Multi-enzyme Cascade Reactions Research Articles

Increasing Multienzyme Cascade Efficiency and Stability of MOF via Partitioning Immobilization.

Enhancing the stability of multienzyme cascade reactions in metal-organic frameworks (MOFs) is a challenging task in the fields of biotechnology and chemistry. However, addressing this challenge could yield far-reaching benefits across the application range in the biomedical, food, and environmental sectors. In this study, multienzyme partitioning immobilization that sequentially immobilizes cascade enzymes with hierarchical MOFs is proposed to reduce substrate diffusion resistance. Conversion results of ginsenosides indicate that this strategy improves the cascade efficiency up to 1.26 times. The substrate diffusion model is used to investigate the dual-interenzyme mass transfer behavior of substrates in the restricted domain space and evaluate the substrate channeling effect under partitioning immobilization. Molecular docking and kinetic simulations reveal that the MOFs effectively limit the conformational changes of cascade enzymes at high temperatures and in organic solvents while maintaining a large pocket of active centers. This phenomenon increased efficient substrate docking to the enzyme molecules, further optimizing cascade efficiency. The results of the immobilization of GOX and horseradish peroxidase as model enzymes indicate that the partitioned MOF immobilization strategy could be used for universal adaptation of cascade enzymes.

Engineering cascade biocatalysis in whole cells for syringic acid bioproduction

BackgroundSyringic acid (SA) is a high-value natural compound with diverse biological activities and wide applications, commonly found in fruits, vegetables, and herbs. SA is primarily produced through chemical synthesis, nonetheless, these chemical methods have many drawbacks, such as considerable equipment requirements, harsh reaction conditions, expensive catalysts, and numerous by-products. Therefore, in this study, a novel biotransformation route for SA production was designed and developed by using engineered whole cells.ResultsAn O-methyltransferase from Desulfuromonas acetoxidans (DesAOMT), which preferentially catalyzes a methyl transfer reaction on the meta-hydroxyl group of catechol analogues, was identified. The whole cells expressing DesAOMT can transform gallic acid (GA) into SA when S-adenosyl methionine (SAM) is used as a methyl donor. We constructed a multi-enzyme cascade reaction in Escherichia coli, containing an endogenous shikimate kinase (AroL) and a chorismate lyase (UbiC), along with a p-hydroxybenzoate hydroxylase mutant (PobA**) from Pseudomonas fluorescens, and DesAOMT; SA was biosynthesized from shikimic acid (SHA) by using whole cells catalysis. The metabolic system of chassis cells also affected the efficiency of SA biosynthesis, blocking the chorismate metabolism pathway improved SA production. When the supply of the cofactor NADPH was optimized, the titer of SA reached 133 μM (26.2 mg/L).ConclusionOverall, we designed a multi-enzyme cascade in E. coli for SA biosynthesis by using resting or growing whole cells. This work identified an O-methyltransferase (DesAOMT), which can catalyze the methylation of GA to produce SA. The multi-enzyme cascade containing four enzymes expressed in an engineered E. coli for synthesizing of SA from SHA. The metabolic system of the strain and biotransformation conditions influenced catalytic efficiency. This study provides a new green route for SA biosynthesis.Graphical

A novel strategy for detoxification of deoxynivalenol via modification of both toxic groups

Deoxynivalenol (DON) is the most abundant mycotoxin in cereal crops and derived foods and is of great concern in agriculture. Bioremediation strategies have long been sought to minimize the impact of mycotoxin contamination, but few direct and effective enzyme-catalyzed detoxification methods are currently available. In this study, we established a multi-enzymatic cascade reaction and successfully achieved detoxification at double sites: glutathionylation for the C-12,13 epoxide group and epimerization for the C-3 hydroxyl group. This yielded novel derivatives of DON, 3-epi-DON-13-glutathione (3-epi-DON-13-GSH) as well as its by-product, 3-keto-DON-13-GSH, for which precise structures were validated via liquid chromatography–high-resolution tandem mass spectrometry (LC-HRMS) and nuclear magnetic resonance (NMR) spectroscopy. Both cell viability and DNA synthesis assays demonstrated dramatically decreased cytotoxicity of the double-site modified product 3-epi-DON-13-GSH. These findings provide a promising and urgently needed novel method for addressing the problem of DON contamination in agricultural and industrial settings.

