Enzymatic Cascade Reaction Research Articles

Combined cross-linked enzyme aggregates of cyclodextrin glucanotransferase and maltogenic amylase from Bacillus lehensis G1 for maltooligosaccharides synthesis

Maltooligosaccharides (MOS) are functional oligosaccharides that can be synthesized through enzymatic cascade reaction between cyclodextrin glucanotransferase (CGTase) and maltogenic amylase (Mag1) from Bacillus lehensis G1. To address the problems of low operational stability and non-reusability of free enzymes, both enzymes were co-immobilized as combined cross-linked enzyme aggregates (Combi-CLEAs-CM) with incorporation of bovine serum albumin (BSA) and Tween 80 (Combi-CLEAs-CM-add). Combi-CLEAs-CM and Combi-CLEAs-CM-add showed activity recoveries of 54.12 % and 69.44 %, respectively after optimization. Combi-CLEAs-CM-add showed higher thermal stability at higher temperatures (40 °C) with longer half-life (46.20 min) as compared to those of free enzymes (36.67 min) and Combi-CLEAs-CM (41.51 min). Both combi-CLEAs also exhibited higher pH stability over pH 5 to pH 9, and displayed excellent reusability with >50 % of initial activity retained after four cycles. The reduction in Km value of about 22.80 % and 1.76-fold increase in starch hydrolysis in comparison to Combi-CLEAs-CM attested the improvement of enzyme-substrate interaction by Tween 80 and pores formation by BSA in Combi-CLEAs-CM-add. The improved product specificity of Combi-CLEAs-CM-add also produced the highest yield of MOS (492 mg/g) after 3 h. Therefore, Combi-CLEAs-CM-add with ease of preparation, excellent reusability and high operational stability is believed to be highly efficacious biocatalyst for MOS production.

A portable and quantitative detection of xanthine and xanthine oxidase based on cascade enzymatic reactions of Fe3O4@MWCNTs/Hemin

A portable and quantitative detection of xanthine and xanthine oxidase based on cascade enzymatic reactions of Fe3O4@MWCNTs/Hemin

Screening the Thermotoga maritima genome for new wide-spectrum nucleoside and nucleotide kinases

Enzymes from thermophilic organisms are interesting biocatalysts for a wide variety of applications in organic synthesis, biotechnology, and molecular biology. Next to an increased stability at elevated temperatures, they were described to show a wider substrate spectrum than their mesophilic counterparts. To identify thermostable biocatalysts for the synthesis of nucleotide analogs, we performed a database search on the carbohydrate and nucleotide metabolism of Thermotoga maritima. After expression and purification of 13 enzyme candidates involved in nucleotide synthesis, these enzymes were screened for their substrate scope. We found that the synthesis of 2'-deoxynucleoside 5'-monophosphates (dNMPs) and uridine 5'-monophosphate from nucleosides was catalyzed by the already known wide-spectrum thymidine kinase and the ribokinase. In contrast, no NMP-forming activity was detected for adenosine-specific kinase, uridine kinase, or nucleotidase. The NMP kinases (NMPKs) and the pyruvate-phosphate-dikinase of T.maritima exhibited a rather specific substrate spectrum for the phosphorylation of NMPs, while pyruvate kinase, acetate kinase, and three of the NMPKs showed a broad substrate scope with (2'-deoxy)nucleoside 5'-diphosphates as substrates. Based on these promising results, TmNMPKs were applied in enzymatic cascade reactions for nucleoside 5'-triphosphate synthesis using four modified pyrimidine nucleosides and four purine NMPs as substrates, and we determined that base- and sugar-modified substrates were accepted. In summary, besides the already reported TmTK, NMPKs of T.maritima were identified to be interesting enzyme candidates for the enzymatic production of modified nucleotides.

