Kcat Research Articles

Calibration-Free and Highly Sensitive Potentiometric Enzyme-Based Biosensors.

Enzyme-based amperometric biosensors have become popular for healthcare applications. However, they have been under constant pressure for technological innovation to improve their sensitivity and usability. An ideal biosensor has high sensitivity and calibration-free characteristics. This study aims to report enzyme-based glucose and lactate sensors that utilize a proposed "time-derivative of potential (dOCP/dt)" method, with a further aim being to prove theoretically and experimentally that dOCP/dt values are proportional to substrate concentration. High sensitivity is obtained regardless of the electrode size because the electrode potential is independent of the electrode area in the biosensor. Importantly, because the substrate diffusion determines the enzyme reaction rate on the sensors, the dOCP/dt biosensors can essentially eliminate external influences such as temperature and pH. The result is the successful realization of a biosensor that is calibration-free, making it a much more practical option.

Rapid determination of the antimicrobial properties of surfaces using an enzymatic activity surrogate

AbstractTypical approaches for assessing the antimicrobial activity of metals‐based surfaces involve the contact of a bacterial culture with the surface for a period of time, followed by culturing on agar plates to assess the decrease in microbial viability versus controls. This is a time‐consuming methodology requiring at least 24 h to produce a set of results, which can be a bottleneck for productivity in novel materials development. An enzyme‐based method was shown to be a satisfactory and much more rapid surrogate test for this application. A β‐galactosidase solution was applied to copper, silver, and zinc‐based antimicrobial surfaces for up to 1 h, and then the rate of colour development at 578 nm was monitored for a few minutes after addition of the chromogenic enzyme substrate chlorophenol red‐β‐d‐galactopyranoside (CPRG). Highly active antimicrobial surfaces were detected by a lack of colour development, due to enzyme inhibition by the metals. The enzymatic reaction rates were quantified and compared, demonstrating that the copper sample showed the greatest inhibition effect followed by the silver and zinc samples. The antimicrobial activity was quantified using bacteria and the plate count method, and the results correlated well with this enzyme assay, demonstrating that the metals‐based antimicrobial activities of both hard and soft (textile) surfaces could be quickly assessed with this enzyme‐based methodology.

Excellent performance of Ag6Si2O7 oxidase mimic endowed by self-released ions for rapid colorimetric determination with reduced operational interference

Increasing attention has been directed towards oxidase mimics due to their catalytic ability toward enzyme substrates in the absence of H2O2, offering great colorimetric sensing potential in assays and diagnostics. However, slow electron-transfer and unstable absorbance over time often lead to unsatisfactory enzymatic reaction rates and unreliable readouts, significantly reducing detection efficiency and accuracy. Herein, we report on an Ag6Si2O7 nanozyme utilizing 3,3′,5,5′-tetramethylbenzidine (TMB) as a chromogenic substrate, demonstrating excellent oxidase-like activity and long-term stable colorimetric signals. Experimental results reveal that the excellent catalytic kinetics of Ag6Si2O7 (Km = 0.197 mM, Vmax = 5.1 × 10−7 M/s) are attributed to ultrafast electron transfer between Ag+ and TMB, mediated by Ag2O intermediates formed from self-released alkaline ions. Additionally, self-released H2SiO42− improves the stability of the oxidized substrate, resulting in a long-term stable colorimetric signal (∼20 min), which exhibits reliable accuracy regardless of the time point at which analyte is added within 20 min, thus eliminating the interference of individual operation time errors. Ascorbic acid (AA) is selected as the target molecule to assess the performance of the Ag6Si2O7-TMB colorimetric sensing system. This approach demonstrates a wide detection range from 5 to 300 μM and a detection limit of 0.452 μM. The developed colorimetric sensing strategy exhibits good stability, specificity, rapidity, and potential application in real sample detection.