Biosynthetic production of anticoagulant heparin polysaccharides through metabolic and sulfotransferases engineering strategies

Heparin is an important anticoagulant drug, and microbial heparin biosynthesis is a potential alternative to animal-derived heparin production. However, effectively using heparin synthesis enzymes faces challenges, especially with microbial recombinant expression of active heparan sulfate N-deacetylase/N-sulfotransferase. Here, we introduce the monosaccharide N-trifluoroacetylglucosamine into Escherichia coli K5 to facilitate sulfation modification. The Protein Repair One-Stop Service-Focused Rational Iterative Site-specific Mutagenesis (PROSS-FRISM) platform is used to enhance sulfotransferase efficiency, resulting in the engineered NST-M8 enzyme with significantly improved stability (11.32-fold) and activity (2.53-fold) compared to the wild-type N-sulfotransferase. This approach can be applied to engineering various sulfotransferases. The multienzyme cascade reaction enables the production of active heparin from bioengineered heparosan, demonstrating anti-FXa (246.09 IU/mg) and anti-FIIa (48.62 IU/mg) activities. This study offers insights into overcoming challenges in heparin synthesis and modification, paving the way for the future development of animal-free heparins using a cellular system-based semisynthetic strategy.

Paper-based enzyme-linked biosensor combined with smartphone for simultaneous colorimetric sensing of xanthines and sarcosine

A portable paper-based enzyme-linked biosensor that integrates a multi-enzyme cascade reaction, natural enzyme, and nanozyme with filter paper, combined with smartphones, is proposed for the detection of xanthine (XA) and sarcosine (SA) in urine. The primary biological component of the sensor is horseradish peroxidase (HRP). By chemically modifying cellulose filter paper (CFP) the HRP in horseradish crude extract is specifically immobilized through boron affinity and metal chelation techniques. Furthermore, the incorporation of UiO-66 on CFP had a good synergistic effect on the electron transfer of HRP and the detection of H2O2, significantly increasing the sensor's sensitivity. Under optimized experimental conditions, the sensor demonstrated a dynamic response range of 8–400 μmol L−1 for SA with a minimum detection limit (LOD) of 5 μmol L−1, and a dynamic response range of 10–400 μmol L−1 with an LOD of 8 μmol L−1 for XA. Additionally, the well-designed detection area of the portable test strip simplifies the detection operation, reduces the detection time, and enables rapid on-site analysis. UV-Vis analysis further confirmed the sensor's accuracy in detecting disease markers in urine. The sensing platform has good reproducibility, specificity and stability, suitable for the detection of SA and XA in real human urine samples, and the sensor has the advantages of simple manufacturing, easy operation, strong portability, low reagent consumption, and low cost benefit. Therefore, the development of this paper-based enzyme-linked biosensor offers a novel approach to the detection of disease markers in urine and has potential applications in medical testing.

Coencapsulating Anionic Cofactor and Silica Nanoparticle-Bound Enzymes in Cationic Microgel for Multienzyme Cascade Reaction with Cofactor Recycling

Coencapsulating Anionic Cofactor and Silica Nanoparticle-Bound Enzymes in Cationic Microgel for Multienzyme Cascade Reaction with Cofactor Recycling

Development of a Thermodynamically Favorable Multi-enzyme Cascade Reaction for Efficient Sustainable Production of ω-Amino Fatty Acids and α,ω-Diamines.

Invited for this issue's cover is the group of Huilei Yu at the East China University of Science and Technology. The image shows a sustainable biosynthesis route to nylon monomers from bio-based substrate α, ω-dicarboxylic acids. The Research Article itself is available at 10.1002/cssc.202301477.

Wool keratin-based colorimetric detection of organophosphorus pesticides via a multi-enzyme cascade reaction

Wool keratin-based colorimetric detection of organophosphorus pesticides via a multi-enzyme cascade reaction

Targeting and Microenvironment-Activated Nanoreactor for Diabetic Chronic Wound Healing via Multienzyme Cascade Reactions.

The development of cell-like nanoreactors with the ability to initiate biocatalytic cascades under special conditions holds tremendous potential for therapeutic applications. Herein, conformationally gated nanoreactors that respond to the acidic microenvironment of infected diabetic wounds were developed by cucur[8]bituril (CB[8])-based supramolecular assembly. The bioinspired nanoreactors exhibit not only self-regulated permeability and selectivity to control internal enzyme activities by substance exchange but also distinct binding specificities toward Gram-positive and Gram-negative bacteria via noncovalent modification with different ligands. The encapsulation of glucose oxidase (GOx), Fe3O4 nanozyme, and l-arginine (l-Arg) into the nanocarriers enables intelligent activation of multienzyme cascade reactions upon glucose (Glu) uptake to produce gluconic acid (GA) and hydrogen peroxide (H2O2), which is further converted into highly toxic hydroxyl radicals (·OH) for selective antibacterial activity. Moreover, acidic H2O2 promotes the oxidization of l-Arg, leading to the release of nitric oxide (NO). Consequently, this nanoreactor provides a multifunctional and synergistic platform for diabetic chronic wound healing by combining enzyme dynamic therapy with NO gas therapy to combat bacterial infections and inflammation under high blood Glu levels.