Electrochemical biomimetic enzyme cascade amplification combined with target-induced DNA walker for detection of thrombin

Fe-N-doped carbon nanomaterials (Fe-N/CMs) were designed as a novel biomimetic enzyme with excellent peroxidase-like activity to achieve high-efficient enzyme cascade catalytic amplification with the aid of glucose oxidase (GOx), which was further combined with target-induced DNA walker amplification to develop a sensitive electrochemical biosensor for thrombin detection. Impressively, massive output DNA was transformed from small amounts of target thrombin by highly effective DNA walker amplification as protein-converting strategy, which could then induce the immobilization of functionalized nanozyme on the electrode surface to achieve the high-efficient electrochemical biomimetic enzyme cascade amplification. As a result, an amplified enzyme cascade catalytic signal was measured for thrombin detection ranging from 0.01pM to 1nM with a low detection limit of 3 fM. Importantly, the new biomimetic enzyme cascade reaction coupled the advantages of natural enzyme and nanozyme, which paved an avenue to construct varied artificial multienzymes amplification systems for biosensing, bioanalysis, and disease diagnosis applications.

Epoxide Hydrolases: Multipotential Biocatalysts.

Epoxide hydrolases are attractive and industrially important biocatalysts. They can catalyze the enantioselective hydrolysis of epoxides to the corresponding diols as chiral building blocks for bioactive compounds and drugs. In this review article, we discuss the state of the art and development potential of epoxide hydrolases as biocatalysts based on the most recent approaches and techniques. The review covers new approaches to discover epoxide hydrolases using genome mining and enzyme metagenomics, as well as improving enzyme activity, enantioselectivity, enantioconvergence, and thermostability by directed evolution and a rational design. Further improvements in operational and storage stabilization, reusability, pH stabilization, and thermal stabilization by immobilization techniques are discussed in this study. New possibilities for expanding the synthetic capabilities of epoxide hydrolases by their involvement in non-natural enzyme cascade reactions are described.

Highly Stable Fe/Co-TPY-MIL-88(NH2) Metal-Organic Framework (MOF) in Enzymatic Cascade Reactions for Chemiluminescence-Based Detection of Extracellular Vesicles.

Metal-Organic Frameworks (MOFs) can deliver many advantages when acting as enzyme mimics to assist with signal amplification in molecular detection: they have abundant active catalytic sites per unit volume of the material; their structures and elemental compositions are highly tunable, and their high specific surface area and porous property can assist with target separation and enrichment. In the present work, we have demonstrated that, by adding the pore partition agent, 2,4,6-tris(4-pyridyl)pyridine (TPY) during synthesis of the bimetallic Fe/Co-MIL-88(NH2) MOF to block the open metal sites, a highly porous MOF of Fe/Co-TPY-MIL-88(NH2) can be produced. This material also exhibits high stability in basic solutions and biofluids and possesses high peroxidase-mimicking activity, which can be utilized to produce long-lasting chemiluminescence (CL) from luminol and H2O2. Moreover, acting as the peroxidase-mimic, the Fe/Co-TPY-MIL-88(NH2) MOF can form the enzymatic cascade with glucose oxidase (GOx) for biomarker detection. When applied to detect extracellular vesicles (EVs), the MOF material and GOx are brought to the proximity on the EVs through two surface proteins, which triggers the enzyme cascade to produce high CL from glucose and luminol. EVs within the concentration range of 5 × 105 to 4 × 107 particles/mL can be detected with an LOD of 1 × 105 particles/mL, and the method can be used to analyze EV contents in human serum without sample preparation and EV purification. Overall, our work demonstrates that the high versatility and tunability of the MOF structures could bring in significant benefits to biosensing and enable ultrasensitive detection of biomarkers with judicious material designs.

