transgenic tobacco plants using an in-vivo technique with a bio-electrochemical sensor. This study reports on the detection of the plant’s water deficit status as indicated by its reporter gene expression, caused due to drought stress. Plant-based in-vivo sensing provides real-time information generated by the actual hydration/dehydration status of the plant; It complements the information provided by ex-vivo sensors sampling the plant’s surroundings. In the method described here, the expression of the β-D-glucuronidase enzyme, expressed under drought conditions in genetically modified tobacco plants, is monitored using the enzyme-substrate (4-nitrophenyl β-D-glucuronide) reaction generating an electrochemically active product. The generated product is oxidized on the working electrode of a three-electrode electrochemical cell-on-chip mounted on the leaves of Nicotiana tabacum plants. The transport mechanisms and the real-time behavior of enzyme activity was studied using the Michaelis-Menten kinetic diffusion model. The obtained electrochemical signals fit the Michaelis-Menten diffusion kinetic model with the mean absolute error below 5%. The proposed bioelectrochemical sensor detected drought stress earlier than other conventional methods. The sensor applicability under dehydration periods up to 3 weeks is demonstrated showing the highlight and problems of this method.

Modelling the Spread of SARS-CoV2 and its variants. Comparison with Real Data. Relations that have to be Satisfied to Achieve the Total Regression of the SARS-CoV2 Infection.

A severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ap- peared in the Chinese region of Wuhan at the end of 2019. Since then, the virus spread to other countries, including most of Europe and USA. This work provides an overview on deterministic and stochastic models that have previously been proposed by us to study the transmission dy- namics of the Coronavirus Disease 2019 (COVID-19) in Europe and USA. Briefly, we describe realistic deterministic and stochastic models for the evolution of the COVID-19 pandemic, subject to the lockdown and quar- antine measures, which take into account the time-delay for recovery or death processes. Realistic dynamic equations for the entire process are derived by adopting the so-called kinetic-type reactions approach. The lockdown and the quarantine measures are modelled by some kind of in- hibitor reactions where susceptible and infected individuals can be trapped into inactive states. The dynamics for the recovered people is obtained by accounting people who are only traced back to hospitalised infected people. To model the role of the Hospitals we take inspiration from the Michaelis-Menten’s enzyme-substrate reaction model (the so-called MM reaction) where the enzyme is associated to the available hospital beds, the substrate to the infected people, and the product to the recovered peo- ple, respectively. In other words, everything happens as if the hospitals beds act as a catalyser in the hospital recovery process. The statistical properties of the models, in particular the relevant correlation functions and the probability density functions, have duly been evaluated. We val- idate our theoretical predictions with a large series of experimental data for Italy, Germany, France, Belgium and United States, and we also compare data for Italy and Belgium with the theoretical predictions of the logistic model. We have found that our predictions are in good agreement with the real world since the onset of COVID 19, contrary to the logistics model that only applies in the first days of the pandemic. In the final part of the work, we can find the (theoretical) relationships that should be satisfied to obtain the disappearance of the virus (corresponding to a value of the effective reproduction number of the infection lower than 1).

Modelling the spreading of the SARS-CoV-2 in presence of the lockdown and quarantine measures by a kinetic-type reactions approach.

We propose a realistic model for the evolution of the COVID-19 pandemic subject to the lockdown and quarantine measures, which takes into account the timedelay for recovery or death processes. The dynamic equations for the entire process are derived by adopting a kinetic-type reactions approach. More specifically, the lockdown and the quarantine measures are modelled by some kind of inhibitor reactions where susceptible and infected individuals can be trapped into inactive states. The dynamics for the recovered people is obtained by accounting people who are only traced back to hospitalized infected people. To get the evolution equation we take inspiration from the Michaelis Menten’s enzyme-substrate reaction model (the so-called MM reaction) where the enzyme is associated to the available hospital beds, the substrate to the infected people, and the product to the recovered people, respectively. In other words, everything happens as if the hospitals beds act as a catalyzer in the hospital recovery process. Of course, in our case, the reverse MM reaction has no sense in our case and, consequently, the kinetic constant is equal to zero. Finally, the ordinary differential equations (ODEs) for people tested positive to COVID-19 is simply modelled by the following kinetic scheme n}{}S+IRightarrow 2I with n}{}IRightarrow R or n}{}IRightarrow D, with n}{}S, n}{}I, n}{}R and n}{}D denoting the compartments susceptible, infected, recovered and deceased people, respectively. The resulting kinetic-type equations provide the ODEs, for elementary reaction steps, describing the number of the infected people, the total number of the recovered people previously hospitalized, subject to the lockdown and the quarantine measure and the total number of deaths. The model foresees also the second wave of infection by coronavirus. The tests carried out on real data for Belgium, France and Germany confirmed the correctness of our model.

Classic Pentachlorophenol Hydroxylating Phenylalanine 4-Monooxygenase from Indigenous Bacillus tropicus Strain AOA-CPS1: Cloning, Overexpression, Purification, Characterization and Structural Homology Modelling.

