Genome-scale metabolic models (GEMs) have been widely used for phenotypic prediction of microorganisms. However, the lack of other constraints in the stoichiometric model often leads to a large metabolic solution space inaccessible. Inspired by previous studies that take allocation of macromolecule resources into account, we developed a simplified Python-based workflow for constructing enzymatic constrained metabolic network model (ECMpy) and constructed an enzyme-constrained model for Escherichia coli (eciML1515) by directly adding a total enzyme amount constraint in the latest version of GEM for E. coli (iML1515), considering the protein subunit composition in the reaction, and automated calibration of enzyme kinetic parameters. Using eciML1515, we predicted the overflow metabolism of E. coli and revealed that redox balance was the key reason for the difference between E. coli and Saccharomyces cerevisiae in overflow metabolism. The growth rate predictions on 24 single-carbon sources were improved significantly when compared with other enzyme-constrained models of E. coli. Finally, we revealed the tradeoff between enzyme usage efficiency and biomass yield by exploring the metabolic behaviors under different substrate consumption rates. Enzyme-constrained models can improve simulation accuracy and thus can predict cellular phenotypes under various genetic perturbations more precisely, providing reliable guidance for metabolic engineering.

The present study was conducted to evaluate the effects of dietary supplementation of tea polyphenol （TP） on serum hormone, serum enzyme activity, antioxidant-related and immune-related gene expression of laying hens under heat stress. A total of 288 Chinese yellow chicken (186 days old) were randomly distributed among two treatments, each of which included 6 replicates of 24 hens. Dietary treatments were that the basal diet was supplemented with 200 mg / kg tea polyphenol. The study lasted for 7 weeks, including 1 week of adaptation and 6 weeks of the formal test. The content of high-density lipoprotein cholesterol (HDL-C) and total protein (TP) in serum significantly decreased by dietary supplementation with tea polyphenol. Dietary tea polyphenol supplementation improved serum superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) enzyme activity and decreased serum malondialdehyde (MDA) content in treatments compared to the control. However, supplementation of tea polyphenol did not affect the activity of serum catalase (CAT). The results indicated that long-term feeding of tea polyphenols help to increase the amount of hormones (FSH, E2) associated with reproduction in laying hens and thus improve egg production. It also improved the immune function of laying hens in high temperature environments. Adding tea polyphenols to the diet can significantly increase the serum IgG, IgM content of the laying hens and can upregulate the IgA content. Dietary supplementation of tea polyphenols in the laying hens significantly increased the expression of antioxidant enzyme-related genes (SOD, CAT and GPX1) in the liver. Moreover, the addition of tea polyphenols significantly increased the expression of immune-related genes (Interferon-γ (INF-γ), Interleukin 2 (IL-2) and Interleukin 4 (IL-4)) in the spleen. It is concluded that addition of tea polyphenols has a positive effect on antioxidant activity and immune function of laying hens.

β-Lactams are pharmacologically important compounds because of their various biological uses, including antibiotic and so on. β-Lactams were synthesized from benzylidene-inden derivatives and acetoxyacetyl chloride. The inhibitory effect of these compounds was also examined for human carbonic anhydrase I and II (hCA I, and II) and acetylcholinesterase (AChE). The results reveal that β-lactams are inhibitors of hCA I, II and AChE. The K_i values of β-lactams (2a-k) were 0.44-6.29 nM against hCA I, 0.93-8.34 nM against hCA II, and 0.25-1.13 nM against AChE. Our findings indicate that β-lactams (2a-k) inhibit both CA isoenzymes and AChE at low nanomolar concentrations.

The increasing global demand for biodiesel is due to the urgent need to replace mineral diesel with a fuel based on renewable energy sources. In this scenario, biocatalysis stands out as an efficient and environmentally friendly alternative to chemical catalysis. When associated with the use of immobilized enzymes, which allow their reuse in the process of obtaining biofuel, this approach makes production sustainable and reduces costs. In this sense, we investigated the use of the lipase produced by an Amazonian endophytic fungus in immobilized form as a biocatalyst in the transesterification of waste cooking oil for biodiesel production. The fungus Endomelanconiopsis endophytica QAT_7AC demonstrated high production of lipase. The lipolytic extract was precipitated in ethanol, which increased the specific enzyme activity by 28%. The lipolytic extract and the precipitated lipolytic extract were immobilized in calcium alginate beads. Immobilization efficiency was over 89%. The immobilized biocatalysts showed thermal stability (30-50 °C) and were used in the production of biodiesel using waste cooking oil and ethanol. It was possible to reuse them for up to four reaction cycles, with yields greater than 70%. These results prove the efficiency of immobilized biocatalysts in the production of ethyl esters from waste oils.

Protein–nanoparticle hybridization can ideally lead to novel biological entities characterized by emerging properties, which can sensibly differ from those of the parent components. Herein, the effect of ionic strength on the biological functions of recombinant, His-tagged spermine oxidase (i.e., SMOX) was studied for the first time. Moreover, SMOX was integrated to colloidal surface active maghemite nanoparticles (SAMNs) by direct self-assembly, leading to a biologically active nano-enzyme (i.e., SAMN@SMOX). The hybrid was subjected to an in-depth chemical-physical characterization highlighting that protein structure was perfectly preserved. The catalytic activity of the nanostructured hybrid (SAMN@SMOX) was assessed by extracting kinetics parameters using spermine as substrate and compared to the soluble enzyme as a function of ionic strength. Results revealed that the catalytic function was dominated by electrostatic interactions and that they were drastically modified upon hybridization with colloidal ɣ-Fe2O3. Noteworthy, the affinity of SMOX toward spermine was significantly higher for the nanohybrid at low salinity. The present study supports the vision of using protein–nanoparticle conjugation as a mean for modulating biological functions.

BACKGROUND: Thromboembolic disorders including Myocardial infarction[ MI] and stroke represent the leading causes of death worldwide. Fibrinolytic enzymes extracted from microbial sources showed high efficacy in the dissolution of different blood clots. AIM OF THE STUDY: Screening and the bacterial recombinant DNA manufacture of bacterial fibrinolytic enzymes from different soil environments in Egypt. METHODOLOGY: A total of 50 soil samples were screened for their fibrinolytic enzyme production. The potent fibrinolytic enzyme-producing bacterial isolates were evaluated for their thrombolytic activities using the plasma plate method. The characterization of the enzyme activity and thermostability were carried out; also, the determination of molecular mass using a mass spectrometer and western blot technique was performed. The purification of the fibrinolytic enzymes was done through the affinity chromatography technique. On the other hand, the production of the purified bacterial fibrinolytic enzyme was achieved via bacterial recombinant DNA technology. Formulation of fibrinolytic enzymes in different dosage forms was tried. RESULTS: In the present study, the purified bacterial fibrinolytic enzyme was approximately 83% pure through affinity chromatography and SDS-PAGE assay. The predominating bacterial isolate secreting fibrinolytic enzymes was found to be Bacillus cereus. The fibrinolytic enzyme activities of 50 soil samples were found to be in the range of 0.167-1.853 U/ ml. The fibrinolytic activity of the extracellular enzyme refined from the culture supernatant of Bacillus cereus was relatively stable over the PH range from 7.4-10 for 18 hours and also it showed stability with the highest productivity at a temperature of 43 0C for nearly 60 minutes. The enzyme was called fibrin-proteinase[ FP] in the present study. The FP degraded the fibrin clots via direct thrombolysis. The specific activity and the molecular mass of the FP were estimated to be nearly 40.71 Units/ mg protein and nearly 30 KDa respectively. Protease activity was noticed to be slightly increased in the presence of Fe+3; While it was inhibited in the presence of EDTA. On the other hand, the FP fibrinolytic activity was enhanced greatly after the addition of monovalent cations such as K+1 and Na +1. The enzyme was also active over the temperature range of 20- 65 0C with the highest activity at 55 0C. The optimum temperature and PH of protease production was at 35-43 0C and PH 8.0-10. Conclusion: During the present study, the thermostable fibrinolytic enzyme FP purified from Bacillus cereus in different soil environments in Egypt was a promising fibrinolytic enzyme because it showed a broader efficacious fibrinolytic activity as well as less adverse effects than the current fibrinolytic enzymes globally. It is recommended in the future advanced exploration of the optimization of suitable formulation of bacterial fibrinolytic enzymes to aid in the dissolution of various blood clots and as a consequence overcoming the high rate of mortality globally.

Thromboembolic conditions, such as stroke and myocardial infarction [MI], are the primary causes of mortality globally. High efficiency was demonstrated by fibrinolytic enzymes isolated from microbial sources in dissolving various blood clots. The study aimed to investigate and produce bacterial fibrinolytic enzymes from various Egyptian soil conditions using recombinant DNA technology. Fifty soil samples were examined to determine the production of fibrinolytic enzymes. The plasmin plate technique assessed the thrombolytic activities of the strong fibrinolytic enzyme-producing bacterial isolates. In addition to determining the molecular mass using a mass spectrometer and the western blot method, the activity and thermostability of the enzymes were characterized. An affinity chromatography approach was used to purify the fibrinolytic enzymes. However, bacterial recombinant DNA technology produced the pure bacterial fibrinolytic enzyme. Several dosage formulations for the formulation of fibrinolytic enzymes were tested. By using affinity chromatography and the SDS-PAGE test, the isolated bacterial fibrinolytic enzyme in this investigation was almost 83% pure. Bacillus cereus ATCC 4342 was identified as the predominant bacterial isolate secreting fibrinolytic enzymes. It was discovered that the fibrinolytic enzyme activity of fifty soil samples ranged from 0.167 U/ml to 1.853 U/ml. The extracellular enzyme extracted from Bacillus cereus culture supernatant demonstrated a relatively stable fibrinolytic activity over 18 hours at a pH range of 7.4–10, as well as stability with maximum productivity at 43℃ for over 60 minutes. In this investigation, the enzyme was referred to as Fibrin-proteinase[ FP]. The fibrin clots were directly thrombolyzed by the FP. The calculated values for the specific activity and molecular mass of the FP were approximately 40.71 units/mg of protein and approximately 30 KDa, respectively. It was observed that the presence of Fe+3 marginally boosted protease activity, but the presence of EDTA decreased it. On the other hand, adding monovalent cations like K+1 and Na+1 significantly increased the fibrinolytic activity of FP. The enzyme exhibited activity throughout the temperature range of 20 to 65℃, peaking at 55℃. The ideal range for the generation of protease was 35–43℃ and 8.0–10. The thermostable fibrinolytic enzyme FP, which was isolated from Bacillus cereus ATCC 4342 in various soil environments in Egypt during the current study, was a promising fibrinolytic enzyme because it demonstrated a more extensive and effective fibrinolytic activity and fewer side effects than the fibrinolytic enzymes that are currently available worldwide. It is advised that further research be done on optimizing the right formulation of bacterial fibrinolytic enzymes in the future to help dissolve different types of blood clots and, as a result, lower the high death rate that exists worldwide.

BACKGROUND: Thromboembolic disorders including Myocardial infarction[ MI] and stroke represent the leading causes of death worldwide. Fibrinolytic enzymes extracted from microbial sources showed high efficacy in the dissolution of different blood clots. AIM OF THE STUDY: Screening and the bacterial recombinant DNA manufacture of bacterial fibrinolytic enzymes from different soil environments in Egypt. METHODOLOGY: A total of 50 soil samples were screened for their fibrinolytic enzyme production. The potent fibrinolytic enzyme-producing bacterial isolates were evaluated for their thrombolytic activities using the plasma plate method. The characterization of the enzyme activity and thermostability were carried out; also, the determination of molecular mass using a mass spectrometer and western blot technique was performed. The purification of the fibrinolytic enzymes was done through the affinity chromatography technique. On the other hand, the production of the purified bacterial fibrinolytic enzyme was achieved via bacterial recombinant DNA technology. Formulation of fibrinolytic enzymes in different dosage forms was tried. RESULTS: In the present study, the purified bacterial fibrinolytic enzyme was approximately 83% pure through affinity chromatography and SDS-PAGE assay. The predominating bacterial isolate secreting fibrinolytic enzymes was found to be Bacillus cereus. The fibrinolytic enzyme activities of 50 soil samples were found to be in the range of 0.167-1.853 U/ ml. The fibrinolytic activity of the extracellular enzyme refined from the culture supernatant of Bacillus cereus was relatively stable over the PH range from 7.4-10 for 18 hours and also it showed stability with the highest productivity at a temperature of 43 0C for nearly 60 minutes. The enzyme was called fibrin-proteinase[ FP] in the present study. The FP degraded the fibrin clots via direct thrombolysis. The specific activity and the molecular mass of the FP were estimated to be nearly 40.71 Units/ mg protein and nearly 30 KDa respectively. Protease activity was noticed to be slightly increased in the presence of Fe+3; While it was inhibited in the presence of EDTA. On the other hand, the FP fibrinolytic activity was enhanced greatly after the addition of monovalent cations such as K+1 and Na +1. The enzyme was also active over the temperature range of 20- 65 0C with the highest activity at 55 0C. The optimum temperature and PH of protease production was at 35-43 0C and PH 8.0-10. Conclusion: During the present study, the thermostable fibrinolytic enzyme FP purified from Bacillus cereus in different soil environments in Egypt was a promising fibrinolytic enzyme because it showed a broader efficacious fibrinolytic activity as well as less adverse effects than the current fibrinolytic enzymes globally. It is recommended in the future advanced exploration of the optimization of suitable formulation of bacterial fibrinolytic enzymes to aid in the dissolution of various blood clots and as a consequence overcoming the high rate of mortality globally.

Natural montane peatlands generally are not a significant source of nitrous oxide (N₂O) due to environment limitation including phosphorus (P) scarcity and temperature lowness. Phosphorus enrichment and warming caused by global change are altering this limitation, likely to increase the source function of N₂O. However, the combined effects of P addition and warming on N₂O fluxes and biotic/abiotic factor in peatlands are still uncertain. To address this, we investigated the long-term (12 yrs) effects of P addition (5 and 10 kg ha^-1 yr^-1) and its interaction with warming on N₂O fluxes in a peatland. The results showed that although long-term P addition did not significantly affect the source/sink function of N₂O in the peatland, it stimulated enzyme activities, promoted peat decomposition. However, warming amplified the effect of P addition to increase N₂O emissions by stimulating enzyme activities and changing soil stoichiometry, even turned the peatland into a significant source of N₂O with an emission of approximate 100 g m^-2 during the growing season. Our study suggests that P enrichment under the current background of global warming will enhance the possibility of strong N₂O emissions in montane peatlands, which may increase the risk to further aggravate global warming.

β-glucosidases (BGLs) play a crucial role in the degradation of lignocellulosic biomass as well as in industrial applications such as pharmaceuticals, foods, and flavors. However, the application of BGLs has been largely hindered by issues such as low enzyme activity, product inhibition, low stability, etc. Many approaches have been developed to engineer BGLs to improve these enzymatic characteristics to facilitate industrial production. In this article, we review the recent advances in BGL engineering in the field, including the efforts from our laboratory. We summarize and discuss the BGL engineering studies according to the targeted functions as well as the specific strategies used for BGL engineering.

The study addresses the management of kitchen waste by transforming it into biofertilizer formulations utilizing an effective in-house developed multi-enzyme preparation. A separate hydrolysis and fermentation bioprocessing approach which in particular relates to the application of a separate enzyme preparation from Aspergillus niger P-19 to separately hydrolyze kitchen waste, followed by the growth of Klebsiella pneumoniae AP-407 in the liquid hydrolysate as well with simultaneous production of carrier-based biofertilizer. In liquid and carrier-based biofertilizers, the viable cell count reaches 3.00 × 1012 CFU/ml and 3.00 × 1012 CFU/g, respectively. The current study adopts a novel process technique for the manufacturing of both carrier and liquid biofertilizers, adopting a zero-waste approach to the management of kitchen waste.