Enzyme‐Encapsulated Protein Trap Engineered Metal–Organic Framework‐Derived Biomineral Probes for Non‐Invasive Prostate Cancer Surveillance

AbstractA paper‐based naked‐eye recognition assay with enzyme‐encapsulated protein engineered metal–organic framework‐derived biominerals is developed for direct quantification of sarcosine in urine samples for screening of prostate cancer individuals. The detection strategy stems from the successful construction of a cascade response model, which involves the introduction of a cascade enzymatic catalytic reaction on Pt nanoparticles (NPs)‐loaded porous CeO2 by integrating a sarcosine oxidase as a special recognition unit and a chromogenic substrate as a signal molecule reporter. Pt NPs‐loaded CeO2 is subjected to a one‐step thermal treatment based on multilayered mesoporous Ce‐based metal–organic framework, and the calcined CeO2 exhibits the same distinct porous graded structure. Importantly, introduction of Pt NPs sharply enhances the peroxidase‐like activity of CeO2, which is considered to be caused by the difference in the adsorption behavior of hydrogen peroxide on the CeO2 surface and Pt/CeO2 obtained by density functional theory calculations. On the basis of this, the probe is used on a mass‐producible paper‐based working platform and 3D‐printed device to specifically screen for minor differences in sarcosine between urine samples from cancer patients and normal individuals. Enzyme‐assisted cascade catalytic reaction can be extended by replacing different recognition units for multiple analytes.

A Cascade Enzymatic Reaction Scheme for Irreversible Transpeptidative Protein Ligation.

Enzymatic peptide ligation holds great promise in the study of protein functions and development of protein therapeutics. Owing to their high catalytic efficiency and a minimal tripeptide recognition motif, peptidyl asparaginyl ligases (PALs) are particularly useful tools for bioconjugation. However, as an inherent limitation of transpeptidases, PAL-mediated ligation is reversible, requiring a large excess of one of the ligation partners to shift the reaction equilibrium in the forward direction. Herein, we report a method to make PAL-mediated intermolecular ligation irreversible by coupling it to glutaminyl cyclase (QC)-catalyzed pyroglutamyl formation. In this method, the acyl donor substrate of PALs is designed to have glutamine at the P1' position of the Asn-P1'-P2' tripeptide PAL recognition motif. Upon ligation with an acyl acceptor substrate, the acyl donor substrate releases a leaving group in which the exposed N-terminal glutamine is cyclized by QC, quenching the Gln Nα-amine in a lactam. Using this method, PAL-mediated ligation can achieve near-quantitative yields even at an equal molar ratio between the two ligation partners. We have demonstrated this method for a wide range of applications, including protein-to-protein ligations. We anticipate that this cascade enzymatic reaction scheme will make PAL enzymes well suited for numerous new uses in biotechnology.

Glycopolymer-Based Hydrogels Impair Energy Metabolism via Delivering Mannose and Depleting Glucose for Tumor Suppression

Carbohydrates serve as a key biomacromolecule, not only as the major building blocks in living organisms, but also as intermediates in energy metabolism via glycolysis. The quest for developing a carbohydrate-based macromolecular system to interfere with aberrant energy metabolism of tumors is a challenging topic. Here we report a class of artificial glycopolymers containing phenolic hydroxyl-functionalized segments that can be cross-linked to form hydrogels by the enzymatic cascade reaction using glucose oxidase (GOD) and horseradish peroxidase (HRP). Glycopolymer-based hydrogels can deliver and sustainably release the antiglycolytic agent mannose at the tumor site; meanwhile, glucose is exhausted gradually during the course of gelation. Glucose uptake and hyperactive glycolysis of tumor cells are concurrently blocked by the enzyme cascade and mannose, respectively, as validated by synergistic tumor suppression in vitro. Finally, the superiority of glycopolymer-based hydrogels loading mannose and enzyme cascade is evaluated in a murine tumor model, demonstrating the ability to impair energy metabolism and retard tumor growth. This work allows glycopolymer-based biomaterials to be promising in intervening energy metabolism for tumor eradication.

Artificial receptor-mediated phototransduction toward protocellular subcompartmentalization and signaling-encoded logic gates.

