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The advent of machine learning (ML) has significantly advanced enzyme engineering, particularly through zero‐shot (ZS) predictors that forecast the effects of amino acid mutations on enzyme properties without requiring additional labeled data for the target enzyme. This review comprehensively summarizes ZS predictors developed over the past decade, categorizing them into predictors for enzyme kinetic parameters, stability, solubility/aggregation, and fitness. It details the algorithms used, encompassing traditional ML approaches and deep learning models, emphasizing their predictive performance. Practical applications of ZS predictors in engineering specific enzymes are discussed. Despite notable advancements, challenges persist, including limited training data for ZS predictors and the necessity to incorporate environmental factors (e.g., pH, temperature) and enzyme dynamics into these models. Future directions are proposed to advance ZS prediction‐guided enzyme engineering, thereby enhancing the practical utility of these predictors. [ABSTRACT FROM AUTHOR]

In this study, we aimed to investigate the inhibitory mechanisms of β‐glucuronidase by flavonoids derived from Alhagi graecorum through both experimental and computational approaches. The activity of β‐glucuronidase was assessed using an in vitro enzyme inhibition assay, where myricetin and chrysoeriol were identified as potent inhibitors based on their low IC50 values. Kinetic studies were conducted to determine the inhibition type, revealing that both compounds exhibit noncompetitive inhibition of β‐glucuronidase‐catalyzed hydrolysis of PNPG. Molecular docking was employed to explore the binding affinities of the flavonoids, showing that myricetin formed the highest number of polar interactions with the enzyme. Additionally, molecular dynamics (MD) simulations were performed to evaluate the stability of the enzyme‐inhibitor complexes, demonstrating consistent trajectory behavior for both compounds, with significant energy stabilization. Interaction energy analyses highlighted the dominant role of electrostatic forces in myricetin′s inhibition mechanism, while Van der Waals forces were more prominent for chrysoeriol. The MM/PBSA method was used to calculate the binding free energies, with myricetin and chrysoeriol exhibiting the lowest values. Potential energy landscape analysis further revealed that β‐glucuronidase adopts a more closed conformation when bound to these inhibitors, limiting substrate access. These findings suggest that flavonoids from Alhagi graecorum hold promise for clinical applications, particularly in managing drug‐induced enteropathy. [ABSTRACT FROM AUTHOR]

A series of thiadiazole derivatives were designed and synthesized as dual inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) for the treatment of Alzheimer's disease (AD). Furthermore, the compounds were assayed for their inhibitory activity to AChE and BuChE in vitro, and the results indicated that most of the compounds had moderate inhibitory activity to AChE and BuChE. Among them, compound 11 u showed the best inhibitory activity against AChE and BuChE (eeAChE, IC50=2.73 μM; eqBuChE, IC50=0.65 μM), which showed approximately 2‐fold more activity against AChE enzyme than galantamine and approximately 28‐fold more activity against BuChE enzyme than galantamine. In addition, molecular docking studies have shown that compound 11 u could bind to the catalytic active site (CAS) and peripheral anion site (PAS) of AChE and BuChE. Among them, compound 11 u combined with BuChE and exhibited a mixed inhibition pattern consistent with enzyme kinetics studies. The interaction's stability of 11 u‐AChE/BuChE were also assessed using a conventional atomistic 100 ns dynamics simulation study, which revealed the conformational stability of representative compound 11 u in the cavity of the AChE/BuChE. Moreover, the molecular properties of all compounds were predicted by online through the SwissADME, and the best active compound 11 u matched the properties of most orally administered drugs. All these suggested that 11 u could be considered as a lead compound for the development of drugs for AD. [ABSTRACT FROM AUTHOR]

To develop the drugs as a second line of preventing a serious form of illness, blocking the interaction between a receptor‐binding domain (RBD) in the SARS‐CoV‐2 S‐protein (spike protein) with human ACE2 (Angiotensin converting enzyme 2) can potentially prevent SARS‐CoV‐2 S‐protein from interacting with host cells. In this research, 20 drug compounds are examined using docking to identify potential drugs that can bind at the common level of the RBD‐ACE2 complex and compared the results with two standard drugs offered (Favipiravir, Arbidol). Among 20 drugs, Ozanimod and Prazosin are selected as the best drug compounds by reviewing the docking scores and drug interaction with the active position of RBD‐ACE2. The results of molecular dynamics simulation showed that Ozanimod with binding energy of −14.24 kcal mol−1 has a higher binding capability than Prazosin with binding energy of −9.55 kcal mol−1 to block the interaction between spike protein RBD and human ACE2 enzyme. Ozanimod effectively binds to the S‐protein RBD and inhibits residues critical to the spike and ACE2 protein interaction. This drug compound is expected to be a potentially effective inhibitor of the interaction between the S‐ RBD and the human ACE2 enzyme. [ABSTRACT FROM AUTHOR]

Background and Purpose: Demand for new antidepressants has resulted in a re‐evaluation of the therapeutic potential of psychedelic drugs. Several tryptamines found in psilocybin‐containing "magic" mushrooms share chemical similarities with psilocybin. Early work suggests they may share biological targets. However, few studies have explored their pharmacological and behavioural effects. Experimental Approach: We compared baeocystin, norbaeocystin and aeruginascin with psilocybin to determine if they are metabolized by the same enzymes, similarly penetrate the blood–brain barrier, serve as ligands for similar receptors and modulate behaviour in rodents similarly. We also assessed the stability and optimal storage and handling conditions for each compound. Key Results: In vitro enzyme kinetics assays found that all compounds had nearly identical rates of dephosphorylation via alkaline phosphatase and metabolism by monoamine oxidase. Further, we found that only the dephosphorylated products of baeocystin and norbaeocystin crossed a blood–brain barrier mimetic to a similar degree as the dephosphorylated form of psilocybin, psilocin. The dephosphorylated form of norbaeocystin was found to activate the 5‐HT2A receptor with similar efficacy to psilocin and norpsilocin in in vitro cell imaging assays. Behaviourally, only psilocybin induced head twitch responses in rats, a marker of 5‐HT2A‐mediated psychedelic effects and hallucinogenic potential. However, like psilocybin, norbaeocystin improved outcomes in the forced swim test. All compounds caused minimal changes to metrics of renal and hepatic health, suggesting innocuous safety profiles. Conclusions and Implications: Collectively, this work suggests that other naturally occurring tryptamines, especially norbaeocystin, may share overlapping therapeutic potential with psilocybin, but without causing hallucinations. [ABSTRACT FROM AUTHOR]

Re‐vegetation types and the application of arbuscular mycorrhizal fungi (AMF) play a crucial role in regulating soil conditions and enhancing vegetation cover during ecological restoration in the northern arid mining area. However, the impact of AMF on the balance of soil–plant–microbial ecosystems through its influence on microbial activities remains unclear. Therefore, we studied the influence of AMF on soil nutrient limitation and the soil fungal community in five different vegetation types in the Daliuta reclamation area. Through the measurement of soil extracellular enzyme activity and the analysis of soil fungal communities by high‐throughput sequencing, we found that the ratio of lnBG:ln(NAG + LAP):lnAP in the AMF sample was closer to 1:1:1, and the soil ecological enzyme stoichiometry was more balanced. The results of enzyme vector showed that AMF inoculation could alleviate the restriction of soil nitrogen nutrients. The results of high‐throughput sequencing showed that AMF improved the diversity of soil fungal community by regulating the composition of soil fungal community, and finally alleviated the nitrogen restriction during decomposition. Functional prediction of soil fungi using FUNGuild indicated that the soil fungi present were mainly saprophytic, saprophytic–symbiotic, and pathotroph–saprophytic–symbiotic trophic types. AMF reduced the percentage of pathotroph fungi and also tended to transform saprophytic into saprophytic–symbiotic type. The findings suggest that mycorrhizal reclamation can enhance the homeostasis of extracellular enzyme dynamics by optimizing fungal community structure and modulating fungal functionality, thereby alleviating microbial nutrient constraints and improving plant adaptability to arid environments. This article provides a theoretical underpinning for eco‐friendly mining construction and sustainable ecological development in arid mining areas. [ABSTRACT FROM AUTHOR]