Food production heavily depends on ammonia-containing fertilizers to improve crop yield and profitability. However, ammonia production is challenged by huge energy demand, the release of ~2% of global CO2, and political instability. To mitigate this challenge, many research efforts have developed bioprocessing technologies to make biological ammonia. This review presents three different biological approaches that drive the biochemical mechanisms to convert nitrogen gas, bioresources, or waste to biological ammonia. The use of advanced technologies— enzyme immobilization and microbial bioengineering – enhanced bio-ammonia production. This review also highlighted some challenges and research gaps that require researchers’ attention for bio-ammonia to be industrially pragmatic.

Several reviews of inhibitors of topoisomerase II literature have been published covering research before 2018. Therefore, this review is focused primarily on more recent publications with relevant points from the earlier literature. Topoisomerase II is an established target for anticancer drugs, that are further subdivided into poisons and catalytic inhibitors. Whereas most of the topoisomerase II-based drugs in clinical use are mostly topoisomerase II poisons, their mechanism of action has posed severe concern due to DNA damaging potential, including development of multi drug resistance. As a result, we are beginning to see a gradual paradigm shift towards a non-DNA damaging agents, such as the lesser studied topoisomerase II catalytic inhibitors. In addition, this review will describe some novel selective catalytic topoisomerase II inhibitors. The ultimate goal is to bring researchers up to speed by curating and delineating new scaffolds as leads for optimization and development to new potent, safe and selective agents for the treatment of cancer.

To get a better knowledge of the effects of residual chlorothalonil on soil characteristics and soil microbial communities, we evaluated the dissipation of chlorothalonil and the effects of different chlorothalonil concentrations on soil respiration, enzyme activities, and microbial community structure in yellow-brown loam soils. Bacterial and fungal soil communities were examined using traditional plate counting and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR–DGGE) methods. Soil properties and the results of DGGE band analysis were both used to estimate the status of the soil microbial ecosystem. The results show that residual chlorothalonil has considerable effects on soil respiration, enzymatic activities, and microbial community structure. In particular, soil respiration and phosphatase activities were increased, while saccharase activity, microbial biomass, and microbial community diversity were decreased by increasing levels of chlorothalonil treatment. Correlation analyses revealed that the application of chlorothalonil was significantly correlated with the change of the soil respiration, urease activity, sucrase activity, soil culturable bacteria and culturable fungi biomass. We conclude that residual chlorothalonil is directly related to soil respiration, enzyme activities, and microbial community structure.

Natural fibers are a gift from nature that we yet fully utilized until now. It can be classified into several groups and bast fibers are the group having the most promising performance when reinforced in polymer composites. However, numerous factors have been reported that influences mechanical properties of fiber reinforcements in the composite. In this review, bast fiber retting process and the effect of enzymatic retting on fiber and fibers reinforced polymer composites have been discussed and reviewed for the latest researches. Retting precedes mechanical processing (i.e. scutching) of the fiber from the stem and is essential for reduction of fiber breakage. All retting methods except chemical retting process are involving secretes of enzymes by bacteria or fungi under controlled (enzymatic retting) or random conditions (water and dew retting). Besides, enzymatic retting is claimed to have more environmentally friendly wastewater products, shorter retting period and controllable fiber biochemical components under mild incubation conditions. This review comprehensively assesses the enzymatic retting process for producing high-quality bast fiber and will become a reference for future development on bast fiber reinforced polymer composites.

To investigate the effect of montmorillonite on nonylphenol enrichment in a zebrafish model. The AB strain zebrafish were used as the animal subjects and three concentration gradients were set for both nonylphenol and montmorillonite, according to their actual concentrations in aquaculture water in Huzhou city. A group treated with nonylphenol alone was also set, adding up to 12 experimental groups. Concentrations of nonylphenol enriched in the liver, muscle, and gill of zebrafish were detected by solid phase microextraction–high performance liquid chromatography at Day 7, 15, and 30, respectively. Besides, the relative enzymatic activity of Superoxide dismutase (SOD) and the Glutathione S-transferase (GST) were also been detected, and the data were statistically analyzed. The results showed that the concentrations of nonylphenol in zebrafish peaked at Day 7 and gradually decreased afterwards for all the experimental groups. And the montmorillonite reduces short-term accumulation of nonylphenol in gills, and the high concentration of nonylphenol facilitates its enrichment in liver and muscle while the low concentration of nonylphenol doesn’t. Meanwhile, the low concentration of nonylphenol in liver exerts an influence on the inductive effect of SOD and GST while the high concentration of nonylphenol shows the inhibiting effect of SOD and GST.

A recent breakthrough in preparation of immobilized enzyme based biocatalysts achieving highly enhanced enzymatic activities and stabilities has become a great alternative to conventional immobilization techniques. The functional hybrid nanobiocatalysts (FHNs) fabricated in this immobilization composed of organic components (amino acid, peptide, protein, enzyme and plant extract) and inorganic components (various metal ions) give flower-like morphology with narrow size distribution and porous structure. The enzyme incorporated FHNs exhibite greatly enhanced catalytic activities and stabilities compared to free and conventionally immobilized enzymes under various experimental conditions. In addition to that, the FHNs consisting of other organic components act as Fenton-like reagents and show peroxidase-like activity owing to presence of metal ions and porous structure in the FHNs. This report basically focuses on preparation, characterization, and bioanalytical applications of the FHNs and explain mechanism of the FHNs formation and thier enhanced activities and stabilities.

This study was aimed to investigate the effect of xylan depolymerizing enzyme namely endo-xylanase (Xyn) combined with debranching enzymes namely arabinofuranosidase (Afd) and feruloyl esterase (FE) on digestion, growth performance and intestinal volatile fatty acid profile of piglets. The in vitro experiments were firstly conducted to examine the enzymological properties of Xyn, Afd and FE, the synergy among these enzymes, together with the effect of combination of these enzymes on digestion of piglet diet. The in vivo experiment was then implemented by allocating 270 35-d-old postweaning piglets into 3 treatment groups: control group, Xyn group and (Xyn+Afd+FE) group. Each group had 6 replicates (15 piglets/replicate). The results revealed a satisfying thermostability and pH stability of Xyn, Afd and FE. Combination of Xyn, Afd and FE had a superiority (P < 0.05) over Xyn alone and its combination with Afd or FE in promoting degradation of different bran fibers rich in arabinoxylan (Abx). Treatment with combination of Xyn, Afd and FE had advantages over Xyn alone to induce increasing trends (P < 0.10) of in vitro digestibility of dietary nutrients (dry matter, crude protein, crude ash and gross energy) and piglet growth performance (average daily gain, final body weight and feed efficiency), concurrent with a reduction (P < 0.05) of diarrhea rate and increases (P < 0.05) in cecal acetic acid, butyric acid and total volatile fatty acids concentrations as well as pH value of piglets. Collectively, combination of Xyn, Afd and FE was efficient in benefiting degradation of Abx in brans, as well as improving digestion, growth performance and intestinal volatile fatty acid profile of piglets.

Metabolomics studies require a deep understanding of enzyme dynamics and their responses to environmental disturbances within a complex metabolic network. This paper presents a novel approach to modeling enzyme activity as a dynamic topological manifold, where each enzyme is represented as a node, and their interactions are described by differential equations. These equations account for both compensatory interactions and external perturbations, simulating how enzymes dynamically stabilize their activities in response to varying conditions. We explore the application of this model to visualize enzyme interactions, using graph theory to represent the network structure and color gradients to illustrate interaction intensities. Multiple disturbances are introduced to analyze the network's resilience and adaptability over time. The results provide insights into the compensatory mechanisms within enzymatic networks, offering a comprehensive visualization through both static and dynamic representations. Our approach allows for a better understanding of how enzymatic systems buffer against mutations and environmental stressors, contributing to the broader field of systems biology and metabolomics.

Constructed wetlands (CWs) is a kind of green environmental protection technology, which is widely used in sewage treatment. The traditional CWs is faced with the problem of low treatment effect of high concentration sewage. In recent years, biochar, as a new type of adsorption material, has been used in CWs because of its advantages of large specific surface area, strong adsorption capacity and wide material sources. This paper systematically summarized the characteristics of biochar and the preparation of biochar by studying the changes of microorganisms added to CWs, and compared the effects of different treatment methods coupled with biochar on the treatment performance of CWs, the effects of biochar coupled with CWs on enzyme activity, functional genes, metabolites and microbial communities were investigated. This review summarizes how different preparation methods affect the properties of biochar and how these biochar cause changes in the microorganisms added to CWs.

Genome-scale metabolic models (GEMs) play an important role in the phenotype prediction of microorganisms, and their accuracy can be further improved by integrating other types of biological data such as enzyme concentrations and kinetic coefficients. Enzyme-constrained models (ecModels) have been constructed for several species and were successfully applied to increase the production of commodity chemicals. However, there was still no genome-scale ecModel for the important model organism Bacillus subtilis prior to this study. Here, we integrated enzyme kinetic and proteomic data to construct the first genome-scale ecModel of B. subtilis (ecBSU1) using the ECMpy workflow. We first used ecBSU1 to simulate overflow metabolism and explore the trade-off between biomass yield and enzyme usage efficiency. Then, we simulated the growth rate on eight previously published substrates and found that the simulation results of ecBSU1 were in good agreement with the literature. Finally, we identified target genes that enhance the yield of commodity chemicals using ecBSU1, most of which were consistent with the experimental data, and some of which may be potential novel targets for metabolic engineering. This work demonstrates that the integration of enzymatic constraints is an effective method to improve the performance of GEMs. The ecModel can predict overflow metabolism more precisely and can be used for the identification of target genes to guide the rational design of microbial cell factories.

Genome-scale metabolic model (GEM) is a powerful tool for interpreting and predicting cellular phenotypes under various environmental and genetic perturbations. However, GEM only consid-ers stoichiometric constraints, and the simulated growth and product yield values will show a monotonic linear increase with increasing substrate uptake rate, which deviates from the experi-mentally measured values. Recently, the integration of enzymatic constraints into stoichiometry-based GEMs was proven to be effective in making novel discoveries and predicting new engineer-ing targets. Here we present the first genome-scale enzyme-constrained model (eciCW773) for Corynebacterium glutamicum reconstructed by integrating enzyme kinetic data from various sources using ECMpy workflow based on the high-quality GEM of C. glutamicum (obtained by modifying the iCW773 model). The enzyme-constrained model improved the prediction of pheno-types and simulated overflow metabolism, while also recapitulating the trade-off between biomass yield and enzyme usage efficiency. Finally, we used eciCW773 to identify several gene modifica-tion targets for L-lysine production, most of which agree with previously reported genes. This study shows that incorporating enzyme kinetic information into the GEM enhances the cellular phenotypes prediction of C. glutamicum, which can help identify key enzymes and thus provide reliable guidance for metabolic engineering.

In this work, PMMA(Poly methyl methacrylate) microfluidic system was used as a micro-reactor for urea hydrolysis by urease enzyme with use conductivity principle and utilize sound level meter(SLM) App in smartphone as a novel detector by considering the peak height in the App as an indicate for urea concentration. the advantage of use small volume and how the reaction carried on in the microfluidic system with simple and low cost are discussed, and the results were analyzed and statically determine. The linearity, detection limit (3×noise )and Correlation Coefficient, 62.5-500 ppm, 31.25 ppm and 0.992 respectively also, recovery studied were between(98.5-100.13%).

Immobilization of enzymes is a good field of study to expand the life of enzyme in and lowering the cost of the chemical processes such as separation processes. Urease is an important enzyme with medical and industrial applications. The aim of the present study is to prepare an immobilized urease on a strong cation exchange resin (Amberlite IR120 -Na) and study of its activity and stability. We monitored the liberation of Na ions in the collected fractions and searching for protein in the fractions as an indicators of immobilization by ion exchange phenomenon. Sodium is measured using atomic absorption spectroscopy techniques, while protein tested by Bradford’s method. Immobilized urease activity was evaluated by salicylate-hypochlorite method. The results indicated a complete immobilization of urease enzyme on the resin surface with reserving 92% of the activity of free enzyme. The immobilized urease enzyme on resin showed good stability and it has a 62% of its activity after 154 days of storage at room temperature. It is concluded that a new immobilized urease enzyme system is prepared with good enzyme activity and stability.

Soil proteases are involved in the transformation of organic matter and thus influence the nutrient turnover in the ecosystem. Phytohormones, similarly to proteases, are synthesized and secreted into the soil by fungi and microorganisms and regulating their activity in the rhizosphere. The aim of our work was to find out how the presence of auxins, cytokinins, ethephone and chlorocholine chloride affects the activity of native soil proteases at the spruce tree stand. Auxins stimulated the native proteolytic activity in the spruce tree stand. Synthetic auxins most stimulated the activity of 2-naphthoxyacetic acid and the naturally occurring auxins of indole-3-acetic acid in the organic horizon of the spruce forest. Cytokinins, ethephone and chlorocholine chloride inhibited the activity of native soil proteases in the spruce tree stand. The highest inhibitory effect was found in ethephone and chlorocholine chloride. Overall, the negative effect of phytohormones on the activity of the native proteolytic activity may slow down the decomposition of organic matter and thus make plant nutrition more difficult. The outcomes of our work assist with understanding of the effect of substances produced by the rhizosphere on the activity of soil microorganisms and the soil nitrogen cycle.

The chemical investigation of the anti-yeast methanol extract from the stem bark of Terminalia mantaly led to the isolation of seven compounds: 3-O-methyl-4-O-α-rhamnopyranoside ellagic acid (1), 3-O-mehylellagic acid (2), arjungenin or 2,3,19,23-tetrahydroxyolean-12-en-28-oïc acid (3), arjunglucoside or 2,3,19,23-tetrahydroxyolean-12-en-28-oïc acid glucopyranoside (4), 2α,3α,24-trihydroxyolean-11,13(18)-dien-28-oïc acid (5), stigmasterol (6), stigmasterol 3-O-β-D-glucopyranoside (7). Their structures were established by means of spectroscopic analysis and comparison with published data. Compounds 1-5 were tested in vitro for activity against three pathogenic yeast isolates, Candida albicans, Candida parapsilosis and Candida krusei. The activity of compounds 1, 2 and 4 were comparable to that of the reference compound fluconazole (MIC values below 32 µg/ml) against the three tested yeast isolates. They were also tested for inhibitory properties against four enzymes of metabolic significance: Glucose-6-Phosphate Deshydrogenase (G6PD), human erythrocyte Carbonic anhydrase I and II (hCA I and hCA II), Glutathione S-transferase (GST). Compound 4 showed highly potent inhibitory property against the four tested enzymes with overall IC₅₀ values below 4 µM and inhibitory constant (Ki) <3 µM.

Angiotensin-converting enzyme (ACE) inhibitors are used primarily in the treatment of hypertension, heart failure, and in the acute phase of myocardial infarction. Lisinopril [N2-[(1S)-1-car-boxy-3-phenylpropyl]-L-lysyl-L-proline], enalapril [(S)-1-[N-[1-(ethoxycarbonyl)-3-phenylpropyl]-L-alanyl]-L-proline] and ramipril [2-aza-bicyclo-[3.3.0]-octane-3-carboxylic acid] are all five-membered heterocycles and three of the most prevalent ACE inhibitors in clinical use worldwide. ACEi-induced angioedema (ACEi-AE) is clinically characterized by self-limited edema of the dermis and subcutaneous lipid tissue, localized on face skin, oral mucosa and tongue in most cases. However, severe episodes of intestinal AE misdiagnosed as acute appendicitis and laryngeal AE requiring incubation have been reported. The pathophysiology of ACEi induced-AE is attributed to the accumulation of bradykinin, which is a potent vasodilator with proinflammatory activity that is normally degraded by angiotensin-converting enzyme (ACE) and aminopeptidase P; however, a small proportion of treated patients is affected. Given that patients do not respond to anti-H1 antihistamines and steroids, the early clinical recognition and the ACEi discontinuation are the treatment of choice for the long term management of ACEi-AE.