Using ion exchange chromatography for the purification of glyoxylate reductase from corn leaves and studying its characteristics

The purpose of this study was to obtain a purified preparation of the studied enzyme from corn leaves and to study its characteristics. In this work, kinetic and regulatory parameters of glyoxylate reductase were calculated for leaves of 14-day-old corn (Zea mays) seedlings grown hydroponically at 25°C. The following methods were used during the study: sample homogenisation, four-step purification including ammonium sulphate for desalting, gel filtration on G-25 columns, and ion exchange chromatography using DEAE-sephacel, as well as electrophoresis on polyacrylamide gels and quantitative analysis of protein. To study the properties of the enzyme, we used electrophoretically homogenous preparations. The influence of pH, substrate concentration, and cofactor on the rate of the enzymatic reaction was determined by a series of measurements with different values of the enzymatic reaction rate. As a result of four-stage purification, we obtained a homogeneous preparation with a specific activity of167 E/mg of protein. Ion exchange chromatography was important for purification; we obtained 1 peak of enzyme activity upon desorption in 104 mM sodium chloride. Studying the properties of glyoxylate reductase showed a significant dependence of activity on pH. The optimal pH value was 6.5 units.

Mushroom Tyrosinase: Six Isoenzymes Catalyzing Distinct Reactions.

"Mushroom tyrosinase" from the common button mushroom is the most frequently used source of tyrosinase activity, both for basic and applied research. Here, the complete tyrosinase family from Agaricus bisporus var. bisporus (abPPO1-6) was cloned from mRNA and expressed heterologously using a single protocol. All six isoenzymes accept a wide range of phenolic and catecholic substrates, but display pronounced differences in their specificity and enzymatic reaction rate. AbPPO3 ignores γ-l-glutaminyl-4-hydroxybenzene (GHB), a natural phenol present in mM concentrations in A. bisporus, while AbPPO4 processes 100 μM GHB at 4-times the rate of the catechol l-DOPA. All six AbPPOs are biochemically distinct enzymes fit for different roles in the fungal life cycle, which challenges the traditional concept of isoenzymes as catalyzing the same physiological reaction and varying only in secondary properties. Transferring this approach to other enzymes and organisms will greatly stimulate both the study of the in vivo function(s) of enzymes and the application of these highly efficient catalysts.

KINETIC MODELLING OF BIOCHEMICAL REACTIONS USING MATHСAD ANALYTICAL TOOLKIT

Background. The study of the kinetics of biochemical reactions provides a better understanding of how biological processes occur in living organisms. Understanding the peculiarities of such reactions is important for the development of new technologies, in particular for the production of biologically active substances and for the synthesis of drugs. A powerful tool for solving problems in biochemical reaction kinetics is mathematical modelling, which can be carried out using computer mathematical systems, in particular the MATHCAD analytical toolkit. Aim: to substantiate the feasibility and effectiveness of using the MATHCAD analytical toolkit to solve problems of kinetic modelling of biochemical reactions in pharmaceutical research, and to review the capabilities of MATHCAD for computer modelling in pharmacy. Materials and methods. In the context of studying the rate of enzymatic reactions and developing models, such as the Michaelis-Menten model, to describe reactions in which enzymes catalyze the transformation of substrates, the use of a computer mathematical system (CMS) is considered. CMS is a software package and environment for performing mathematical computations, modelling and visualization. The possibilities of using the MATHCAD system to create mathematical models of biochemical reactions based on kinetic equations are demonstrated. This involves the creation of differential equations describing changes in reagent concentrations over time. These equations were solved using numerical methods in MATHCAD. In addition, the results obtained are visualized using 3D graphics in MATHCAD. The stages of using the MATHCAD analytical toolkit in the kinetic modelling of biochemical reactions have been determined. Results. The use of MATHCAD in the kinetic modelling of biochemical reactions is effective for the study of: (1) the kinetics of enzymatic reactions, e.g. reactions in which an enzyme catalyzes the conversion of a substrate into a product; (2) biochemical reactions that take place in reaction vessels in which reagents mix and interact; (3) modelling of reactions in reaction vessels based on the solution of differential equations of reaction kinetics; (4) the effect of inhibitors or activators on enzymatic reactions; (5) scenarios of interaction of reagents to determine changes in the kinetics of reactions that occur when different active substances are introduced; (6) kinetics of biochemical reactions in cases where reactions are accompanied by diffusion of reagents through membranes or other semi-permeable barriers; (7) modelling the effect of diffusion processes on the kinetics of biochemical reactions; (8) models describing the kinetics of decomposition of substances, for example the decomposition of biologically active compounds in the body or in the environment; (9) predicting the effect of changes in the conditions of the reaction medium (temperature, pH, concentration of reagents) on the kinetics of biochemical reactions. It is substantiated that model descriptions of the kinetics of biochemical reactions are important for forming an understanding of the functions of biological systems, including metabolism, enzymatic reactions, and other physiological phenomena. Tools have been used to visualize the modelling results in the form of three-dimensional MATHCAD graphics, which improves the understanding of the reaction mechanism and allows a more thorough analysis of its kinetics. Conclusion. MATHCAD provides an optimized environment for kinetic modelling of biochemical reactions through its ergonomic interface. Particular advantages are the ability to work with symbolic expressions and to use a wide range of built-in functions and tools for exploring mathematical models and visualizing results. The obtained results may be important both for further scientific pharmaceutical research and for implementation in the training of future Masters of Pharmacy in the discipline of "Computer Modelling in Pharmacy" in higher medical education institutions.

