Enzyme Mimic Based on a Self-Assembled Chitosan/DNA Hybrid Exhibits Superior Activity and Tolerance

Nature has evolved enzymes with exquisite active sites that catalyze biotransformations with high efficiency. However, the exploitation of natural enzymes is often hampered by poor stability, and natural enzyme production and purification are costly. Supramolecular self-assembly allows the construction of biomimetic active sites, although it is challenging to produce such artificial enzymes with catalytic activity and stability that rival those of natural enzymes. We report herein a strategy to produce a horseradish peroxidase (HRP) mimic based on the assembly of chitosan with a G-quadruplex DNA (G-DNA)/hemin complex. A network-like morphology of the assembled nanomaterial was observed together with a remarkable enhancement of peroxidase activity induced by the chitosan and G-DNA components. The turnover frequency and catalytic efficiency of the enzyme-mimicking material reached or even surpassed those of HRP. Moreover, the catalytic complex exhibited higher tolerance than HRP to harsh environments, such as extremely low pH or high temperatures. In accord with the experimental and simulated results, it is concluded that the spatial distribution of the G-DNA and chitosan components and the exposure of the catalytic center may facilitate the coordination of substrates by the hemin iron, leading to the superior activity of the material. Our work provides a simple and affordable avenue to produce highly active and robust enzyme-mimicking catalytic nanomaterials.     

Recent progress in carbon-dots-based nanozymes for chemosensing and biomedical applications

Nanozymes are nanomaterials with enzyme-like activities that efficiently overcome the drawbacks of natural enzymes in biosensing, detection, and biomedical fields, and they are the most widely used artificial enzymes. Owing to their excellent catalytic characteristics, biocompatibility, and environmental favorability, carbon-dots-based (CDs) nanozymes have inspired a research upsurge. However, no review focusing on CDs nanozymes has been published, even though substantial advances have been achieved. Herein, the advances, catalytic activities, and applications of CDs nanozymes are highlighted and summarized. In addition, the critical issues and challenges of researching nanozymes are discussed. We hope that this review will broaden the horizons of nanozymes and CDs nanozymes, as well as promote their development.     

Catalytic,theoretical, and biological investigation of an enzyme mimic model

Artificial catalyst studies were always stayed at the kinetics investigation level, in this work bioactivity of designed catalyst were shown by the induction of biomineralization of the cells, indicating the possible use of enzyme mimics for biological applications. The development of artificial enzymes is a continuous quest for the development of tailored catalysts with improved activity and stability. Understanding the catalytic mechanism is a replaceable step for catalytic studies and artificial enzyme mimics provide an alternative way for catalysis and a better understanding of catalytic pathways at the same time. Here we designed an artificial catalyst model by decorating peptide nanofibers with a covalently conjugated catalytic triad sequence. Owing to the self-assembling nature of the peptide amphiphiles, multiple action units can be presented on the surface for enhanced catalytic performance. The designed catalyst has shown an enzyme-like kinetics profile with a significant substrate affinity. The cooperative action in between catalytic triad amino acids has shown improved catalytic activity in comparison to only the histidine-containing control group. Histidine is an irreplaceable contributor to catalytic action and this is an additional reason for control group selection. This new method based on the self-assembly of covalently conjugated action units offers a new platform for enzyme investigations and their further applications. Artificial catalyst studies always stayed at the kinetics investigation level, in this work bioactivity of the designed catalyst was shown by the induction of biomineralization of the cells, indicating the possible use of enzyme mimics for biological applications.     

Self-Assembling Catalytic Peptide Nanomaterials Capable of Highly Efficient Peroxidase Activity

The self-assembly of short peptides gives rise to versatile nanomaterials capable of promoting efficient catalysis. We have shown that short, seven-residue peptides bind hemin to produce functional catalytic materials which display highly efficient peroxidation activity, reaching a catalytic efficiency of 3×10⁵ m ⁻¹ s⁻¹. Self-assembly is essential for catalysis as non-assembling controls show no activity. We have also observed peroxidase activity even in the absence of hemin, suggesting the potential to alter redox properties of substrates upon association with the assemblies. These results demonstrate the practical utility of self-assembled peptides in various catalytic applications and further support the evolutionary link between amyloids and modern-day enzymes.     