A recent report from the American Heart Association in 2018 shows that over 103 million American adults have hypertension. The angiotensin-converting enzyme (ACE) (EC 3.4.15.1) is a dipeptidyl carboxylase that, when inhibited, can reduce blood pressure through the renin-angiotensin system. ACE inhibitors are used as a first-line medication to be prescribed to treat hypertension, chronic kidney disease, heart failure among others. It has been suggested that ACE inhibitors can reduce the symptoms in mouse models. Despite the benefits of ACE inhibitors, previous studies also have suggested that alterations in the ACE gene are risk factors for Alzheimer’s disease (AD) and other neurological diseases. In mice, overexpression of ACE in the brain reduces symptoms of the AD-model systems. Thus, we find opposing effects of ACE on health. To clarify the effects, we dissect the functions of ACE as follows: (1) angiotensin-converting enzyme that hydrolyzes angiotensin I to make angiotensin II in the renin-angiotensin system; (2) amyloid-degrading enzyme that can hydrolyze beta-amyloid and reduce amyloid toxicity. The efficacy of the ACE inhibitors is well established in humans, while the knowledge specific to AD remains to be open for further research. We provide an overview of ACE and inhibitors that link a wide variety of age-related comorbidities from hypertension to Alzheimer’s disease to aging. ACE also serves as an example of the middle-life crisis theory that assumes deleterious events during the midlife, leading to age-related later events.

Celiac disease is an autoimmune disease triggered by oral ingestion of gluten, with certain gluten residues resistant to digestive tract enzymes. Within the duodenum, the remaining peptides incite immunogenic responses, including the generation of autoantibodies and inflammation, leading to irreversible damage. Our previous exploration unveiled a glutenase called Bga1903 derived from the Gram-negative bacterium Burkholderia gladioli. The cleavage pattern of Bga1903 indicated its moderate ability to mitigate the toxicity of pro-immunogenic peptides. The crystal structure of Bga1903, along with the identification of subsites within its active site, was determined. To improve its substrate specificity toward prevalent motifs like QPQ within gluten peptides, the active site of Bga1903 underwent site-directed mutagenesis according to structural insights and enzymatic kinetics. Among the double-site mutants, E380Q/S387L exhibits an approximately 38-fold increase in its specificity constant toward the QPQ sequence, favoring glutamines at the P1 and P3 positions compared to the wild type. Not only does the heightened specificity of E380Q/S387L improve the hydrolytic activity against pro-immunogenic peptides, but also holds this version of the enzyme as a promising candidate as an oral therapy enzyme for celiac disease.

A new pathway leading to the n-10 fatty acid series has been recently evidenced, starting from sapienic acid - a monounsaturated fatty acid (MUFA) resulting from the transformation of palmitic acid by delta-6 desaturase. Sapienic acid attracts attention as novel marker of cancer cell plasticity. Here, we analyzed fatty acids including the n-10 fatty acid contents, and compared for the first time cell membranes and the corresponding extracellular vesicles (EV) of two human prostatic adenocarcinoma cell lines of different aggressiveness (PC3 and LNCaP). The n-10 components were 9-13% of the total fatty acids in both cancer cell lines and EVs, with total MUFA levels significantly higher in EVs of the most aggressive cell type (PC3). High sapienic/palmitoleic ratios indicated the preference for delta-6 vs. delta-9 desaturase enzymatic activity in these cell lines. The expressions analysis of enzymes involved in desaturation and elongation by qRT-PCR showed a higher desaturase activity in PC3 and a higher elongase activity toward polyunsaturated fatty acids than toward saturated fatty acids, compared to LNCaP cells. Our results improve the present knowledge in cancer fatty acid metabolism and lipid phenotypes, highlighting EV lipidomics to monitor positional fatty acid isomer profiles and MUFA levels in cancer.

The kiwifruit cultivars include fruit having a diversity of flesh colors in the range red to yellow to green, associated with specific pigment accumulations. In the yellow-fleshed cultivars, chlorophyll degradation is necessary to unmask the underlying carotenoid pigments, which are responsible for the yellow color. This study aimed to conform the enzymatic activity involved in chlorophyll degradation using different degreening temperatures in the yellow-fleshed kiwifruit (cv. ‘Kiss,’ Actinidia chinensis var. chinensis) and in the contrasting green-flesh kiwifruit (cv. ‘Hayward’, Actinidia chinensis var. deliciosa). ‘Kiss’ was harvested at hue angle 106.9 ° and degreening was carried out at 0, 5, 10 or 15 °C for 41 d. Additionally, one week later, a second batch of kiwifruit was subsequently treated at 30 °C for one week to explore the inhibitory effect on degreening and to elucidate the roles of chlorophyllase and magnesium dechelatase enzymes in the process. Fruit degreened at 30 °C was maintained at 15°C for 23 d afterward and was compared with fruit stored at 0 °C or at 15 °C for 30 d. Similar degreening treatments were applied to cv. ‘Hayward’ at commercial maturity. Every seven days, flesh color, pigment concentrations and the activities of chlorophyllase and magnesium dechelatase in the flesh were evaluated. Degreening of ‘Kiss’ kiwifruit was enhanced by the 15 °C treatment or by 30 °C treatment for seven days and was associated with increasing of carotenoids and decreasing of chlorophyll a. The magnesium dechelatase activity was greater in fruit degreening at 15°C, which was consistently maintained throughout the degreening period; however, the activity remained low at very high temperature (30 °C). Regardless of the above, degreening at 30 °C was not inhibited, showing faster color change and high chlorophyllase activity during the exposure time. In contrast, ‘Hayward’ maintained its green color with high concentrations of the chlorophylls and carotenoids. However, the high chlorophyllase and the low magnesium dechelatase activities found in ‘Hayward’, suggest the chlorophyllase enzymes are not the only ones involved in flesh degreening.

β-hydroxybutyrate (BHB) is generated as one of the ketone bodies in the liver under fasted and hypoglycemic conditions. While BHB is utilized as an alternative energy substrate in extrahe-patic tissues/organs, this ketone body has been reported to exert anti-inflammatory effects via modulating inflammatory signaling cascades. Several bacterial pathogens that originated in hy-gienic litter conditions consecutively threaten chickens, and its harmful constituent, lipopoly-saccharide (LPS), induces intestinal and systemic inflammation. Therefore, the management of inflammation is a significant concern in poultry production. However, the therapeutic effects of BHB on inflammation have yet to be investigated in chickens. This study demonstrated that BHB administration can alleviate LPS-induced inflammation in broiler chickens, and the effects could be dependent on gene expression levels of ketolytic enzymes, BHB dehydrogenase-1 and suc-cinyl-CoA:3-ketoacid CoA transferase, in extrahepatic tissues/organs. This study is the first to show the therapeutic effects of BHB on LPS-induced inflammation in chickens, possibly via an involvement of BHB utilization.

Glycerol represents a useful functional component of heat-transfer fluids. Glycerol is known to be triboelectrically active. We report how the flow of glycerol, stopped in a cylindrical coiled pipe, influences horseradish peroxidase (HRP) enzyme. Namely, glycerol flow was pumped through the coil and then stopped. After that, we incubated the 0.1 µM solution of HRP near the side of the coil, and then adsorbed HRP onto mica. This operation markedly increased the HRP aggregation on mica in comparison with the control sample. We explain this phenomenon by the influence of triboelectrically induced electromagnetic fields. Our results can be useful in the development of flow-based systems for biosensors and bioreactors.

Schiff bases attract research interest due to their applications in chemical synthesis and medicinal chemistry. In recent years, benitrobenrazide and benserazide containing imine moiety, have been synthesized and characterized as promising inhibitors of hexokinase 2 (HK2) an enzyme overexpressed in most cancer cells. Both compounds possess a common structural fragment, a 2,3,4-trihydroxybenzaldehyde moiety connected through hydrazone or hydrazine linker with acylated on N’-nitrogen atom by serine or 4-nitrobenzoic acid fragment. To avoid presence of toxicophoric nitro group in benitrobenrazide molecule we introduced common pharmacophores 4-fluorophenyl or 4-aminophenyl substituents. Modification of benserazide regards an introduction of other endogenous amino acids instead of serine. Herein we report the synthesis of benitrobenrazide and benserazide analogues and preliminary results of inhibitory activity against HK2 evoked by these structural changes. The derivatives contain a fluorine atom or amino group instead of a nitro group in BNB, exhibited the most potent inhibitory effects against HK2 at a concentration of 1 µM, with HK2 inhibition rates of 60% and 54%, respectively.

Wolfenden and his coworkers observed the astronomical numbers for the catalytic proficiency of some enzymes. We connected that pinnacle of biological evolution to the universal thermodynamic evolution. We added or multiplied a random noise with chosen rate constants to explore the correlation between dissipation and enzyme efficiency for ten enzymes: beta-galactosidase, glucose isomerase, β-lactamases from three bacterial strains, ketosteroid isomerase, triosephosphate isomerase, and carbonic anhydrase I, II, and T200H. The turnover number kcat and catalytic efficiency kcat/KM are proportional to the overall entropy production – the main parameter from irreversible thermodynamics. For most enzymes with the Michaelis-Menten type cycle kinetics, the best increase in the forward kcat/KM follows after increasing the equilibrium constant of substrate-enzyme association. The Discussion section emphasizes the role of biological evolution in harvesting order (high enzyme efficiency) from disorder (high noise and dissipation). It also connects the applications of the maximum partial entropy production theorem in optimizing enzyme kinetics (D. Juretić "Bioenergetics - A Bridge across Life and Universe") with the present total entropy production role analysis. De novo enzyme design and various attempts to speed up the rate-limiting catalytic steps may profit from our theoretical insights.

Enzyme immobilization is known to enhance polypeptide resistance towards environmental changes, particularly in terms of their stability. The Y509E mutant of β-xylosidase from Geobacil-lus stearothermophilus (XynB2Y509E) (which also bears xylanase activity) has been immobilized in chitosan spheres through either entrapment or covalent bond formation methods. In the immo-bilization by entrapment, besides observing a total loss of xylanase activity, it was found that chitosan concentration affects the process efficiency. Additionally, the maturation time of the spheres and the alkaline pH used to precipitate the spheres affect the β-xylosidase activity of the enzyme. In covalent bond immobilization, it was found that the pH during the modification with glutaraldehyde, as well as the subsequent protein binding, were key factors in preserving the dual enzymatic activity. The activity expressed after immobilization by covalent bonding was 23% higher compared to the activity expressed following entrapment immobilization, with values of 122.3 and 99.4 U. g-1, respectively. Both biocatalysts showed increased thermal and pH stability, along with an improved storage capacity, as they retained 40% and 88% of their activi-ty after being stored at 4 C for two months. Moreover, XynB2Y509E immobilized by covalent binding also exhibited outstanding reusability, retaining a 92% of activity after ten cycles of re-use.

The TGACG motif-binding factor1 (TGA1) transcription factor, a significant protein in the bZIP transcription factor family, exhibits a myriad of functions, contributes to numerous biological events, and holds substantial application potential. In this study, our analysis revealed a diverse range of photoregulatory and hormone regulatory elements within the MtTGA1 promoter region. The expression profile of MtTGA1 indicated its highest expression in the root, with its regulation influenced by SA, ABA, BR, and GA. Under salt stress conditions, transgenic plants demonstrated significantly longer root lengths and heightened activities of antioxidant enzymes such as ascorbic acid catalase (APX), peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) in their roots and leaves compared to control plants. Simultaneously, the levels of endogenous hormones ABA and BR escalated in transgenic plants, with a marked change in the morphology of their leaf cells. Transcriptome analysis identified a total of 193 differentially expressed genes, implicating a wide array of biological processes. Furthermore, we found that MtTGA1 is able to interact with Medicago truncatula SPX domain containing protein 1 (SPX1). In conclusion, the MtTGA1 transcription factor plays a crucial role in enhancing salt tolerance, which provides new insights for improving plant survival under salt stress conditions.

Soil enzyme activity can be affected by both production and degradation, as enzymes can be degraded by proteases. However, the impact of nutrient addition on enzyme activity is often solely attributed to changes in enzyme production without fully considering degradation. Here, we demonstrated that the activities of β-1,4-glucosidase (BG), β-D-cellobiohydrolase (CBH), β-1,4-Xylosidase (BX), and β-1,4-N-acetyl-glucosaminnidase (NAG) were comparable in nitrogen (N) and phosphorus (P) fertilized soils and the unfertilized control under field conditions, but the reduction in activity was substantially greater in the fertilized soils during short-term laboratory incubation. The results show that the interruption of the natural, continuous supply of organic matter or non-soil microbial-derived enzymes, which typically occurs under field conditions, leads to a more significant reduction in apparent enzyme activities in fertilized soils compared to unfertilized control. This may be attributed to the higher abundance of protease in fertilized soils, resulting in faster enzyme degradation. Interestingly, P fertilization alone did not have a similar effect, indicating that N fertilization is likely the main cause of the larger decreases in enzyme activity during incubation in fertilized soils compared to unfertilized control soils, despite our study site being poor in P and rich in N. These findings highlight the importance of considering enzyme degradation when investigating material dynamics in forest ecosystems, including the impact of nutrient addition on enzyme activity, as enzyme production alone may not fully explain changes in apparent enzyme activity.

Black horehound (Ballota nigra L.) is one of the important medicinal plants, which is a rich source of health-promoting essential oils. Salinity stress affects plant development and alters the quality and quantity of plants extracts and their composition. This study was aimed to investigate the effect of salinity on morphological, physiological characteristics, and secondary metabolites of B. nigra under greenhouse, and in vitro culture conditions. The plants were treated with different concentrations of NaCl (25, 50, 75, 100 mM) and fresh and dry weight of leaf and stem were measured as well as morphological characteristics of the plant. Plant growth was reduced with the increased salinity concentrations. The results showed that all growth-related traits and SPAD were decreased both in vivo and in vitro. Additionally, increased salt concentration affected the cell membrane integrity. Total phenolics content of plants growing in the greenhouse, increased by 21% at 50 mM NaCl, but at higher stress levels (100 mM NaCl), the amounts were decreased significantly. Total flavonoids contents followed similar patterns, with a slight difference. In addition, the maximum and minimum total phenolics contents of plants growing under in vitro condition were observed at 50 mM NaCl and control treatments, respectively. Increasing the salt concentration significantly affected the total flavonoids content, and as a result, the highest amount was observed in 50 and 75 mM NaCl treatments. Antioxidant activity was also measured. Among the NaCl treatments, the highest DPPH scavenging activities (IC50) under greenhouse and in vitro conditions were detected at 50 mM and 25 mM concentrations, respectively. In general, based on the results, with increasing the salinity level to 75 mM, the activities of CAT and APX were significantly upregulated in both greenhouse and in vitro culture conditions. A correlation between total phenolics and flavonoids contents as well as antioxidant activity were obtained. With shifting salinity stress, the type and the amount of the identified essential oil compounds changed. Compounds such as styrene, tridecanol, germacrene-D, beta-Ionone, beta-bisabolene, and caryophyllene oxide increased compared to the controlled treatment.

