Native Enzyme Research Articles

Pathway-Dependent Catalytic Activity of Short-Peptide-Based Metallozyme: From Promiscuous Activity to Cascade Reaction.

Many natural enzymes contain metal ions as cofactors in the active site for biological activity. However, the pathway of the introduction of metal ions in the earliest protein folds for the emergence of higher catalytic activity remains an intriguing open question. Herein, we demonstrate that pathway-dependent self-assembly of short-peptide-based metallozymes results in differences in catalytic activity. Short-peptide-based amyloids with solvent exposed arrays of colocalized catalytic units are able to bind highly soluble Cu2+ ions to demonstrate oxidase-like and RNase-like activity (promiscuity). Further, the metallozyme was able to exhibit higher hydrolase-oxidase cascade activity compared to the mixture of natural enzymes, esterase, and laccase. The collaboration between short-peptide-based amyloid microphases and metal ions suggests that metallozymes might have played a pivotal role in early metabolic processes and biopolymer evolution on the prebiotic earth.

NADH-dependent CO2 reductase on graphite for capacitive electrocatalytic interfacing mediated by solid-binding peptide.

NAD+/NADH-dependent CO2 reductase (CR) adapted from Candida methylica(E.C. 1.17.1.9) was introduced with a non-native graphite-specific peptide (Gr; IMVTESSDYSSY) as molecular binder to modify the native enzyme (CR-WT) with peptide insertion at N, C and NC terminus (CR-GrN, CR-GrC and CR-GrNC) to assess the influence of site-specific fusion on electrode binding. Graphite surface-binding activity relative to the electrode topography was evaluated for both native and synthetic CRs to establish the enzyme-electrode interfacing potentiality for efficient electron channelling. Impact of site-specific peptide fusion and amino-acids positioning was assessed for the active site binding availability and adsorption/desorption capability towards competent CO2-based redox catalysis. Solid-binding peptide and graphite surface interactive ability on direct electron transfer was studied with structural, enzymatic and electrochemical characterizations for efficient CO2 electrosynthesis. Overall, enzymatic CO2 reduction to formate based on interactive potentiality of enzyme-electrode complex with peptide modifications and graphite surface towards possibility of bioelectronics upscaling was depicted.

Nanozyme-based wearable biosensors for application in healthcare.

Recent years have witnessed tremendous advances in wearable sensors, which play an essential role in personalized healthcare for their ability for real-time sensing and detection of human health information. Nanozymes, capable of mimicking the functions of natural enzymes and addressing their limitations, possess unique advantages such as structural stability, low cost, and ease of mass production, making them particularly beneficial for constructing recognition units in wearable biosensors. In this review, we aim to delineate the latest advancements in nanozymes for the development of wearable biosensors, focusing on key developments in nanozyme immobilization strategies, detection technologies, and biomedical applications. The review also highlights the current challenges and future perspectives. Ultimately, it aims to provide insights for future research endeavors in this rapidly evolving area.

Directed Evolution's Selective Use of Quantum Tunneling in Designed Enzymes─A Combined Theoretical and Experimental Study.

Natural enzymes are powerful catalysts, reducing the apparent activation energy for reactions and enabling chemistry to proceed as much as 1015 times faster than the corresponding solution reaction. It has been suggested for some time that, in some cases, quantum tunneling can contribute to this rate enhancement by offering pathways through a barrier inaccessible to activated events. A central question of interest to both physical chemists and biochemists is the extent to which evolution introduces mechanisms below the barrier, or tunneling mechanisms. In view of the rapidly expanding chemistries for which artificial enzymes have been created, it is of interest to see how quantum tunneling has been used in these reactions. In this paper, we study the evolution of possible proton tunneling during C-H bond cleavage in enzymes that catalyze the Morita-Baylis-Hillman (MBH) reaction. The enzymes were generated by theoretical design, followed by laboratory evolution. We employ classical and centroid molecular dynamics approaches in path sampling computations to determine whether there is a quantum contribution to lowering the free energy of the proton transfer for various experimentally generated protein and substrate combinations. These data are compared to experiments reporting on the observed kinetic isotope effect (KIE) for the relevant reactions. Our results indicate the modest involvement of tunneling when laboratory evolution has resulted in a system with a higher classical free energy barrier to chemistry (that is, when optimization of processes other than chemistry results in a higher chemical barrier).