Spatiotemporal dynamics of self-assembled structures in enzymatically induced agonistic and antagonistic conditions

Predicting and designing systems with dynamic self-assembly properties in a spatiotemporal fashion is an important research area across disciplines ranging from understanding the fundamental non-equilibrium features of life to the fabrication of next-generation materials with life-like properties. Herein, we demonstrate a spatiotemporal dynamics pattern in the self-assembly behavior of a surfactant from an unorganized assembly, induced by adenosine triphosphate (ATP) and enzymes responsible for the degradation or conversion of ATP. We report the different behavior of two enzymes, alkaline phosphatase (ALP) and hexokinase (HK), towards adenosine triphosphate (ATP)-driven surfactant assembly, which also results in contrasting spatiotemporal dynamic assembly behavior. Here, ALP acts antagonistically, resulting in transient self-assemblies, whereas HK shows agonistic action with the ability to sustain the assemblies. This dynamic assembly behavior was then used to program the time-dependent emergence of a self-assembled structure in a two-dimensional space by maintaining concentration gradients of the enzymes and surfactant at different locations, demonstrating a new route for obtaining ‘spatial’ organizational adaptability in a self-organized system of interacting components for the incorporation of programmed functionality.

We have shown ATP-driven spatiotemporally distinct self-organization pattern of a surfactant in a two-dimensional space using enzymes, demonstrating a new route for obtaining ‘spatial’ organizational adaptability among interacting components.     

Modification of Cyclodextrins for Use as Artificial Enzymes

The magical powers of enzymes have been attributed to their ability to bind specific substrates and catalyze reactions of the bound substrate. Artificial enzymes synthetically mimic the binding and the catalytic site to produce molecules that are not only smaller in size but also potentially have similar activity to the real enzymes. The main objective of our research is to create artificial redox enzymes by using cyclodextrins as binding sites and attaching flavin derivatives as the catalytic site. We have developed a strategy to attach a catalytic site to cyclodextrin exclusively at the 2-, 3- or the 6-position. The evaluation of the artificial enzyme in which flavin is attached to the 2-position gives a 647-fold acceleration factor. Although this is modest compared to those of real enzymes (which can have acceleration factors of a trillion), the artificial enzymes allow us to understand the elements that contribute to the incredible catalytic power of enzymes.     

Enzyme-Driven,Switchable Catalysis Based on Dynamic Self-Assembly of Peptides

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.     

Deciphering a Nanocarbon‐Based Artificial Peroxidase: Chemical Identification of the Catalytically Active and Substrate‐Binding Sites on Graphene Quantum Dots

The design and construction of efficient artificial enzymes is highly desirable. Recent studies have demonstrated that a series of carbon nanomaterials possess intrinsic peroxidase activity. Among them, graphene quantum dots (GQDs) have a high enzymatic activity. However, the catalytic mechanism remains unclear. Therefore, in this report, we chose to decipher their peroxidase activity. By selectively deactivating the ketonic carbonyl, carboxylic, or hydroxy groups and investigating the catalytic activities of these GQD derivatives, we obtained evidence that the ? C?O groups were the catalytically active sites, whereas the O?C? O? groups acted as substrate‐binding sites, and ? C? OH groups can inhibit the activity. These results were corroborated by theoretical studies. This work should not only enhance our understanding of nanocarbon‐based artificial enzymes, but also facilitate the design and construction of other types of target‐specific artificial enzymes.     

Catalytically Active Peptide–Gold Nanoparticle Conjugates: Prospecting for Artificial Enzymes

The self-assembly of peptides onto the surface of gold nanoparticles has emerged as a promising strategy towards the creation of artificial enzymes. The resulting high local peptide density surrounding the nanoparticle leads to cooperative and synergistic effects, which result in rate accelerations and distinct catalytic properties compared to the unconjugated peptide. This Minireview summarizes contributions to and progress made in the field of catalytically active peptide–gold nanoparticle conjugates. The origin of distinct properties, as well as potential applications, are also discussed.     

