Preorganization boosts the artificial esterase activity of a self-assembling peptide

The creation of artificial enzymes to mimic natural enzymes remains a great challenge owing to the complexity of the structural arrangement of the essential amino acids in catalytic centers. In this study, we used the phosphatase-based enzyme-instructed self-assembly(EISA) to supervise artificial esterases' final structures and catalytic activities. We reported that peptide precursors containing different phosphorylation sites could preorganize into alternated nanostructures and undergo dephosphorylation in the presence of alkaline phosphatase(ALP) with variation in kinetic and thermodynamic profiles. Although identical self-assembly compositions were formed after dephosphorylation, precursors with more enhanced preorganized states tended to better promote ALP dephosphorylation, facilitate further self-assembly, and strengthen the catalytic activities of the final assemblies. We envisioned that our strategy would be useful for further construction and manipulation of various artificial enzymes with superior catalytic activities.     

A study of gelled enzyme ultrafiltration reactors

Experiments were carried out in UF reactors with dynamically formed gelled enzyme artificial membranes. Unstirred batch systems and UF reactors with continuous recirculation of the substrate on the membrane were investigated. A significant increase in enzyme stability was tested in both systems. The enzymes used were acid phosphatase, urease, and β-glucosidase. The agreement between the experimental results and the predictions of a simple analytical model for the two classes of UF heterogeneous enzymatic reactors is generally satisfactory.     

Novel enzyme activities and functional plasticity revealed by recombining highly homologous enzymes

BACKGROUND: Directed evolution by DNA shuffling has been used to modify physical and catalytic properties of biological systems. We have shuffled two highly homologous triazine hydrolases and conducted an exploration of the substrate specificities of the resulting enzymes to acquire a better understanding of the possible distributions of novel functions in sequence space. RESULTS: Both parental enzymes and a library of 1600 variant triazine hydrolases were screened against a synthetic library of 15 triazines. The shuffled library contained enzymes with up to 150-fold greater transformation rates than either parent. It also contained enzymes that hydrolyzed five of eight triazines that were not substrates for either starting enzyme. CONCLUSIONS: Permutation of nine amino acid differences resulted in a set of enzymes with surprisingly diverse patterns of reactions catalyzed. The functional richness of this small area of sequence space may aid our understanding of both natural and artificial evolution.     

Protein-imprinted materials: rational design, application and challenges

Protein imprinting is a promising tool for generating artificial biomimetic receptors with antibody-like specific recognition sites. Recently, protein-imprinted materials, as potential antibody substitutes, have attracted much attention in many fields, for example chemical sensors, chromatographic stationary phases, and artificial enzymes, owing to their long-term storage stability, potential re-usability, resistance to harsh environment, and low cost. In this critical review, we focus our discussion on the rational preparation of protein-imprinted materials in terms of choice of template, functional monomer, crosslinker, and polymerization format. In addition, several highlighted applications of protein-imprinted materials are emphasized, not only in well-known fields but also in some unique fields, for example proteomics and tissue engineering. Finally, we propose challenges arising from the intrinsic properties of protein imprinting, for example obtaining the template, heterogeneous binding, and extrinsic competition, for example immobilized aptamers.     

金属配合物-寡聚核苷酸定位断裂剂研究进展

核酸切割试剂与寡聚核苷酸(ODN)偶联制得的人工核酸酶能在特定位点断裂DNA或RNA,为人工核酸酶的分子设计提供了一种新方法.本文综述了金属配合物-ODN识别切割试剂的偶联方式及其与靶分子的作用机制,并指出了今后的研究方向.     

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.     

人工模拟酶的研究与应用进展

人工模拟酶具有性质稳定、易于制备、环境耐受性强等优点,在某种程度上解决了天然酶易失活、难制备的缺点。本文按照人工模拟酶的分类,综述对比了传统模拟酶与纳米材料模拟酶的研究现状,对人工模拟酶优缺点进行总结分析,并对其应用前景进行了展望。     

Co‐immobilized Phosphorylated Cofactors and Enzymes as Self‐Sufficient Heterogeneous Biocatalysts for Chemical Processes

Enzyme cofactors play a major role in biocatalysis, as many enzymes require them to catalyze highly valuable reactions in organic synthesis. However, the cofactor recycling is often a hurdle to implement enzymes at the industrial level. The fabrication of heterogeneous biocatalysts co‐immobilizing phosphorylated cofactors (PLP, FAD⁺, and NAD⁺) and enzymes onto the same solid material is reported to perform chemical reactions without exogeneous addition of cofactors in aqueous media. In these self‐sufficient heterogeneous biocatalysts, the immobilized enzymes are catalytically active and the immobilized cofactors catalytically available and retained into the solid phase for several reaction cycles. Finally, we have applied a NAD⁺‐dependent heterogeneous biocatalyst to continuous flow asymmetric reduction of prochiral ketones, thus demonstrating the robustness of this approach for large scale biotransformations.     