Engineering artificial cellular systems capable of perceiving and transmitting external signals across membranes to activate downstream targets and coordinate protocellular responses is key to build cell-cell communications and protolife. Here, we report a synthetic photoreceptor-mediated signaling pathway with the integration of light harvesting, photo-to-chemical energy conversion, signal transmission, and amplification in synthetic cells, which ultimately resulted in protocell subcompartmentalization. Key to our design is a ruthenium-bipyridine complex that acts as a membrane-anchored photoreceptor to convert visible light into chemical information and transduce signals across the lipid membrane via flip-flop motion. By coupling receptor-mediated phototransduction with biological recognition and enzymatic cascade reactions, we further develop protocell signaling-encoded Boolean logic gates. Our results illustrate a minimal cell model to mimic the photoreceptor cells that can transduce the energy of light into intracellular responses and pave the way to modular control over the flow of information for complex metabolic and signaling pathways.

Enhancing Multistep Reactions: Biomimetic Design of Substrate Channeling Using P22 Virus-Like Particles.

Many biocatalytic processes inside cells employ substrate channeling to control the diffusion of intermediates for improved efficiency of enzymatic cascade reactions. This inspirational mechanism offers a strategy for increasing efficiency of multistep biocatalysis, especially where the intermediates are expensive cofactors that require continuous regeneration. However, it is challenging to achieve substrate channeling artificially in vitro due to fast diffusion of small molecules. By mimicking some naturally occurring metabolons, nanoreactors are developed using P22 virus-like particles (VLPs), which enhance the efficiency of nicotinamide adenine dinucleotide (NAD)-dependent multistep biocatalysis by substrate channeling. In this design, NAD-dependent enzyme partners are coencapsulated inside the VLPs, while the cofactor is covalently tethered to the capsid interior through swing arms. The crowded environment inside the VLPs induces colocalization of the enzymes and the immobilized NAD,which shuttles between the enzymes for in situ regeneration without diffusing into the bulk solution. The modularity of the nanoreactors allowsto tune their composition and consequently their overall activity, and also remodel them for different reactions by altering enzyme partners. Given the plasticity and versatility, P22 VLPs possess great potential for developing functional materials capable of multistep biotransformations with advantageous properties, including enhanced efficiency and economical usage of enzyme cofactors.

Quantifying effects of stochastic focusing and defocusing on noise propagation in biochemical reaction networks

Biochemical reaction networks are intrinsically stochastic. Such stochastic effects are pronounced when molecules have low copy numbers. In an enzymatic cascade reaction network, the formation of product molecules is dictated by the enzymatic signal. Here the noise level in the signaling molecules can have counterintuitive effects. Specifically, increased noise level can reduce the uncertainty in amplified responses of product formation. This phenomenon has been termed Stochastic Focusing. Of particular interest is the sensitivity of product formation to changes in the distribution of signaling enzyme molecules. However, this sensitivity between stochastic noise in signaling molecules and the product formation is yet to be quantified. In this study, we use the Accurate Chemical Master Equation (ACME) algorithm to (1) characterize the stochastic behavior of the enzymatic cascade reaction network and (2) define the system sensitivity of production formation to perturbation in distribution of the signaling molecule. Our approach is based on solving the discrete Chemical Master Equation (dCME) to obtain the exact probability landscape of this network. By perturbing the signal molecule distribution after reaching steady state, we define noise change as the difference in signal distribution widths before and after the shift. We find that the sensitivity of the network correlates exponentially with the level of signaling noise in both the stochastic focusing (increase in system sensitivity) and defocusing (decrease in system sensitivity) regimes. We further studied noise propagation in this enzymatic reaction network by examining the effects of different number of intermediate species required for forming the product molecule. Our findings provide a mechanistic understanding on how signaling noise controls product formation in biological reaction networks, enabling the design of better control mechanisms in cellular systems.

MOF-Immobilized Two-in-One Engineered Enzymes Enhancing Activity of Biocatalytic Cascade for Tumor Therapy.