Fluorescence detection of adenosine-5′-triphosphate and alkaline phosphatase based on the generation of CdS quantum dots

We have developed an analytical method to detect adenosine-5′-triphosphate (ATP) and alkaline phosphatase (ALP) based on the generation of CdS quantum dots (QDs). We demonstrated that Cd²⁺ cation reacts with S²⁻ anion to generate fluorescent CdS QDs in the presence of some certain amount of ATP. With increase in the ATP concentration, the fluorescence intensity of CdS QDs was also enhanced. ATP can be converted into adenosine by the dephosphorylation of ALP, so that the generation of CdS QDs would be inhibited in the presence of ALP. Therefore, this novel analysis system could be applied to assay ATP and ALP based on the growth of fluorescent CdS QDs.     

Artificial Esterase Based on Self-assembly Gel Microspheres Constructed from Chitosan and Amino Acids

A novel artificial esterase based on chitosan and amino acids was synthesized in the present study. The Fmoc-His and Glu were linked to chitosan by active ester method(AEM). The hydroxide radical in chitosan, imidazole group of Fmoc-His and carboxyl from Glu formed a catalytic center of natural esterase. Gel microspheres were coated with a protective layer and a supporting layer by seiassembly construction function in carboxymethylcellulose sodium(CMCS) solution. As for catalytic activity, chitosan-His-Glu was found to be more efficient than chitosan- His and chitosan-Glu in mimicking the core catalytic sites of natural esterase, and the best ratio(mass ratio) of chitosan-His-Glu:CMCS was 1:3. Furthermore, metal ions, such as Ca^2+, Mg^2+, Fe^2+, etc., were able to improve the catalytic efficiency of artificial esterase. And tlie Lineweaver-Burk plot indicated that the catalytic kinetics of artificial esterase conformed to Michaelis-Menten equation.     

Metal-directed self-assembly of cavitand frameworks

The self-assembly between bidentate cavitand ligands and mono/dinuclear metal precursors to give cavitand frameworks has been explored. For this purpose, two new cavitands bearing AB and AC phenylpyridyl moieties at the upper rim have been synthesized. A series of self-assembled molecular dimers featuring fac-Re(CO)(3)Br as metal corners have been prepared and characterized. Two possible dimeric structures (C-shaped and S-shaped) are possible when AB cavitand 2 is used in the self-assembly reaction; only one is obtained in the case of AC cavitand 3. In addition, the self-assembly of AB-dibridged cavitand 2 with dinuclear Pd/Pt metal precursors 5a and 5b has been studied. At this level of complexity, the self-assembly can lead to more than one structure. Several different final structures have been envisioned and their formation analyzed in silico and in solution. Out of the three possible cyclic structures (dimer, trimer, and tetramer), only the entropically favored dimer 6a (6b) is formed, as predicted from molecular modeling and demonstrated by PGSE NMR experiments.     

Precisely Designed Isopeptide Bridge‐Crosslinking Endows Artificial Hydrolases with High Stability and Catalytic Activity under Extreme Denaturing Conditions

Enzymes normally lose their activities under extreme conditions due to the dissociation of their active tertiary structure. If an enzyme could maintain its catalytic activity under non‐physiological or denaturing conditions, it might be used in more applications in the pharmaceutical and chemical industries. Recently, we reported a coiled‐coil six‐helical bundle (6HB) structure as a scaffold for designing artificial hydrolytic enzymes. Here, intermolecular isopeptide bonds were incorporated to enhance the stability and activity of such biomolecules under denaturing conditions. These isopeptide bridge‐tethered 6HB enzymes showed exceptional stability against unfolding and retained or even had increased catalytic activity for a model hydrolysis reaction under thermal and chemical denaturing conditions. Thus, isopeptide bond‐tethering represents an efficient route to construct ultrastable artificial hydrolases, with promising potential to maintain biocatalysis under extreme conditions.     