Design Strategy of PepNzymes-SH for an Emerging Catalyst with Serine Hydrolase-Like Functionality.

Serine hydrolases, as a class of green catalysts with hydrolytic and dehydrating activities, hold significant application value in the fields of biosynthesis and organic synthesis. However, practical applications face numerous challenges, including maintaining enzyme stability and managing usage costs. PepNzymes-SH, an emerging green catalytic material with enzyme-like activity, overcomes the operational limitations of natural enzymes and holds great promise as a substitute for hydrolases. Unfortunately, a systematic review of the design strategies for PepNzymes-SH is currently lacking. The core significance of this report lies in providing researchers with a comprehensive understanding and theoretical guidance through the summarization and performance evaluation of different design strategies of PepNzymes-SH. This review summarizes strategies for simulating and enhancing the stability of serine hydrolase active sites, oxyanion holes, and hydrophobic environmental structures. By comparing their catalytic activities, we assess the performance changes brought about by different strategies. Furthermore, the applications of PepNzymes-SH in the chemical, biomedicine, and environmental fields are also discussed.

In vitro gastrointestinal digestion simulation screening of novel ACEI peptides from broccoli: mechanism in high glucose-induced VSMCs dysfunction

Many natural angiotensin-converting enzyme inhibitory (ACEI) peptides have been widely studied. However, their stability in vivo is poor in most cases. In this study, peptides were initially digested from broccoli in vitro, and absorption was simulated by Caco2 cells transport and then analyzed by Peptideomics and molecular docking. Subsequently, the mechanisms were verified using a high glucose-induced vascular smooth muscle cells (VSMCs) dysfunction model. Results showed that ACEI activity of broccoli crude peptide increased by 70.73 ± 1.42% after digestion. The enzymatic hydrolysates of crude broccoli peptides before and after digestion were detected by HPLC. The digested crude peptides were highly stable (with a stability level > 90%) in the intestine and possessed a strong absorptive potential. Five peptides with high stability and strong permeability were first identified, including HLEVR, LTEVR, LEHGF, HLVNK, and LLDGR, which exhibited high activity with IC50 values of 3.19 ± 0.23 mM, 17.07 ± 1.37 mM, 0.64 ± 0.02 mM, 0.06 ± 0.01 mM, and 2.81 ± 0.12 mM, respectively. When the VSMCs model was exposed to Ang II, the expressions of PCNA, MMP2, and Bcl2 were increased, while the expression of BAX was inhibited. When the VSMCs was exposed to high glucose (HG), the Ang II concentration significantly increased. This indicates that HG elevated Ang II levels. Finally, five peptides significantly attenuated Ang II-induced VSMCs proliferation and migration by down-regulating AT1R expression and inhibiting ERK and p38 MAPK phosphorylation. Notably, in exploring VSMCs dysfunction on a high glucose-induced model, ACEI peptides resulted in down-regulation of ACE and up-regulation of ACE2 expression. Therefore, it can be further referenced for the functional food against hypertension and cardiovascular diseases.

Transaminases: high-throughput screening via a ketone-fluorescent probe and applications

Transaminases are a class of enzymes that catalyze the transfer of amino between amino acids and keto acids, playing an important role in the biosynthesis of organic amines and the corresponding derivatives. However, natural enzymes often have low catalytic efficiency against non-natural substrates, which limits their widespread applications. Enzyme engineering serves as an effective approach to improve the catalytic properties and thereby expand the application scope of transaminases. In this study, a high-throughput screening method for transaminases was established based on the fluorescent color reaction between methoxy-2-aminobenzoxime (PMA) and ketones. According to the changes in fluorescence intensity, the concentration changes of ketones could be easily monitored. The efficiency, sensitivity, and accuracy of the screening method were improved by optimization of the system. With 4-hydroxy-2-butanone as the substrate, the mutant library of the transaminase from Actinobacteria sp. was established and a mutant with increased activity was successfully obtained, which improved the production efficiency of (R)-3-aminobutanol by enzyme-catalyzed synthesis. This study laid an important foundation for efficient screening, modification, and application of transaminase.