Biomolecular condensates are membraneless organelles that orchestrate various metabolic pathways in living cells. Understanding how these crowded structures regulate enzyme reactions remains yet challenging due to their dynamic and intricate nature. Coacervate microdroplets formed by associative liquid‐liquid phase separation of oppositely charged polyions have emerged as relevant condensate models to study enzyme catalysis. Enzyme reactions within these droplets show altered kinetics, influenced by factors such as enzyme and substrate partitioning, crowding, and interactions with coacervate components; it is often challenging to disentangle the contributions of each. Here, we investigate the peroxidase activity of a de novo enzyme within polysaccharide‐based coacervates. By comparing the reaction kinetics in buffer, in a suspension of coacervates and in the bulk coacervate phase collected after centrifugation of the droplets, we show that the coacervate phase significantly increases the enzyme catalytic efficiency. We demonstrate that the main origin of this enhanced activity lies in macromolecular crowding coupled to changes in the conformational dynamics of the enzyme within the coacervate environment. Altogether, these findings underline the crucial role of the coacervate matrix in enzyme catalysis, beyond simple partitioning effects. The observed boost in enzyme activity within the coacervate phase provides insights for designing biocatalytically active synthetic organelles. [ABSTRACT FROM AUTHOR]

In the pentose phosphate pathway, dehydroepiandrosterone (DHEA) uncompetitively inhibits glucose‐6‐phosphate dehydrogenase (G6PD), reducing NADPH production and increasing oxidative stress, which can influence the onset and/or progression of several diseases, including cancer. 2‐Deoxy‐D‐glucose (2‐DG), a glucose mimetic, competes with glucose for cellular uptake, inhibiting glycolysis and competing with glucose‐6‐phosphate (G‐6‐P) for G6PD activity. In this study, we report that DHEA‐α‐2‐DG (5), an α‐covalent conjugate of DHEA and 2‐DG, exhibits better anticancer activity than DHEA, 2‐DG, DHEA +2‐DG, and polydatin in MCF‐7 cells, and reduces NADPH/NADP+ ratio in cellular assays. In vitro enzyme kinetics and molecular docking studies showed that 5 uncompetitively inhibits human G6PD activity and binds to the structural NADP+ site but not to the catalytic NADP+ site. Further combining 5 with the FDA‐approved drug tamoxifen enhanced its cytotoxicity against MCF‐7 cells, suggesting that it could serve as a candidate for combination of drug strategies. [ABSTRACT FROM AUTHOR]

Biological soil crusts (biocrusts) are crucial elements of desert ecosystems, exhibiting patchy distribution patterns across the soil surface and significantly impacting surface soil nutrient dynamics. However, the influence of biocrust patch units, serving as fundamental functional entities, on microbial nutrient restriction remains underexplored. This study conducted measurements on moss crust patches of varying sizes and subcrust soils. Stoichiometric analysis of extracellular enzyme activities (EEAs) related to carbon, nitrogen, and phosphorus was performed, along with vector and redundancy analyses to evaluate microbial nutrient limitation and key influencing factors. The findings reveal that both patch size and soil layer of biocrusts collectively influence soil nutrients and enzyme dynamics, with heightened enzyme activity observed in the crust layer. Vector analysis based on EEA stoichiometry indicates that moss crust patch size insignificantly impacts microbial nutrient restriction within the crust layer. However, microbial nitrogen restriction in the subcrust layer demonstrates a “single-peak” trend, initially increasing before gradually declining. This suggests that microorganisms in medium-sized crust patches exhibit peak activity, intensifying nutrient competition. This research underscores the pivotal role of biocrust patch units as fundamental functional entities, offering comprehensive insights into microbial metabolic constraints under crust cover. The findings underscore the significant implications of enzyme stoichiometric characteristics for desert land management and conservation. [ABSTRACT FROM AUTHOR]

Ciliates are a well‐defined, species‐rich, versatile group of heterotrophic and mixotrophic protists. Their response to increased temperature during global warming is critical for the structure and functioning of freshwater food webs.I conducted a meta‐analysis of the literature from field studies and experimental evidence to assess the parameters characterising the thermal response of freshwater ciliates. The shape of the thermal performance curve predicts the ciliates' survival at supraoptimal temperatures (i.e., the width of the thermal safety margin).The ciliates' typical thermal safety margin is c. 5°C. More than two‐thirds of the freshwater ciliates dwelling permanently or occasionally in the pelagial cannot survive at temperatures exceeding 30°C. Likewise, cold‐stenothermic species, which represent a significant fraction of euplanktonic ciliates, cannot survive by evolutionary adaptation to rapidly warming environments.The statistical analysis revealed that the ciliates' thermal performance is affected by their planktonic lifestyle (euplanktonic vs. tychoplanktonic), ability to form cysts and nutritional ecology. Bactivorous ciliates have the widest temperature niche, and algivorous ciliates have the narrowest temperature niche.Phenotypic plasticity and genetic variation, favouring the selection of pre‐adapted species in a new environment, are widespread among freshwater ciliates. However, the lack of evidence for the temperature optima and imprecisely defined tolerance limits of most species hampers the present analysis. The extent of acclimation and adaptation requires further research with more ciliate species than the few chosen thus far.Recent eco‐evolutionary experimental work and modelling approaches have demonstrated that the ciliates' thermal responses follow general trends predicted by the metabolic theory of ecology and mechanistic functions inherent in enzyme kinetics. The present analysis identified current knowledge gaps and avenues for future research that may serve as a model study for other biota. Thermal adaptation may conflict with adaptation to other stressors (predators, food availability, pH), making general predictions on the future role of freshwater ciliates in a warmer environment difficult, if not impossible, at present. [ABSTRACT FROM AUTHOR]

BACKGROUND: The industrial‐scale application of enzymes faces obstacles due to elevated costs and difficulties in stability and reuse. In this study, magnetic spent coffee grounds, an ecotoxic waste, have been utilized successfully for the first time to immobilize β‐glucosidase to overcome these challenges. RESULTS: The spent coffee grounds were magnetized and amine‐functionalized, followed by characterization using various techniques. Under optimized conditions, forming an imine bond between the functionalized support and β‐glucosidase resulted in a 62% immobilization yield (92.81 mg g−1 enzyme loading) and 12.5 U mg−1 activity after immobilization. A relatively small kinetic change was observed in the Km value (902 to 946 μmol L−1) after immobilization, suggesting minimal hindrance by AMSCG3 on substrate access or product release. Moreover, Glu@AMSCG3 showed exceptional stability (>90% residual activity) within a pH range of 3 to 6 after 2 h of incubation at 25 °C. A residual activity of 87.94% was maintained even at 80 °C and pH 5 after 2 h of incubation compared to the free β‐glucosidase, which showed only 6.5% residual activity at the same temperature. When cellobiose was hydrolyzed using Glu@AMSCG3 under optimum conditions, 91.33% cellobiose conversion was achieved initially, and over 79% conversion was maintained for 10 reusability cycles. CONCLUSION: The improved stability of β‐glucosidase after covalent immobilization on amine‐modified magnetically separable spent coffee grounds indicates their potential as a support matrix for application in enzyme immobilization. © 2024 Society of Chemical Industry (SCI). [ABSTRACT FROM AUTHOR]

Peroxyacid synthesis is the first step in Prilezhaev epoxidation, which is an industrial method to form epoxides. Motivated by the development of a kinetic model as a tool for solvent selection, the effect of solvent type and acid chain length on the lipase‐catalyzed peroxyacid synthesis was studied. A thermodynamic activity‐based ping‐pong kinetic expression was successfully applied to predict the effect of the reagent loadings in hexane. The activity‐based reaction quotients provided a prediction of solvent‐independent equilibrium constants. However, this strategy did not achieve satisfactory estimations of initial rates in solvents of higher polarity. The lack of compliance with some assumptions of this methodology could be confirmed through molecular dynamics calculations i.e. independent solvation energies and lack of solvent interaction with the active site. A novel approach is proposed combining the activity‐based kinetic expression and the free binding energy of the solvent with the active site to predict kinetics upon solvent change. Di‐isopropyl ether generated a strong interaction with the enzyme's active site, which was detrimental to kinetics. On the other hand, toluene or limonene gave moderate interaction with the active site rendering improved catalytic yield compared with less polar solvents, a finding sharpened when peroctanoic acid was produced. [ABSTRACT FROM AUTHOR]