Biocatalytic systems based on enzyme cascade reactions have attracted growing interest in the field of biocatalytic medicine. However, it is a major challenge to reasonably construct enzyme cascade reactions with high stability, selectivity, and catalytic efficiency for the in vivo biocatalytic application. Herein, two-in-one engineered glucose oxidase (GOx-Fe0 ) isfabricated by a biomineralization strategy, through which a nanozyme (Fe0 NP) is anchored within the inner cavity of GOx. Then, GOx-Fe0 is immobilized in a pH-sensitive metal-organic framework (MOF) zeolitic imidazolate framework-8 (ZIF-8) to establish a stable and effective MOF-immobilized two-in-one engineered enzyme, GOx-Fe0 @ZIF-8. In vitro studies show that GOx-Fe0 @ZIF-8 exhibits excellent stability and high pH/glucose selectivity, and the shorter spacing between cascade enzymes can increase the cascade throughput and effectively improve the reaction efficiency of the enzyme cascade. In vivo experiments exhibit that GOx-Fe0 @ZIF-8 solves the instability and systemic toxicity of free enzymes, and achieves deep tumor penetration and significant chemodynamic therapeutic efficacy through a pH/glucose-selective enzyme cascade reaction in tumor site. Taken together, such an orchestrated enzyme engineering strategy can effectively improve enzyme stability, selectivity, and enzyme cascade reaction efficiency via chemical transformations, and also provide a promising strategy for the application of biocatalytic cascade reactions in vivo.

A chemoenzymatic process for preparation of highly purified dehydroepiandrosterone in high space-time yield

Dehydroepiandrosterone (DHEA) is an important neurosteroid hormone to keep human hormonal balance and reproductive health. However, DHEA was always produced with impurities either by chemical or biological method and required high-cost purification before the medical use. To address this issue, a novel chemoenzymatic process was proposed and implemented to produce DHEA. An acetoxylated derivate of 4-androstene-3,17-dione (4-AD) was generated by chemical reaction and converted into DHEA by an enzyme cascade reaction combining a hydrolysis reaction with a reduction reaction. The hydrolysis reaction was catalyzed by a commercial esterase Z03 while the reduction reaction was catalyzed by E. coli cells co-expressing a 3β-hydroxysteroid dehydrogenase SfSDR and a glucose dehydrogenase BtGDH. After the condition optimization, DHEA was synthesized at a 100 mL scale under 100 mM of substrate loading and purified as white powder with the highest space–time yield (4.80 g/L/h) and purity (99 %) in the biosynthesis of DHEA. The successful attempt in this study provides a new approach for green synthesis of highly purified DHEA in the pharmaceutical industry.

Enhancing the activity of a monomeric alcohol dehydrogenase for site-specific applications by site-directed mutagenesis.

Gene fusion or co-immobilization are key tools to optimize enzymatic reaction cascades by modulating catalytic features, stability and applicability. Achieving a defined spatial organization between biocatalysts by site-specific applications is complicated by the involvement of oligomeric enzymes. It can lead to activity losses due to disturbances of the quaternary structures and difficulties in stoichiometric control. Thus, a toolkit of active and robust monomeric enzymes is desirable for such applications. In this study, we engineered one of the rare examples of monomeric alcohol dehydrogenases for improved catalytic characteristics by site-directed mutagenesis. The enzyme from the hyperthermophilic archaeon Thermococcus kodakarensis naturally exhibits high thermostability and a broad substrate spectrum, but only low activity at moderate temperatures. The best enzyme variants showed an ~5-fold (2-heptanol) and 9-fold (3-heptanol) higher activity while preserving enantioselectivity and good thermodynamic stability. These variants also exhibited modified kinetic characteristics regarding regioselectivity, pH dependence and activation by NaCl.