Mechanistic Investigation of Enzyme Triggered Release from a Xyloglucan Matrix Tablet for Controlled Colonic Drug Delivery

The objective of this study was to investigate the mechanisms underlying drug release from a controlled colonic release (CCR) tablet formulation based on a xyloglucan polysaccharide matrix and identify the factors that control the rate of release for the purpose of fundamentally substantiating the concept and demonstrating its robustness for colonic drug delivery. Previous work demonstrated in vitro limited release of 5-aminosalicylic acid (5-ASA) and caffeine from these tablets in small intestinal environment and significant acceleration of release by xyloglucanase, an enzyme of the colonic microbiome. Targeted colonic drug delivery was verified in an animal study in vivo. In the present work, interaction of the xyloglucan matrix tablets with aqueous dissolution media containing xyloglucanase was found to lead to the spontaneous formation of a hydrated highly viscous gummy layer at the surface of the matrix which had a reduced drug content compared to the underlying regions and persisted with a nearly constant thickness that was inversely correlated to the enzyme concentration throughout the duration of the release process. Enzymatic hydrolysis of xyloglucan was determined to take place at the surface of the matrix leading to matrix erosion and a relation for the rate of enzymatic reaction as a function of bulk enzyme concentration and the concentration of dissolved xyloglucan in the gummy layer was derived. A mathematical model was developed encompassing aqueous medium ingress, matrix metamorphosis due to xyloglucan dissolution and matrix swelling, enzymatic hydrolysis of the polysaccharide and concomitant drug release due to matrix erosion and simultaneous drug diffusion. The model was fitted to data of reducing sugar equivalents in the medium reflecting matrix erosion and released drug amount. Enzymatic reaction parameters and reasonable values of medium ingress velocity, xyloglucan dissolution rate constant and drug diffusion coefficient were deduced that provided an adequate approximation of the data. Erosion was shown to be the overwhelmingly dominant drug release mechanism while the role of diffusion marginally increased at low enzyme concentration and high drug solubility. Changing enzyme concentration had a rather weak effect on matrix erosion and drug release rate as demonstrated by model simulations supported by experimental data, while xyloglucan dissolution was slow and had a stronger effect on the rate of the process. Therefore, reproducible colonic drug delivery not critically influenced by inter- and intra-individual variation of microbial enzyme activity may be projected.

The transcription factor WRKY22 modulates ethylene biosynthesis and root development through transactivating the transcription of ACS5 and ACO5 in Arabidopsis.