Enzyme kinetics and inhibition studies are crucial in biochemistry education and research. Conventional methods often require expensive equipment and reagents, potentially limiting their accessibility in limited resource settings. Our approach sought to develop a cost‐effective experimental design for studying enzyme kinetics and inhibition. Lactase was chosen as a protein model and its activity was investigated by measuring glucose production from lactose hydrolysis. In the study, commercially available lactase pills were used as an enzyme source, while milk was used as a substrate. Instead of scientific equipment, glucometers were used to measure lactase activity. Enzyme kinetics were evaluated using Michaelis–Menten and Lineweaver–Burk plots. In the study, the effects of temperature, pH, and inhibitors were also investigated. The results of our study aligned with established enzyme kinetics theories and previous studies. Lactase showed temperature and pH‐dependent activity, with decreased activity observed at both low and high extremes. Results also showed that galactose acts as a competitive inhibitor of lactase. The approach presented here offers a cost‐effective procedure for studying enzyme kinetics and inhibition. It can act as a valuable tool for educational purposes and for preliminary research in settings with limited resources. [ABSTRACT FROM AUTHOR]

Here we investigate the evolutionary dynamics of five enzyme superfamilies (CYPs, GSTs, UGTs, CCEs and ABCs) involved in detoxification in Helicoverpa armigera. The reference assembly for an African isolate of the major lineages, H. a. armigera, has 373 genes in the five superfamilies. Most of its CYPs, GSTs, UGTs and CCEs and a few of its ABCs occur in blocks and most of the clustered genes are in subfamilies specifically implicated in detoxification. Most of the genes have orthologues in the reference genome for the Oceania lineage, H. a. conferta. However, clustered orthologues and subfamilies specifically implicated in detoxification show greater sequence divergence and less constraint on non‐synonymous differences between the two assemblies than do other members of the five superfamilies. Two duplicated CYPs, which were found in the H. a. armigera but not H. a. conferta reference genome, were also missing in 16 Chinese populations spanning two different lineages of H. a. armigera. The enzyme produced by one of these duplicates has higher activity against esfenvalerate than a previously described chimeric CYP mutant conferring pyrethroid resistance. Various transposable elements were found in the introns of most detoxification genes, generating diverse gene structures. Extensive resequencing data for the Chinese H. a. armigera and H. a. conferta lineages also revealed complex copy number polymorphisms in 17 CCE001s in a cluster also implicated in pyrethroid metabolism, with substantial haplotype differences between all three lineages. Our results suggest that cotton bollworm has a versatile complement of detoxification genes which are evolving in diverse ways across its range. [ABSTRACT FROM AUTHOR]

Fructose bisphosphate aldolases (FBAs) catalyze the reversible cleavage of fructose 1,6‐bisphosphate into dihydroxyacetone phosphate and glyceraldehyde 3‐phosphate. We analyzed two previously uncharacterized cytosolic Arabidopsis FBAs, AtFBA4 and AtFBA5. Based on a recent report, we examined the interaction of AtFBA4 with calmodulin (CaM)‐like protein 11 (AtCML11). AtFBA4 did not bind AtCML11; however, we found that CaM bound AtFBA5 in a Ca2+‐dependent manner with high specificity and affinity (KD ~ 190 nm) and enhanced its stability. AtFBA4 and AtFBA5 exhibited Michaelis–Menten kinetics with Km and Vmax values of 180 μm and 4.9 U·mg−1 for AtFBA4, and 6.0 μm and 0.30 U·mg−1 for AtFBA5, respectively. The flavonoid morin inhibited both isozymes. Our study suggests that Ca2+ signaling and flavanols may influence plant glycolysis/gluconeogenesis. [ABSTRACT FROM AUTHOR]

Hydrogen‐bonded organic frameworks (HOF) represent an emerging category of organic structures with high crystallinity and metal‐free, which are not commonly observed in alternative porous organic frameworks. These needle‐like porous structure can help in stabilizing enzymes and allow transfer of molecules between enzymes participating in cascade reactions for enhanced substrate channelling. Herein, we systematically synthesized and investigated the stability of HOF at extreme conditions followed by one‐pot encapsulation of single and bi‐enzyme systems. Firstly, we observed HOF to be stable at pH 1 to 14 and at high temperatures (up to 115 °C). Secondly, the encapsulated glucose oxidase enzyme (GOX) showed 80 % and 90 % of its original activity at 70 °C and pH 11, respectively. Thirdly, transient time close to 0 seconds was observed for HOF encapsulated bi‐enzyme cascade reaction system demonstrating a 4.25‐fold improvement in catalytic activity when compared to free enzymes with enhanced substrate channelling. Our findings showcase a facile system synthesized under ambient conditions to encapsulate and stabilize enzymes at extreme conditions. [ABSTRACT FROM AUTHOR]

The ability to finely tune/balance the structure and rigidity of enzymes to realize both high enzymatic activity and long‐term stability is highly desired but highly challenging. Herein, we propose the concept of the "silicazyme", where solid inorganic silica undergoes controlled hybridization with the fragile enzyme under moderate conditions at the single‐enzyme level, thus enabling simultaneous structure augmentation, long‐term stability, and high enzymatic activity preservation. A multivariate silicification approach was utilized and occurred around individual enzymes to allow conformal coating. To realize a high activity–stability trade‐off the structure flexibility/rigidity of the silicazyme was optimized by a component adjustment ternary (CAT) plot method. Moreover, the multivariate organosilica frameworks bring great advantages, including surface microenvironment adjustability, reversible modification capability, and functional extensibility through the rich chemistry of silica. Overall silicazymes represent a new class of enzymes with promise for catalysis, separations, and nanomedicine. [ABSTRACT FROM AUTHOR]

Nanozymes have demonstrated significant potential in combating malignant tumor proliferation through catalytic therapy. However, the therapeutic effect is often limited by insufficient catalytic performance. In this study, we propose the utilization of strain engineering in metallenes to fully expose the active regions due to their ultrathin nature. Here, we present the first report on a novel tensile strain‐mediated local amorphous RhRu (la‐RhRu) bimetallene with exceptional intrinsic photothermal effect and photo‐enhanced multiple enzyme‐like activities. Through geometric phase analysis, electron diffraction profile, and X‐ray diffraction, it is revealed that crystalline‐amorphous heterophase boundaries can generate approximately 2 % tensile strain in the bimetallene. The ultrathin structure and in‐plane strain of the bimetallene induce an amplified strain effect. Both experimental and theoretical evidence support the notion that tensile strain promotes multiple enzyme‐like activities. Functioning as a tumor microenvironment (TME)‐responsive nanozyme, la‐RhRu exhibits remarkable therapeutic efficacy both in vitro and in vivo. This work highlights the tremendous potential of atomic‐scale tensile strain engineering strategy in enhancing tumor catalytic therapy. [ABSTRACT FROM AUTHOR]

A novel and versatile approach called "physical imprinting" is introduced to modulate enzyme conformation using mesoporous materials, addressing challenges in achieving improved enzyme activity and stability. Metal–organic frameworks with tailored mesopores, precisely matching enzyme size and shape, are synthesized. Remarkably, enzymes encapsulated within these customized mesopores exhibit over 1670% relative activity compared to free enzymes, maintaining outstanding efficiency even under harsh conditions such as heat, exposure to organic solvents, wide‐ranging pH extremes from acidic to alkaline, and exposure to a digestion cocktail. After 18 consecutive cycles of use, the immobilized enzymes retain 80% of their initial activity. Additionally, the encapsulated enzymes exhibit a substantial increase in catalytic efficiency, with a 14.1‐fold enhancement in kcat/KM compared to native enzymes. This enhancement is among the highest reported for immobilized enzymes. The improved enzyme activity and stability are corroborated by solid‐state UV–vis, electron paramagnetic resonance, Fourier‐transform infrared spectroscopy, and solid‐state NMR spectroscopy. The findings not only offer valuable insights into the crucial role of size and shape complementarity within confined microenvironments but also establish a new pathway for developing solid carriers capable of enhancing enzyme activity and stability. [ABSTRACT FROM AUTHOR]