Extracellular enzymes catalyze biogeochemical reactions in soil, cycling carbon and nutrients in agricultural systems. Enzymes respond quickly to soil management, including organic amendment inputs, such as biochar, a charcoal-like solid byproduct of bioenergy production. In a previous agricultural field trial, a pine biochar amendment caused an approximately 40% decrease in the enzyme activities of β-glucosidase (BG) and phosphatase (PHOS). The large surface area of the pine biochar has the potential to sorb nutrients and other organic molecules. To test if sorption caused decreased enzyme activity, we used a laboratory assay to quantify the activity of two sorbed enzymes: BG and acid PHOS, involved in the cycling of carbon and phosphorous. The enzymes were incubated with three solid phases: (1) the high surface area pine biochar, (2) the agricultural soil, and (3) a low surface area grass biochar, for an additional comparison. We quantified the sorbed enzymes at pH 6, 7, and 8, using a Bradford protein assay, and measured the immobilized enzyme activities via high-throughput fluorometric analysis. After sorption onto pine biochar, detectable BG and PHOS activity levels dropped by over 95% relative to the soil, supporting direct sorption as one mechanism that reduces enzyme activity in biochar amended soil. This laboratory assay demonstrated that sorption could account for the lack of priming of native soil organic matter and changes in soil phosphorous cycling after pine biochar addition.

COX II containing a dual core CuA active site is one of the three core subunits of mitochondrial Cco, which plays a significant role in the physiological process. In this report, the full-length cDNA of COXⅡ gene was cloned from Sitophilus zeamais, which had an ORF of 684 bp encoding 227 amino acids residues. The predicted COXⅡ protein had a molecular mass of 26.2 kDa with pI value of 6.37, and multiple sequence alignment and phylogenetic analysis indicated that Sitophilus zeamais COXⅡ had high sequence identity 78.51% with the COXⅡ of other insect species, especially similarity to sitophilus oryzae. This gene was subcloned into the prokaryotic expression vector pET-32a, and induced by IPTG in E.coli Transetta (DE3) expression system. Finally the COXⅡ with 6-His tag was purified using affinity chromatography with Ni²⁺-NTA agarose. WB showed the recombinant COXⅡ was about 44 kD, and the concentration of fusion protein was 50μg/mL. UV-spectrophotometer and infrared spectrometer analysis showed that recombinant COXⅡ could catalyze the oxidation of substrate Cytc, and influenced by AITC. It was found that AITC could form a hydrophobic region with COXⅡ protein via molecular docking, besides, a sulfur atom of AITC structure could form a length of 2.9 Å hydrogen bond with Leu-31. These results will provide valuable information for elucidating the role of COXⅡ in Sitophilus zeamais responses to AITC, meanwhile, it will helpful to carry out a point mutation in AITC binding sites for the future research.

Plant extracts are widely used in sustainable agriculture practices to enhance crop production and reduce chemical usage in agriculture. Using various extraction solutions of different plant extracts, this study synthesized planting and spraying techniques and extracted the persistence efficacy of rice. The influence of four water extracts on rice plant secondary compounds applied at various planting times. Psidium guajava, Aloe vera, Allium sativum, and Medicago sativa extracts with concentrations of 0.1 and 0.5% produced better growth promotion rates than urea 0.6%. Water extracts were more effective than foliar extracts in promoting rice growth when applied as soil. When direct seeding and transplanting methods were used, plants treated with these four selected extracts showed increases in shoot fresh weight of 48 and 68%, respectively. Thus, transplanted plants were more suitable for determining the influence of plant extracts on rice growth. The highest shoot fresh weight of two rice varieties was obtained seven days after treatment (DAT), followed by 14, 21, and 28 DAT. This study suggests that photosynthesis and antioxidant activities may play crucial roles in plant growth. These extracts can be used to promote plant growth in organic farming.

Nowadays circular economy trend drives researchers in the recovery of various bioactive compounds from agri-food by-products. Enzyme-assisted extraction (EAE) is shown as an innovative green technology for the effective extraction of various phytochemicals from agri-food section by-products, therefore this study aimed to evaluate the application of EAE as green technology to obtain extracts from olive leaves (Olea europaea) for potential industrial production. EAE was conducted under various enzyme dose combinations, and incubation time (120 min). Obtained extracts were characterized in terms of total polyphenols (TP) and Total Antioxidant Activity (AA). Firstly, the enzyme synergistic effect in enzymatic extraction of polyphenols was evaluated. TP optimal extraction conditions (468.19 mg GAE/L) were achieved after EAE (50-50%) and for AA (69.85 AA%). According to the above results, a second experimental part occurred for the incubation time (min.) and enzyme dose (mL) of optimal extraction conditions of olive leaves. Results indicated EAE as an excellent choice for green extraction of polyphenols from the olive leaves.

The shikimate pathway is crucial for the biosynthesis of aromatic amino acids in plants and represents a promising target for developing new herbicides. This work aimed to identify inhibitors of shikimate dehydrogenase (SDH), a key enzyme of the shikimate pathway that catalyzes the conversion of 3-dehydroshikimate to shikimate. Virtual screening and molecular docking were performed on the SDH active site of Arabidopsis thaliana (AtSDH), and 6-nitroquinazoline-2,4-diol (NQD) was identified as a potential inhibitor. In vitro assays showed that NQD decreased the activity of AtSDH by reducing Vmax while keeping KM unchanged, indicating noncompetitive inhibition. In vivo, hydroponic experiments revealed that NQD reduced the root length of soybean and maize. Additionally, NQD increased the total protein content and certain amino acids. Soybean roots uptake NQD more efficiently than maize roots. Furthermore, NQD reduced shikimate accumulation in glyphosate-treated soybean roots, suggesting its potential to restrict the flow of metabolites along the shikimate pathway in soybean. Simultaneous treatment of maize seedlings with glyphosate and NQD accumulated gallic acid in the roots, indicating that NQD inhibits SDH in vivo. Overall, the data indicate that NQD inhibits SDH both in vitro and in vivo, providing valuable insights into the potential development of herbicides targeting SDH.

Nereis as a mudflat organism has a variety of uses, in the history of China as a Chinese herbal record, because of its body contains rich amino acids, fatty acids and other nutrients, it can be used for therapeutic treatment and treatment can also be used as health care products. acids, fatty acids and other nutrients, it can be used for therapeutic treatment and treatment can also be used as health care products. In modern times, more and more researchers have studied the protease extract of Nereis as a mudflat organism. In modern times, more and more researchers have studied the protease extracted from the body of nereis and found that there are a variety of functional proteins in nereis, including superoxide dismutase, superoxide dismutase, superoxide dismutase and superoxide dismutase. More and more researchers have studied the proteases extracted from the body of nereis and found that there are a variety of functional proteins in nereis, including superoxide dismutase, adenylate cyclase, glycosidase, and plasminase. In this paper, the functional proteases in the extracts of nereis were summarized, and the functions and characteristics of proteases in nereis were discussed from the perspective of molecular structure.

Flower color is an important ornamental feature, often modulated by the contents of flavonoid. Chalcone synthase is the first key enzyme in the biosynthesis of flavonoid, but little is known about the role of R. delavayi CHS in flavonoid biosynthesis. In this paper, three CHS genes (RdCHS1-3) were successfully cloned from R. delavayi flowers. According to multiple sequence alignment and phylogenetic analysis, only RdCHS1 contained all the highly conserved and important residues, and classified into the cluster of bona fide CHSs. Thus RdCHS1 was then subjected for further functional analysis. Real-Time PCR analysis revealed that transcripts of RdCHS1 was the highest in leaves and lowest in root, did not match the anthocyanin accumulation patterns during flower development. Biochemical characterization displayed that RdCHS1 could catalyze p-coumaroyl-CoA and malonyl-CoA molecules to produce naringenin chalcone. Physiological function of RdCHS1 was checked in Arabidopsis mutant and tobacco, the results showed RdCHS1 transgenes could recover the color phenotypes of tt4 mutant and made tobacco flower color from pink to dark pink through modulating the expression of endogenous structural and regulatory genes in tobacco. All these results demonstrated that RdCHS1 is a bona fide CHS and contribute to flavonoid biosynthesis in R. delavayi.

To study the mechanism of light color on a sea urchin (Tripneustes gratilla), blue light (B, λ450nm), yellow light (Y, λ585-590nm), red light (R, λ640nm), green light (G, λ510nm), white light (W, λ400-780nm) and darkness groups (H) were set up in a seawater recirculating aquaculture system. Six different LED light color treatment groups with a photoperiod of 12 L:12 D were tested for 30 d to investigate the effects of different light colors on the feeding, growth and enzyme activities of T. gratilla (142.45±4.36 g). We found that varying LED light colors significantly impacts the feeding, growth, and enzyme activity of T. gratilla. Notably, the sea urchins in group B exhibited better growth with a weight gain rate of 39.26%, while those in group R demonstrated poorer growth with a weight gain rate of only 26%. The feeding differed significantly (P&lt;0.01) between groups B and R, with group B consuming the highest daily intake (6.03±1.69 g) and group R consuming the lowest (4.54±1.26 g). Throughout the three phases, there was no significant change in the viability of the AMS (P&gt;0.05). Conversely, pepsin viability significantly increased (P&lt;0.01) in group B. LPS viability remained consistently at the lowest level, with no notable differences in group W from those observed in group B. In group R, both AMS and pepsin viability remained lower, whereas lipase viability was noticeably higher in each phase compared to group B (P&lt;0.01). Regarding antioxidant enzymes, group R exhibited a trend of initially increasing and then decreasing CAT, SOD, and GSH-PX activities, particularly during the third stage (15-30 d), with a significant decrease observed in antioxidant enzyme activities (P&lt;0.01). The outcomes suggest that blue light positively affects the growth, feeding, digestion, and antioxidant capacity of T. gratilla in comparison to other light environments, whereas the red light has an inhibitory impact.

The adoption of sustainable forest management practices emerged as a contemporary imperative for forest biodiversity preservation, functional integrity, and mitigation of climate change. Within the spectrum of these practices, thinning is recognized as an environmentally sustainable strategy. The primary objective of thinning is the equitable redistribution of aboveground growth, diverting resources from younger trees to promote the accelerated development of more valuable specimens compared to unthinned forests. Thinning, while fostering aboveground changes, induces substantial alterations in soil chemical and biochemical properties. This review addresses the often-debated consequences of thinning on soil characteristics, focusing on the intricate interplay involving different thinning intensities and their impact on soil chemical and biochemical parameters. Specifically, the analysis delves into the modulation of organic carbon levels, microbial biomass, and enzymes crucial to the organic matter cycle. Two key aspects are scrutinized: firstly, the influence of thinning intensity and the temporal dimension post-thinning on soil properties; secondly, the comparative impact of thinning intensity versus the time elapsed from the thinning event on soil properties and associated biological processes. The findings underscore pivotal insights: a) temporal dynamics significantly affect soil organic matter across broadleaved (excluding beech), coniferous, and mixed forests, manifesting as early as 2-7 years post-thinning; b) thinning intensity, up to a threshold of 50%, enhances soil quality by augmenting organic carbon content, bolstering microbial communities, and amplifying associated enzyme activities. This review consolidates valuable information for forest managers, providing guidance to optimize natural processes in tandem with achieving specific management objectives. By illuminating the nuanced interactions between thinning practices and soil dynamics, this knowledge equips stakeholders to make informed decisions in the pursuit of sustainable forest management and ecological resilience.

Electrochemical biosensors based on immobilized enzymes are among the most popular and commercially successful biosensors. Literature in this field suggests that modification of electrodes with nanomaterials is an excellent method for enzyme immobilization, which can greatly improve the stability and sensitivity of the sensor. However, the poor stability, weak reproducibility, and limited lifetime of the enzyme itself still limit the requirements for the development of enzyme electrochemical biosensors for food production process monitoring. Therefore, constructing sensing technologies based on enzyme electrochemical biosensors remains a great challenge. This article outlines the construction principles of four generations of enzyme electrochemical biosensors and discusses the applications of single-enzyme systems, multi-enzyme systems and nano-enzyme systems developed based on these principles. The article further describes methods to improve enzyme immobilisation by combining different types of nanomaterials such as metals and their oxides, graphene-related materials, metal-organic frameworks, carbon nanotubes and conducting polymers. In addition, the article highlights the challenges and future trends of enzyme electrochemical biosensors, providing theoretical support and future perspectives for further research and development of high-performance enzyme chemical biosensors.

Seeds stored for a prolonged period are subject to aging and reduction in germination potential (GP), which will negatively affect seed sales. Rare-earth elements have a synergistic effect on the improvement of seed GP. In this study, we examined the effects of neodymium on biochemical components, the antioxidant protective system, and metabolism-related enzymes during germination of naturally and artificially aged seeds of three wheat cultivars. Seed germination indices, biochemical substance contents, and enzyme activities decreased after seed aging. Soaking seeds in neodymium nitrate solution revived aged wheat seeds at an optimal concentration of 20 µmol/L for 8 h. Soaking in neodymium nitrate solution increased the GP (by 2.25%–60.9%), germination index (by 1.69%–29.2%), and vigor index (by 3.36%–18.7%) of aged seeds. Compared with non-soaked seeds, soaking significantly changed the contents of biochemical substances, and the activities of antioxidant protective enzymes and metabolic enzymes in seedlings were increased. Soaking with neodymium may revive aged seeds by regulating the synthesis of soluble sugars, soluble proteins, chlorophyll, and carotenoids, and decomposing malondialdehyde, in the germinating seed. Root dehydrogenase and amylase showed different responses to the aging modes. The differential responses of root dehydrogenase and amylase may reflect differences in the resistance of enzymes to long-term mild seed aging and short-term severe environmental aging.

The use of low-intensity ultrasound has gotten surprising consideration over the last decade as a method for enhancing the catalytic activity of the enzyme. Ultrasounds have the potential to significantly influence the activity of the enzymatic processes, provided that the energy input is not too high to inactivate the enzyme. By providing the variation in parameters, various physical and chemical effects can be attained that can enhance the enzymatic reaction. Ultrasonic reactors are known for their application in bioprocesses. However, the potential of their applications is still limited broadly due to the lack of proper information about their operational and performance parameters. In this review, the detailed information about ultrasonic reactors is provided by defining the different types of reactors, number and position of ultrasonic transducers. Also, it includes the mechanism of intensification and influence of ultrasonic parameters (intensity, duty cycle and frequency) and enzymatic factors (enzyme concentration, temperature and pH) on the catalytic activity of enzyme during ultrasound treatment.

Objectives Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has high infectivity in humans, attributed to the strong affinity of its spike (S) protein to human angiotensin-converting enzyme 2 (ACE2). Here, we analyzed the structural similarity of the S protein between SARS-CoV-2 and other SARS-related coronaviruses (CoVs). Methods We performed multiple alignment analysis of nine amino acid sequences of CoV S proteins from NCBI with MAFFT web-based software, followed by phylogeny analysis. Three-dimensional structure modeling was performed by SWISS-MODEL. We calculated the template modeling score between the S protein of SARS-CoV-2 and that of other SARS-related CoVs. Results The S1 domain of the unclassified CoV RaTG13 (the host of which is the intermediate horseshoe bat) was structurally very similar to that of SARS-CoV-2, implying that RaTG13 could be the origin of SARS-CoV-2. In addition, the folding property of the entire S protein was nearly the same between SARS-CoV-2 and RaTG13 after the PRRA amino acid insertion was removed from SARS-CoV-2. Conclusions RaTG13 could have a high binding affinity to ACE2, similar to SARS-CoV-2, and it is therefore highly likely to infect other animals. Therefore, massive research and monitoring of CoVs in animals is necessary to prevent future COVID-19-like disasters.