Nanoenzyme-based sensors for the detection of anti-tumor drugs.

Natural enzymes are a class of biological catalysts that can catalyze a specific substrate. Although natural enzymes have catalytic activity, they are susceptible to the influence of external environment such as temperature, and storage requirementsare more stringent. Since the first discovery of magnetic Fe3O4 nanoparticles with peroxidase-like activity in 2007, the research on nanoenzymes has entered a rapid development stage. Nanoenzymes synthesized by chemical methods not only have the catalytic activity of natural enzymes but also are more stable, easy to store, and convenient to prepare. Anthracyclines, as a commonly used anti-tumor chemotherapy drug, will produce many side effects such as myelosuppression and liver function damage after long-term use, which will affect its therapeutic effects. This paper reviews the characteristics, classification, and mechanisms of nanoenzymes. The detection of anti-tumor drugs, especially anthracycline drugs, using a nanoenzyme-based sensor was emphatically introduced. On this basis, the application of nanoenzyme-based sensors in the detection of anti-tumor drugs is prospected.

Nucleic acid joining enzymes: biological functions and synthetic applications beyond DNA.

DNA-joining by ligase and polymerase enzymes has provided the foundational tools for generating recombinant DNA and enabled the assembly of gene and genome-sized synthetic products. Xenobiotic nucleic acid (XNA) analogues of DNA and RNA with alternatives to the canonical bases, so-called 'unnatural' nucleobase pairs (UBP-XNAs), represent the next frontier of nucleic acid technologies, with applications as novel therapeutics and in engineering semi-synthetic biological organisms. To realise the full potential of UBP-XNAs, researchers require a suite of compatible enzymes for processing nucleic acids on a par with those already available for manipulating canonical DNA. In particular, enzymes able to join UBP-XNA will be essential for generating large assemblies and also hold promise in the synthesis of single-stranded oligonucleotides. Here, we review recent and emerging advances in the DNA-joining enzymes, DNA polymerases and DNA ligases, and describe their applications to UBP-XNA manipulation. We also discuss the future directions of this field which we consider will involve two-pronged approaches of enzyme biodiscovery for natural UBP-XNA compatible enzymes, coupled with improvement by structure-guided engineering.

Efficient hydrogen peroxide reduction in glutathione peroxidase cycle using cost-effective FeSe2 nanospheres

IntroductionHydrogen peroxide plays a crucial role in melanogenesis by regulating tyrosinase activity, the key melanin-forming enzyme responsible for the browning of fruits, vegetables, and seafood. The need for effective solutions to mitigate such browning processes highlights the significance of developing advanced catalytic agents.MethodsWe synthesized highly effective FeSe2 nanospheres using a one-step solvothermal process. The nanospheres were characterized through transmission electron microscopy (TEM), energy-dispersive X-ray analysis (EDX), and powder X-ray diffraction (XRD). Enzymatic activity was evaluated by plotting Michaelis-Menten and Lineweaver-Burk graphs to calculate the Vmax and Km parameters. Comparative analyses with a control sample and other known enzymes were performed to assess the catalytic efficiency.Results and discussionFeSe2 nanospheres successfully catalyzed the reduction of hydrogen peroxide to water and alcohol, demonstrating enzyme-like activity. The initial reaction rate was 11 times higher than the control sample and significantly outperformed other enzymes, except for those relying on expensive noble metals. These nanospheres (termed Nanozymes) mimic the enzymatic action of natural antioxidants, such as the glutathione peroxidase (GPx) enzyme, in biological systems. Their exceptional efficiency makes them a strong candidate for practical applications in mitigating early browning caused by melanogenesis.ConclusionsFeSe2 nanozymes exhibit great promise as a biocatalyst for enhancing the shelf life of fruits and vegetables by reducing damage due to early melanogenesis. This cost-effective and efficient alternative to natural or noble metal-based enzymes offers significant potential for applications in food preservation and other industries.