Summary: Obesity is becoming a top global health problem. Inhibition of digestive enzymes is an effective approach in weight management due to the reduction of energy absorption. In this study, the inhibitory effects of luteolin on α‐glucosidase and pancreatic lipase were investigated in vitro and in vivo via enzyme kinetics, fluorescence quenching, molecular docking and rat experiments. The results showed that luteolin inhibited α‐glucosidase and pancreatic lipase in a mixed‐type and uncompetitive manner with IC50 values of 29.31 and 42.74 μg mL−1 respectively. The binding of luteolin with the two enzymes caused the quenching of intrinsic fluorescence of protein. Molecular docking simulation revealed that luteolin could be located in the active pocket of α‐glucosidase and interact with the key amino acid residues. Moreover, a hydrophobic interaction and a van der Waals force were formed between luteolin and the key amino acid residues of pancreatic lipase (His264 and Ser153). In vivo studies further confirmed that luteolin could significantly decrease the absorption of glucose and triacylglyceride in rats by inhibiting α‐glucosidase and pancreatic lipase. [ABSTRACT FROM AUTHOR]

The present work is focused on the study of polyurethane adhesives in the presence of functional fillers (encapsulated enzymes) intended to make them degradable. This is part of a general strategy aiming to recycle multilayer films for packaging, at their end of life, by means of separating the constituent layers as a result of adhesives degradation. Primarily, it was found that the presence of small quantities of functional filler greatly affects both, the quality of the coating process and the kinetics of the adhesive curing. The former, because of the dependency on particle size distribution, leads to high variations in thickness and mechanical properties of multilayers and, the latter, because of a significant catalytic effect (higher curing rates). Indeed, an assessment of the adhesives' kinetics by rheology showed that the pot‐life and the cross‐over point (G′ = G″) for the enzyme‐containing system were obtained three times faster in comparison to the control formulation. These findings were also confirmed by differential scanning calorimetry measurements. On the other hand, by reducing and homogenizing the filler's particle size using separated sieved material (≤50 μm), improvements in coating control as well as in the quality and final properties of the multilayers were evidenced. Highlights: Recycling multilayer plastic films for flexible packaging.Encapsulated enzymes to degrade the polyurethane adhesive in specific conditions.The presence of functional fillers affects the coating process.Process optimization to ensure the coating quality control. [ABSTRACT FROM AUTHOR]

Heterogeneous biocatalysis is highly relevant in biotechnology as it offers several benefits and practical uses. To leverage the full potential of heterogeneous biocatalysts, the establishment of well‐crafted protocols, and a deeper comprehension of enzyme immobilization on solid substrates are essential. These endeavors seek to optimize immobilized biocatalysts, ensuring maximal enzyme performance within confined spaces. For this aim, multidimensional characterization of heterogeneous biocatalysts is required. In this context, spectroscopic and microscopic methodologies conducted at different space and temporal scales can inform about the intraparticle enzyme kinetics, the enzyme spatial distribution, and the mass transport issues. In this Minireview, we identify enzyme immobilization, enzyme catalysis, and enzyme inactivation as the three main processes for which advanced characterization tools unveil fundamental information. Recent advances in operando characterization of immobilized enzymes at the single‐particle (SP) and single‐molecule (SM) levels inform about their functional properties, unlocking the full potential of heterogeneous biocatalysis toward biotechnological applications. [ABSTRACT FROM AUTHOR]

Pestalotiopsis guepinii is a pathogenic fungus that causes grey blight on Camellia pitardii. In this study, we investigated the enzyme activity and kinetics of these cell‐wall‐degrading enzymes (CWDEs) produced by Pestalotiopsis guepinii in both C. pitardii leaves and culture medium. Our enzyme activity experiments revealed that the activities of xylanase, pectin methyl‐galacturonase (PMG), β‐1,4‐endoglucanase (Cx), and β‐glucosidase were high in both C. pitardii leaves and culture medium. These enzymes played a significant role in the pathogenic process. However, the activity of laccase was found to be very low and had a minor impact on the pathogenic process. Furthermore, our enzyme dynamics experiments demonstrated that the optimal reaction temperature for PMG and Cx was 50°C, while for β‐glucosidase and xylanase, it was 60°C. The optimal reaction pH for Cx, β‐glucosidase, and xylanase was 5.0, whereas for PMG, it ranged from 5.0 to 6.0. This indicates that these four enzymes prefer acidic conditions. Moreover, we observed that the activities of Cx, PMG, and xylanase decreased with increasing reaction time. On the other hand, the activity of β‐glucosidase initially increased sharply and then decreased slowly. The maximum reaction rates of the four cell‐wall‐degrading enzymes were ranked as follows: xylanase > PMG > β‐glucosidase > Cx. Additionally, the affinities of these enzymes with substrates were ranked as follows: PMG < Cx < xylanase < β‐glucosidase. [ABSTRACT FROM AUTHOR]

Enzymes are nature's catalysts, mediating chemical processes in living systems. The study of enzyme function and mechanism includes defining the maximum catalytic rate and affinity for substrate/s (among other factors), referred to as enzyme kinetics. Enzyme kinetics is a staple of biochemistry curricula and other disciplines, from molecular and cellular biology to pharmacology. However, because enzyme kinetics involves concepts rarely employed in other areas of biology, it can be challenging for students and researchers. Traditional graphical analysis was replaced by computational analysis, requiring another skill not core to many life sciences curricula. Computational analysis can be time‐consuming and difficult in free software (e.g., R) or require costly software (e.g., GraphPad Prism). We present Enzyme Kinetics Analysis (EKA), a web‐tool to augment teaching and learning and streamline EKA. EKA is an interactive and free tool for analyzing enzyme kinetic data and improving student learning through simulation, built using R and RStudio's ShinyApps. EKA provides kinetic models (Michaelis–Menten, Hill, simple reversible inhibition models, ternary‐complex, and ping‐pong) for users to fit experimental data, providing graphical results and statistics. Additionally, EKA enables users to input parameters and create data and graphs, to visualize changes to parameters (e.g., KM or number of measurements). This function is designed for students learning kinetics but also for researchers to design experiments. EKA (enzyme-kinetics.shinyapps.io/enzkinet_webpage/) provides a simple, interactive interface for teachers, students, and researchers to explore enzyme kinetics. It gives researchers the ability to design experiments and analyze data without specific software requirements. [ABSTRACT FROM AUTHOR]

This review article aims to significantly advance the scientific community's efforts to develop effective nucleoside‐based drugs for treating severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and other emerging infectious diseases. This study concentrates on the main viral protease (Mpro) and explores nucleoside‐based compounds as potential therapeutic agents. This investigation investigated the impact of acylation‐induced modifications on the nucleoside hydroxyl group and subsequent properties. Nucleoside analogs, which are recognized for their diverse biochemical properties, were synthesized and rigorously screened to evaluate their antimicrobial efficacy. In the domain of pharmaceutical research, computational pharmacokinetics has emerged as a critical tool, especially in the pursuit of nucleoside analogs as potential therapeutics. In silico methods aid in predicting pharmacokinetic traits, interactions with crucial enzymes, and the stability of these analogs in biological environments, thereby streamlining drug design and reducing experimental costs. Concurrently, computational studies revealed the intricate interactions between the analogs and the active site of the main protease. The amalgamation of experimental screening and computational insights underscores the emergence of potent nucleoside candidates with inhibitory activity against SARS‐CoV‐2 Mpro. Additionally, this review integrates computational studies that provide valuable insights into the interactions between nucleoside analogs and the main protease of SARS‐CoV‐2. [ABSTRACT FROM AUTHOR]