Development of a Thermodynamically Favorable Multi-enzyme Cascade Reaction for Efficient Sustainable Production of ω-Amino Fatty Acids and α,ω-Diamines.

Aliphatic ω-amino fatty acids (ω-AFAs) and α,ω-diamines (α,ω-DMs) are essential monomers for the production of nylons. Development of a sustainable biosynthesis route for ω-AFAs and α,ω-DMs is crucial in addressing the challenges posed by climate change. Herein, we constructed an unprecedented thermodynamically favorable multi-enzyme cascade (TherFavMEC) for the efficient sustainable biosynthesis of ω-AFAs and α,ω-DMs from cheap α,ω-dicarboxylic acids (α,ω-DAs). This TherFavMEC was developed by incorporating bioretrosynthesis analysis tools, reaction Gibbs free energy calculations, thermodynamic equilibrium shift strategies and cofactor (NADPH&ATP) regeneration systems. The molar yield of 6-aminohexanoic acid (6-ACA) from adipic acid (AA) was 92.3 %, while the molar yield from 6-ACA to 1,6-hexanediamine (1,6-HMD) was 96.1 %, which were significantly higher than those of previously reported routes. Furthermore, the biosynthesis of ω-AFAs and α,ω-DMs from 20.0 mM α,ω-DAs (C6-C9) was also performed, giving 11.2 mM 1,6-HMD (56.0 % yield), 14.8 mM 1,7-heptanediamine (74.0 % yield), 17.4 mM 1,8-octanediamine (87.0 % yield), and 19.7 mM 1,9-nonanediamine (98.5 % yield), respectively. The titers of 1,9-nonanediamine, 1,8-octanediamine, 1,7-heptanediamine and 1,6-HMD were improved by 328-fold, 1740-fold, 87-fold and 3.8-fold compared to previous work. Therefore, this work holds great potential for the bioproduction of ω-AFAs and α,ω-DMs.

Enzyme Cascade Electrode Reactions with Nanomaterials and Their Applicability towards Biosensor and Biofuel Cells.

Nanomaterials, including carbon nanotubes, graphene oxide, metal-organic frameworks, metal nanoparticles, and porous carbon, play a crucial role as efficient carriers to enhance enzyme activity through substrate channeling while improving enzyme stability and reusability. However, there are significant debates surrounding aspects such as enzyme orientation, enzyme loading, retention of enzyme activity, and immobilization techniques. Consequently, these subjects have become the focus of intensive research in the realm of multi-enzyme cascade reactions. Researchers have undertaken the challenge of creating functional in vitro multi-enzyme systems, drawing inspiration from natural multi-enzyme processes within living organisms. Substantial progress has been achieved in designing multi-step reactions that harness the synthetic capabilities of various enzymes, particularly in applications such as biomarker detection (e.g., biosensors) and the development of biofuel cells. This review provides an overview of recent developments in concurrent and sequential approaches involving two or more enzymes in sequence. It delves into the intricacies of multi-enzyme cascade reactions conducted on nanostructured electrodes, addressing both the challenges encountered and the innovative solutions devised in this field.

Tandem-biocatalysis reactors constructed by topological evolution of CaCO3 particles into hollow metal hydroxide spheres

Despite remarkable advances in the design and synthesis of hollow inorganic spheres (HISs), the harsh synthetic conditions have precluded the applications of HISs to biochemical and biological fields. Herein we report a biocompatible strategy for synthesizing metal hydroxide HISs (MH-HISs) by simply mixing CaCO3 particles with metal ions in water. The ion-exchange reaction between Ca2+ and metal ions leads to the structural and chemical evolution from solid CaCO3 particles to hollow MH-HISs via core-shell and yolk-shell structures, while enabling the encapsulation of enzymes to the shells without loss of catalytic activities. The biocompatible protocol makes multienzymatic cascade reactions achievable, with great recyclability due to mechanical durability of MH-HISs.

A cell-free multi-enzyme cascade for the synthesis of CDP-glycerol.