Biofunctionalization of aqueous dispersed, alumina membrane-templated polymer nanorods for use in enzymatic chemiluminescence assays

The noncovalent immobilization of alkaline phosphatase (ALP) onto aqueous dispersed nylon 6 nanorods ( approximately 310 nm mean diameter; approximately 6 microm mean length) prepared by anodic aluminum oxide (AAO) membrane templating was studied. Using multi-stacked layer-by-layer (LBL) assembly with the cationic quaternary ammonium polymer Sapphire II , the amount of ALP enzyme loaded onto the polymer nanostructures was found to be 115+/-7 microg mg(-1) nanorod. The biofunctionalized nanorods were also characterized for their chemiluminescent activity with the dioxetane substrate, CSPD . The results indicate that the kinetic parameters, K(m) and V(max), for the catalytic activity of the nanostructure-bound ALP enzyme are different from those of soluble ('free') ALP. While the K(m) value was measured to be 156 microM for free ALP, the apparent K(m) value determined for the LBL-immobilized ALP is approximately 20% lower (122 microM). Furthermore, despite the relatively high enzyme loading capacity of the nanorods, the specific activity of the bound ALP enzyme was found to be almost nine times lower than that measured for free ALP. Finally, additional experiments revealed that the catalytic activities of both free ALP and nanorod-conjugated ALP are affected similarly by changes in pH, with optimal performance levels occurring under conditions of pH 9.5. To the best of our knowledge, this study represents the first report examining the preparation of aqueous dispersed, AAO-templated polymer nanorods for potential application as enzyme scaffolds in chemiluminescent-based assay systems.     

Polymerization-induced phase separation as a one-step strategy to self-assemble alkanethiol-stabilized gold nanoparticles inside polystyrene domains dispersed in an epoxy matrix

The rational design of nanoparticle (NP)/polymer composites with advanced functional properties is based on controlling the distribution and self-assembly of NPs in the polymer matrix. In this study we report a new one-step strategy to produce the self-assembly of alkanethiol-stabilized Au NPs in one of the phases generated by polymerization-induced phase separation. The polymerization of a formulation composed of stoichiometric amounts of diglycidylether of bisphenol A (DGEBA) and m-xylylenediamine (mXDA), containing polystyrene (PS) and dodecanethiol-stabilized Au NPs as modifiers, produced the phase separation of PS and Au NPs into microdomains dispersed in the epoxy matrix. A subsequent phase separation and self-assembly of Au NPs took place inside the PS domains leading to an increase in their concentration in a region close to the interface as revealed by TEM images. SAXS spectra showed that NPs self-assembled as colloidal crystals with a body-centered cubic (bcc) structure. By an adequate selection of the amount of PS and the nature of the epoxy precursors, different morphologies of the final blend could be generated. This brings the possibility of controlling the dispersion and self-assembly of NPs in the final material.     

Self-assembly of alkyl-substituted cubic siloxane cages into ordered hybrid materials

Siloxane-organic hybrids with well-ordered mesostructures were synthesized through the self-assembly of novel amphiphilic molecules that consist of cubic siloxane heads and hydrophobic alkyl tails. The monoalkyl precursors functionalized with ethoxy groups (C(n)H(2n+1)Si(8)O(12)(OEt)(7), 1 Cn, n=16, 18, and 20) were hydrolyzed under acidic conditions with the retention of the siloxane cages, leading to the formation of two-dimensional hexagonal phases by evaporation-induced self-assembly processes. Analysis of the solid-state (29)Si MAS NMR spectra of these hybrid mesostructures confirmed that the cubic siloxane units were cross-linked to form siloxane networks. Calcination of these hybrids gave mesoporous silica, the pore diameter of which varied depending on the alkyl-chain length. We also found that the precursors that had two alkyl chains formed lamellar phases, thus confirming that the number of alkyl chains per cage had a strong influence on the mesostructures. These results expand the design possibility of novel nanohybrid and nanoporous materials through the self-assembly of well-defined oligosiloxane-based precursors.     