The photoenzyme protochlorophyllide oxidoreductase (POR) is an important enzyme for understanding biological H‐transfer mechanisms. It uses light to catalyse the reduction of protochlorophyllide to chlorophyllide, a key step in chlorophyll biosynthesis. Although a wealth of spectroscopic data have provided crucial mechanistic insight, a structural rationale for POR photocatalysis has proved challenging and remains hotly debated. Recent structural models of the ternary enzyme–substrate complex, derived from crystal and electron microscopy data, show differences in the orientation of the protochlorophyllide substrate and the architecture of the POR active site, with significant implications for the catalytic mechanism. Here, we use a combination of computational and experimental approaches to investigate the compatibility of each structural model with the hypothesised reaction mechanisms and propose an alternative structural model for the cyanobacterial POR ternary complex. We show that a strictly conserved tyrosine, previously proposed to act as the proton donor in POR photocatalysis, is unlikely to be involved in this step of the reaction but is crucial for Pchlide binding. Instead, an active site cysteine is important for both hydride and proton transfer reactions in POR and is proposed to act as the proton donor, either directly or through a water‐mediated network. Moreover, a conserved glutamine is important for Pchlide binding and ensuring efficient photochemistry by tuning its electronic properties, likely by interacting with the central Mg atom of the substrate. This optimal 'binding pose' for the POR ternary enzyme–substrate complex illustrates how light energy can be harnessed to facilitate enzyme catalysis by this unique enzyme. [ABSTRACT FROM AUTHOR]

Diabetes mellitus (DM) is one of the most challenging noncommunicable diseases, as it causes significant costs for medical treatment as well as high morbidity and mortality rates. Dietary plants with antidiabetic properties have been explored as an alternative to synthetic medicines to treat DM because of their safety and nutrition. Hence, the objective of the present study was to determine the inhibitory activities of twenty commonly consumed Thai culinary vegetables against α-glucosidase and α-amylase. All vegetables were extracted using deionized water, ethanol, and hexane at 150 rpm and 30°C for 24 hours. The enzyme inhibitory activities were performed using a colorimetric assay. Diverse results for α-glucosidase and α-amylase inhibitory activities were found for all vegetable extracts. The most potent anti-α-glucosidase activity was obtained from the ethanolic extract of Leucaena leucocephala (Lamk.) de Wit with the half maximal inhibitory concentration (IC50) of 13.39 ± 0.14 μg/mL, followed by the aqueous and ethanolic extracts of Polygonum odoratum Lour with IC50 of 25.60 ± 0.42 and 49.03 ± 0.72 μg/mL, respectively. All the samples exhibited mixed, noncompetitive, and uncompetitive inhibition. It can be concluded that the α-glucosidase and α-amylose inhibitory effects of the investigated extracts may be an indicator of antidiabetic potency, and these extracts might potentially be beneficial as functional components for postprandial hyperglycemia treatment. [ABSTRACT FROM AUTHOR]

Single component flavin‐dependent halogenases (FDHs) possess both flavin reductase and FDH activity in a single enzyme. We recently reported that the single component FDH AetF catalyzes site‐selective bromination and iodination of a variety of aromatic substrates and enantioselective bromolactonization and iodoetherification of styrenes bearing pendant carboxylic acid or alcohol substituents. Given this inherent reactivity and selectivity, we explored the utility of AetF as catalyst for alkene and alkyne C−H halogenation. We find that AetF catalyzes halogenation of a range of 1,1‐disubstituted styrenes, often with high stereoselectivity. Despite the utility of haloalkenes for cross‐coupling and other applications, accessing these compounds in a stereoselective manner typically requires functional group interconversion processes, and selective halogenation of 1,1′‐disubstituted olefins remains rare. We also establish that AetF and homologues of this enzyme can halogenate terminal alkynes. Mutagenesis studies and deuterium kinetic isotope effects are used to support a mechanistic proposal involving covalent catalysis for halogenation of unactivated alkynes by AetF homologues. These findings expand the scope of FDH catalysis and continue to show the unique utility of single component FDHs for biocatalysis. [ABSTRACT FROM AUTHOR]

Computational design advances enzyme evolution and their use in biocatalysis in a faster and more efficient manner. In this study, a synergistic approach integrating tunnel engineering, evolutionary analysis, and force‐field calculations has been employed to enhance the catalytic activity of D‐lactonohydrolase (D‐Lac), which is a pivotal enzyme involved in the resolution of racemic pantolactone during the production of vitamin B5. The best mutant, N96S/A271E/F274Y/F308G (M3), was obtained and its catalytic efficiency (kcat/KM) was nearly 23‐fold higher than that of the wild‐type. The M3 whole‐cell converted 20 % of DL‐pantolactone into D‐pantoic acid (D‐PA, >99 % e.e.) with a conversion rate of 47 % and space‐time yield of 107.1 g L−1 h−1, demonstrating its great potential for industrial‐scale D‐pantothenic acid production. Molecular dynamics (MD) simulations revealed that the reduction in the steric hindrance within the substrate tunnel and conformational reconstruction of the distal loop resulted in a more favourable"catalytic" conformation, making it easier for the substrate and enzyme to enter their pre‐reaction state. This study illustrates the potential of the distal residue on the pivotal loop at the entrance of the D‐Lac substrate tunnel as a novel modification hotspot capable of reshaping energy patterns and consequently influencing the enzymatic activity. [ABSTRACT FROM AUTHOR]

Single‐molecule enzyme activity assay is a platform that enables the analysis of enzyme activities at single proteoform level. The limitation of the targetable enzymes is the major drawback of the assay, but the general assay platform is reported to study single‐molecule enzyme activities of esterases based on the coupled assay using thioesters as substrate analogues. The coupled assay is realized by developing highly water‐soluble thiol‐reacting probes based on phosphonate‐substituted boron dipyrromethene (BODIPY). The system enables the detection of cholinesterase activities in blood samples at single‐molecule level, and it is shown that the dissecting alterations of single‐molecule esterase activities can serve as an informative platform for activity‐based diagnosis. [ABSTRACT FROM AUTHOR]

The study aims at revealing the mechanisms of the viscous medium effects on the kinetic features of NAD(P)H:FMN‐oxidoreductase from luminous bacteria (Red), which are exhibited in a single enzyme assay and in coupling with bacterial luciferase (BLuc). Different concentrations of glycerol and sucrose were used to vary the medium viscosity. The activity of Red, alone and in the presence of BLuc, was analyzed, as well as BLuc activity in the presence of Red, whereas in the absence of BLuc, the Red activity was suppressed in viscous medium, and in the presence of BLuc, the increase in Red activity was observed at low glycerol concentrations (5–20 wt%). The interaction of glycerol and sucrose with Red substrates FMN and NADH was studied using absorption spectroscopy and molecular dynamics. Glycerol was found to form hydrogen bonds with the phosphate groups of the substrates, unlike sucrose. A mechanism for the activation of Red in the presence of BLuc in glycerol solutions through the acceleration of FMN reoxidation was proposed. Thus, it was concluded that, under the conditions used, the weakest link of the coupled enzyme system BLuc‐Red in viscous medium is the FMN concentration, which depends on Red activity and the medium viscosity. [ABSTRACT FROM AUTHOR]

The combination of X‐ray free‐electron lasers (XFELs) with serial femtosecond crystallography represents cutting‐edge technology in structural biology, allowing the study of enzyme reactions and dynamics in real time through the generation of 'molecular movies'. This technology combines short and precise high‐energy X‐ray exposure to a stream of protein microcrystals. Here, the XFEL structure of carbonic anhydrase II, a ubiquitous enzyme responsible for the interconversion of CO2 and bicarbonate, is reported, and is compared with previously reported NMR and synchrotron X‐ray and neutron single‐crystal structures. [ABSTRACT FROM AUTHOR]