Photo‐Regulated Catalysis of Nanozymes

AbstractNanozymes are a class of artificially synthesized enzymes that aim to overcome the limitations of natural enzymes, such as their susceptibility to denaturation and high production costs and are found to possess multiple enzyme activities over the natural enzymes that can be adopted for multi‐feature biomedical use. By harnessing the unique properties of nanomaterials, nanozymes can be tailored to specific applications, making them promising alternatives to natural enzymes. Researchers have adopted various strategies to regulate the activity of nanozymes such as altering the size, morphology, surface modifications, and so on to better manipulate their activities for specific uses. Due to its unique advantages such as non‐invasiveness, ease of adjusting power, and instant on/off capabilities, light regulation has emerged as a popular strategy for regulating the activity of nanozymes in recent years. This article provides a short review of the recent advances in regulating the activities of nanozymes through light modulation and offers insights into their mechanism and promising applications in the biomedical fields.

Computational Investigation of the Role of Metal Center Identity in Cytochrome P450 Enzyme Model Reactivity.

Mononuclear Fe enzymes such as heme-containing cytochrome P450 enzymes catalyze a variety of C-H activation reactions under ambient conditions, and they represent an attractive platform for engineering reactivity through changes to the native enzyme. Using density functional theory, we study both native Fe and non-native group 8 (Ru, Os) and group 9 (Ir) metal centers in an active site model of P450. We quantify how changing the metal changes spin state preferences throughout the catalytic cycle. Our calculations reveal an intermediate-spin ground state for all Fe intermediates while the heavier metals prefer low-spin ground states across most intermediates in the reaction cycle. We also study the rate-determining hydrogen atom transfer (HAT) step and the subsequent rebound step. We observe comparable HAT barriers for Fe and Ru, a much higher barrier for Os, and the lowest HAT barrier for Ir. Rebound steps are barrierless for all metals, and the rebound intermediate for Fe is most significantly stabilized. Examination of ground spin states of all intermediates in the reaction cycle reveals spin-allowed pathways for the group 8 metals and spin-forbidden energetics for the group 9 Ir with potential two-state reactivity. Our work highlights the differences between the group 8 metals and the group 9 Ir, and it suggests that engineered P450 enzymes with Ru in particular result in improved enzyme reactivity toward C-H hydroxylation.

Colorimetric Detection of Dopamine Based on Peroxidase-like Activity of β-CD Functionalized AuNPs.

Catalytically active nanomaterials, or nanozymes, have gained significant attention as alternatives to natural enzymes due to their low cost, ease of preparation, and enhanced stability. Because of easy preparation, excellent biocompatibility, and unique optoelectronic properties, gold nanoparticles (AuNPs) have attracted increasing attention in many fields, including nanozymes. In this work, we demonstrated the applicability of beta-cyclodextrin functionalized gold nanoparticles (β-CD-AuNPs) as enzyme mimics for different substances, including TMB and DA. We found that β-CD-AuNPs can catalyze the H2O2-mediated oxidation of DA. The dopamine signal-off sensor was developed by taking advantage of the peroxidase-like activity of β-CD-AuNPs towards TMB and DA, where both 3,3',5,5'-tetramethylbenzidine (TMB) and dopamine (DA) may compete for the binding sites with β-CD-AuNPs. As a result, the presence of dopamine can be detected even through the naked eye (up to the concentration of 3.75 µM) and using a spectrophotometer (up to the concentration of 1.0 µM) by monitoring the disappearance of the blue color of the oxidized form of TMB in the presence of dopamine. Furthermore, no obvious disappearance of color was observed at lower concentrations of interferences including ascorbic and uric acid. Given the versatility of cyclodextrin to host large numbers of analyte molecules, we envision that a similar principle can be applied for the detection of other analyte molecules of biological, medical, and environmental significance.

A Powerful Tumor Catalytic Therapy by an Enzyme-Nanozyme Cascade Catalysis (ENCAT) System.

