Multi-enzyme Cascade Research Articles

Supramolecular Nanozyme System Based on Polydopamine and Polyoxometalate for Photothermal-Enhanced Multienzyme Cascade Catalytic Tumor Therapy.

The advent of enzyme-facilitated cascade events in which endogenous substrates within the human body are used to generate reactive oxygen species (ROS) has spawned novel cancer treatment possibilities. In this study, a supramolecular cascade catalytic nanozyme system was successfully developed, exhibiting photothermal-enhanced multienzyme cascade catalytic and glutathione (GSH) depletion activities and ultimately triggering the apoptosis-ferroptosis synergistic tumor therapy. The nanozyme system was fabricated using β-cyclodextrin-functionalized polydopamine (PDA) as the substrate, which was then entangled with polyoxometalate (POM) via electrostatic forces and assembled with adamantane-grafted hyaluronic acid and glucose oxidase (GOx) via host-guest supramolecular interaction for tumor targeting and GOx loading. The catalytic function of GOx facilitates the conversion of glucose to H2O2 and gluconic acid. In turn, this process affirms the propitious generation of hydroxyl radical (•OH) through the POM-mediated cascade catalysis. Additionally, the POM species actively deplete the intracellular GSH pool, initiating a cascade catalytic tumor therapy. In addition, the PDA-POM-mediated photothermal hyperthermia boosted the cascade catalytic effect and increased ROS production. This confers considerable promise for photothermal therapy (PTT)/nanocatalytic cancer therapy on supramolecular nanozyme systems. The in vitro and in vivo antitumor efficacy studies demonstrated that the supramolecular cascade catalytic nanozyme system was effective at reducing tumor development while maintaining an acceptable level of biocompatibility. Henceforth, this study is to widen the scope of cascade catalytic nanoenzyme production using supramolecular techniques, as well as endeavor to delineate a prospective pathway for the application of PTT-enhanced nanocatalytic tumor therapy.

A Portable Nanozyme‐Strip Fabricated by Ru‐ZIF‐8 with Dual Nanozymatic Activity for Detection of Acetylcholine

AbstractIn the present study, the incorporation of Ru into the framework of ZIF‐8 resulted in that a Ru‐ZIF‐8 nanozyme with both esterase‐like activity and peroxidase‐like activity. Owing to the dual nanozymatic activities of Ru‐ZIF‐8 nanozyme, the acetylcholine (ACh) detection process utilized only one natural enzyme (choline oxidase), which greatly simplified the detection procedure. In the detection process, nanozyme Ru‐ZIF‐8 showed esterase‐like activity to catalyze the hydrolysis of ACh to choline. Then the generated choline was oxidized by choline oxidase to generate H2O2. Finally, Ru‐ZIF‐8 with peroxidase‐like activity was used to catalyze H2O2 to oxidize 2,2′‐azino‐bis (3‐ethylbenzothiazo‐line‐6‐sulfonic acid) ammonium salt with a color change. As a result, the linear range for ACh was from 0.01 μM‐22 μM with the detection limit (DL) of 5.5 nM. In addition, Ru‐ZIF‐8 was utilized to fabricate a portable detection test strip. Our work provided a simple, portable, low‐cost, highly sensitive and selective assay for the detection of ACh, which paved the way for the development of multi enzyme cascade assays.

An artificial multienzyme cascade for the whole-cell synthesis of rare ketoses from glycerol.

In our previous study, we constructed a one-pot multi-enzyme system for rare ketoses synthesis based on L-rhamnulose-1-phosphate aldolase (RhaD) from accessible glycerol in vitro. To eliminate tedious purification of enzymes, a facile Escherichia coli whole-cell cascade platform was established in this study. To enhance the conversion rate, the reaction conditions, substrate concentrations and expressions of related enzymes were extensively optimized. The biosynthetic route for the cascade synthesis of rare ketoses in whole cells was successfully constructed and three rare ketoses including D-allulose, D-sorbose and L-fructose were produced using glycerol and D/L-glyceraldehyde (GA). Under optimized conditions, the conversion rates of rare ketoses were 85.0% and 93.0% using D-GA and L-GA as the receptor, respectively. Furthermore, alditol oxidase (AldO) was introduced to the whole-cell system to generate D-GA from glycerol, and the total production yield of D-sorbose and D-allulose was 8.2gl-1 only from the sole carbon source glycerol. This study demonstrates a feasible and cost-efficient method for rare sugars synthesis and can also be applied to the green synthesis of other value-added chemicals from glycerol.

