Multienzyme System Research Articles

An artificial pathway for trans-4-hydroxy-L-pipecolic acid production from L-lysine in Escherichia coli

Trans-4-hydroxy-L-pipecolic acid (trans-4-HyPip) is a hydroxylated product of L-pipecolic acid, which is widely used in the pharmaceutical and chemical industries. Here, a trans-4-HyPip biosynthesis module was designed and constructed in Escherichia coli by overexpressing lysine α-oxidase, Δ1-piperideine-2-carboxylase reductase, glucose dehydrogenase, lysine permease, catalase and L-pipecolic acid trans-4-hydroxylase for expanding the lysine catabolism pathway. A total of 4.89 g/L of trans-4-HyPip was generated in shake flasks from 8 g/L of L-pipecolic acid. By this approach, 14.86 g/L of trans-4-HyPip was produced from lysine after 48 h in a 5 L bioreactor. As far as we know, this is the first multi-enzyme cascade catalytic system for the production of trans-4-HyPip using E. coli from L-lysine. Therefore, it can be considered as a potential candidate for the industrial production of trans-4-HyPip in microorganisms.

DNA-directed coimmobilization of multiple enzymes on organic-inorganic hybrid DNA flowers.

The artificial multienzyme systems developed by mimicking nature has attracted much interest. However, precisely controlled compositions and ratios of multienzymatic co-immobilization systems are still limited by the indistinguishable nature of enzymes. Herein, a strategy for fabricating DNA-directed immobilization of horseradish peroxidase (HRP) and glucose oxidase (GOx) on hybrid DNA nanoflowers (GOx-HRP@hDFs) is presented. The preparation of micron-sized hybrid DNA flowers (hDFs) begins with the predetermined repeatable polymer-like DNA sequences which contained two strands. The hDFs structure is generated through one-pot rolling circle amplification (RCA) and self-assembly with magnesium pyrophosphate inorganic crystals. Based on the rigid-base pairing, GOx and HRP conjugated with sequences complementary to strands would be anchored to the predesigned locations, respectively. By adjusting the loading amount/ratio of enzymes properly, the maximal catalytic efficiency can be precisely regulated. The reaction activity of GOx-HRP@hDFs was 7.4 times higher than that of the free GOx-HRP under the optimal mole ratio (GOx/HRP 4:1). In addition, this multienzyme catalyst system exhibits excellent precision, specificity, reproducibility, and long-term storage stability when applied to real human blood samples. The preceding results validate that GOx-HRP@hDFs are promising candidates for personal diabetes detection.

Biocatalytic Production of a Nylon 6 Precursor from Caprolactone in Continuous Flow.

6‐Aminocaproic acid (6ACA) is a key building block and an attractive precursor of caprolactam, which is used to synthesize nylon 6, one of the most common polymers manufactured nowadays. (Bio)‐production of platform chemicals from renewable feedstocks is instrumental to tackle climate change and decrease fossil fuel dependence. Here, the cell‐free biosynthesis of 6ACA from 6‐hydroxycaproic acid was achieved using a co‐immobilized multienzyme system based on horse liver alcohol dehydrogenase, Halomonas elongata transaminase, and Lactobacillus pentosus NADH oxidase for in‐situ cofactor recycling, with >90 % molar conversion (m.c.) The integration of a step to synthesize hydroxy‐acid from lactone by immobilized Candida antarctica lipase B resulted in >80 % m.c. of ϵ‐caprolactone to 6ACA, >20 % of δ‐valerolactone to 5‐aminovaleric acid, and 30 % of γ‐butyrolactone to γ‐aminobutyric acid in one‐pot batch reactions. Two serial packed‐bed reactors were set up using these biocatalysts and applied to the continuous‐flow synthesis of 6ACA from ϵ‐caprolactone, achieving a space‐time yield of up to 3.31 g6ACA h−1 L−1 with a segmented liquid/air flow for constant oxygen supply.

Nanozyme–Cellulose Hydrogel Composites Enabling Cascade Catalysis for the Colorimetric Detection of Glucose

Catalytic cascades obtained from the combination of nanozymes, which are nanomaterials with enzyme-like activity, and natural enzymes have drawn much attention for biosensing and biomedical applications. A key consideration in the development of cascade reaction systems is the integration of nanozymes with enzymes to boost the overall catalytic performance. Here, we report an efficient one-pot approach for the preparation of an enzyme–nanozyme hydrogel composite for cascade catalysis. Prussian blue (PB) nanoparticles (NPs) were prepared by using mild synthetic conditions in a cellulose-based hydrogel network in the presence of glucose oxidase (GOx), resulting in the simultaneous immobilization of PB NPs and active GOx in the hydrogel. This integrated system not only displays peroxidase-like activity relative to the PB NPs but also reveals an enhanced cascade catalytic performance for the colorimetric detection of glucose due to the proximity effect of the enzyme–nanozyme system within the hydrogel matrix. Compared to the analogue mixture with GOx in solution, the composite hydrogel shows enhanced glucose detection and improved stability. The developed colorimetric assay was successfully applied for the analysis of glucose in human serum samples, demonstrating its potential in clinical diagnosis. The versatility of this one-pot protocol holds promise for the development of different multienzyme systems, leading to efficient cascade catalysis for sensing applications.