Litter enzyme dynamics are strongly shaped by litter, soil and microbial attributes during decomposition; however, the enzyme dynamics of leaf and root litter remain unresolved due to contrasting differences in rates and controls on leaf and root litter decomposition.Herein, we conducted a 784‐day field experiment to evaluate the relative importance of litter, alkaline soil and microbial attributes to enzyme activities and their C:N:P stoichiometry of leaf and root litter during decomposition under subtropical land use change in China.We found that only the C‐ and N‐acquiring enzyme activities of shrub leaves were greater than those of wood and crop, and there was no significant difference in P‐acquiring enzyme activity among the three species of leaves. Both the C‐ and P‐acquiring enzyme activities of crop roots were significantly lower than those of afforested lands (i.e. woodland and shrubland). The N‐acquiring activities of wood roots were significantly lower than those of shrub and crop. At the temporal dynamics, the C‐, N‐ and P‐acquiring enzyme activities of the leaves decreased with mass loss, which was affected by the shift in litter nutrients (e.g. N and P) and soil moisture during decomposition. In contrast, the three enzyme activities of roots increased with mass loss, largely due to the increase in microbial biomass of bacteria regulated by litter stoichiometry. The enzymatic C:nutrient (N and P) ratios declined with mass loss, but the enzymatic P:N ratios remained relatively constant with mass loss during the leaf litter decomposition. Whereas, both of the enzymatic C:nutrient ratios and enzymatic P:N ratios decreased with mass during the root litter decomposition. Our results showed that the enzymatic C:N:P stoichiometry of decaying leaves and roots was predominantly predicted by microbial biomass and bacterial biomass, respectively.Overall, we outlined the pattern of contrasting contributions of litter, soil and microbial attributes to enzyme dynamics during decomposition, which provided a framework for better understanding litter C, N and P dynamics in relation to microbial resource allocation strategy during decomposition. Read the free Plain Language Summary for this article on the Journal blog. [ABSTRACT FROM AUTHOR]

Cold Plasma (CP) is an emerging non‐thermal technology that preserves and decontaminates food. Kiwifruit juice spoilage enzymes polyphenol oxidase (PPO) and peroxidase (POD) were studied at 24‐36 kV, 2‐10 mm juice depth, and 2‐10 min treatment time using log‐linear, Weibull distribution, and logistic models. The Weibull and logistic model explained enzyme inactivation kinetics well based on goodness of fit (Coefficient of determination: 0.97‐0.99 & root mean square error: 0.11‐2.15) and validation indices (accuracy factor: 1‐1.06 and bias factor: 0.96‐1.04). The logistic model suited PPO and POD inactivation curves well, with the least Akaike weight in 71.29% and 61.45%, respectively. POD had a half‐maximal activity of 11.37, 5.22, and 3.60 min at 24 kV, 30 kV, and 36 kV at 6 mm juice depth, compared to 8.71, 4.41, and 2.51 min for PPO. This shows that POD is more CP‐resistant than PPO, making its inactivation in kiwifruit juice crucial. Practical Application: Kinetic modeling helps to comprehend the fundamental process and is suitable for improving the inactivation process and enhancing its efficacy. As a relatively primary method, mathematical models facilitate a greater comprehension of the mechanism and factors influencing the inactivation processes. This study involves the inactivation of PPO and POD to produce a stable kiwifruit juice. The inactivation of PPO and POD enzymes will prevent the undesirable enzymatic browning of juice. The kinetic data related to enzyme inactivation will help in deciding the process parameters like plasma voltage, juice depth, and treatment time for producing enzymatically stable kiwifruit juice. [ABSTRACT FROM AUTHOR]

In this study, with an aim to develop novel heterocyclic hybrids as potent enzyme inhibitors, we synthesized a series of 10 novel 2‐(4‐(4‐ethyl‐5‐(2‐(substitutedphenyl)‐2‐oxo‐ethylthio)‐4H‐1,2,4‐triazol‐3‐yl)‐phenyl)‐5,6‐dimethyl‐1H‐benzimidazole (5a–5j) derivatives and characterized by 1H‐NMR, 13C‐NMR, and HRMS. These compounds were evaluated for their inhibitory activity against hCA I and hCA II. All the compounds exhibited good hCA I and hCA II inhibitory activities with IC50 values in range of 1.288 μM–3.122 μM. Among all these compounds, compound 5e, with an IC50 value of 1.288 μM is the most active against carbonic hCA I. Compound 5h with an IC50 value of 1.532 μM is the most active against carbonic hCA‐II. Compounds 5a–5j were also evaluated for their cytotoxic effects on the L929 mouse fibroblast (normal) cell line. The compounds were also analyzed for their antioxidant capacity by TAS, FRAP, and DPPH activity. Enzyme inhibition kinetics showed all compounds 5a–5j to inhibit the enzyme by non‐competitive. The most active compound 5e for hCA I and compound 5h for hCA‐II were subjected to molecular docking, which revealed their binding interactions with the enzyme's active site, confirming the experimental findings. [ABSTRACT FROM AUTHOR]

Cochliobolus hawaiiensis Alcorn Assiut University Mycological Centre 8606 was chosen from the screened 20 fungal species as the potent producer of fibrinolytic enzyme on skimmed‐milk agar plates. The greatest enzyme yield was attained when the submerged fermentation (SmF) conditions were optimized, and it was around (39.7 U/mg protein). Moreover, upon optimization of fibrinolytic enzyme production under solid‐state fermentation (SSF), the maximum productivity of fibrinolytic enzyme was greatly increased recorded a bout (405 U/mg protein) on sugarcane bagasse, incubation period of 5 days, moisture level of 100%, initial pH of salt basal medium 7.8, incubation temperature at 35°C, and supplementation of the salt basal medium with corn steep liquor (80％, v/v). The yield of fibrinolytic enzyme by C. hawaiiensis under SSF was higher than that of SmF with about 10.20‐fold. The purification procedures of fibrinolytic enzyme by ammonium sulfate (70%), gel filtration, and ion‐exchange columns chromatography caused a great increase in its specific activity to 2581.6 U/mg protein with an overall yield of 55.89%, 6.37 purification fold and molecular weight of 35 kDa. Maximal activity was recorded at pH 7 and 37°C. Significant pH stability was recorded at pH 6.6–7.2, and thermal stability was recorded at 33–41°C. The enzyme showed the highest affinity toward fibrin, with Vmax of 240 U/mL and an apparent Km value of 47.61 mmol. Mg2+ and Ca2+ moderately induced fibrinolytic activity, whereas Cu2+ and Zn2+ greatly suppressed the enzyme activity. The produced enzyme is categorized as serine protease and non‐metalloprotease. The purified fibrinolytic enzyme showed efficient thrombolytic and antiplatelet aggregation activities by completely prevention and dissolution of the blood clot which confirmed by microscopic examination and amelioration of blood coagulation assays. These findings suggested that the produced fibrinolytic enzyme is a promising agent in management of blood coagulation disorders. [ABSTRACT FROM AUTHOR]

For the green fabrication of silver nanoparticles (AgNPs), scientists are favoring herbal sources to avoid the toxic effects of synthetic sources. Thus, in this study, the AgNPs were generated using the herbal extract of the whole plant Hippeastrum hybridum L. (HH) and 1 mM AgNO3 aqueous solution. These HH‐AgNPs were characterized via UV–Vis Spectroscopy, which showed a maximum absorbance of 1.61 at a 432 nm sharp peak; FT‐R analysis, which confirmed the functional groups in HH extract and their AgNPs; XRD analysis, which gives 4‐Bragg's reflections at 2θ and confirmed the HH‐AgNPs crystal structure with 13.3 nm average size; SEM analysis, which confirmed the irregular‐shaped morphology with 40 nm mean size; and EDX analysis, which confirmed the elemental composition and reported that Ag was present in 22.75%. After characterization, these AgNPs were tested as anti‐Alzheimer agents against acetylcholinesterase (AChE) in ex vivo activity using rat brain homogenate as a source of AChE enzyme. AChE showed 48 ± 0.03 and 42 ± 0.05% activity at 150 μg concentration of HH plant and HH‐AgNPs, respectively, with 145 ± 0.24 and 124.2 ± 0.14 μg IC50 concentration. According to enzyme kinetics results, the plant extract inhibits AChE in competitive mode (Km increased from 166.2% to 1,379.2% and Vmax not affected), while HH‐AgNPs showed the mixed mode of inhibition (Km increased from 0.029 to 0.048, and Vmax decreased from 9 to 5.4). KIapp and Vmaxiapp were also calculated (KIapp increased from 39 to 95.1 μg, and Vmaxiapp remained constant for the plant), while for HH‐AgNPs, both Kmaxippand Vmaxiapp increased from 122.32 to 325 μg and 8.15 to 9.8 μg, respectively. The Km, Ki, and KI were also calculated and were found to be 0.017 mM (HH‐plant) and 0.2 mM (HH‐AgNPs); 98 μg (HH‐plant); and 129 μg (HH‐AgNPs), 53 μg (HH‐plant), and 179 μg (HH‐AgNPs), respectively. γKm (18 mM) was calculated for HH‐AgNPs, while for HH‐plant, it is not applicable. In conclusion, it could be said that HH‐plant plays a significant role in the generation of small‐size HH‐AgNPs. These AgNPs exhibited significant anti‐AChE potential as compared to HH‐plant extract and proved to be an herbal source for the treatment of AD. [ABSTRACT FROM AUTHOR]