Multi-Enzymatic Cascade for Efficient Deracemization of dl-Pantolactone into d-Pantolactone.

d-pantolactone is an intermediate in the synthesis of d-pantothenic acid, which is known as vitamin B5. The commercial synthesis of d-pantolactone is carried out through the selective resolution of dl-pantolactone catalyzed by lactone hydrolase. In contrast to a kinetic resolution approach, the deracemization of dl-pantolactone is a simpler, greener, and more sustainable way to obtain d-pantolactone with high optical purity. Herein, an efficient three-enzyme cascade was developed for the deracemization of dl-pantolactone, using l-pantolactone dehydrogenase from Amycolatopsis methanolica (AmeLPLDH), conjugated polyketone reductase from Zygosaccharomyces parabailii (ZpaCPR), and glucose dehydrogenase from Bacillus subtilis (BsGDH). The AmeLPLDH was used to catalyze the dehydrogenated l-pantolactone into ketopantolactone; the ZpaCPR was used to further catalyze the ketopantolactone into d-pantolactone; and glucose dehydrogenase together with glucose fulfilled the function of coenzyme regeneration. All three enzymes were co-expressed in E. coli strain BL21(DE3), which served as the whole-cell biocatalyst. Under optimized conditions, 36 h deracemization of 1.25 M dl-pantolactone d-pantolactone led to an e.e.p value of 98.6%, corresponding to productivity of 107.7 g/(l·d).

Synthesis of Bioactive Hydroxytyrosol Esters via Multienzyme Cascade on Electroactive Melanin Lignin Nanoparticles: A One‐Pot Approach without Extra Reducing Agents

AbstractA one‐pot strategy for the synthesis of bioactive hydroxytyrosol esters from tyrosol and lipophilic carboxylic acids has been developedviamultienzyme cascade immobilized on electroactive melanin lignin nanoparticles. The novel catalyst involved the co‐immobilization of lipase and tyrosinase on the polyphenolic support and was operative in the presence of sustainable 2‐methyltetrahydrofuran as organic reaction solvent. The system did not require the use of extra reducing agents for the selective synthesis of catechols. Optoelectronic and electrochemical studies suggested the role played by melanin lignin nanoparticles in the regeneration of the active site of tyrosinase from unproductivemet‐form to reversibledeoxy/oxy‐form. This effect improved the overall activity of tyrosinase in the multienzyme cascade favouring the reduction of undesired quinones to corresponding catechols.

Engineering a Carboxyl Methyltransferase for the Formation of a Furan-Based Bioplastic Precursor.

FtpM from Aspergillus fumigatus was the first carboxyl methyltransferase reported to catalyse the dimethylation of dicarboxylic acids. Here the creation of mutant R166M that can catalyse the quantitative conversion of bio-derived 2,5-furandicarboxylic acid (FDCA) to its dimethyl ester (FDME), a bioplastics precursor, was reported. Wild type FtpM gave low conversion due to its reduced catalytic efficiency for the second methylation step. An AlphaFold 2 model revealed a highly electropositive active site, due to the presence of 4 arginine residues, postulated to favour the binding of the dicarboxylic acid over the intermediate monoester. The R166M mutation improved both binding and turnover of the monoester to permit near quantitative conversion to the target dimethyl ester product. The mutant also had improved activity for other diacids and a range of monoacids. R166M was incorporated into 2 multienzyme cascades for the synthesis of the bioplastics precursor FDME from bioderived 5-hydroxymethylfurfural (HMF) as well as from poly(ethylene furanoate) (PEF) plastic, demonstrating the potential to recycle waste plastic.

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.