The WRKY transcription factor (TF) genes form a large family in higher plants, with 72 members in Arabidopsis (Arabidopsis thaliana). The gaseous phytohormone ethylene (ET) regulates multiple physiological processes in plants. It is known that 1-aminocyclopropane-1-carboxylic acid (ACC) synthases (ACSs, EC 4.4.1.14) limit the enzymatic reaction rate of ethylene synthesis. However, whether WRKY TFs regulate the expression of ACSs and/or ACC oxidases (ACOs, EC 1.14.17.4) remains largely elusive. Here, we demonstrated that Arabidopsis WRKY22 positively regulated the expression of a few ACS and ACO genes, thus promoting ethylene production. Inducible overexpression of WRKY22 caused shorter hypocotyls without ACC treatment. A qRT-PCR screening demonstrated that overexpression of WRKY22 activates the expression of several ACS and ACO genes. The promoter regions of ACS5, ACS11, and ACO5 were also activated by WRKY22, which was revealed by a dual luciferase reporter assay. A follow-up chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) and electrophoretic mobility shift assay (EMSA) showed that the promoter regions of ACS5 and ACO5 could be bound by WRKY22 directly. Moreover, wrky22 mutants had longer primary roots and more lateral roots than wild type, while WRKY22-overexpressing lines showed the opposite phenotype. In conclusion, this study revealed that WRKY22 acts as a novel TF activating, at least, the expression of ACS5 and ACO5 to increase ethylene synthesis and modulate root development.

Proximity Effects on the Reactivity of a Nonheme Iron (IV) Oxo Complex in C-H Oxidation.

Precise control of substrate positioning and orientation (its proximity to the reactive unit) is often invoked to rationalize the superior enzymatic reaction rates and selectivities when compared to synthetic models. Artificial nonheme iron (IV) oxo (Fe(IV)=O) complexes react with C(sp3)-H bonds via a biomimetic Hydrogen Atom Transfer/Hydroxyl Rebound mechanism, but rates, site-selectivity and even hydroxyl rebound efficiency (ligand rebound versus substrate radical diffusion) are smaller than in oxygenases. Herein, we quantitatively analyze how substrate binding modulates nonheme Fe(IV)=O reactivity by comparing rates and outcomes of C-H oxidation by a pair of Fe(IV)=O complexes that share the same first coordination sphere but only one contains a crown ether receptor that recognizes the substrate. Substrate binding makes the reaction intramolecular, exhibiting Michaelis-Menten kinetics and increased reaction rates. In addition, C-H oxidation occurs with high site selectivity for remote sites. Analysis of Effective Molarity reveals that the system operates at its maximal theoretical capability for the oxidation of these remote sites. Remarkably, substrate positioning also affects Hydroxyl Rebound, whose efficiency only increases on the sites placed in proximity by recognition. Overall, these observations provide evidence that supramolecular control of substrate positioning can effectively modulate the reactivity of oxygenases and its models.

Proximity Effects on the Reactivity of a Nonheme Iron (IV) Oxo Complex in C−H Oxidation

AbstractPrecise control of substrate positioning and orientation (its proximity to the reactive unit) is often invoked to rationalize the superior enzymatic reaction rates and selectivities when compared to synthetic models. Artificial nonheme iron (IV) oxo (Fe(IV)=O) complexes react with C(sp3)−H bonds via a biomimetic Hydrogen Atom Transfer/Hydroxyl Rebound mechanism, but rates, site‐selectivity and even hydroxyl rebound efficiency (ligand rebound versus substrate radical diffusion) are smaller than in oxygenases. Herein, we quantitatively analyze how substrate binding modulates nonheme Fe(IV)=O reactivity by comparing rates and outcomes of C−H oxidation by a pair of Fe(IV)=O complexes that share the same first coordination sphere but only one contains a crown ether receptor that recognizes the substrate. Substrate binding makes the reaction intramolecular, exhibiting Michaelis–Menten kinetics and increased reaction rates. In addition, C−H oxidation occurs with high site selectivity for remote sites. Analysis of Effective Molarity reveals that the system operates at its maximal theoretical capability for the oxidation of these remote sites. Remarkably, substrate positioning also affects Hydroxyl Rebound, whose efficiency only increases on the sites placed in proximity by recognition. Overall, these observations provide evidence that supramolecular control of substrate positioning can effectively modulate the reactivity of oxygenases and its models.