The metabolically promiscuous pentachlorophenol (PCP) hydroxylating Phe4MO (represented as CpsB) was detected, amplified (from the genome of Bacillus tropicus strain AOA-CPS1), cloned, overexpressed, purified and characterized here. The 1.755-kb gene cloned in the pET15b vector expressed a ≅ 64kDa monomeric protein which was purified to homogeneity by single-step affinity chromatography, with a total yield of 82.1%. The optimum temperature and pH of the enzyme were found to be 30°C and 7.0, respectively. CpsB showed functional stability between pH 6.0-7.5 and temperature 25-30°C. The enzyme-substrate reaction kinetic studies showed the allosteric nature of the enzyme and followed pre-steady state using NADH as a co-substrate with apparent vmax, Km, kcat and kcat/Km values of 0.465μM.s-1, 140μM, 0.099s-1 and 7.07 × 10-4μM-1.s-1, respectively, for the substrate PCP. The in-gel trypsin digestion experiments and bioinformatic tools confirmed that the reported enzyme is a Phe4MO with multiple putative conserved domains and metal ion-binding site. Though Phe4MO has been reported to have a diverse catalytic function, here we report, for the first time, that it functions as a PCP dehalogenase or PCP-4-monooxygenase by hydroxylating PCP. Hence, the use of this enzyme may be further explored in the bioremediation of PCP and other related xenobiotics.

Paper-Based Analytical Device for the On-Site Detection of Nerve Agents.

We report a colorimetric paper-based microfluidic device based on an enzyme inhibition assay that allows the on-site detection of nerve agents by sampling and wicking. The sample and reagents are automatically transported through the channel where an enzyme inhibition reaction is conducted, followed by an enzyme-substrate reaction and a color reaction. This device can detect 0.1 μg/mL of the nerve agent VX in a 2.5 μL drop and is nerve agent selective and robust against temperature, pH, and several liquids. We confirmed that sampling procedures (dilution and wiping) are applicable to this device. Furthermore, the fabrication procedure is easy, and the cost is at most a few tens of cents. Thus, the present device provides a practical method for the urgent detection of nerve agents in suspected chemical terrorism incidents.

Esterase, Glutathione S-Transferase and NADPH-Cytochrome P450 Reductase Activity Evaluation in Cacopsylla pyri L. (Hemiptera: Psyllidae) Individual Adults.

Simple SummaryThe resistance of pest insects to insecticides is a growing problem in all types of crops. Cacopsylla pyri (L.) (Hemiptera: Psyllidae) is a key pest of pear orchards in Spain, and the large number of insecticide treatments necessary for its control may contribute to the emergence of resistance. Laboratory insecticide toxicity (mortality assays) and biochemical assays (to know the enzymatic mechanisms of the insects in acquiring resistance) are necessary to confirm the existence of this resistance. All the previous methodologies developed to evaluate enzyme activity in C. pyri to date have been performed using “pools” of adults. In this study, we determined the optimal working conditions for the evaluation of the enzymatic activities in individual insects. Determining the frequency of resistant individuals within a population could be used as an indicator for the evolution of insecticide resistance over time. Two laboratory strains, one of them selected with cypermethrin, and two field populations were analyzed for this purpose. The data obtained revealed that one of the three resistance mechanisms studied had a high level of activity and was present in a high proportion of insects in the population selected with the insecticide as well as and in the field populations. These results validated the applied methodology.Cacopsylla pyri (L.) (Hemiptera: Psyllidae) is a key pest of pear orchards in Spain. The large number of insecticide treatments necessary for control may be an important contributor to the emergence of resistance. Laboratory toxicity and biochemical assays are necessary to validate the existence of insecticide resistance and establish the underlying mechanisms. All the methodologies developed to evaluate enzyme activity in C. pyri to date have incorporated “pools” of adults to detect minimum activity ranges. In this study, we determined the optimal working conditions for evaluation of the activities of esterase, glutathione S-transferase and NADPH-cytochrome P450 reductase in individual insects via colorimetric methods using a microplate reader. The main factors affecting enzymatic analysis activity, such as enzyme source and substrate concentration, filter wavelength, buffer pH, reaction time and additives, were evaluated for optimization. Determining the frequency of resistant individuals within a population could be used as an indicator for the evolution of insecticide resistance over time. Two laboratory strains, one of them selected with cypermethrin, and two field populations were analyzed for this purpose. The data obtained revealed high values and great variation in the activity ranges of esterase (EST) in the insecticide-selected population as well as in the field populations validating the applied methodology.

Production and immobilization of β-galactosidase isolated from Enterobacter aerogenes KCTC2190 by entrapment method using agar-agar organic matrix.

In the present study, Enterobacter aerogenes KCTC2190 was isolated from soil around a cattle shed area, which was capable of producing intracellular β-galactosidase. Partially purified β-galactosidase was immobilized by entrapment method in agar-agar gel matrix. Agar-agar entrapped beads were prepared by dropping the enzyme-agar solution to ice-cooled toluene-chloroform ((3:1 (v/v)). 45.88±0.11% activity of partially purified β-galactosidase was retained after immobilization (bead shape). Maximum immobilization yield was observed in the presence of 2.5% agar-agar concentration. After immobilization, optimum temperature required for the enzyme-substrate reaction was shifted from 50 to 60 °C and the optimum reaction time was shifted from 15 to 25 min. The optimum pH for both free and immobilized β-galactosidase was pH 7. Free enzyme showed lower activation energy in comparison with the immobilized one. For free as well as immobilized β-galactosidase thermal deactivation, rate constant (kd) increased with increasing temperature while the values of decimal reduction time (D-values) and half-lives (t1/2) decreased. Immobilization process increased the t1/2 and D-values of β-galactosidase while it decreased the kd. Thermostability of immobilized β-galactosidase was higher as they showed higherenthalpy (ΔΗ0) and Gibb's free energy (ΔG0)value than those of the free β-galactosidase. The negative entropy (ΔS0) of free and immobilized β-galactosidase established that both were in a more ordered state within the temperature range (50 to 70 °C) studied. Immobilized β-galactosidase was able to retain 51.65±1.61% of its initial activity after 7 batches of enzyme-substrate reaction. Immobilized β-galactosidase showed 78.09±3.69% of its initial activity even after 40 days of storage at 4 °C.