In situ and layer-by-layer immobilization of ternary enzyme cascades based on CaCO3 microspheres

The efficiency of multi-enzyme immobilization is often influenced by the carrier and immobilization techniques employed. In this work, we proposed a ternary enzyme cascade reaction system composed of β-glucosidase (β-G), glucose oxidase (GOD) and horseradish peroxidase (HRP). CaCO3 microspheres served as a template for in situ immobilization, while poly (allylamine hydrochloride) (PAH) and sodium poly (4-styrene sulfonate) (PSS) were utilized to create PAH-CaCO3 and PSS-CaCO3 templates for layer-by-layer multi-enzyme immobilization. The effects of template structure, glutaraldehyde (GA) addition, ethylenediaminetetraacetic acid (EDTA) concentration, and the structure of layer-by-layer assembly were systematically investigated. Our results indicated that an assembly of PSS-CaCO3--PAH--β-G--HRP--GOD presented simultaneous high enzyme loading and relative activity. The loadings of β-G, GOD and HRP were 75.2 ± 2.0%, 87.7 ± 4.4% and 85.5 ± 3.2%, respectively, while the relative activities were 81.8 ± 2.7%, 93.6 ± 4.7% and 90.6 ± 3.1%, respectively. Kinetic parameters confirmed that the reaction rate of the immobilized ternary enzymes in this assembly was close to that of the free enzymes. This work provides insights for constructing other multi-enzyme cascade reaction systems using immobilization techniques.

Spatial distribution and proximity effect-dependent multi-enzyme cascades catalysis operating in dendritic mesoporous silica nanoparticles

Enzymatic cascades catalysis engenders complex and selective intracellular transformations due to the organization of the enzymes in confined cellular environments. In this work, a biomimetic strategy is employed to develop a synthetic cell analogue for operating multi-enzymatic cascade catalysis in vitro. The glucose peroxidase (GOx) and chloroperoxidase (CPO) are co-encapsulated in the channel of dendritic mesoporous silica nanoparticles (DMSP) modified by phase-transitioned lysozyme (PTL@GOx&CPO@DMSP). The cascade efficiency is substantially enhanced compared with the diffusion mixtures of the free enzymes in bulk solution due to some nanoscale construction, such as confinement effect, proximity effect and special distribution mode of the enzymes. The high specific surface area and high concentration of mesoporous cages in DMSP is beneficial for encapsulation of enzyme. The cross-linked cavities ensure directional transfer of the intermediate from GOx to CPO, known as “substrate channel”. PTL plays multiple functions including inhibiting leaking of enzyme from DMSP, concentrating intermediate H2O2 at DMSP domains and keeping this biocomposite stable in aqueous environment by improving its dispersibility. The prepared biocomposite PTL@GOx & CPO@DMSP is very stable at harsh conditions. It can retain 42% of original catalytic activity at 90°C with incubation for 3 h while the retained activity of free enzymes is only 5% under the same conditions. Meanwhile, it can keep 92.4% of its original catalytic activity after 10 reuses. These characteristics ensure practical application of this biocatalytic material. The active HClO generated by GOx-CPO cascade has an efficient anti-tumor effect and is effectively applied to preserve fruit.

Efficient biosynthesis of high-value 5‑Hydroxytryptophan using a multienzyme cascade

5-Hydroxytryptophan (5-HTP) is a precursor of serotonin. The increasing demand for 5-HTP is driving the search for a more efficient and cost-effective production method. In this study, a new in vitro multi-enzyme cascade system (MECCS) was developed to convert L-tryptophan (L-trp) to 5-HTP in a single container while achieving the cycling and regeneration of cofactors and coenzymes. The rate-limiting enzyme tryptophan hydroxylase (TPH) was truncated to improve the overall efficiency of the cascade. After screening, the mutant M1 (NΔ143/CΔ26) showed excellent enzymatic performance, with a 2.3-fold increase in catalytic activity. Molecular dynamics simulations showed that the increase in active center binding force and the improvement of substrate tunnel in M1 improved the overall catalytic efficiency. In addition, the rigidity enhancement of the ligand binding pocket surrounding the region improved the thermal stability of the enzyme. Under optimized conditions, the MECCS converted 50 mM L-trp to 36.9 mM 5-HTP, with a conversion rate of 73.8%. This study provides an alternative pathway for the efficient production of 5-HTP.