β‐eudesmol is a major bioactive component of Atractylodes lancea (AL). AL has been developed as the capsule formulation of standardized AL extract for treating cholangiocarcinoma (CCA). However, the complex constituents of herbal products increase the risk of adverse drug interactions. β‐eudesmol has demonstrated inhibitory effects on rCYP2C19 and rCYP3A4 in the previous research. This study aimed to identify the cytochrome P450 (CYP) isoforms responsible for the metabolism of β‐eudesmol and determine the enzyme kinetic parameters and the metabolic stability of β‐eudesmol metabolism in the microsomal system. Reaction phenotyping using human recombinant CYPs (rCYPs) and selective chemical inhibitors of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 was performed, and enzyme kinetics and metabolic stability were investigated using human liver microsome (HLM). The results suggest that CYP2C19 and CYP3A4 play significant roles in β‐eudesmol metabolism. The disappearance half‐life (t1/2) and intrinsic clearance (CLint) of β‐eudesmol were 17.09 min and 0.20 mL/min·mg protein, respectively. Enzyme kinetic analysis revealed the Michaelis–Menten constant (Km) and maximum velocity (Vmax) of 16.76 μM and 3.35 nmol/min·mg protein, respectively. As a component of AL, β‐eudesmol, as a substrate and inhibitor of CYP2C19 and CYP3A4, has a high potential for drug–drug interactions when AL is co‐administered with other herbs or conventional medicines. [ABSTRACT FROM AUTHOR]

Postharvest losses in fruits and vegetables pose a key challenge to the global horticulture industry. Improved storage stability of horticultural commodities through irradiation is a technological intervention which has greater compliance over more than 60 foods and food products. The present study was intended to explore the effect of γ-irradiation doses on the postharvest quality of fresh guava fruits stored under ambient (temperature 20 ± 2 °C, RH 85%) conditions. Various storage specific quality attributes including physiological weight loss, respiration rate (CO2), ethylene estimation, ripening index, fruit firmness, ascorbic acid, nutraceutical potential, and enzyme kinetics were evaluated during postharvest storage at four days of interval. The results indicated that the fruits irradiated at doses (0.8 kGy and 1.0 kGy) exhibited significant reduction in physiological parameters in contrast with untreated fruit samples. Firmness, total phenolic compounds, flavonoids, and radical scavenging activity were successfully retained in irradiated fruit samples up to twenty days of storage. The respiration and ethylene production rates were suppressed in irradiated guava fruits compared to control. Moreover, it has also been discovered that the fruits exposed to 1.0 kGy had considerably decreased the polyphenol oxidase (PPO) activity compared to nonirradiated fruits, thus resulting in lowering the enzymatic browning. Aside from that, irradiation guava fruits had considerably enhanced antioxidant enzyme activity (P ≤ 0.05) in terms of catalase, peroxidase, and superoxide dismutase. In conclusion, the study revealed that γ -rays irradiation doses up to 1.0 kGy might be considered effective to improve the postharvest storage of native guava fruits variety with intact nutritional attributes. [ABSTRACT FROM AUTHOR]

The European catfish Silurus glanis is attracting growing interest as an object of fisheries and aquaculture, which is reinforced by the expansion of its natural range under climate change. Shaping the effective exploitation strategy for this valuable species requires detailed knowledge of its biology, including feeding and digestion processes, especially near the natural limits of the species range. Meanwhile, the digestion physiology of the European catfish remains poorly explored, including the activity of major digestive enzymes and the possible effect of intestinal parasites on this activity. In this regard, the activity of proteinases and α‐amylase in the intestinal mucosa of the catfish was studied. Adult catfish were collected in the Rybinsk reservoir (Upper Volga) located close to the northern limit of the species range. It was shown that all subclasses of intestinal digestive proteinases, including serine proteinases, metalloproteases and cysteine (thiol) proteinases, function in the gut mucosa of the catfish. The mucosal levels of total proteolytic activity depended on fish size, in contrast to those of trypsin, chymotrypsin and α‐amylase. The level of chymotrypsin activity was significantly higher than that of trypsin activity. It was also found that the incubation medium and extract of the cestodes Silurotaenia siluri parasitizing the catfish gut had a significant inhibitory effect on the activity of serine proteases (trypsin and chymotrypsin) operating in the intestines of the host fish. [ABSTRACT FROM AUTHOR]

An understanding of structure–function relationships in proteins is essential for modern biochemical studies. The integration of common freely accessible bioinformatics tools available online with the knowledge of protein‐engineering tools provide a fundamental understanding of the application of protein structure–function for biochemical research. In order for students to apply their prior knowledge of recombinant protein technology into the understanding of protein structure–function relationships, we developed a semester‐long project‐oriented biochemistry laboratory experience that is the second laboratory course of a series. For easier integration of knowledge and application, we organized this course into four sequential modules: protein structure visualization/modification, mutagenesis target identification, site‐directed mutagenesis, and mutant protein expression, purification, and characterization. These tasks were performed on the protein small laccase (SLAC) that was cloned and characterized by students in the previous semester during the first biochemistry laboratory course of the series. This goal‐oriented project‐based approach helped students apply their prior knowledge to newly introduced techniques to understand protein structure–function relationships in this research‐like laboratory setting. A student assessment before and after the course demonstrated an overall increase in learning and enthusiasm for this topic. [ABSTRACT FROM AUTHOR]

Covalent regulatory systems of enzymes are widely used to modulate biological enzyme activities. Inspired by the regulation of reactive‐site phosphorylation in organisms, we developed peptide‐based catecholase mimetics with switchable catalytic activity and high selectivity through the co‐assembly of nanofibers comprising peptides and copper ions (Cu2+). Through careful design and modification of the peptide backbone structure based on the change in the free energy of the system, we identified the peptide with the most effective reversible catalytic activity. Kinase/phosphatase switches were used to control the reversible transition of nanofiber formation and depolymerization, as well as to modulate the active‐site microenvironment. Notably, the self‐assembly and disassembly processes of nanofibers were simulated using coarse‐grained molecular dynamics. Furthermore, theoretical calculations confirmed the coordination of the peptide and Cu2+, forming a zipper‐like four‐ligand structure at the catalytically active center of the nanofibers. Additionally, we conducted a comprehensive analysis of the catalytic mechanism. This study opens novel avenues for designing biomimetic enzymes with ordered structures and dynamic catalytic activities. [ABSTRACT FROM AUTHOR]

Fungal unspecific peroxygenases (UPOs) have gained substantial attention for their versatile oxyfunctionalization chemistry paired with impressive catalytic capabilities. A major drawback, however, remains their sensitivity towards their co‐substrate hydrogen peroxide, necessitating the use of smart in situ hydrogen peroxide generation methods to enable efficient catalysis setups. Herein, we introduce flavin‐containing protein photosensitizers as a new general tool for light‐controlled in situ hydrogen peroxide production. By genetically fusing flavin binding fluorescent proteins and UPOs, we have created two virtually self‐sufficient photo‐enzymes (PhotUPO). Subsequent testing of a versatile substrate panel with the two divergent PhotUPOs revealed two stereoselective conversions. The catalytic performance of the fusion protein was optimized through enzyme and substrate loading variation, enabling up to 24300 turnover numbers (TONs) for the sulfoxidation of methyl phenyl sulfide. The PhotUPO concept was upscaled to a 100 mg substrate preparative scale, enabling the extraction of enantiomerically pure alcohol products. [ABSTRACT FROM AUTHOR]