Nicotine is a pharmacologically active component of tobacco which adversely affects the male reproductive system and fertility and Icariin (ICA) is the main active ingredient of Epimedium herba which has been used to treat several male reproductive problems. This study was aimed at investigating the protective or ameliorative effect of ICA against reproductive toxicity induced by intraperitoneal injection of nicotine in mice. Forty male mice were randomly divided into 4 groups: control, nicotine (0.75 mg/kg intraperitoneally), icariin (ICA, 75 mg/kg), and icariin plus nicotine (ICA + nicotine) group. After 35 days of treatment, the mice were weighed, sacrificed, and their reproductive organs were collected and examined for further studies. In the nicotine-treated group, epididymal sperm density and serum testosterone concentrations significantly decreased relative to the control group. Nicotine also caused oxidative damage as shown by significant reduction in the activities of antioxidant enzymes and an elevation in Malondialdehyde (MDA) levels. Icariin on the other hand, improved the reduction in sperm characteristics, hormone levels, and activities of antioxidant enzymes alterations observed in the nicotine treated mice. These findings indicate that the nicotine-induced reproductive toxicity and oxidative damages on male reproductive tissues can be effectively attenuated by icariin.

Cyanobacterial mass developments in eutrophic ponds and lakes are a major concern for lake management, as many cyanobacteria produce a huge variety of toxic secondary metabolites, e.g. microcystins. The aim of this research was to culture a strain of the cyanobacterium Microcystis sp strain BM25, to observe its biomass production and to isolate and purify protease inhibitors from this cyanobacterial biomass. Different secondary metabolites were isolated following a standard bioassay-guideline. Isolation was performed, with an enzymatic protease assay as bioassay. High performance liquid chromatography was used to identify different fractions of secondary metabolite from the strain BM25. Moreover, protease homogenates were isolated from Daphnia magna in order to test the inhibitors against naturally occurring major digestive proteases trypsin and chymotrypsin. It was measured that 60% MeOH and the 80% MeOH C18-SPE fraction inhibits chymotrypsin activity 98% (6 nmol pNA min^-1 mg^-1) and 99 % (4 nmol pNA min^-1 mg^-1), respectively. In contrast, trypsin activity was not inhibited by methanolic extracts of this cyanobacterium strain.

The antioxidant, antimicrobial, antiproliferative, and enzyme inhibitory properties of five extracts from aerial parts of Salvia pachyphylla were examined to assess the prospective of this plant as a source of natural products with therapeutic potential. Those properties were analyzed performing a set of standard assays. The extract obtained with dichloromethane showed the most variety of components, as yielded promising results in all completed assays. Furthermore, the extract obtained with ethyl acetate exhibited that greatest antioxidant activity as well as the best xanthine oxidase inhibitory activity. Remarkably, both extracts obtained with n-hexane or dichloromethane revealed significant antimicrobial activity against the Gram-positive bacteria; also, they showed greater antiproliferative activity against three representative cell lines of the most common types of cancers in women worldwide, and against a cell line that exemplifies cancers that typically develop drug resistance. Despite that other extracts were less active, such as the methanolic or aqueous, their results are promising for the isolation and identification of novel bioactive molecules.

Carbonic Anhydrases (CAs) are a superfamily of metalloenzymes widespread in all life kingdoms, classified into seven genetically different families (α-θ). These enzymes catalyse the reversible hydration of carbonic anhydride (CO2), generating bicarbonate (HCO3-) and protons (H+). Fifteen isoforms of human CA (hCA I-XV) have been isolated, their presence being fundamental for the regulation of many physiological processes. In addition, overexpression of some isoforms has been associated with the outbreak or the progression of several diseases. For this reason, for a long time CA inhibitors (CAIs) are used in the control of glaucoma and as diuretics. Furthermore, the search for new potential CAIs for other pharmacological applications is a very active field. Amino acids constitute the smallest fundamental monomers of protein and, due to their useful bivalent chemical properties, are widely used in organic chemistry. Both proteinogenic and non-proteinogenic amino acids have been extensively used to synthesize CAIs. This article provides an overview of the different strategies that have been used to design new CAIs containing amino acids, and how these bivalent molecules influence the properties of the inhibitors.

G protein-coupled receptors (GPCRs) comprise the largest family of membrane receptors that control many cellular processes and consequently often serve as drug targets. These receptors undergo a strict regulation by mechanisms such as internalization and desensitization, which are strongly influenced by posttranslational modifications. Ubiquitination is a posttranslational modification with a broad range of functions that is currently gaining increased appreciation as a regulator of GPCR activity. The role of ubiquitination in directing GPCRs for lysosomal degradation has already been well-established. Furthermore, this modification can also play a role in targeting membrane and endoplasmic reticulum-associated receptors to the proteasome. Most recently, ubiquitination was also shown to be involved in GPCR signaling. In this review, we present current knowledge on the molecular basis of GPCR regulation by ubiquitination, and highlight the importance of E3 ubiquitin ligases, deubiquitinating enzymes and β-arrestins. Finally, we discuss classical and newly-discovered functions of ubiquitination in controlling GPCR activity.

ω-Transaminases have been immobilized on macrocellular silica monoliths and used as heterogeneous biocatalysts in a continuous flow mode enantioselective transamination reaction. The support was prepared by a sol-gel method based on emulsion templating. The enzyme was immobilized on the structured silica monoliths both by adsorption, and by covalent grafting using amino-functionalized silica monoliths and glutaraldehyde as a coupling agent. A simple reactor set-up based on the use of a heat-shrinkable Teflon tube is presented and successfully used for the continuous flow kinetic resolution of a chiral amine, 4-bromo-α-methylbenzylamine. The porous structure of the supports ensures effective mass transfer and the reactor works in the plug flow regime without preferential flow paths. When immobilized in the monolith and used in the flow reactor, transaminases retain their activity and their enantioselectivity. The solid biocatalyst is also shown to be stable both on stream and during storage. These essential features pave the way to the successful development of an environmentally friendly process for chiral amines production.

Cholest-4-en-3-one Δ1-dehydrogenase (AcmB) from Sterolibacterium denitrificans is successfully immobilized on 3-aminopropyltrimethoysilane functionalized MCF and SBA-15 silica supports using adsorption or covalently with glutaraldehyde or divinyl sulfone linkers. The best catalyst, AcmB on MCF linked covalently with glutaraldehyde, retains the specific activity of the homogenous enzyme while exhibiting a substantial increase of the operational stability. The immobilized enzyme was used continuously in the fed-batch reactor for 27 days, catalyzing 1,2-dehydrogenation of androst-4-en-3-one to androst-1,4-dien-3-one with a final yield of 29.9 mM (8.56 g/L) and 99% conversion. The possibility of reuse of the immobilized catalyst was also demonstrated and resulted with a doubling of the product amount compared to that in the reference homogenous reactor. Finally, it was shown that molecular oxygen from the air can efficiently be used as an electron acceptor either reoxidizing directly the enzyme or the reduced DCPIPH2. Keywords: 3-ketosteroid D1-dehydrogenase; KSTD; KSDH; AcmB; 1,2-dehydrogenation; cholest-4-en-3-one Δ1-dehydrogenase; enzyme immobilization, FAD-dependent enzymes; enzyme immobilization;

In this study, we have designed a promising platform based on metal-organic frameworks (MOFs) to develop paper-based analytical devices (PADs) to immobilize biomolecules for application in biosensors. PADs have become increasingly popular in sensing applications in recent years due to their portability, low cost, simplicity, efficiency, fast detection capability, excellent sensitivity, and selectivity. In addition, MOFs are an excellent choice for developing highly sensitive and selective sensors. Their ability to reversibly and reproducibly adsorb and desorb specific molecules allows using these materials as sensors for various applications. They also offer the possibility to modify their structure and properties, making them highly versatile and adaptable to different environments and sensing needs. In this research, we synthesized and characterized three different amino-functionalized MOFs: UiO66-NH2 (Zr), MIL125-NH2 (Ti), and MIL101-NH2 (Fe). These MOFs were used to fabricate PADs capable of sensitive and portable monitoring of alkaline phosphatase (ALP) enzyme activity by laser-induced fluorescence (LIF). Overall, amino-derivated MOF platforms demonstrate significant potential for integration into biosensor PADs, offering key properties that enhance their performance and applicability in analytical chemistry and diagnostics.

Abstract: Background: Gender dimorphism and marital status on colorectal cancer mortality have 9 been previously documented, but the relationship between these factors and DNA mismatch repair 10 protein (MMRP) expression status is unknown. Methods: Colectomy specimens were reviewed ret- 11 rospectively for patients between 2018 to 2023 with demographics including race/ethnicity, gender, 12 marital status, faith, body mass index, pathologic staging, and MMRP expression status. Statistical 13 analyses were performed by using baseline characteristics tables and various programs in R-pack- 14 age. Results: A total 1018 colectomies were reviewed, and the tumor stages were significantly higher 15 in right colon (stage 3 and 4) than those in left colon and rectosigmoid colon (p<0.01). Marital status 16 was significantly associated with patients’ gender, age, tumor size and tumor stages (all p<0.01). 17 MMRP status were available in 775 cases, and 139 (17.9%) were MMRP deficient, and 636 (82%) 18 MMRP proficient. MMRP deficiency was significantly associated with older female patients, larger 19 tumor in size, higher tumor stages, higher histologic grade and more common in right colon (all 20 p<0.01). In addition, MMRP deficiency was statistically associated with higher percentage of di- 21 vorced and widowed patients (p<0.01). Multivariate linear regression analysis revealed persistent 22 association of MMRP deficiency with tumor size, tumor grade, tumor stage and nodal metastasis, 23 but the associations with gender and marital status no longer existed. Conclusion: The differences 24 in prevalence of CRC by gender and marital status and tumor MMRP status illustrate the im- 25 portance of these factors on tumor stages and nodal metastasis but these associations were more 26 complex with other confounding factors.

The inclusion of exogenous phytase in broiler P and Ca deficient diets in response to heightened concerns over excessive P excretion into the environment over the years has remarkably been documented. However, responses among these studies have been inconsistent because of the several factors affecting P utilization. For this reason, a systematic review with meta-analysis of results from forty-one studies published from 2000 to February 2024 was evaluated to: 1) quantitatively summarize the size of phytase effect on growth performance, bone strength, and mineralization in broilers fed P and Ca deficient basal diets and 2) estimate and explore the heterogeneity of the effect sizes of outcomes using sub-group and meta-regression analyses. The quality of the included studies was assessed using the Cochrane Collaboration’s SYRCLE Risk of Bias checklists for animal studies. Additionally, applying the random-effects models, Hedges’ g effect size of supplementation with phytase was calculated using the R software to determine the standardized mean difference (SMD) at a 95% confidence interval. Sub-group analysis and meta-regression further explored heterogeneity (P_SMD ≤ 0.05, I² > 50%, n ≥ 10). The meta-analysis showed that supplemental phytase increases ADFI and BWG and reduces FCR at each time point of growth (< 0.0001). Additionally, phytase supplementation consistently increased tibia ash, P and Ca, and bone strength (< 0.0001) of broilers fed P and Ca deficient diets. Results of the subgroup and meta-regression analyses showed that age and strain of broiler, dietary P source, and duration of phytase exposure significantly influence the effect size of phytase on growth and bone parameters. In conclusion, phytase can attenuate the effect of reducing dietary available phosphorus and calcium and improve ADFI, BWG, and reduced FCR, especially when added to starter diets. It further enhances bone ash, bone mineralization, and bone-breaking strength of broilers, even though the effects of bone ash and strength can be maximized in the starter phase of growth. However, the effect sizes of phytase were related to the age and strain of the broiler, dietary P source, and duration of phytase exposure rather than the dosage.

There are several well-known methods for determining enzyme catalytic activity, including spectral techniques and electrochemical approaches. However, at present, there is no definitive solution to the challenges associated with the fluorescent approach in, for instance, systems of reverse micelles, non-aqueous media, and model biomembranes. We propose the use of the fluorescent resonant energy transfer (FRET) technique, which can enhance the selectivity of fluorescent detection of enzyme activity by shifting the maximum emission wavelength to the longer-wavelength range and reducion the influence of medium components. As fluorescence donors, we have used derivatives of 4-methylumbelliferone (MUmb) and 7-amino-4-methylcoumarin (AMC), which also serve as substrates for hydrolases such as chymotrypsin, phosphatase, and asparaginase. During the catalytic depletion of substrates, an accumulation of a highly fluorescent product is observed. This product can be specifically detected using the fluorescent marker rhodamine 6G (R6G), which emits light in the red region of the spectrum (550–600 nm) with high quantum yield. A significant FRET effect is observed when forming AOT reverse micelles. This is due to the proximity of fluorophores within the hydrophobic core of the micelle, which is shorter than in a buffer solution. This approach have practical implications for visualizing intracellular processes. The FRET technique can be used to determine intracellular enzymatic activity by detection with confocal laser scanning microcopy (CLSM), as demonstrated with L-asparaginase, a clinically important anti-leukemia medication. The approach suggested will allow researchers to study enzyme activity within cells. This has the potential to revolutionize medicine by improving drug development and shedding light on previously unknown aspects of cellular metabolism.

There is growing interest in the discovery of novel xanthine oxidase inhibitors for gout prevention and treatment with fewer side effects. This study aimed to identify the xanthine oxidase (XO) inhibitory potential and drug-likeness of the metabolites present in the methanolic leaves extract of Anastatica (A.) hierochuntica L. using in vitro and in silico models. The extract-derived metabolites were identified by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). Molecular docking predicted the XO inhibitory activity of the identified metabolites and validated the best scored in vitro XO inhibitory activities for experimental verification as well as predictions of their anticancer, pharmacokinetic and toxic properties, oral bioavailability, and endocrine disruption using SwissADMET, PASS, ProTox-II and Endocrine Disruptome web servers. A total of 12 metabolites with a majority of flavonoids were identified. Rutin, quercetin and luteolin flavonoids demonstrated the highest ranked docking score of -12.39, -11.15 and -10.43, respectively, while the half-maximal inhibitory concentration (IC50) values of these metabolites against XO activity were 11.35 µM, 11.1 µM and 21.58 µM, respectively. In addition, SwissADMET generated data related to physicochemical properties and drug-likeness of the metabolites. Similarly, PASS, ProTox-II and Endocrine Disruptome prediction models stated the safe and potential use of these natural compounds. However, in vivo studies are necessary to support the development of prominent and promising therapeutic use of A. hierochuntica methanolic leaves extract-derived metabolites as XO inhibitors for the prevention and treatment of hyperuricemic and gout patients. Furthermore, the predicted findings of the present study open a new paradigm for these extract-derived metabolites by revealing novel oncogenic targets for the potential treatment of human malignancies.

The present study aims to elucidate the effect of melatonin (MEL) on dexamethasone (DEX)-induced reproductive impairments in rats. Rats were procured and acclimatized with adequate food and water ad libitum. Twenty-four rats were divided into four groups (six in each) of Control, DEX (70µg/100g bw), DEX plus MEL, and MEL (200µg/100g bw) for 21 days. After completion, rats were sacrificed, and testes were dissected, weighed, and processed for biochemical, and receptor expression. Serum was collected for hormonal assay. DEX-treated rats showed a significant increase in lipid peroxidation (TBARS), a decrease in weight of the body and testes, and the level of antioxidative enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione oxidase (GSH), glutathione reductase (GR), glucose 6-phosphate dehydrogenase (G6PDH), glutathione S-transferase (GST)), total cellular protein content, serum testosterone, corticosterone, LH, FSH, melatonin levels, AR, MT1, and MT2 receptor expression. Marked cellular alterations were noted in the testis histology of the DEX group. Melatonin treated in DEX rats showed recovery and restoration in gravimetric, serum hormonal, biochemical parameters, receptor expression, and cellularity of testes. Therefore, findings suggest that melatonin improved defence against DEX-induced testicular damages suggesting its therapeutic potential for the management of male reproductive health.