Hydrogenase-based oxidative biocatalysis without oxygen

Biocatalysis-based synthesis can provide a sustainable and clean platform for producing chemicals. Many oxidative biocatalytic routes require the cofactor NAD+ as an electron acceptor. To date, NADH oxidase (NOX) remains the most widely applied system for NAD+ regeneration. However, its dependence on O2 implies various technical challenges in terms of O2 supply, solubility, and mass transfer. Here, we present the suitability of a NAD+ regeneration system in vitro based on H2 evolution. The efficiency of the hydrogenase-based system is demonstrated by integrating it into a multi-enzymatic cascade to produce ketoacids from sugars. The total NAD+ recycled using the hydrogenase system outperforms NOX in all different setups reaching up to 44,000 mol per mol enzyme. This system proves to be scalable and superior to NOX in terms of technical simplicity, flexibility, and total output. Furthermore, the system produces only green H2 as a by-product even in the presence of O2.

In Situ Immobilization of Multi-Enzymes for Enhanced Substrate Channeling of Enzyme Cascade Reactions: A Nanoarchitectonics Approach by Directed Metal-Organic Frameworks.

Rationally tailoring a controlled spatial organization of enzymes in a nanoarchitecture for multi-enzyme cascade reactions can enhance the catalytic efficiency via substrate channeling. However, attaining substrate channeling is a grand challenge, requiring sophisticated techniques. Herein, we report facile polymer-directed metal-organic framework (MOF)-based nanoarchitechtonics for realizing a desirable enzyme architecture with significantly enhanced substrate channeling. The new method involves the use of poly(acrylamide-co-diallyldimethylammonium chloride) (PADD) as a modulator in a one-step process for simultaneous MOF synthesis and co-immobilization of enzymes (GOx and HRP). The resultant enzymes-PADD@MOFs constructs showed a closely packed nanoarchitecture with enhanced substrate channeling. A transient time close to 0 s was observed, owing to a short diffusion path for substrates in a 2D spindle-shaped structure and their direct transfer from one enzyme to another. This enzyme cascade reaction system showed a 3.5-fold increase in catalytic activity in comparison to free enzymes. The findings provide a new insight into using polymer-directed MOF-based enzyme nanoarchitectures to improve catalytic efficiency and selectivity.

Peroxygenase‐Promoted Enzymatic Cascades for the Valorisation of Fatty Acids Front Cover: (ChemCatChem 11/2023)

The Front Cover shows enzymatic cascades for the valorisation of fatty acids with peroxygenase-catalysed hydroxylation as key step. Functionalisation of fatty acids generally relies on pre-existing functional groups such as C=C-double bonds. In their Research Article, F. Hollmann and co-workers demonstrated the synthetic potential of a peroxygenase mutant AaeUPO for selective fatty acid oxyfunctionalisation. The primary products (i.e., hydroxy fatty acid (esters)) are interesting building blocks for lactone- and polyester synthesis. Furthermore, the ω-1 hydroxy fatty acid (esters) produced were transformed into alcohols, esters and amines via multi-enzyme cascades thereby paving the way for new fatty acid valorisation pathways. More information can be found in the Research Article by F. Hollmann and co-workers.

Highly efficiency production of D-allulose from inulin using curli fiber multi-enzyme cascade catalysis