Polyphenol oxidase mediates (−)-epigallocatechin gallate to inhibit endogenous cathepsin activity in Apostichopus japonicus

Apostichopus japonicus (A. japonicus) has rich nutritional value and is an important economic crop. Due to its rich endogenous enzyme system, fresh A. japonicus is prone to autolysis during market circulation and storage, resulting in economic losses. In order to alleviate this phenomenon, we investigated the effect of polyphenol oxidase (PPO) mediated (−)-epigallocatechin gallate (EGCG) on the activity and structure of endogenous cathepsin series protein (CEP) from A. japonicus. Research on cathepsin activity showed that PPO mediated EGCG could significantly reduce enzyme activity, resulting in a decrease in enzymatic reaction rate. SDS-PAGE and scanning electron microscopy results showed that PPO mediates EGCG could induce CEP aggregation to form protein aggregates. Various spectral results indicated that EGCG caused changes in the structure of CEP. Meanwhile, the conjugates formed by PPO mediated EGCG had lower thermal stability. In conclusion, PPO mediated EGCG was an effective method to inhibit the endogenous enzyme activity.

Enzymatic Performance of Aspergillus oryzae α-Amylase in the Presence of Organic Solvents: Activity, Stability, and Bioinformatic Studies.

Enzymatic reactions can be modulated by the incorporation of organic solvents, leading to alterations in enzyme stability, activity, and reaction rates. These solvents create a favorable microenvironment that enables hydrophobic reactions, facilities enzyme-substrate complex formation, and reduces undesirable water-dependent side reactions. However, it is crucial to understand the impact of organic solvents on enzymatic activity, as they can also induce enzyme inactivation. In this study, the enzymatic performance of Aspergillus oryzae α-amylase (Taka-amylase) in various organic solvents both experimentally and computationally was investigated. The results demonstrated that ethanol and ether sustain Taka-amylase activity up to 20% to 25% of the organic solvents, with ether providing twice the stability of ethanol. Molecular dynamics simulations further revealed that Taka-amylase has a more stable structure in ether and ethanol relative to other organic solvents. In addition, the analysis showed that the loop located near the active site in the AB-domain is a vulnerable site for enzyme destabilization when exposed to organic solvents. The ability of Taka-amylase to preserve the secondary loop structure in ether and ethanol contributed to the enzyme's activity. In addition, the solvent accessibility surface area of Taka-amylase is distributed throughout all enzyme structures, thereby contributing to the instability of Taka-amylase in the presence of most organic solvents.

Human growth hormone produced with recombinant DNA technology

Hormones affect the metabolism by stimulating the genetic apparatus of the cell, activating enzymes and changing the rate of enzymatic reactions. They increase the formation of informative ribonucleic acid, which determines the structure of the protein, and affect the biosynthesis of proteins. Somatotropin hormone (STG) is a peptide hormone consisting of 192 amino acids and is secreted from the anterior lobe of the pituitary gland. The deficiency of this hormone causes hypophysis. The demand for recombinant somatotropin is likely to increase in the near future. Because current production technologies cannot meet the demand for cheap somatotropin due to limited production capacity and high production cost. As a result, it is necessary to study the mechanisms of production of therapeutic recombinant proteins. Recombinant somatotropin is mainly synthesized using E. coli lemo strain and is used for therapeutic purposes.

Membrane binding and lipid-protein interaction of the C2 domain from coagulation factor V

Anchoring of coagulation factors to anionic regions of the membrane involves the C2 domain as a key player. The rate of enzymatic reactions of the coagulation factors is increased by several orders of magnitude upon membrane binding. However, the precise mechanisms behind the rate acceleration remain unclear, primarily because of a lack of understanding of the conformational dynamics of the C2-containing factors and corresponding complexes. We elucidate the membrane-bound form of the C2 domain from human coagulation factor V (FV–C2) by characterizing its membrane binding the specific lipid-protein interactions. Employing all-atom molecular dynamics simulations and leveraging the highly mobile membrane-mimetic (HMMM) model, we observed spontaneous binding of FV-C2 to a phosphatidylserine (PS)-containing membrane within 2–25 ns across twelve independent simulations. FV-C2 interacted with the membrane through three loops (spikes 1–3), achieving a converged, stable orientation. Multiple HMMM trajectories of the spontaneous membrane binding provided extensive sampling and ample data to examine the membrane-induced effects on the conformational dynamics of C2 as well as specific lipid-protein interactions. Despite existing crystal structures representing presumed “open” and “closed” states of FV-C2, our results revealed a continuous distribution of structures between these states, with the most populated structures differing from both “open” and “closed” states observed in crystal environments. Lastly, we characterized a putative PS-specific binding site formed by K23, Q48, and S78 located in the groove enclosed by spikes 1–3 (PS-specificity pocket), suggesting a different orientation of a bound headgroup moiety compared to previous proposals based upon analysis of static crystal structures.