Amylin, also known as islet amyloid polypeptide (IAPP), is a metabolic homeostasis-related hormone that is produced and released by the β cells of the pancreas, the same cells that produce insulin. Insulin-degrading enzyme (IDE) is a protease enzyme that plays an essential role in the breakdown and degradation of various peptides, including insulin and amylin. Direct binding & interaction between amylin and IDE is inextricably linked to the degradation of amylin, and research and development effort in this area is crucial to understand the potential therapeutic implications of disrupting the IDE-amylin interaction in the context of conditions where metabolic homeostasis needs to be regulated, such as diabetes and obesity. Here, this article incorporates currently available experimental complex structure of amylin and IDE, and delves deep into the interstructural biophysics underlying the binding interface of the two interacting partners. With a set of comprehensive structural biophysical analysis, this article identified an intriguing region of high electrostatic potential indicative of strong binding sites between the first N-terminal lysine (Lys1, K1) residue of amylin and Glu341 (E341) of IDE. This unique electrostatic hotspot presented herein paves the way for the rational design of drug-like small molecules that can selectively disrupt this interaction, offering a targeted therapeutic strategy for improved metabolic homeostasis, particularly for patients with diabetes and obesity.

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) utilizes Angiotensin Converting Enzyme 2 (ACE2) as its main receptor for cell entry. We have bioengineered a soluble ACE2 protein termed ACE2 618-DDC-ABD that has increased binding to SARS-CoV-2 and prolonged duration of action. Here we investigated the protective effect of this protein when administered intranasally to k18-hACE2 mice infected with the aggressive SARS-CoV-2 delta variant. k18-hACE2 mice were infected with the SARS-CoV-2 delta variant by inoculation of a lethal dose (2x104 PFU) and followed for up to 14 days. ACE2-618-DDC-ABD (10mg/kg) or PBS was administered intranasally six hours prior and 24 and 48 hours post viral inoculation. All animals in the PBS-control group had succumbed to the disease on day 7 post infection (0% survival) whereas, by contrast, there was only one casualty in the group that received ACE2-618-DDC-ABD (90% survival). Mice in the ACE2-618-DDC-ABD group had minimal disease as assessed by a clinical score and stable weight and both brain and lung viral titers were markedly reduced. These findings demonstrate the efficacy of a bioengineered soluble ACE2 decoy with extended duration of action in protecting against the aggressive delta SARS-CoV-2 variant. Together with previous work these findings demonstrate the universal protective potential against current and future emerging SARS-CoV-2 variants.

Fluorescent labels, especially FRET probes, are promising tools for studying a number of bio-chemical processes. In this paper, we promoted 2 important applications of FRET probes (MUTMAC-R6G and FITC-R6G): i) the formation of micelles from surfactants of various natures and polymers (chitosan – fatty acid), as well as ii) monitoring of enzymatic activity with improved parameters (increased analytical signal and improved selectivity due to shift to the long-wavelength region). The formation of micelles is accompanied by the convergence of fluorophores in the hydrophobic micelle core by a distance closer than in the buffer solution, thus r/R0 (where R0 – Foerster radius) is chosen as an analytical signal of micelle formation, including critical micelle concentration (CMC) and critical pre-micelle concentration (CPMC). The CMC values calculated using FRET probes are in a good agreement with literature data. At the same time, the r/R0 function provides valuable information about the nature and mechanism of micelle formation. With the second analytical application of FRET probes, we considered the optimization of techniques for studying enzymatic activity. The enzyme catalyses the reaction with the release of a fluorescent product, the signal from which may not be enough for detection, or it may be quenched, for example, in the reverse micelles (AOT-octane). Here we have proposed a solution – a FRET probe containing a rhodamine 6G (R6G) acceptor, which allows us to monitor the enzymatic reaction selectively (in the red region, 550-600 nm), and obtain a significantly higher fluorescence yield (potentially from 10 to 250 times). Thus, we have demonstrated a high potential for the FRET probes application as indicators of micelle formation as well as for the study of the enzyme catalytic activity. In the future, the method developed has prospects for application in the visualization of the enzyme functioning in cells due to the shift of the fluorescence signal to the long-wavelength region with an increase in the signal selectivity to suppress autofluorescence.

In light of the hazardous effects of global warming brought on by climate change, bioethanol production is one of the key alternatives in which the world has expressed a great deal of interest. Two factors—indigenous production and price—are crucial for bioethanol to be appealing in Africa and, for that matter, Ghana. The objective of this research is to produce bioethanol from cheap lignocellulosic materials using onsite enzymes produced from cassava peels degrading fungi. Due to Ghana's prominent position as one of the leading cassava producers in the world, the study focused on utilizing cassava peels, which are underutilized in the country. The research was conducted in multiple stages. In the first stage, the peels were prepared by washing, drying, and manually crushing them using mortar and pestle. The second stage involved analyzing the chemical composition of the peels, including starch and insoluble fibre, which were determined as Acid Detergent Fiber (ADF), Neutral Detergent Fiber (NDF), and Acid Detergent Lignin (ADL). The results showed significant levels of cellulose (39.78%) and starch (31.21%), indicating that cassava peels are valuable raw materials for bioethanol production. The content of hemicellulose (21.11%) and lignin (3.84%) were also determined. In the third stage, using a Petri dish, three fungi (ICPF1, ICPF2, and ICPF3) were isolated from two different cassava peels. ICPF1 was only identified from fresh cassava peels (FCP), while all three fungi were identified from decayed cassava peels (DCP). Morphologically, these fungi were identified as Aspergillus niger, Aspergillus flavus, and Rhizopus stolonifer. The fourth stage focused on optimizing the enzyme activity of the three isolates for potential applications, with A. niger demonstrating the highest enzyme activity with a diameter of zone of clearance of 16 mm. Stage five involved optimizing the production of the onsite enzyme in a 50ml flask using A. niger, basal salt medium (BSM), and cassava peels as a substrate. The DNSA method was used to measure the absorbance of maltose and glucose at 540 nm for various substrate concentrations (1%, 3%, 5%, 8%, and 10%) at specific intervals of 2, 4, 6, 8, and 10 days with a spore concentration of 2.1 x 105 cells/ml. The maltose and glucose concentrations were calculated as 7.138mg/ml and 6.398 mg/ml, respectively, and the corresponding enzyme activity was determined as 4.759U/ml and 4.265U/ml. The optimal conditions of Day 4 and a substrate concentration of 10%, along with a fixed temperature of 30°C and a pH of 5.5, were used to prepare the onsite enzymes in a 500 ml flask for the fermentation process. The onsite enzymes were used for saccharification and Saccharomyces cerevisiae for fermentation under simultaneous saccharification and fermentation (SSF) process. A mixture of 20 ml of onsite enzymes and 1.5 g of S. cerevisiae were added to substrate concentrations of 5%, 10%, and 20%, and the ethanol concentration was analyzed daily for 7 days using Gas Chromatography (GC). The highest ethanol concentration (1.316%) was observed on Day 5 with a substrate concentration of 20%, while the lowest concentration (0.123%) was recorded on Day 1 with a substrate concentration of 5%.

Excess weight, which is seen as the background cause of most diseases, is gradually becoming the most important problem in the world. The cafeteria diet is robust diet that mimics the western diet and provides rapid weight gain associated with the obesity, diabetes, and cancer. The relationship between obesity and cancer is well known. For this reason, it is of great importance to reveal the first molecular and metabolic alterations that occur before entering the obesity process with any factor to understand the pathology of many diseases and to treat them in the early period. The beneficial effects of probiotics are attributed to their metabolite content with antioxidant properties or the postbiotic metabolites produced by them. In this study, the effects of different dietary practices on the hepatic antioxidant system of rats in developmental period were investigated by real-time PCR, enzyme activity measurements, and quantitative measurements of metabolites. After all, it was observed that CAF caused serious impairments by affecting the hepatic antioxidant system at gene and protein levels, and these impairments were relatively reversed with SCD Probiotics supplementation. In addition, regardless of the CAF diet, the regulatory effect of SCD Probiotics supplementation on the hepatic antioxidant system is remarkable.

Biochars are often proposed as a strategy for long-term carbon sequestration. Nevertheless, application of pyrolysed feedstock, particularly along with exogenous organic matter, may affect carbon dynamics in soil through introduction of labile carbon pools and stimulation of extracellular enzymes activity. The main aim of this research was to evaluate the influence of biochars and unprocessed organic amendments addition in two agricultural soils on the dissolved organic carbon (DOC) content and activity of three enzymes involved in carbon turnover. In the incubation experiment, activity of dehydrogenase, β-glucosidase, cellulase and DOC content were measured on day 30, 60, 90, 180 and 360. Addition of biochars stimulated the activity of dehydrogenase and β-glucosidase, while cellulase was suppressed. Fresh biomass amendment enhanced activity of the enzymes through priming effect. DOC content tended to be the highest in treatments with high enzyme activity, suggesting that DOC introduced with amendments acted as a source of energy for microbes. Our findings support the hypothesis that biochar properties and presence of exogenous organic matter affect microbial response in soil, what might be crucial for carbon sequestration potential of biochar. However, long-term studies are recommended to fully understand the mechanisms that determine response of soil biota to biochar addition.

No-till and cereal-legume intercropping have been recognized as favorable cropping practices to increase crop yields while maintaining soil quality in arid and semiarid environment, but the bio-logical mechanisms are poorly understood. The present study was to determine the response of soil properties, enzyme activities, and microbial community diversity and composition in mono- and inter-cropping under conventional and no-tillage conditions. We initiated a field experiment in Wuwei, a typical arid area of China, in 2014. Soil was sampled in August 2022 and, yields, soil properties, enzyme activities, and the microbial community diversity and composition were de-termined in the maize and pea strips in inter- and mono-cropping systems. Results revealed that the maize and pea strips in the no-till intercropping significantly increased yields, total and or-ganic carbon stocks, decreased NO3--N, and obtained the highest total and organic P in the soil. The α- and β-diversity of archaea and eukaryotes were significantly affected by planting patterns, while α- and β-diversity of the bacterial community were significantly affected by tillage practices. Both no-tillage and intercropped maize significantly increased the abundance of archaea phylum Thaumarchaeota and bacterial phylum Nitrospirae, benefiting nitrogen fixation of intercropped pea from the atmosphere under the no-tillage cereal/legume intercropping. No-till intercropping was conducive to the accumulation of organic carbon, while decreasing the abundance of Prote-obacteria, Acidobacteria, and Verrucomicrobia. Limited soil enzyme activities (ACP, ALP, DP, NAG, BG, AG, CB) led to decreases in organic carbon turnover and utilization. Intercropping al-tered soil microbial community diversity and composition due to changes in soil properties and enzyme activities. These findings suggest that no-tilled cereal-legume intercropping is a sustaina-ble cropping practice for improving soil properties and enhancing microbial (archaea, bacterial, Eukaryota) diversity, but the long-term persistence is not conducive to rapid turnover of soil nu-trients due to limited enzyme activities.

Industrial effluents contain contaminants that pose threats to the environment and human health. Bioremediation is an environmentally friendly, sustainable, and cost-effective approach to degrade or transform pollutants using microbial enzymes. Laccase is a versatile enzyme that oxidizes a wide range of substrates, such as dyes, phenolic compounds, pesticides, and polycyclic aromatic hydrocarbons (PAHs), with the consumption of molecular oxygen. Laccase can also remove heavy metals by forming complexes with organic ligands or reducing them to fewer toxic forms. However, the application of laccase in bioremediation is limited by its low production, stability, and specificity. Recombinant technology has been employed to enhance the expression, activity, and stability of laccase in various bacterial hosts. Immobilization techniques have been developed to improve the reusability and stability of laccase in different environmental conditions. This review summarizes the recent developments in the production optimization of recombinant laccase enzymes and their role in the bioremediation of industrial effluents. It also discusses the challenges of laccase-based biocatalytic systems for environmental cleanup.

Cervi cornu has been extensively used in traditional medicine as an antioxidant and for the treatment of various disorders, including osteoporosis. However, since it is not easy to separate the active ingredient, absorption is not performed well. In this study, we extracted the low molecular weight (843Da) collagen NP-2007 from cervi cornu by enzyme hydrolyzation, and evaluated the therapeutic effect in a monosodium iodoacetate (MIA)-induced rat osteoarthritis model, and identified their molecular mechanisms. NP-2007 was orally administered at 50, 100, and 200 mg/kg for 21 d. Efficacy was evaluated based on serum parameters, inflammatory mediators, and mor-phological and histopathological analyses of knee joints. Production of matrix metalloproteinases, including MMP-2, MMP-3, and MMP-9, in the serum was higher in MIA-treated rats; however, these levels decreased after NP-2007 treatment. Serum levels of tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and prostaglandin E2 were lower in the NP-2007 group than in the MIA group. Furthermore, the administration of NP-2007 resulted in effective preservation of both the synovial membrane and knee cartilage, and significantly decreased the transformation of fibrous tissue. This study indicates that NP-2007 can prevent and alleviate symptoms of osteoarthritis and have applications as a novel arthritis treatment.

The Severe acute respiratory syndrome-Corona virus-2 (SARS-CoV-2) which is responsible for recurring pandemics takes advantage of host-cell processes including the glycosylation pathway. The heavily glycosylated spike protein assists viruses in attachment and penetration. The N-glycans of N-terminal domain N165 and N234 play a significant role in conformational dynamics of the receptor-binding domain (RBD) with angiotensin-converting enzyme 2 (ACE2). In addition, the deletion of N-glycan sites N331 and N334 have been associated with reduced infectivity. This signifies the importance of targeting the N-glycans making them unavailable for interacting with the ACE2 receptor, ultimately leading to reduced infectivity. These glycans can be specifically targeted and can be used for designing SARS specific drugs or neutralizing molecules. In the current study, lectins Griffithsin, Cyanovirin-N, Cyanovirin homolog, and BanLec H84T were used to target the spike protein's N-glycans. Molecular docking programs AutoDock Vina and HADDOCK were used to study lectin-glycan interactions. The interactions look convincible in accordance with the lowest interaction energies best-fit approach but the characteristic feature (scoring functions) of the docking programs are questionable concerning Lectin-Glycan interactions exist in nature. There is a high need of the hour for developing specific algorithms for docking glycans with a preferential selection of terminal residues.

Clean, fresh, and safe drinking water is essential to human health and well-being. Occasionally, chemical pollutants taint surface water quality used for consumption. Microcystins (MCs) are toxic heptapeptides produced by freshwater cyanobacteria. These secondary metabolites can reach hazardous concentrations, impairing surface drinking water supplies. Inconsistent screening of MCs is not uncommon in Florida waters as no provisional guidance value is established to protect public health. The occurrence of MCs in Lake Manatee and Lake Washington was monitored over the potential peak algae bloom season (June-August). An indirect competitive enzyme-linked immunosorbent assay (icELISA) quantified total MCs in two drinking water systems. Varied concentrations occurred between June and July, whereas concentrations peaked in August. Overall, MC prevalence was higher in Lake Manatee than Lake Washington. Colorimetric assays measured phosphate and nitrite in environmental water samples. Phosphate and nitrite concentrations strongly correlated with total MCs (p < 0.01). The results indicate the intrinsic nature of environmental MCs in surface drinking water supplies and the need to examine hepatotoxin dynamics to preserve drinking water quality in community served areas.

The apicomplexan parasite Theileria haneyi is one of two known causative agents of equine theileriosis. It causes milder clinical disease than its more virulent counterpart, Theileria equi, in experimentally infected horses, and can superinfect T. equi-positive horses. The current EMA1-based competitive ELISA used in the U.S. to detect equine theileriosis detects T. equi but not T. haneyi, and the complexity of molecular assays precludes widespread use for epidemiologic studies. In order to facilitate urgently needed studies on the prevalence of T. haneyi, the goal of this study was to develop a sensitive and specific serologic assay for the diagnosis of T. haneyi based on the equi mero-zoite antigen 11 (ThEMA11). To achieve this objective, ThEMA11 was recombinantly expressed in eukaryotic cells and its antigenicity assessed using sera from T. haneyi-experimentally infected horses. Confirmation of sera reactiv-ity enabled design and optimization of an indirect ELISA. Specificity of the ELISA for T. haneyi was assessed using a cohort of sera from horses experimentally infected and confirmed PCR-positive for either T. equi or T. haneyi. Data from field samples further demonstrate that the ThEMA11 ELISA is capable of identifying T. haneyi antibodies in horses from multiple continents around the world.