Self-assembly on natural cellulose: Towards high-efficient catalysts

Nanoarchitectures based on the layer-by-layer self-assembly technique hold great potential for the availability and applicability of bio-inspired functional materials. The introduction of various specific functional building blocks onto the nanofibers of natural cellulose substances (e.g., commercial filter paper, cotton, etc.) through the self-assembly approach provides a facile strategy for the fabrication of artificial nanomaterials. This review summarizes a series of cellulose-based catalytic materials fabricated by utilizing the natural cellulose substance as the structural scaffolds or templates through the LbL self-assembly process. The unique three-dimensional network porous structures and high surface areas of the cellulose substances were maintained by the resultant cellulose-derived catalysts, while the excellent mechanical strength of the cellulose-based membrane catalysts was inherited from the initial cellulose substrates. When employed for the photodegradation of organic dyes, the photocatalytic hydrogen production from water splitting, and the antibiosis, these cellulose-based catalysts exhibited high activities and excellent cycling stabilities.     

Nitric oxide producing artificial enzymes based on metalloporphyrins

Artificial enzymes are synthetic materials or molecules that exhibit enzyme-like activities. Herein, we report artificial enzymes based on metalloporphyrins (MPs) that can mimic nitric oxide synthase (NOS) to produce nitric oxide (NO) from l-arginine (Arg) using hydrogen peroxide (H₂O₂) as an oxidant, and we determine the microenvironmental influence on their activity and stability. An assortment of structurally diverse water-soluble MPs was synthesized, and their NO production was compared. Improved catalytic activity and stability of the lead MP were observed when it was conjugated to the hydrophilic homopolymer poly(2-carboxyethyl acrylate) (PCEA). Micelles assembled from the amphiphilic block copolymer poly(cholesteryl methacrylate)-block-PCEA functionalized with the lead MP, and MP-conjugated PCEA polymer chains immobilized on silica particles resulted in moieties with ～3× higher catalytic activity compared to the monomeric MP. Finally, the PCEA-coated silica particles could use the MPs as motor units to exhibit enhanced diffusion in the presence of the required fuel molecules Arg and H₂O₂. Taken together, the microenvironments of the artificial enzymes have a marked impact on their activity and stability, and these NO-producing MPs offer an interesting synthetic alternative to their natural counterparts.     

Stable N-heterocyclic carbene (NHC)-palladium(0) complexes as active catalysts for olefin cyclopropanation reactions with ethyl diazoacetate

The Pd(0) complexes [(NHC)PdL(n)] (NHC=N-heterocyclic carbene ligand; L=styrene for n=2 or PR(3) for n=1) efficiently catalyse olefin cyclopropanation by using ethyl diazoacetate (EDA) as the carbene source with activities that improve on previously described catalytic systems based on this metal. Mechanistic studies have shown that all of these catalyst precursors deliver the same catalytic species in solution, that is, [(IPr)Pd(sty)], a 14e(-) unsaturated intermediate that further reacts with EDA to afford [(IPr)Pd(=CHCO(2)Et)(sty)], from which the cyclopropane is formed.     

The designing strategies of graphene-based peroxidase mimetic materials

Natural enzymes have been praised highly as ideal catalysts, presumably owing to their remarkable advantages of high efficiency, high selectivity, and mild reaction conditions. The reports of chemical simulation and systematic synthesis of natural enzymes such as peroxidase (POD) are rare because of their complex biological structures. POD represents a large family of oxidoreductases and offers a wide range of applications in many fields of science. Recent advance in the fusion of nanomaterial, catalysis, and biochemistry has inspired the development of artificial enzymes implemented with desired catalytic features of natural enzymes. Herein, we review the redox chemistry of POD and compare its catalytic performance to graphene-based nanomaterials (G-NMs) as POD mimetic nanoenzymes bases on catalytic center, binding site, and carrier function. Based on the viewpoints of stereo chemistry and molecular kinetic and dynamics in heterogeneous system, we evaluate and compare the suitability of different NMs as artificial enzyme constituent. We propose that reevaluates design strategies of graphene-based peroxidase (G-POD) mimetic materials and emphasizes on their selectivity (role as catalytic center, binding site, or carrier) is of uttermost.