Dynamic Assembly of Cascade Enzymes by the Shape Transformation of a DNA Scaffold

AbstractWithin cells, the close spatial arrangement of cascade enzymes facilitates the channeling of intermediates and enhances cascade reaction efficiency. Reconfigurable DNA nanostructures, owing to their structural controllability and precise spatial addressability, are promising tools for mimicking such processes. In this study, a 3D DNA origami scaffold, with a dynamic shape transformation from its open boat form to a closed hexagonal prism induced by toehold‐mediated strand displacement, is designed to investigate the enzyme cascade reaction of xylose reductase and xylitol dehydrogenase from D‐xylose metabolic pathway. Enzymes are assembled on the DNA scaffold in its open state, which is subsequently closed by the assistance of DNA sequence‐specific closing keys. The enzyme cascade efficiency is much higher in the static encapsulated closed state than in the open state due not only to the enzyme proximity but also the environmental factors of 3D DNA structure. These results provide novel insights into controlling enzyme cascade reactions by inducing the shape transformation of DNA nanostructures and how environmental factors affect the action of multi‐enzyme complexes in the cell.

Expanding the Synthetic Toolbox through Metal-Enzyme Cascade Reactions.

The combination of metal-, photo-, enzyme-, and/or organocatalysis provides multiple synthetic solutions, especially when the creation of chiral centers is involved. Historically, enzymes and transition metal species have been exploited simultaneously through dynamic kinetic resolutions of racemates. However, more recently, linear cascades have appeared as elegant solutions for the preparation of valuable organic molecules combining multiple bioprocesses and metal-catalyzed transformations. Many advantages are derived from this symbiosis, although there are still bottlenecks to be addressed including the successful coexistence of both catalyst types, the need for compatible reaction media and mild conditions, or the minimization of cross-reactivities. Therefore, solutions are here also provided by means of catalyst coimmobilization, compartmentalization strategies, flow chemistry, etc. A comprehensive review is presented focusing on the period 2015 to early 2022, which has been divided into two main sections that comprise first the use of metals and enzymes as independent catalysts but working in an orchestral or sequential manner, and later their application as bionanohybrid materials through their coimmobilization in adequate supports. Each part has been classified into different subheadings, the first part based on the reaction catalyzed by the metal catalyst, while the development of nonasymmetric or stereoselective processes was considered for the bionanohybrid section.

A ratiometric fluorescence sensor for determination of choline based on gold nanoclusters and enzymatic reaction

Choline (ChO) is a biomarker of many diseases. Its accurate and sensitive detection is of great significance for the diseases diagnosis. Gold nanoclusters with bovine serum albumin as template (BSA-AuNCs) were synthesized by microwave-assisted method. A ratiometric fluorescence sensor was constructed based on BSA-AuNCs and 2,3-diaminophenazine, which was the oxidation product of o-phenylenediamine. Combined with cascade enzymatic reaction, it was used for the detection of choline. In the detection system, choline oxidase improved the selectivity of detection, and the peroxidase-like activity of molybdenum disulfide nanosheets shortened the detection time. The linear ranges of choline detection by ratiometric fluorescence sensor were 0.5–20 μM and 20–100 μM. The detection limit was 0.27 μM. The ratiometric fluorescence sensor was easy to operate and had good selectivity. Compared with single signal sensor, it can effectively reduce the error interference such as environmental change, and the detection result was more accurate.

Enzyme Cascade Reactions on DNA Origami Scaffold.

The protocols for constructing, characterizing, and analyzing enzyme cascade reaction systems on the DNA scaffold are described. Two-step and three-step enzyme cascade reactions were adapted from the xylose metabolic pathway as the example of natural metabolic pathway and were assembled on the DNA scaffold by using the DNA binding adaptors.

Mechanistic modeling, parametric study, and optimization of immobilization of enzymatic cascades in porous particles

A framework for simulating and optimizing reaction processes catalyzed by enzymes immobilized in porous particles was developed. The conditions under which four different spatial immobilization distributions are advantageous were determined.