We hypothesized that rumen fluid with yeast producing cellulase enzyme can occur and also produces a high biomass compared to S. cerevisiae. Therefore, the aim of this study was to screen and isolate yeast from rumen fluids with an experimental design method. We optimized a fermentation medium containing sugarcane molasses as a carbon source and urea as a nitrogen source to measure the efficiency of biomass production and cellulase activity. Two fistulated-crossbred Holstein Friesian steers, averaging 350 ± 20 kg body weight, were used to screen and isolate ruminal yeast. The two experiments were designed. A 12 × 3 × 3 factorial was used in a completely randomized design to determine biomass and carboxymethyl cellulase activity. Factor A was isolated yeasts and S. cerevisiae. Factor B was sugarcane molasses (M) concentration. Factor C was urea (U) concentration. Potential yeast was selected for identified and analyzed as a 4 × 3 factorial use in a completely randomized design including. Factor A was incubation times. Factor B was isolated yeast strains including code H-KKU20 (as P. kudriavzevii-KKU20), I-KKU20 (C. tropicalis-KKU20), and C-KKU20 (as Galactomyces sp.-KKU20). Isolation was under aerobic conditions, resulting in a total of 11 different colonies. We noted two appearances of colonies including, asymmetric colonies of isolated yeast (indicated as A, B, C, E, and J) and ovoid colonies (coded as D, F, G, H, I, and K). The highest biomass was observed in three yeasts including codes H, I, and C-KKU20 when inoculated in 25% molasses with 1% urea (M25+U1) (p <0.01). The highest CMCase activity was observed in yeast code H-KKU20 when inoculated in all media solutions (p <0.01). Ruminal yeasts strains H-KKU20, I-KKU20, and C-KKU20 were selected for their ability to produce biomass and their CMCase enzyme synthesis. Identification of isolates H-KKU20 and I-KKU20 revealed that those isolates belonged to Pichia kudriavzevii-KKU20 and Candida tropicalis-KKU20, while C-KKU20 was identified as Galactomyces sp.-KKU20. Two strains provided maximum cell growth: P. kudriavzevii-KKU20 (9.78 and 10.02 Log cell/ml) and C. tropicalis-KKU20 (9.53 and 9.6 Log cells/ml) at 60 and 72 h of incubation time, respectively. The highest ethanol production was observed in S. cerevisiae: 76.4, 77.8, 78.5, and 78.6 g/L at 36, 48, 60, and 72 h of incubation time, respectively (p <0.01). The P. kudriavzevii-KKU20 yielded the least reducing sugar about 30.6 and 29.8 g/L at 60 and 72 h of incubation time, respectively. It could be concluded that screening and isolating yeast from rumen fluids resulted in 11 different characteristics of yeasts. The first novel yeasts discovered in the rumen fluid of cattle were Pichia kudriavzevii-KKU20, Candida tropicalis-KKU20, and Galactomyces sp.- KKU20. P. kudriavzevii-KKU20 had higher results than the other yeasts in terms of biomass production, cellulase enzyme activity, and cell number.

The outbreak of a novel coronavirus (SARS-CoV2) associated with acute respiratory disease called COVID-19 marked the introduction of the third spillover of an animal CoV to humans in the last 2 decades. The SARS-CoV2 genome analysis with various bioinformatics tools revealed that it belongs to beta CoVs genera, with highly similar genome as bat coronavirus and receptor binding domain (RBD) of spike glycoprotein as Malayan pangolin coronavirus. Based on its genetic proximity, SARS-CoV2 is likely to be originated from bat derived CoV and transmitted to humans via an unknown intermediate mammalian host, probably Malayan pangolin. Further spike protein S1/S2 cleavage site of SARS-CoV2 has acquired polybasic furin cleavage site which is absent in bat and pangolin suggesting natural selection either in an animal host before zoonotic transfer or in humans following zoonotic transfer. In the current review, we recapitulate a preliminary opinion about the disease, origin and life cycle of SARS-CoV2, roles of virus proteins in pathogenesis, commonalities and differences between different corona viruses. We have also highlighted the evidences regarding the potential drugs and vaccine candidates with their modes of action to cope with this viral outbreak. Our review provides comprehensive up-dated information on molecular aspects of the SARS-CoV2.

We hypothesize that the reason severe COVID-19 is closely linked to a limited number of conditions such as obesity, hypertension, older age and diabetes is that these conditions are characterized by a pro-inflammatory state in general and an angiotensin converting enzyme-1-bias in particular. We review recent evidence that this pro-inflammatory state can be reversed via regular exercise and a healthy diet. Studies are suggested that could test this hypothesis.

This study was conducted to determine the dynamic effects of dietary crude protein (CP) intake on nitrogen (N) balance, ileal amino acid digestibility, and gene expression levels of digestive enzymes at three stages in pigs. In Experiment 1, 18 growing pigs (average body weight (BW) = 9.5 kg) were randomly assigned to one of three treatments (n = 6/treatment group), including normal (20% CP), low (17% CP), and very low (14% CP) protein intake. In Experiment 2, 18 growing pigs (average BW = 30 kg) were allotted randomly to one of three treatments (n = 6/treatment group), including normal (18% CP), low (15% CP), and very low (12% CP) protein intake. In Experiment 3, 18 growing pigs (average BW = 45 kg) were assigned randomly to one of three treatments (n = 6/treatment group), including normal (16% CP), low (13% CP), and very low (10% CP) protein intake. Growing pigs fed the 14% CP and 17% CP diets had lower final BW (P < 0.05) and average daily gain (ADG) (P < 0.05) compared to pigs fed the 20% CP diet. Reducing the dietary CP level from 20 to 14% decreased urinary N excretion by 52.8% (P < 0.001) in Experiment 1. Reducing the dietary CP level from 18 to 12% decreased urinary N excretion by 55.3% (P < 0.001) and reduced fecal N excretion by 34% (P < 0.05) in Experiment 2. Reducing the dietary CP level from 16 to 10% decreased urinary N excretion by 56.4% (P < 0.001) and fecal N excretion by 47.1% (P < 0.001) in Experiment 3. Pigs fed the very low (14%, 12%, and 10% CP) diets showed higher digestibility for CP (P < 0.05), His (P < 0.05), Ile (P < 0.05), Phe (P < 0.05), Thr (P < 0.05), Trp (P < 0.05), Glu (P < 0.05), and Ser (P < 0.05) compared to pigs fed the normal (20%, 18%, and 16% CP) diets among the three experiments. Pigs fed the very low (14%, 12%, and 10% CP) diets showed higher mRNA levels for chymotrypsin C (P < 0.01 in Experiment 1 and 2; P < 0.05 in Experiment 3) compared to pigs fed the normal (20%, 18%, and 16% CP) diets among the three experiments. These results indicated that a reduction in dietary CP by 6% limited the growth performance of growing pigs, and a reduction of dietary CP by 3% supplemented with essential amino acids could reduce the excretion of N into the environment without affecting weight gain.

Field cricket (Gryllus bimaculatus) is newly emerged as an edible insect in several countries. Anti-inflammatory effect of glycosaminoglycan derived this cricket was not fully investigated on chronic disease animal model such as diabetic mouse. For potential therapeutic agents, anti-diabetic activities of field cricket glycosaminoglycan (GbG) was evaluated in diabetic mice based on their abilities to reduce glucose, ALT, AST, LDL-cholesterol, and BUN levels, compared with dung beetle (Catharsius molossus) glycosaminoglycan (CaG) as a positive control glycosaminoglycan. Db mice were orally administered for one month according to their groups: Db Hetero (normal), Db Homo (type-2 diabetic), CaG (5 mg/kg), GbG (5 mg/kg), and metformin (10 mg/kg). Blood glucose level was decreased after 1^st week treatment with GbG. It also inhibited LDL-cholesterol and alkaline phosphatase levels. Regarding oxidative damage of diabetic state, levels of hepatocellular biomarkers levels and protein carbonyl content were reduced in db mice treated with GbG. Especially anti-oxidative activities of catalase, superoxide dismutase, and glutathione peroxidase were significantly increased in GbG treated group compared to those in the control. GbG was composed of heparin disaccharides and main N-glycan was identified as Hex₉GlcNAc₂ (m/z 1905.7) of with neutral mono-sugar mainly comprising of hexose, L (+) rhamnose by mass spectroscopy. These results from sero-biochemical, hepatocellular anti-oxidant assay in db mice data suggest cricket (G. bimaculatus) glycosaminoglycan might play a role in its anti-diabetic action.

Puerarin (PUE) is a compound isolated from the roots of Pueraria lobata. We studied the pharmacokinetics and tissue distribution kinetics of PUE in Sprague-Dawley rats following intraperitoneal administration of three concentrations. Indirect competitive ELISA based on an anti-PUE monoclonal antibody was used to determine the concentration of PUE in the blood, heart, liver, spleen, lung, kidney, hippocampus, cerebral cortex, and striatum. The plasma and tissue distribution kinetic characteristics following a single injection of PUE (20, 40, and 80 mg/kg) were calculated using a non-compartment model. In the high-dose (80 mg/kg) and medium-dose (40 mg/kg) groups, the kinetic profile of PUE in blood and kidney samples showed two absorption peaks, while that of the other tissues showed only one peak. In the low-dose (20 mg/kg) group, there was only one peak, irrespective of the sample type. Pharmacokinetic parameters such as the area under the curve, C_max, and T_max varied according to the administered dose. AUC and C_max values increased dose-dependently. PUE was widely distributed in areas of the brain such as the hippocampus, cerebral cortex, and striatum, providing a foundation for guiding the use of PUE in the treatment of cerebral ischaemic stroke and neurodegenerative diseases.

Immunosorbent turnip vein clearing virus (TVCV) particles displaying the IgG-binding domains D and E of Staphylococcus aureus protein A (PA) on every coat protein (CP) subunit (TVCVPA) were purified from plants via optimized and new protocols. The latter used polyethylene glycol (PEG) raw precipitates, from which virions were selectively re-solubilized in reverse PEG concentration gradients. This procedure improved the integrity of both TVCVPA and the wildtype subgroup 3 tobamovirus. TVCVPA could be loaded with more than 500 IgGs per virion, which mediated the immunocapture of fluorescent dyes, GFP and active enzymes. Bi-enzyme ensembles of cooperating glucose oxidase and horseradish peroxidase were tethered together on the TVCVPA carriers by a single antibody type, with one enzyme conjugated chemically to its Fc region, and the other one bound as target, yielding synthetic multienzyme-complexes. In microtiter plates, the TVCVPA-displayed sugar-sensing system possessed a considerably increased reusability upon repeated testing, compared to the IgG-bound enzyme pair in the absence of the virus. A high coverage of the viral adapters was achieved also on Ta2O5 sensor chip surfaces coated with a polyelectrolyte interlayer, as a prerequisite for durable, TVCVPA-assisted electrochemical biosensing through modularly IgG-assembled sensor enzymes.

Hydrogen is the lightest atom and composes roughly half of the atomic content in macromolecules, yet their location can only be inferred or predicted in most macromolecular structures. This is because hydrogen can rarely be directly observed by the most common structure determination techniques (such as X-ray crystallography and electron cryo-microscopy). However, knowledge of hydrogen atom positions, especially for enzymes, can reveal protonation states of titratable active site residues, hydrogen bonding patterns, and the orientation of water molecules. Though we know they are present, this vital layer of information, which can inform a myriad of biological processes, is frustratingly invisible to us. The good news is that, even at modest resolution, neutron crystallography (NC) can reveal this layer and has emerged this century as a powerful tool for elucidating enzyme catalytic mechanisms. Due to its strong and coherent scattering of neutrons, incorporation of deuterium into the protein crystal amplifies the power of NC. This is especially true when solvation and the specific participation of key water molecules are crucial for catalysis. Neutron data allow the modeling of all three atoms in water molecules and have even revealed previously unobserved and unique species such as hydronium (D3O+) and deuteroxide (OD-) ions as well as lone deuterons (D+). Herein, we briefly review why neutrons are ideal probes for identifying catalytically important water molecules and these unique water-like species, limitations in interpretation, and four vignettes of enzyme success stories from disparate research groups. One of these groups was that of Dr. Chris G. Dealwis, who died unexpectedly in 2022. As a memorial appreciation of his scientific career, we will also highlight his interest and contributions to the neutron crystallography field. As both the authors were mentored by Chris, we feel we have a unique perspective on his love of molecular structure and admiration for neutrons as a tool to query those structures.

Litter decomposition is a crucial biochemical process regulated by microbial activities in forest ecosystem. However, the dynamic response of fungal community during litter decomposition to vegetation changes is not well understood. Here, we investigated the litter decomposition rate, extracellular enzyme activities, fungal community, and nutrient cycling-related genes in leaf and twig litters over a three-year decomposition period in a pure Liquidamabar formosana forest and a mixed L. formosana / Pinus thunbergii forest. The result showed that during the three-year decomposition, twig litter decomposed faster in the mixed forest than that in the pure forest. In both leaf litter and twig litter, β-cellobiosidase, N-acetyl-glucosamidase exhibited higher activities in the mixed forest, whereas phosphatase, β-glucosidase and β-xylosidase were higher in the pure forest. The fungal α-diversity were higher in both litters in the pure forest compared to mixed forest, with leaf litter showing higher α-diversity than twig litter. Fungal species richness and α-diversity within leaf litter increased as decomposition progressed. Within leaf litter, Basidiomycota dominated in the mixed forest while Ascomycota dominated in the pure forest. Funguild analysis revealed that symbiotroph and ectomycorrhizal fungi were more abundant in the mixed forest compared to the pure forest. In the third-year decomposition, genes related to phosphorus cycling was most abundant in both forests, with the pure forest having a higher abundance of cex and gcd genes. Fungal community structure, predicted functional structure, and gene composition differed between the two forest types and between the two litter types. Notably, the fungal functional community structure during the first-year decomposition was distinct from that in the subsequent two-year. These findings suggest that both dominant tree species, litter quality and decomposition time significantly influence the litter decomposition by recruiting different fungal community, thereby affecting the entire decomposition process.

Because we observed a reduced humoral immune response to the COVID-19 vaccine and subsequently discovered a lower susceptibility to COVID-19 in individuals carrying the ALDH2 rs671 variant, we hypothesized that rs671 is beneficial for cellular immunity against COVID-19. Using the IFN-γ enzyme-linked immunospot (ELISPOT) assay, we evaluated cellular immunity before and after COVID-19 vaccination in two subcohorts of a previously reported cohort. Subcohort 1 (26 participants) had six repeated observations at baseline and after one to three doses, whereas subcohort 2 (19 participants) had two observations, before and after the third dose. ELISPOT counts at six months after the second dose increased from baseline in carriers of the rs671 variant, but not in non-carriers. A positive effect of rs671 on ELISPOT counts was estimated using a mixed model, including the random effect of subcohort, repeated measures, and fixed effects of vaccine type, age, sex, height, lifestyle, steroid use, and allergic disease. There was no association between ELISPOT counts and specific IgG levels, suggesting a limitation in estimating protective potential by humoral response. There is lower humoral and higher cellular immunity against SARS-CoV-2 in rs671 variant carriers compared to non-carriers, providing a potential basis for optimizing preventive measures and drug discovery.