Complexity of tumor and its microenvironment as obstacles often restrict traditional tumor therapies. Enzyme/nanozyme-mediated catalytic therapy has been emerged, but the efficacy of single catalytic therapy is still moderate. Inspired by the concepts of catalytic and synergetic therapy, an enzyme-nanozyme cascade catalysis (ENCAT)-enhanced tumor therapy is developed. First, metal-organic framework (MOF) PCN222-Mn (PM) and glucose oxidase (GOx) are chosen as nanozyme and natural enzyme, respectively. Then two assembled together to form enzyme-nanozyme complex PCN222-Mn@GOx (PMG). To achieve tumor targeting and GOx protection, hyaluronic acid (HA) is modified on PMG to obtain PCN222-Mn@GOx/HA (PMGH). Both cellular and animal studies demonstrate a cascade catalysis-enhanced tumor therapy by PMGH. Specifically, a cascade catalysis-enhanced PDT is achieved based on enzyme-nanozyme mediated cascade-catalyzed O2 generation; an enhanced synergistic therapy is demonstrated by combining PM-mediated PDT, GOx-mediated starvation therapy, and activated/promoted immunotherapy together. Additionally, the designed tumor catalytic therapy is explored in a tumor bearing mouse model, where it exhibits powerful anti-tumor effects against both primary and metastatic tumors. This strategy has the potential to broaden tumor therapeutic approaches.

Intervening with nanozymes in aging-related diseases: Strategies for restoring mitochondrial function.

The decline in mitochondrial function has been identified as one of the central pathological mechanisms underlying a variety of aging-related diseases. Nanozymes are nanomaterials with intrinsic enzyme-like properties and are important alternatives to natural enzymes. As emerging biocatalysts, nanozymes exhibit significant potential in mimicking the activity of natural enzymes, enhancing mitochondrial function, and offering novel therapeutic strategies for aging-related conditions. This review provides an overview of various approaches to modulate the catalytic activity of nanozymes, considering factors such as particle size, shape, surface modifications, and constituent elements. It then examines the role of nanozymes in mitigating aging-related diseases by preserving mitochondrial health, with a particular focus on their ability to regulate three critical aspects: mitochondrial energy metabolism, quality control, and antioxidant capacity. By improving mitochondrial energy generation, supporting mitochondrial integrity, and eliminating excess reactive oxygen species (ROS), nanozymes offer new therapeutic possibilities for neurodegenerative diseases, bone-related disorders, and diabetes. Finally, this article discusses the major challenges faced in this field, including issues such as the scalability, biocompatibility, and targeting ability of nanozymes. It also emphasizes that future research should focus on enhancing clinical translation to ensure that nanozymes can play an effective role in practical therapeutic applications.

Nanozyme-based synergistic therapeutic strategies against tumors.

Cancer remains a global health threat, with traditional treatments limited by adverse effects and drug resistance. Nanozyme-based catalytic therapy with high stability and controllable activity provides targeted and specific in situ tumor treatment to address these challenges. More intriguingly, the tremendous advances in nanotechnology have enabled nanozymes to rival the catalytic activity of natural enzymes, presenting an exciting opportunity for innovating antitumor nanodrugs. This review systematically summarizes the latest progresses in nanozyme-based anticancer catalytic therapy, with a particular focus on various synergistic antitumor strategies, including other functional enzymes, drugs, exogenous stimuli and radiotherapy. These combinations not only enhance the efficacy of cancer treatment and reduce systemic toxicity but also offer insights into the development of potent antitumor nanodrugs.

Strand-Swapped SH3 Domain Dimer with Superoxide Dismutase Activity.

The design of metalloproteins allows us to better understand metal complexation in proteins and the resulting function. In this study, we incorporated a Cu2+-binding site into a natural protein domain, the 58 amino acid c-Crk-SH3, to create a miniaturized superoxide dismutase model, termed SO1. The resulting low complexity metalloprotein was characterized for structure and function by circular dichroism and UV spectroscopy as well as EPR spectroscopy and X-ray crystallography. The miniprotein was found to be a strand-swapped dimer with an unusual coupled binuclear Type 2-like copper center in each protomer. SO1-Cu was found to be SOD-active with an activity only 1 order of magnitude lower than that of natural SOD enzymes and 1 to 2 orders of magnitude higher than that of other low-complexity SOD protein models of similar size. This serendipitous design provides us with a new structural template for future designs of binuclear metalloproteins with different metal ions and functions.