CDP-glycerol is a nucleotide-diphosphate-activated version of glycerol. In nature, it is required for the biosynthesis of teichoic acid in Gram-positive bacteria, which is an appealing target epitope for the development of new vaccines. Here, a cell-free multi-enzyme cascade was developed to synthetize nucleotide-activated glycerol from the inexpensive and readily available substrates cytidine and glycerol. The cascade comprises five recombinant enzymes expressed in E. coli that were purified by immobilized metal affinity chromatography. As part of the cascade, ATP is in-situ regenerated from polyphosphate to reduce synthesis costs. The enzymatic cascade was characterized at laboratory scale and the products analyzed by high performance anion exchange chromatography (HPAEC)-UV and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). After the successful synthesis was confirmed, a design of experiments approach was used to screen for optimal operation conditions (temperature, pH value and MgCl2 concentration). Overall, a substrate conversion of 89% was achieved with respect to the substrate cytidine.

Co-encapsulating Cofactor and Enzymes in Hydrogen-Bonded Organic Frameworks for Multienzyme Cascade Reactions with Cofactor Recycling.

Use of hydrogen-bonded organic frameworks (HOFs) for enzyme immobilization faces challenges in the improvement of enzyme activity recovery and the assembly of cofactor-dependent multienzyme systems. Herein, we report a polyelectrolyte-assisted encapsulation approach (PAEA) that enables two cascades with four oxidoreductases and two nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) cofactors co-encapsulated in BioHOF-1 with excellent cargo loading and over 100 % cascade activity. The key role of the polyelectrolyte is to coat enzymes and tether NAD(P)H, thus interacting with HOF monomers in place of enzymes, avoiding the destruction of enzymes by HOF monomers. The versatility and efficiency of PAEA are further illustrated by an HOF-101-based bio-nanoreactor. Moreover, the immobilization by PAEA makes enzymes and NAD(P)H display excellent stability and recyclability. This study has demonstrated a facile and versatile PAEA for fabricating cofactor-dependent multienzyme cascade nanoreactors with HOFs.

Co‐encapsulating Cofactor and Enzymes in Hydrogen‐Bonded Organic Frameworks for Multienzyme Cascade Reactions with Cofactor Recycling

AbstractUse of hydrogen‐bonded organic frameworks (HOFs) for enzyme immobilization faces challenges in the improvement of enzyme activity recovery and the assembly of cofactor‐dependent multienzyme systems. Herein, we report a polyelectrolyte‐assisted encapsulation approach (PAEA) that enables two cascades with four oxidoreductases and two nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) cofactors co‐encapsulated in BioHOF‐1 with excellent cargo loading and over 100 % cascade activity. The key role of the polyelectrolyte is to coat enzymes and tether NAD(P)H, thus interacting with HOF monomers in place of enzymes, avoiding the destruction of enzymes by HOF monomers. The versatility and efficiency of PAEA are further illustrated by an HOF‐101‐based bio‐nanoreactor. Moreover, the immobilization by PAEA makes enzymes and NAD(P)H display excellent stability and recyclability. This study has demonstrated a facile and versatile PAEA for fabricating cofactor‐dependent multienzyme cascade nanoreactors with HOFs.

A multi-enzymatic cascade reaction for the synthesis of bioactive C-oligosaccharides

C-Oligosaccharides are rare in nature and possess diverse bioactivities. However, their chemical synthesis faces many challenges. In this work, enzymatic introduction of C-linked sugar chains to target aglycones was successfully achieved by multi-enzymatic cascade reactions. A C-glycosyltransferase from Aloe barbadensis was employed to introduce the first C-linked glucose and then a cyclomaltodextrin glucanotransferase from Bacillus licheniformis was used to extend the sugar chain. A total of twenty C-oligosaccharides with 2–6 sugars were synthesized from scale-up reactions and exhibited good water solubility and sodium-dependent glucose transporter 2 (SGLT2) inhibitory activity. Furthermore, a glucoamylase was used to control the length of the sugar chain and the C-maltosides were efficiently synthesized. These findings not only expanded the structural diversity of C-oligosaccharides, but also provided a strategy for the modification of C-glycoside drugs to improve the druggability.

Synthesis of cello-oligosaccharides which promotes the growth of intestinal probiotics by multi-enzyme cascade reaction