Environmentally friendly industrial and biotech processes greatly benefit from enzyme‐based technologies. Their use is often possible only when the enzyme‐catalytic mechanism is thoroughly known. Thus, atomic‐level knowledge of a Michaelis enzyme‐substrate complex, revealing molecular details of substrate recognition and catalytic chemistry, is crucial for understanding and then rationally extending or improving enzyme‐catalyzed reactions. However, many known enzymes sample huge protein conformational space, often preventing complete structural characterization by X‐ray crystallography. Moreover, using a cognate substrate is problematic since its conversion into a reaction product in the presence of the enzyme will prevent the capture of the enzyme‐substrate conformation in an activated state. Here, we outlined how to deal with such obstacles, focusing on the recent discovery of a Renilla‐type bioluminescence reaction mechanism facilitated by a combination of engineered ancestral enzyme and the availability of a non‐oxidizable luciferin analogue. The automated ancestral sequence reconstructions using FireProtASR provided a thermostable enzyme suited for structural studies, and a stable luciferin analogue azacoelenterazine provided a structurally cognate chemical incapable of catalyzed oxidation. We suggest that an analogous strategy can be applied to various enzymes with unknown catalytic mechanisms and poor crystallizability. [ABSTRACT FROM AUTHOR]

Alkaline proteases from microbial sources have been found suitable for diverse industrial applications, with serine proteases being the most common enzymes used in the detergent industry. In the present study, we have purified and characterized an extracellular alkaline serine protease from Microbacterium paraoxydans sp. SKS10. The protease was purified using ammonium sulfate precipitation followed by different chromatography techniques (fold purification 6.919). Km and Vmax for the protease were determined to be 0.183 mg/mL and 4.904 U/mL, respectively. This enzyme is a thermostable high molecular weight (∼109.4 kDa) protease which has maximal activity at 60°C, and above pH 10. Inhibitor assays revealed the enzyme to be a serine protease whose activity increased by 2.5‐fold in the presence of EDTA. This enzyme remained active in the presence of various metal salts and organic solvents and was compatible with commercially available laundry detergents highlighting its potential for use in the detergent industry. [ABSTRACT FROM AUTHOR]

The amino acid residues at the entrance of the catalytic pocket may impose steric hindrance on the substrate to enter the active center of the enzyme. Based on the analysis of the three‐dimensional structure of the Saccharomyces cerevisiae old yellow enzyme 3 (OYE3), four bulky residues were chosen and mutated to small amino acids. The results showed that mutation of the W116 residue had interesting impacts on the catalytic performance. All four variants became inactive for the reduction of (R)‐carvone and (S)‐carvone, but inverted the stereoselectivity for the reduction of (E/Z)‐citral. The mutation of the F250 residue had a more positive effect on the activity and stereoselectivity. Two variants, F250A and F250S, showed excellent diastereoselectivity and activity for the reduction of (R)‐carvone (de > 99%, c > 99%) and increased diastereoselectivity and activity for the reduction of (S)‐carvone (de > 96%, c > 80%). One variant of the P295 residue, P295G, displayed excellent diastereoselectivity and activity only for the reduction of (R)‐carvone (de > 99%, c > 99%). Mutation of the Y375 residue had a negative impact on the activity of the enzyme. These findings provide some solutions for rational enzyme engineering of OYE3. [ABSTRACT FROM AUTHOR]

Cystathionine β‐synthase (CBS) catalyzes the condensation of l‐serine and l‐homocysteine to give l‐cystathionine in the transsulfuration pathway. Recently, a few O‐acetylserine (l‐OAS)‐dependent CBSs (OCBSs) have been found in bacteria that can exclusively function with l‐OAS. CBS from Toxoplasma gondii (TgCBS) can efficiently use both l‐serine and l‐OAS to form l‐cystathionine. In this work, a series of site‐specific variants substituting S84, Y160, and Y246 with hydrophobic residues found at the same positions in OCBSs was generated to explore the roles of the hydroxyl moieties of these residues as determinants of l‐serine/l‐OAS preference in TgCBS. We found that the S84A/Y160F/Y246V triple mutant behaved like an OCBS in terms of both substrate requirements, showing β‐replacement activity only with l‐OAS, and pH optimum, which is decreased by ~1 pH unit. Formation of a stable aminoacrylate upon reaction with l‐serine is prevented by the triple mutation, indicating the importance of the H‐bonds between the hydroxyl groups of Y160, Y246, and S84 with l‐serine in formation of the intermediate. Analysis of the independent effect of each mutation on TgCBS activity and investigation of the protein–aminoacrylate complex structure allowed for the conclusion that the hydroxyl group of Y246 has a major, but not exclusive, role in controlling the l‐serine preference by efficiently stabilizing its leaving group. These studies demonstrate that differences in substrate specificity of CBSs are controlled by natural variations in as few as three residue positions. A better understanding of substrate specificity in TgCBS will facilitate the design of new antimicrobial compounds. [ABSTRACT FROM AUTHOR]

Background: The addition of organic solvents to an aqueous medium for enzymatic reactions offers several advantages, as they can increase the solubility of substrates but can also lead to enzyme inactivation and/or aggregation. Results: The effect of adding 30% of several water‐soluble organic solvents on the catalytic activity of lipase B from Candida antarctica (CalB) was studied and the results showed that the highest activity was obtained with the addition of t‐butanol. t‐Butanol and acetonitrile were selected and the kinetic parameters, determined to deepen their effect on CalB activity, showed that the addition of acetonitrile improved the enzyme–substrate affinity, while water–t‐butanol mixtures led to a more than ninefold increase in kcat. To rationalize at a molecular level the kinetic results, molecular dynamic simulations were performed. Analysis of the accessibility of the active‐site cavity, solvent occupancy in the site and in the oxyanion hole, and the stability of the catalytic triad in the two solvent mixtures, provided insight into their effects on the catalytic properties of CalB. Conclusion: The lower occupancy in the oxyanion hole of water molecules and a shorter residence time in the active site of acetonitrile molecules in the acetonitrile–water mixture contribute to the higher enzyme–substrate affinity found experimentally. Conversely, the higher kcat in the t‐butanol mixture is explained by the higher stability of the catalytic triad and by an increase in the nucleophilicity of the catalytic serine due to the persistent presence of t‐butanol molecules in the active site. © 2023 The Authors. Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI). [ABSTRACT FROM AUTHOR]

In this study, a new series of benzoxazolone−thiosemicarbazide/1,2,4‐triazole/1,3,4‐thiadiazole hybrids were designed and synthesized. The structures of novel compounds were elucidated by spectral methods (IR, 1H‐NMR, 13C‐NMR, ESI‐MS) and elemental analyses. Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibition activity of the compounds were investigated using in vitro Ellman method using donepezil and tacrin as the standards. Although none of compounds showed significant inhibitor activity on BuChE, compounds 2 c, 3 a, 4 a and 5 b displayed good inhibitory activities on AChE with IC50 values of 0.031, 0.049, 0.117 and 0.085 μg/mL, respectively. Molecular docking studies were carried out performed to the interactions with the active site of the enzyme (PDB:4EY7) and enzyme kinetics studies were performed for the most potent AChE inhibitor compound 2 c. [ABSTRACT FROM AUTHOR]

A thermophilic amylase produced by a newly isolated Geobacillus thermoleovorans TTIO4 from a hot spring in India is investigated. The isolate TTIO4 optimally produces extracellular amylase at 55 °C, pH 7, and 24 h of growth. The amylase is purified by 42 fold with a yield of 9.7%. The enzyme has a molecular weight of 40 kDa. The purified amylase catalyzes the reaction optimally at 60 °C and pH 7. Besides, it displays maximum thermal stability at 50 °C for 18 h. While the catalyst is improved by Ca+2, it is wholly inhibited by Hg+2. Furthermore, the chelators, inhibitors and solvents adversely affect the enzyme activity. The amylase is stabilized by SDS, while other surfactants negatively affect the enzyme. Km, Vmax, Kcat, and Kcat/Km of the amylase are 0.26 mg mL−1, 192.3 µg mL−1 min−1, 1.92 s−1, and 7.383 s−1 mg−1, respectively. The deactivation constant, half‐life, and change in Gibb's free energy, enthalpy, and entropy are determined at 50–70 °C, which supports the thermal stability and substrate affinity of the amylase. The washing quality of commercial detergents is distinctly enhanced when supplemented with this enzyme. Therefore, this amylase possesses significant thermal and surfactant stability has significant commercial potential. [ABSTRACT FROM AUTHOR]