Evaluation of the Effects of Some Metal Chlorides on the Initial Reaction Rate of Crude Peroxidase from Watermelon Peels

Aims: This study evaluates the effect of some chloride on the initial reaction rates of crude peroxidase from watermelon peels. Study Design: In vitro enzyme assay. Place and Duration of Study: Department of Biochemistry, Faculty of Life Sciences, Ambrose Alli University, Ekpoma, Edo State, Nigeria between April 2021 and June 2021. Methodology: The kinetics of crude peroxidase catalyzed oxidation of 3,5,3′,5′-tetramethylbenzidine (TMB) in the presence of varying concentrations of different chloride salts and hydrogen peroxidase was determined spectrophotometrically at 655nm. The assay mixture contained 2.3 mL of sodium phosphate buffer of pH 7.0, 0.1 mL of a crude enzyme from the watermelon peels, 0.2 mL of varying concentration of the respective chloride salts, 0.2 mL of 0.02 mM TMB, and 0.2 mL of 2 mM hydrogen peroxidase added last to start the reaction. Results: Results showed that chloride salts of Pb2+ Hg2+ Fe2+ and Na+ had peroxidase activating effects at low salt concentration (1 mM). The activating effect increased proportionately up to their respective optimum salt concentration. Further increment of salt concentration above their respective optimum value lead to a decrease in initial reaction rate of the crude enzyme. Within a chloride concentration range of 0.025 mM to 3 mM, the highest activating effect recorded was in the order Pb2+ > Fe2+ > Na+ > Hg2+.. Results show that Pb2+ had the highest enzyme activating effect at the lowest concentration (0.5 mM) of the salt when compared with other salts within the same concentration range. Conclusion: These findings are of great significance in research to understanding the mechanism of action of peroxidase from the peels of watermelon, especially as the search for cheap and alternative sources of peroxidases continues.

Enzymatic degradation of poly(ethylene terephthalate) (PET): Identifying the cause of the hypersensitive enzyme kinetic response to increased PET crystallinity

Plastic pollution poses a significant environmental challenge, with poly(ethylene terephthalate) (PET) being a major contributor due to its extensive use in single use applications such as plastic bottles and other packaging material. Enzymatic degradation of PET offers a promising solution for PET recycling, but the enzyme kinetics in relation to the degree of crystallinity (XC) of the PET substrate are poorly understood. In this study, we investigated the hypersensitive enzyme kinetic response on PET at XC from ∼8.5–12% at 50 °C using the benchmark PET hydrolysing enzyme LCCICCG. We observed a substantial reduction in the maximal enzymatic reaction rate (invVmax) with increasing XC, corresponding to a 3-fold reduction in invVmax when the XC of PET increased from 8.6% to 12.2%. The kinetic analysis revealed that the level of the Mobile Amorphous Fraction (XMAF) was a better descriptor for the enzymatic degradation rate response than XC (or (100%-XC)). By continuous monitoring of the enzymatic reaction progress, we quantified the lag phase prolongation in addition to the steady-state kinetic rates (vss) of the reactions and found that the duration of the lag phase of a reaction could be predicted from the vss and XC by multiple linear regression modeling. The linear correlation between the duration of the lag phase and the vss of the enzymatic PET degradation affirmed that the LCCICCG worked via a random/endo-type enzymatic attack pattern. The longer lag phase at increased XC of PET is proposed to be due to increased substrate entanglement density as well as unproductive enzyme binding to the crystalline regions of PET. The findings enhance our understanding of PET enzymatic degradation kinetics and its dependence on substrate composition, i.e., XMAF and XC.