Soluble cello-oligosaccharide with 2-6 oligosaccharide units is a kind of oligosaccharide with various biological functions, which can promote the proliferation of intestinal probiotics such as Bifidobacteria and Lactobacillus paracei. Therefore, it has a regulatory effect on human intestinal microbiota. In this study, a Cc 01 strain was constructed by expressing cellodextrin phosphorylase (CDP) in Escherichia coli. By combining with a previously constructed COS 01 strain, a three-enzyme cascade reaction system based on strains COS 01 and Cc 01 was developed, which can convert glucose and sucrose into cello-oligosaccharide. After optimization, the final titer of soluble cello-oligosaccharides with 2-6 oligosaccharide units reached 97 g/L, with a purity of about 97%. It contained cellobiose (16.8 wt%), cellotriose (49.8 wt%), cellotetrose (16.4 wt%), cellopentaose (11.5 wt%) and cellohexose (5.5 wt%). When using inulin, xylo-oligosaccharide and fructooligosaccharide as the control substrate, the biomass (OD600) of Lactobacillus casei (WSH 004), Lactobacillus paracei (WSH 005) and Lactobacillus acidophilus (WSH 006) on cello-oligosaccharides was about 2 folds higher than that of the control. This study demonstrated the efficient synthesis of cello-oligosaccharides by a three-enzyme cascade reaction and demonstrated that the synthesized cello-oligosaccharides was capable of promoting intestinal microbial proliferation.

Recent Advances on Key Enzymes of Microbial Origin in the Lycopene Biosynthesis Pathway.

Lycopene is a common carotenoid found mainly in ripe red fruits and vegetables that is widely used in the food industry due to its characteristic color and health benefits. Microbial synthesis of lycopene is gradually replacing the traditional methods of plant extraction and chemical synthesis as a more economical and productive manufacturing strategy. The biosynthesis of lycopene is a typical multienzyme cascade reaction, and it is important to understand the characteristics of each key enzyme involved and how they are regulated. In this paper, the catalytic characteristics of the key enzymes involved in the lycopene biosynthesis pathway and related studies are first discussed in detail. Then, the strategies applied to the key enzymes of lycopene synthesis, including fusion proteins, enzyme screening, combinatorial engineering, CRISPR/Cas9-based gene editing, DNA assembly, and scaffolding technologies are purposefully illustrated and compared in terms of both traditional and emerging multienzyme regulatory strategies. Finally, future developments and regulatory options for multienzyme synthesis of lycopene and similar secondary metabolites are also discussed.

Construction of multi-enzyme cascade reactions and its application in the synthesis of bifunctional chemicals

The synthesis of fine chemicals using multi-enzyme cascade reactions is a recent hot research topic in the field of biocatalysis. The traditional chemical synthesis methods were replaced by constructing in vitro multi-enzyme cascades, then the green synthesis of a variety of bifunctional chemicals can be achieved. This article summarizes the construction strategies of different types of multi-enzyme cascade reactions and their characteristics. In addition, the general methods for recruiting enzymes used in cascade reactions, as well as the regeneration of coenzyme such as NAD(P)H or ATP and their application in multi-enzyme cascade reactions are summarized. Finally, we illustrate the application of multi-enzyme cascades in the synthesis of six bifunctional chemicals, including ω-amino fatty acids, alkyl lactams, α, ω-dicarboxylic acids, α, ω-diamines, α, ω-diols, and ω-amino alcohols.

Construction a starving therapy induced photothermal enhanced cascade nanoreactor for imaging guided catalytic synergistic therapy of tumor

Multienzyme cascade reaction has shown great potential in tumor therapy and photothermal was usually introduced to nanozyme to improve the catalytic efficiency which is limited by tumor microenvironment. However, it is difficult to determine the optimal treatment window in photothermal enhanced catalytic therapy. In order to solve the problem, a trackable-imaging multifunctional cascade reactor was designed with glucose oxidase (GOx) loaded bimetallic Fe/Cu-MOF-199 as core and polydopamine (PDA) as shell, with the pH/GSH targeting of the shell PDA, Fe/Cu-MOF-199/GOx@PDA can be enriched in tumor cells. With the release of GOx from the core Fe/Cu-MOF-199, the glucose in tumor can be catalyzed to supply H2O2, reduce heat resistance, and with the self-cyclic valence alteration of Fe3+/Fe2+ and Cu2+/Cu+, GSH can be constantly consumed, greatly reducing the anti-oxidation defense effect of tumor. Through the activities of catalase and peroxidase of Fe/Cu-MOF-199, O2 and hydroxyl radicals (•OH) are generated to kill cancer cells and compensate for O2 consumed in glucose catalytic reaction, respectively. Furthermore, under MRI guiding, near-infrared light excitation is performed at the optimal concentration of Fe/Cu-MOF-199/GOx@PDA in the tumor. Promoting the multienzyme activity in TME to the greatest extent. Cytotoxicity proved the selectivity, and in vivo experiments confirmed that the catalytic synergistic treatment of multienzyme cascade reaction and photothermal therapy was satisfactory. The study provides new promising ideas for designing multifunctional cascade nanoreactor for efficient cancer treatment.