Enhanced Antibacterial Activity of Hydrophobic Modified Lysozyme Against Gram-Negative Bacteria Without Accumulated Resistance.

Macromolecule bactericides present challenges such as low biocompatibility and not being biodegradable, so broad-spectrum bactericides without accumulated bacteria resistance are now in urgent demand all over the world. Lysozyme, a kind of wide-spread natural enzyme easily extracted from nature, has become attractive for agriculture and medicine use. However, Gram-negative bacterial strains are highly resistant to natural lysozymes, which limits their practical application. In this study, rather than directly modifying antibacterial-active substance with lysozyme, we show an effective way to improve antibacterial performance by altering the hydrophobic functional groups of natural lysozymes and synthesize a type of hydrophobic modified lysozyme (HML). Compared with other modification methods, the antibacterial performance has been increased by over 50%. We investigated its antibacterial mechanism against Gram-negative bacteria and showed that HML could be used to treat pathogenic bacteria without obvious accumulated resistance appearance, which is a great advantage over commercial antibiotics. Overall, it is anticipated that HML could be potentially applied to food safety, infection therapy, and enzyme-medicine applications.

Allzyme® Spectrum supplementation improves apparent ileal phosphorus digestibility and retention in older laying hens

Abstract Phosphorus (P) is required by laying hens for various biological functions, and it is usually provided in excess, in a readily digestible inorganic form in diet formulations. By adding enzymes to diets, the availability of phytate-bound P can be increased, allowing for the reduced inclusion of inorganic P in the diet formulation. The following study was conducted to examine the effect of a natural enzyme complex that contains phytase and xylanase on P digestibility in laying hens. Novogen Brown Classic layers (n=314, 95 weeks of age, approximately 14 weeks into second lay) were fed one of two diets; either a basal control diet or a the basal control diet plus a natural mixed-enzyme product (Allzyme® Spectrum; ASC, Alltech Inc, KY, USA) containing phytase (1000 SPU/g) and xylanase (7,500 EPU/g). The experimental diets were provided for 15 days, and excreta was collected from each pen at day 13–15 of the trial to determine total tract retention and apparent ileal digestibility of P and gross energy. At the end of the trial, birds were euthanised and ileal contents were collected. Productivity parameters, including egg production and feed conversion ratio, were recorded over the course of the study. The amount of P in the excreta from birds in the control group was significantly higher than the ASC enzyme-supplemented group. Both P retention, its ileal digestibility and gross energy digestion (ileum and total tract) were significantly higher in the ASC enzyme-supplemented birds. This study showed that the ASC enzyme supplement can improve the total retention and ileal digestibility of P and gross energy in older layers.

Substrate Specificity of Adenine-Cu-PO4 Nanozyme: Ascorbic Acid Oxidation and Selective Cytotoxicity.

Though nanozymes are becoming promising alternatives to natural enzymes due to their superior properties, constructing nanozyme with high specificity is still a great challenge. Herein, with Cu2+ as an active site and adenine as a ligand, Adenine-Cu-PO4 is synthesized in phosphate-buffered saline. As an oxidase mimic, Adenine-Cu-PO4 could selectively catalyze oxidation of ascorbic acid (AA) to dehydroascorbic acid, but not universal substrates (3,3',5,5'-tetramethylbenzidine (TMB), 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and 2,4-dichlorophenol (2,4-DP)), small biomolecules (dopamine, glutathione, glucose, galactose), other vitamins (vitamin A acid, vitamin B1, vitamin K1) and even dithiothreitol (a common interference of AA). Such the specific AA catalytic oxidation is revealed that Adenine-Cu-PO4 selectively binds with AA through hydrogen bonds, accompanied with catalyzing AA oxidation, and concurrently O2 transferring to H2O2 via O2⋅-, further to H2O via ⋅OH. Based on the produced reactive oxygen species, with AA as a pro-oxidant, Adenine-Cu-PO4 nanozyme efficiently triggers severe intratumor oxidative stress to induce tumor cell death. This work opens a new avenue to design intrinsic nanozymes with high specificity, and also presents a promising application in the field of AA oxidation induced cancer therapy.