Co-immobilized Phosphorylated Cofactors and Enzymes as Self-Sufficient Heterogeneous Biocatalysts for Chemical Processes

Enzyme cofactors play a major role in biocatalysis, as many enzymes require them to catalyze highly valuable reactions in organic synthesis. However, the cofactor recycling is often a hurdle to implement enzymes at the industrial level. The fabrication of heterogeneous biocatalysts co-immobilizing phosphorylated cofactors (PLP, FAD⁺, and NAD⁺) and enzymes onto the same solid material is reported to perform chemical reactions without exogeneous addition of cofactors in aqueous media. In these self-sufficient heterogeneous biocatalysts, the immobilized enzymes are catalytically active and the immobilized cofactors catalytically available and retained into the solid phase for several reaction cycles. Finally, we have applied a NAD⁺-dependent heterogeneous biocatalyst to continuous flow asymmetric reduction of prochiral ketones, thus demonstrating the robustness of this approach for large scale biotransformations.     

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.     

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.     

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.     

High-performance,synergistically catalytic luminescent nanozyme for the degradation and detection of endocrine-disrupting chemical diethylstilbestrol

A synergistically catalytic luminescent nanozyme was designed and synthesized for the degradation and enzymatic fluorescence detection of diethylstilbestrol, an endocrine-disrupting environmental pollutant. Because of the integration of cocatalytic Cu²⁺ ion and CeO₂ particle, luminescent Tb³⁺ ion, and functional ligand dipicolinic acid through flexible metal-organic framework structure, this nanozyme has not only the dual functions of luminescence and multienzyme such as laccase and horseradish peroxidase but also synergistically catalytic effect via a regeneration of Cu²⁺ oxidized by CeO₂. The synergistically catalytic effect of nanozyme greatly enhances the degradation of diethylstilbestrol. The resultants sensitized the luminescence of Tb³⁺ ions, which was used to sense the pM level of diethylstilbestrol in environmental samples. Such a high-performance catalytic luminescent nanozyme can be used to replace natural enzymes for the enzyme-based degradations and ultrasensitive assays. The strategy of constructing artificial enzymes directly from functional units provides a new way for developing fit-for-purpose multifunctional artificial enzymes.     

Stereodivergent Evolution of Artificial Enzymes for the Michael Reaction

Enzymes are valuable biocatalysts for asymmetric synthesis due to their exacting stereocontrol. Changing the selectivity of an existing catalyst for new applications is, however, challenging. Here we show that, in contrast, the stereoselectivity of an artificial enzyme created by design and directed evolution is readily tunable. We engineered a promiscuous artificial retro‐aldolase into four stereocomplementary catalysts for the Michael addition of a tertiary carbanion to an unsaturated ketone. Notably, this selectivity is also preserved with alternative Michael nucleophiles. Complete stereodiversification of other designer enzymes should similarly be possible by extension of these approaches.     

Artificial Photosynthesis(AP): from Molecular Catalysts to Heterogeneous Materials

The development of green and renewable energy sources is in high demand due to energy shortage and productivity development. Artificial photosynthesis(AP) is one of the most effective ways to address the energy shortage and the greenhouse effect by converting solar energy into hydrogen and other carbon-based high value-added products through the understanding of the mechanism, structural analysis, and functional simulation of natural photosynthesis. In this review, the development of AP from natural catalysts to artificial catalysts is described, and the processes of oxygen production, hydrogen production, and carbon fixation are sorted out to understand the properties and correlations of the core functional components in natural photosynthesis, to provide a better rational design and optimization for further development of advanced heterogeneous materials.     