D-Allulose is a rare sugar with numerous physiological benefits and low caloric content, which can be obtained from inulin through enzymatic catalysis. In this study, we combined D-allulose 3-epimerase, exo- and endo-inulinases (EXINU and ENINU) with the NGTag/NGCatcher/CsgA system to accelerate D-allulose accumulation from inulin. Molecular dynamics simulations were used to screen linkers of appropriate length for ENINU. In vitro, we successfully observed the assembled NGCatcher_ENINU_CsgA, NGTag_EXINU, and DAERK fibers using fluorescent labelling with GFP, YFP, and mCherry. The optimal pH and temperature of the tagged variants were comparable to those of the wild-type, and the MD simulations showed that NGCatcher_ENINU_CsgA had improved stability in the working environment of EXINU. D-Allulose accumulation rate of the assembled enzymes cascade (NGCatcher_ENINU_CsgA/NGTag_EXINU_CsgA/DAERK) reached 0.25 g/L min−1 (1.25 mgD-allulose mgDAERK−1 min−1) at an inulin concentration of 100 g/L. The assembled system greatly improves the high-valued productions of rare sugars from cheap biomass.

Yolk-shell structured nanoreactor Au@Co3O4/CeO2@mSiO2 with superior peroxidase-like activity as nanozyme for ultra-sensitive colorimetric biosensing

For yolk-shell structured nanoreactors, multiple active components can be precisely positioned on core and/or shell that can afford more exposed accessible active sites, and the internal voids can guarantee sufficient contact of reactants and catalysts. In this work, a unique yolk–shell structured nanoreactor Au@Co3O4/CeO2@mSiO2 was fabricated and applied as nanozyme for biosensing. The Au@Co3O4/CeO2@mSiO2 exhibited superior peroxidase-like activity with a lower Michaelis constant (Km) and a higher affinity to H2O2. The enhanced peroxidase-like activity was attributed to the unique structure and the synergistic effects between the multiple active components. Colorimetric essays were developed based on Au@Co3O4/CeO2@mSiO2 for the ultra-sensitive sensing of glucose in the range of 3.9 nM–1.03 mM with the limit of detection as low as 3.2 nM. In the detection of glucose-6-phosphate dehydrogenase (G6PD), the cooperation between G6PD and Au@Co3O4/CeO2@mSiO2 triggered the redox cycling between NAD+ and NADH, thereby achieving the amplification of the signal and enhancing the sensitivity of the assay. The assay showed superior performance as compared to other methods with linear response of 5.0 × 10−3–15 mU mL−1 and lower detection limit of 3.6 × 10−3 mU mL−1. The fabricated novel multi-enzyme catalytical cascade reaction system allowed rapid and sensitive biodetection, demonstrating its potential in biosensors and biomedical applications.

A multi-enzyme cascade microneedle reaction system for hierarchically MRSA biofilm elimination and diabetic wound healing

The existence of biofilm remains a significant impediment to the healing of chronic wounds, including diabetic wounds. The dense extracellular polymeric substance (EPS) barrier of biofilms leads to poor drug permeability, making biofilms eradication a challenging task. Additionally, high glucose levels hinder the process of wound healing. To address these issues, we developed a multi-enzyme cascade microneedle system (MN) by combining enzymatic biofilm disruption with GOx-based chemodynamic therapy (CDT) for the first time. The microneedle tip dissolves quickly and delivers α-amylase and glucose oxidase-loaded metal–organic framework MIL-101 (M@G) into the biofilm’s interior. The α-amylase disintegrates the EPS structure, making the bacteria vulnerable and producing glucose as a substrate for the next cascade reaction. Next, GOx uses the glucose produced from EPS degradation and present at the wound site to produce significant amounts of H2O2, which is then transformed to reactive oxygen species (ROS) through MIL-101. The system eradicates methicillin-resistant Staphylococcus aureus (MRSA) biofilms by sequentially destroying EPS, consuming glucose at the wound, and self-supplying H2O2 to produce ROS, thereby reducing glucose concentrations and concurrently decreasing bacterial infection and inflammatory responses. Additionally, the pro-angiogenic peptide functionalized hydrogel (Gel-Q-M) serves as a backing to promote collagen deposition and angiogenesis, thereby accelerating the healing of chronic wound. In conclusion, the constructed MN platform showed excellent anti-biofilm and angiogenic properties in vitro, which could effectively eliminate biofilm, reduce inflammation and promote diabetic wound repair.

A Multi-Enzyme Cascade for Efficient Production of Pyrrolidone from l-Glutamate.