The toxic cadmium (Cd) poses a serious threat to plant growth and human health. Populus euphratica CPK21 has previously been shown to attenuate Cd toxicity by reducing Cd accumulation, enhancing antioxidant defense and improving water balance in transgenic Arabidopsis. Here, we confirmed a protein-protein interaction between PeCPK21 and AtNF-YC3 by yeast two-hybrid, HaloTag pull-down and bimolecular fluorescence complementation assays. AtNF-YC3 was induced by Cd and strongly expressed in PeCPK21-overexpressed plants. Overexpression of AtNF-YC3 in Arabidopsis reduced salt inhibition of root length, fresh weight and membrane stability under Cd stress conditions (100 µM, 7 d), suggesting that AtNF-YC3 appears to contribute to the improvement of Cd stress tolerance. AtNF-YC3 improved Cd tolerance by limiting Cd uptake and accumulation, activating antioxidant enzymes, and reducing H2O2 production under Cd stress. We conclude that PeCPK21 interacts with AtNF-YC3 to limit Cd accumulation, enhance the ROS scavenging system, and thereby positively regulate plant adaptation to Cd environments. This study highlights the interaction between PeCPK21 and AtNF-YC3 under Cd stress conditions, which can be utilized to improve Cd tolerance in higher plants.

The long-term use of fertilizers and pesticides in conventional cultivation has resulted in a decrease in soil productivity and vegetable yields in greenhouses. However, only a limited number of studies have explored the impacts of organic cultivation on greenhouse soil. Specifically, there is a scarcity of research examining the alterations in soil aggregate mediated organic carbon and microbial communities, which are known to enhance soil productivity. This study investigated the properties of soil aggregates, including the levels of organic carbon fractions, microbial community, and enzyme activity with the three aggregate classes microaggregates (&lt;0.25 mm), small macroaggregates (2-0.25 mm) and large macroaggregates (&gt;2 mm). These investigations were conducted under three different cultivation methods in a greenhouse: conventional cultivation (CC), integrated cultivation (IC), and organic cultivation (OC) in greenhouse. The results showed that (1) OC and IC promoted the formation of small macro-aggregates and enhanced aggregate stability compared to CC, (2) SOC in the three size fractions of OC increased by 98.99%-92.06% compared to CC; EOC increased by 98.47%-117.59%; POC increased by 138.59%-208.70%; MBC increased by 104.71%-230.61%; DOC increased by 21.93%-40.90%, respectively; (3) Organic cultivation significantly increased enzyme activity in all three particle size aggregates and increased the relative abundance of bacteria in micro-aggregates as well as the relative abundance of fungi in small macro-aggregates. Structural equation model (SEM) analysis revealed that organic farming practices fostered the development of smaller macro-aggregates, elevated microbial and enzyme activities within soil aggregates, and facilitated the conversion of soil nutrients and carbon sequestration. consequently, this led to an increase in soil organic carbon content and improved vegetable yield in greenhouse environments. In conclusion, the soil carbon content and vegetable yield are enhanced through long-term organic cultivation in greenhouses, primarily by increasing the proportion of small macro-aggregates.

Antibacterial bone cements (ABCs), such as antibiotic-loaded bone cements (ALBCs), have been widely utilized in clinical treatments. Currently, bone cements loaded with vancomycin, gentamicin, tobramycin, or clindamycin are approved by the US Food and Drug Administration. However, traditional ALBCs exhibit drawbacks like burst release and bacterial resistance. Therefore, there is a demand for the development of antibacterial bone cements containing novel agents. In this review, we summarize the latest advancements in ABCs. More importantly, we provide an overview and outlook on novel antibacterial agent-loaded bone cements, including metallic antibacterial agents, pH-switchable antibacterial agents, cationic polymers, N-halamines, non-leaching acrylic monomers, antimicrobial peptides, etc. Offering novel perspectives for designing ABCs and treating bone infections.

: Protease-producing yeasts were isolated from potato wastes and screened for protease production on skim milk agar plates The best producer of protease isolate was identified as Clavispora lusitaniae. The strain showed better enzyme production using tomato pomace and bread waste mix as a solid fermentation substrate. The optimized conditions improved enzyme activity and showed a maximal production of 33450±503 IU/g compared with the initial activity of 11205,78 ±360 without medium optimization. A threefold increase in protease activity after medium optimization proved the reliability of using the PBD and CCD design. A 19.76-fold purified enzyme and a yield of 32.94% were obtained after purification. The protease showed maximum activity at pH 4 and 60 °C and was resistant to Tween 20, Tween 40, SDS, and β-mercaptoethanol, Ca2+, and Mg2+ stimulated it. The protease activity was strongly inhibited in the presence of urea, and EDTA. The results revealed Clavispora lusitaniae protease's ability to degrade wheat seeds and flour gluten by 98.7% and 97% respectively under pH 4 for 24 hours at 40 °C. According to this study, this enzyme could be a potential candidate for the food industry, particularly for treating wheat seed and flour to reduce the immunogenicity of gluten.

Transcription of glycine-rich RNA-binding protein 2 (PeGRP2) transiently increased in root and shoot of Populus euphratica (salt-resistant poplar) upon initial salt exposure and tended to decrease after long-term NaCl stress (100 mM, 12 days). PeGRP2 overexpression in the hybrid Populus tremula × P. alba ‘717-1B4’ (P. × canescens) increased salt sensitivity, which was reflected in plant growth and photosynthesis. PeGRP2 contains a conserved RNA recognition motif domain at the N-terminus, and RNA affinity purification (RAP) sequencing was developed to enrich target mRNAs that physically interact with PeGRP2 in P. × canescens. RAP sequencing combined with RT-qPCR revealed that NaCl decreased the transcripts of PeGRP2-interacting mRNAs encoding photosynthetic proteins, antioxidative enzymes, ATPases, and Na+/H+ antiporters in transgenic poplar. Particularly, PeGRP2 negatively affected the stability of target mRNAs encoding photosynthetic proteins (PETC and RBCMT), antioxidant enzymes (SOD[Mn], CDSP32 and CYB1-2), ATPases (AHA11, ACA8 and ACA9), and Na+/H+ antiporter (NHA1). This resulted in (i) a greater reduction in Fv/Fm, YII, ETR and Pn, (ii) a less pronounced salt activation of antioxidative enzymes, and (iii) a reduced ability to maintain Na+ homeostasis in transgenic poplars during long-term salt stress, leading to a lowered ability to tolerate salinity stress.

CD44 is a type I transmembrane glycoprotein, and possesses various isoforms which are largely classified into CD44 standard and CD44 variant (CD44v) isoforms. Some variant-encoded regions play critical roles in tumor progression. However, the function of CD44 variant 4 (CD44v4)-encoded region has not been fully understood. Using peptide immunization, we developed an anti-CD44v4 mAb, C₄₄Mab-108, which is useful for flow cytometry, western blotting, and immunohistochemistry. In this study, we determined the critical epitope of C₄₄Mab-108 by enzyme-linked immunosorbent assay (ELISA). We used the alanine (or glycine)-substituted peptides of the CD44v4-encoded region (amino acids 271-290 of human CD44v3-10), and found that C₄₄Mab-108 did not recognize the alanine-substituted peptides of D280A and W281A. Furthermore, these peptides could not inhibit the recognition of C₄₄Mab-108 in flow cytometry and immunohistochemistry. The results indicate that the critical binding epitope of C₄₄Mab-108 includes Asp280 and Trp281 of CD44v3-10.

The adsorption of proteins on polymer is widely used in biosensors. Here, molecular dynamics (MD) simulation was used to study the immobilization of angiotensin converting enzyme II (ACE2) with six initial orientations proposed on polystyrene (PS) at the ambient conditions of pH (4.5, 6, 7, 8, 9.5) and NaCl (0.01, 0.05, 0.1, 0.15, 0.2, 0.25 M). ACE2 immobilization under favorable ambient conditions was characterized by minimum distance (short), settlement time (fast), interaction energy (substantial) and protein configuration (stable). ACE2 orientations proposed in 0.15M NaCl were respectively preferable to (90, 0, 0), (0, 0, 0) and (0, 270, 0), (180, 0, 0) and (0, 90, 0), (90, 0, 0) at pH 4.5, 6, 7, 9.5. ACE2 immobilization was further evaluated at pH 7 by optimizing NaCl concentration. Its proposed orientations of (i) (0, 270, 0), (ii) (0, 0, 0) and (90, 0, 0), and (iii) (0, 90, 0) and (90, 0, 0) were preferable in 0.05, 0.1 and 0.2 M NaCl, respectively. The great significance of cubic-orientation settlement mode provides tangible improvement for the microfabrication of biochips for rapid diagnosis of severe acute respiratory syndrome coronavirus II (SARS-CoV-2).

The aim of this study was to investigate the potential of converting potato peel waste into bio ethanol using an in-house produced multi-enzyme preparation. To achieve this, a variety of pre-treatment strategies, including thermal, chemical, and thermo-chemical methods, were applied to the potato peels. These included boiling for 30 and 60 min, steam at varying pressures and times, and different concentrations of chemicals such as H2SO4, HNO3, CH3COOH, HCl, NaOH, Ca(OH)2, KOH, NH3, and H2O2, either alone or in combination with steam. The pre-treated potato peels were then enzymatically hydrolyzed using a crude multi-enzyme cocktail derived from solid-state fermentation of wheat bran by a natural variety of Aspergillus niger P-19, containing cellulases, hemicellulases, pectinase, and amylases. The most effective pre-treatment combination was found to be 3% H2SO4 followed by steam under pressure and enzymatic hydrolysis using the crude multi-enzyme preparation. This combination resulted in the highest yield of reducing sugars (141.04 ±12.31 g/l) with a 98.49% carbohydrate conversion at a 20% substrate loading, giving an ethanol yield of 43.2 g/l. Furthermore, supplementing the medium with peptone, (NH4)(H2PO4), and ZnSO4 at 0.1%w/v each with 22% and 24% solid, respectively, resulted in appreciable yields of 51.67 and 54.75 g/l, further enhancing the ethanol yield.

Encephalartos spp. establish symbioses with nitrogen (N)-fixing bacteria that contribute to soil nutrition and improve plant growth. Despite the Encephalartos mutualistic symbioses with N-fixing bacteria, the identity of other bacteria and their contribution to soil fertility and eco-system functioning are not well understood. This limited information presents a challenge in developing comprehensive conservation and management strategies for these cycad species. Therefore, this study identified the nutrient cycling bacteria in Encephalartos natalensis coral-loid roots, rhizosphere, and non-rhizosphere soils. Additionally, the soil characteristics and soil enzyme activities of the rhizosphere and non-rhizosphere soils were assayed. The coral-loid roots, rhizosphere, and non-rhizosphere soils of E. natalensis were collected from a popu-lation of >500 E. natalensis in a disturbed savanna woodland at Edendale in KwaZulu-Natal (South Africa) for nutrient analysis, bacterial identification, and enzyme activity assays. Nu-trient cycling bacteria such as Lysinibacillus xylanilyticus; Paraburkholderia sabiae, and Novo-sphingobium barchaimii were identified in the coralloid roots, rhizosphere, and non-rhizosphere soils of E. natalensis. Phosphorus (P) cycling (alkaline and acid phosphatase) and N cycling (β-(D)-Glucosaminidase and nitrate reductase) enzyme activities showed a pos-itive correlation with the P and N concentrations in the rhizosphere and non-rhizosphere soils of E. natalensis. Nutrient cycling bacteria identified in E. natalensis coralloid roots, rhizo-sphere, and non-rhizosphere soils and associated enzymes assayed may contribute to soil nu-trient inputs of E. natalensis plants growing in acidic and nutrient-poor savanna woodland ecosystems.

We investigated whether a functional insertion/deletion (I/D) polymorphism of angiotensin-converting enzyme (ACE) influenced antipsychotic treatment. At baseline, and after 8 weeks of treatment with various antipsychotic medications, we assessed patients’ Positive and Negative Syndrome Scale (PANSS) scores, PANSS factors, and metabolic syndrome-related parameters (fasting plasma lipid and glucose levels, and body mass index). A total of 186 antipsychotic-naïve first-episode psychosis patients or nonadherent chronic psychosis individuals (99 males and 87 females) were genotyped by polymerase chain reaction analysis. The ACE-I/D polymorphism was significantly associated with changes in PANSS psychopathology only (p < 0.05). Compared to ACE-II homozygous males, ACE-DD homozygous and ACE-ID heterozygous males manifested significantly greater decreases in PANSS positive score, PANSS excitement factor, and PANSS cognitive factor. ACE-DD homozygous females manifested higher decreases in PANSS depression factor compared to ACE-II homozygous and ACE-ID heterozygous females. The polymorphism’s effect size was estimated as moderate to strong, while its contribution to the PANSS psychopathology ranged from ~5.4–8.7%, with the lowest contribution observed for PANSS positive score changes and the highest for PANSS depressive factor changes. Our results indicated that ACE-I/D polymorphism had a statistically significant but weak gender-specific impact on psychopathology data and showed no association between ACE-I/D polymorphism and metabolic syndrome-related parameters.

Proteases or peptidases are hydrolases that catalyze the breakdown of polypeptide chains into smaller peptide subunits. Proteases exist in all life forms, including archaea, bacteria, protozoa, insects, animals, and plants, due to their vital functions in cellular processing and regulation. There are several classes of proteases in the MEROPS database based on their catalytic mecha-nisms. This review focuses on the post-proline cleaving enzymes (PPCEs), especially the prolyl endoprotease/oligopeptidase (PEP/POP). To date, most PPCEs studied are of microbial and ani-mal origins. Recently, there are reports of new plant PPCEs. The most common PEP/POP are members of the S9 family that comprise two conserved domains. The substrate-limiting β-propeller domain prevents unwanted digestion, while the α/β hydrolase catalyzes reaction at the carboxyl-terminal of proline residues. PPCEs have diverse applications, are widely used in the beer brewing industry, and have potential as therapeutic agents for Alzheimer’s disease and celiac disease by targeting proline-rich substrates. Protein engineering via mutagenesis has been performed to improve heat resistance, pepsin-resistant capability, specificity, and protein turno-ver of PPCEs for pharmacological applications. This is the first comprehensive review to cover the biotechnological applications of PPCEs and discuss the unique prolyl cleaving activity of dif-ferent enzymes based on the recent structure-function studies from diverse taxa.

L-asparaginase (ASNase) is an aminohydrolase currently used in the pharmaceutical and food industries. Enzyme immobilization is an exciting option for both applications, allowing a more straightforward recovery and increased stability. High surface area and customizable porosity make carbon xerogels (CXs) promising materials for ASNase immobilization. This work describes the influence of contact time, pH, and ASNase concentration on the immobilization yield (IY) and relative recovered activity (RRA) using Central Composite Design methodology. The most promising results were obtained using CX with an average pore size of 4 nm (CX-4), reaching IY and RRA of 100%. At the optimal conditions, the ASNase-CXs biocomposite was characterized and evaluated in terms of kinetic properties and operational, thermal and pH stabilities. The immobilized ASNase onto CX-4 retained 71% of its original activity after six continuous reaction cycles, showed a good thermal stability at 37 °C (RRA of 91% after 90 min) and was able to adapt to both acidic and alkaline environments. Finally, the results indicated a 3.9-fold increase in the immobilized ASNase affinity for the substrate, confirming the potential of CXs as a support for ASNase and as a cost-effective tool for subsequent use in the therapeutic and food sectors.

Enzymes are in high demand for very diverse biotechnological applications. However, natural biocatalysts often need to be engineered for fine-tuning their properties towards the end applications, such as the activity, selectivity, stability to temperature or co-solvents, and solubility. Computational methods are increasingly used in this task, providing predictions that narrow down the space of possible mutations significantly and can enormously reduce the experimental burden. Many computational tools are available as web-based platforms, making them accessible to non-expert users. These platforms are typically user-friendly, contain walk-throughs, and do not require deep expertise and installations. Here we describe some of the most recent outstanding web-tools for enzyme engineering and formulate future perspectives in this field.