Simple cyclodextrin aldehydes as excellent artificial oxidases

Cyclodextrin based oxidases, with a ketone as functional group are well known as good artificial enzyme mimics (Fenger et al. Org Biomol Chem 7:933?C943; Marinescu and Bols Angew Chem Int Ed 45:4590?C4593; Bjerre et al. Eur J Org Chem 704?C710; Marinescu et al. J Am Chem Soc 127:17578?C17579). We here report a series of modified cyclodextrins, having aldehydes as functional groups. The aldehyde based artificial enzymes have, in most cases, better catalysis than the ketones, because of their powerful covalent binding of hydrogen peroxide. Among the modified cyclodextrins studied are mono and di aldehydes on the 6 positions, with or without methylated hydroxyl groups. The aldehyde functionality was also introduced close to the secondary side, by attaching ethoxy-2-al or propoxy-3-al to the 2 position. The modified cyclodextrins showed excellent enzymatic activity towards oxidation of different aminophenols, and 4-methoxy benzyl alcohol with hydrogen peroxide as a stoichiometric oxidant. Rate enhancements up to 4,600 were achieved for oxidation of 4-methoxy benzyl alcohol, where as oxidation of amines gave rate enhancements up to 3,400. The artificial oxidases catalyses oxidations under enzymatic conditions (water, pH 7, 25 °C), following Michaelis?CMenten kinetics. To confirm the enzyme activity, inhibition studies with sodium naphthalene-2-sulfonate were carried out. These studies showed competitive inhibition of the enzymes, verifying the cyclodextrins enzyme like character.     

Artificial redox enzymes. II. A computational chemistry study1

A computational chemistry study of the artificial redox enzyme synthesized by covalently attaching flavin to cyclodextrins explains some of its properties. Calculations indicate that the flavin moiety covalently attached to cyclodextrin is not within the cavity of cyclodextrin. This result is consistent with the UV-vis spectrum of the artificial enzyme. The calculations also indicate hydrogen bonds formed between the carbonyl groups of the catalytic functionality and the hydroxyl groups of cyclodextrin play a role in their most stable conformation. This explains the observed overall stability of these artificial enzymes compared to riboflavin. Electrostatic energies and solvation energies play a major role in the stability of the hosts and the orientation of guests included within the artificial enzymes. The rates of oxidation of various thiols catalyzed by the artificial enzyme can be explained by the relative distances between the sulfur atom of the substrates and C(4a) of the flavin moiety.     

Reactions with enzymes covalently bonded to heterogeneous ultrafiltration membranes

The hydrolysis of urea by urease, a model system for the reaction of enzymes with a low molecular weight substance capable of permeating ultrafiltration membranes without resistance, and the degradation of dextran by dextranase, a substance highly rejected by appropriate membranes, are described. The enzymes are covalently bonded to heterogeneous aminated polysulfone, using glutaraldehyde or diazo compounds as immobilization agents. Preliminary results are given.     

A De Novo Designed Metalloenzyme for the Hydration of CO2

Protein design will ultimately allow for the creation of artificial enzymes with novel functions and unprecedented stability. To test our current mastery of nature’s approach to catalysis, a Zn^II metalloenzyme was prepared using de novo design. α₃DH₃ folds into a stable single‐stranded three‐helix bundle and binds Zn^II with high affinity using His₃O coordination. The resulting metalloenzyme catalyzes the hydration of CO₂ better than any small molecule model of carbonic anhydrase and with an efficiency within 1400‐fold of the fastest carbonic anhydrase isoform, CAII, and 11‐fold of CAIII.     

Supramolecular Coordination Cages for Asymmetric Catalysis

Inspired by the high efficiency and specificity of enzymes in living systems, the development of artificial catalysts intrinsic to the key features of enzyme has emerged as an active field. Recent advances in supramolecular chemistry have shown that supramolecular coordination cages, built from non-covalent coordination bonds, offer a diverse platform for enzyme mimics. Their inherent confined cavity, analogous to the binding pocket of an enzyme, and the facile tunability of building blocks are essential for substrate recognition, transition-state stabilization, and product release. In particular, the combination of chirality with supramolecular coordination cages will undoubtedly create an asymmetric microenvironment for promoting enantioselective transformation, thus providing not only a way to make synthetically useful asymmetric catalysts, but also a model to gain a better understanding for the fundamental principles of enzymatic catalysis in a chiral environment. The focus here is on recent progress of supramolecular coordination cages for asymmetric catalysis, and based on how supramolecular coordination cages function as reaction vessels, three approaches have been demonstrated. The aim of this review is to offer researchers general guidance and insight into the rational design of sophisticated cage containers for asymmetric catalysis.