Pyrrolidone is a high value-added monomer and an important active drug intermediate. However, the efficient enzymatic synthesis of pyrrolidone remains a challenge. Here, we developed and reconstructed a three-enzyme cascade pathway using Escherichia coli BL21(DE3) for the production of pyrrolidone from l-glutamate (l-Glu). The carnitine-CoA ligase from Escherichia coli (EcCaiC) at a low expression level and with a low activity is regarded as the rate-limiting enzyme. Here, we obtained the best EcCaiCF380M/N430D double mutant with a kcat/Km value 1.5 times higher than that of the wild type via mechanism-based protein engineering. For this, we (i) eliminated the steric hindrance of the loop ring to improve the precatalytic conformation of the adenylation intermediate and (ii) fixed the hinge region to stabilize the closed conformation of the enzyme. Furthermore, ribosome-binding site (RBS) optimization led to an increase in the expression level of EcCaiCF380M/N430D, which was then cloned into the plasmid pET-EcCaiCF380M/N430D-DegoPPK2. Finally, under optimal induction and transformation conditions, 16.62 g/L of pyrrolidone was generated from 30 g/L l-Glu (batch feeding) within 24 h with a molar conversion rate of 95.2% and the highest productivity ever obtained, to our knowledge (0.69 g/L/h). Our findings demonstrate a strategy that is potentially attractive for the industrial production of pyrrolidone. IMPORTANCE This study developed a three-enzyme cascade pathway for the production of pyrrolidone from l-Glu. The catalytic efficiency of carnitine CoA ligase from Escherichia coli (EcCaiC) was improved by mechanism-based protein engineering, and the titer of pyrrolidone was further increased by ribosome-binding site (RBS), induction conditions, and conversion conditions optimization. Finally, we efficiently produced pyrrolidone by one pot in vivo with 95.2% conversion and 0.69 g/L/h productivity. Our study provides a new possibility for the industrial production of enzymatic synthesis of pyrrolidone.

Multi-enzyme cascade excluding costly cofactors for pyruvate production from glucose

In vitro enzymatic cascade is a useful biomanufacturing technique, but requirement for costly exogenous cofactors restricts its economic feasibility. In this study, we designed a costly cofactor-free enzymatic cascade to transform glucose into pyruvate, an important platform compound widely used in different industries. Only five enzymes including glucose oxidase from Aspergillus niger, mutant alditol oxidase from Streptomyces coelicolor, 2-keto-3-deoxy-d-gluconate aldolase from Sulfolobus acidocaldarius, catalase from Aspergillus niger, dihydroxy acid dehydratase from Paralcaligenes ureilyticus, and Mg2+ were needed for pyruvate production from glucose. Under the optimal reaction conditions, 30.82 mM pyruvate was produced with a yield of 74.27% of the theoretical value in 8 h. In addition, acetoin, another important platform chemical, can also be produced from glucose through further introducing pyruvate decarboxylase from Zymomonas mobilis and thiamine pyrophosphate into the enzymatic cascade. Acetoin at a concentration of 17.79 mM was produced from glucose with 84.71% of the theoretical yield in 10 h.

Spatially Segregated MOF Bioreactor Enables Versatile Modular Glycoenzyme Assembly for Hierarchical Glycan Library Construction.

The multienzyme cascade has received growing attention to obtain structurally defined glycans in vitro. However, due to poor enzyme stability and low compatibility between glycoenzymes, artificially designed multienzyme pathways to access glycans are often inefficient. Herein, based on the strategy "Modular-Enzymes Assembly by Spatial Segregation" (MASS), we developed a universal immobilization platform to assemble multiple glycoenzymes in compartmentalized MOF particles, inside and outside, significantly reducing the undesired interference and cross-inhibitions. By changing the enzyme modules, a series of glycosyl donor, disaccharides, oligosaccharides, and polysaccharides bearing cofactor regeneration were efficiently prepared. This bioreactor was further successfully applied to the reaction system with high substrate concentration to demonstrate its industrial potential. This robust multienzyme immobilization platform should serve to promote the enzymatic synthesis of more complex glycans.