Electromagnetic Fields: Insight into Sources, and Their Effects on Vital Organs and the Risk of Cancer

Background: Electromagnetic radiation has become an extensive new pollution source in modern civilization. Therefore, the biological effects of electromagnetic radiation have attracted considerable attention worldwide. Objectives: The current review was aimed to highlight on sources of electromagnetic fields and their effects on vital organs and the risk of cancer. Electromagnetic sources can be classified into natural electromagnetic sources (sun, some distant stars, atmospheric discharges like thunder, or human body) and unnatural or human made sources (printers, vacuum cleaners, cellular phones, hair dryers, refrigerators, washing machines, kettles microwaves, cables that carry electrical currents, television and computers, electrical home gadgets, radio and television base stations, mobile phone base stations and phone equipment), home wiring airport, and transformers. Electromagnetic fields (EMFs) are electromagnetic waves less than 300 GHz, that are divided into extremely low frequencies (ELFs; 3–3,000 Hz), involving high-voltage transmission lines and in-house wiring; and radiofrequencies (RFs; 30 kHz to 300 GHz), involving mobile phones, smart devices, base stations, WiFi, and 5G technologies. Cell phone technology is an integral part of everyday life and its use is not only restricted to voice conversations but also conveying news, high-resolution pictures, and the internet. Exposure to electromagnetic fields might produce oxidative stress, sperm damage, DNA damage, changes in the chromatin conformation, formation of micronucleus in different cell types, gene expression, enzyme activity, and changes in the structure and function of cell membrane, stimulated an increase in apoptosis and biosynthesis of plasma metallothionein and corticosterone. It causes headaches, chronic fatigue, heart problems, stress, nausea, chest pain, gastrointestinal issues, pain in the muscles and joints, sweating, neurocognitive disturbances, eye burning, nose, ear, and throat issues, bad effects on reproductive, central nervous, endocrine, cardiovascular, and immune systems. Also, it increases anxiety-related behavior; spatial memory, and learning deficits in male mice offspring, decreased thermal pain perception, induced a sleep disturbance, latency, and day dysfunction especially in females, a change in memory performance, damage to the lens epithelial cells of rabbits after 8 hours of exposure to microwave radiation, produced lens opacity in rats, which is linked to the production of cataracts, and derangement of chicken embryo retinal differentiation. There are a relationship between exposure to electromagnetic fields and the increased incidence of the occurrence of some tumors types, particularly brain cancer and leukemia. EMFs induce damage of tissues by increasing free radicals and changing the antioxidant defense systems of tissues, eventually leading to oxidative stress which leads to behavioral, histopathological and biochemical alterations. Exposure to radar, which uses RF fields above 6 GHz similar to 5 G causes effects on production of cancer at different sites, and other diseases. The possible mechanism proposed of how EMFs lead to cancer is the impact of EMFs on free radical combination rates in certain enzymes, such as coenzyme B12-dependent ethanolamine ammonia lyase. The enzyme reaction rate may be amplified by a factor of up to 100. A case-control studies found an increased risk of gliomas, acoustic neuromas, and temporal lobe tumours in users with highest self-reported cell phone use. Exposure of experimental animals to microwave radiation caused a decrease in learning and memory ability, abnormal hippocampal morphology and abnormal Electroencephalogram. Also, it could lead to a decrease in norepinephrine and epinephrine contents in the brain, leading to neurotransmitter production disorders. Conclusion: It can be concluded that electromagnetic sources classified into natural electromagnetic and human made sources. EMFs) are electromagnetic waves less than 300 GHz are divided into extremely low frequencies and radiofrequencies. Exposure to electromagnetic fields might produce oxidative stress, which leads to histopathological and biochemical alterations in different body organs and increased risk of gliomas, acoustic neuromas, and temporal lobe tumours in users with highest self-reported cell phone use. Further studies are needed to confirm these effects in human and experimental animals.

NADH and NADPH peroxidases as antioxidant defense mechanisms in intestinal sulfate-reducing bacteria

Animal and human feces typically include intestinal sulfate-reducing bacteria (SRB). Hydrogen sulfide and acetate are the end products of their dissimilatory sulfate reduction and may create a synergistic effect. Here, we report NADH and NADPH peroxidase activities from intestinal SRB Desulfomicrobium orale and Desulfovibrio piger. We sought to compare enzymatic activities under the influence of various temperature and pH regimes, as well as to carry out kinetic analyses of enzymatic reaction rates, maximum amounts of the reaction product, reaction times, maximum rates of the enzyme reactions, and Michaelis constants in cell-free extracts of intestinal SRB, D. piger Vib-7, and D. orale Rod-9, collected from exponential and stationary growth phases. The optimal temperature (35 °C) and pH (7.0) for both enzyme’s activity were determined. The difference in trends of Michaelis constants (Km) during exponential and stationary phases are noticeable between D. piger Vib-7 and D. orale Rod-9; D. orale Rod-9 showed much higher Km (the exception is NADH peroxidase of D. piger Vib-7: 1.42 ± 0.11 mM) during the both monitored phases. Studies of the NADH and NADPH peroxidases—as putative antioxidant defense systems of intestinal SRB and detailed data on the kinetic properties of this enzyme, as expressed by the decomposition of hydrogen peroxide—could be important for clarifying evolutionary mechanisms of antioxidant defense systems, their etiological role in the process of dissimilatory sulfate reduction, and their possible role in the development of bowel diseases.

Alanine dehydrogenases from four different microorganisms: characterization and their application in L-alanine production

BackgroundAlanine dehydrogenase (AlaDH) belongs to oxidoreductases, and it exists in several different bacteria species and plays a key role in microbial carbon and nitrogen metabolism, spore formation and photosynthesis. In addition, AlaDH can also be applied in biosynthesis of L-alanine from cheap carbon source, such as glucose.ResultsTo achieve a better performance of L-alanine accumulation, system evaluation and comparison of different AlaDH with potential application value are essential. In this study, enzymatic properties of AlaDH from Bacillus subtilis 168 (BsAlaDH), Bacillus cereus (BcAlaDH), Mycobacterium smegmatis MC2 155 (MsAlaDH) and Geobacillus stearothermophilus (GsAlaDH) were firstly carefully investigated. Four different AlaDHs have few similarities in optimum temperature and optimum pH, while they also exhibited significant differences in enzyme activity, substrate affinity and enzymatic reaction rate. The wild E. coli BL21 with these four AlaDHs could produce 7.19 g/L, 7.81 g/L, 6.39 g/L and 6.52 g/L of L-alanine from 20 g/L glucose, respectively. To further increase the L-alanine titer, competitive pathways for L-alanine synthesis were completely blocked in E. coli. The final strain M-6 could produce 80.46 g/L of L-alanine with a yield of 1.02 g/g glucose after 63 h fed-batch fermentation, representing the highest yield for microbial L-alanine production.ConclusionsEnzyme assay, biochemical characterization and structure analysis of BsAlaDH, BcAlaDH, MsAlaDH and GsAlaDH were carried out. In addition, application potential of these four AlaDHs in L-alanine productions were explored. The strategies here can be applied for developing L-alanine producing strains with high titers.

Analysis of evolutionary and functional features of the bullfrog SULT1 family

We identified the bullfrog Rana catesbeiana sulfotransferase 1 (SULT1) family from the BLAST search tool of the public databases based on the SULT1 families of Nanorana parkeri, Xenopus laevis, and Xenopus tropicalis as queries, revealing the characteristics of the anuran SULT1 family. The results showed that the anuran SULT1 family comprises six subfamilies, four of which were related to the mammalian SULT1 subfamily. Additionally, the bullfrog has two SULT1Cc subfamily members that are consistent with the characteristics of the expanded Xenopus SULT1C subfamily. Several members of the bullfrog SULT1 family were suggested to play important roles in sulfation during metamorphosis. Among these, cDNAs encoding SULT1Cc1 and SULT1Y1 were cloned, and the sulfation activity was analyzed using recombinant proteins. The affinity for 2-naphthol and 3′-phosphoadenosine 5′-phosphosulfate (PAPS) and the enzymatic reaction rate were higher in SULT1Cc1 than in SULT1Y1. Both the enzymes showed inhibitory effect of many thyroid hormones (THs) analogs on the sulfation of 2-naphthol. The potency of sulfation activities of SULT1Cc1 and SULT1Y1 against T4 indicated their possible role in the intracellular T4 clearance during metamorphosis.