Spatial confinement of multi-enzyme for cascade catalysis in cell-inspired all-aqueous multicompartmental microcapsules

Biocatalytic reaction networks in eukaryotic cells is realized by the immobilized and compartmental multi-enzymatic system. Inspired by the spatial localization of natural cells, multiple enzymes were confined within the multicompartmental microcapsules, which were created using a gas-shearing method coupled with surface-triggered in situ gelation strategy. Heterogeneous multicompartmental (two-, three-, four-, six-, or eight-faced) core particles, due to their capacity for positional assembly, were encapsuled in alginate hydrogel shells. The generated microcapsules integrate logic network to access complex digital design through a three-step convergent enzymatic cascade reaction as a model, and the capsules with high stability, recyclability and cytocompatibility are ideal enzymatic reactor systems to be used for biomimetic biocatalysis process.

Engineering substrate specificity of HAD phosphatases and multienzyme systems development for the thermodynamic-driven manufacturing sugars

Naturally, haloacid dehalogenase superfamily phosphatases have been evolved with broad substrate promiscuity; however, strong specificity to a particular substrate is required for developing thermodynamically driven routes for manufacturing sugars. How to alter the intrinsic substrate promiscuity of phosphatases and fit the “one enzyme-one substrate” model remains a challenge. Herein, we report the structure-guided engineering of a phosphatase, and successfully provide variants with tailor-made preference for three widespread phosphorylated sugars, namely, glucose 6-phosphate, fructose 6-phosphate, and mannose 6-phosphate, while simultaneously enhancement in catalytic efficiency. A 12000-fold switch from unfavorite substrate to dedicated one is generated. Molecular dynamics simulations reveal the origin of improved activity and substrate specificity. Furthermore, we develop four coordinated multienzyme systems and accomplish the conversion of inexpensive sucrose and starch to fructose and mannose in excellent yield of 94–96%. This innovative sugar-biosynthesis strategy overcomes the reaction equilibrium of isomerization and provides the promise of high-yield manufacturing of other monosaccharides and polyols.

Multienzyme System in Amorphous Metal-Organic Frameworks for Intracellular Lactate Detection.

Lactate is an important downstream product of glycolysis in living cells, and its level is highly related with diseases. On the basis of amorphous metal-organic frameworks (aMOFs), a multienzyme system consisting of lactate oxidase (LOx) and horseradish peroxidase (HRP) was established for intracellular lactate detection. By coencapsulation in aMOFs with proximity, LOx and HRP were delivered into cells, serving as artificially constructed organelles, exhibiting high activity and selectivity for the intracellular detection of the important metabolite lactate, which improved the signal to noise ratio by ∼650-fold. As demonstrated by both experimental and simulation results, the high efficiency was attributed to the short distance between the two types of enzymes coencapsulated in aMOFs. The concept of constructing multienzyme systems in this study shows promise for the detection of various intracellular metabolites.

Endogenous microenvironmental engineering through targeted alteration of salt bridge network can effectively regulate enzymatic pH adaptation

For a multi-enzyme cascade system, reconciling all enzymes under optimal catalytic conditions is key to improving pathway throughput. However, changing the enzymatic pH adaptation has long been a difficult challenge. To develop an effective and versatile method to alter enzymatic pH adaptation, we investigated members of the PfkB family. We found that one enzyme of this family has markedly different environmental pH requirements when phosphorylating dissimilar substrates, called pH promiscuity. Experiments proved that such promiscuity is a property of the enzyme itself rather than caused by the diversity of physicochemical properties of the substrate. Ancestral sequence reconstruction showed that the ancestral PfkB family enzymes also possessed pH promiscuity. We hypothesized that the pH of the enzyme might be an evolutionarily relevant feature and those regions directly affecting the pH could be found in the amino acid sequence. Sequences analysis revealed a region that was highly variable across genera but relatively conserved within the same genus. The results showed that direct changes in a few amino acids in this region could significantly alter the pH adaptation and optimal pH of the engineered enzymes without affecting the function. Structural analysis indicated that the optimal pH value and pH adaptation of the enzyme may be influenced by changes in the salt bridge network in the pH-affecting region. This leads to the proposal of engineering enzymatic pH adaptation by changing the salt bridge network around the active site. Finally, based on the same strategy, we successfully modified the pH adaptation of an enzyme not related to the PfkB function. Our work should provide an ingenious strategy for the engineering of enzymatic pH adaptation.

A Novel Acyl-AcpM-Binding Protein Confers Intrinsic Sensitivity to Fatty Acid Synthase Type II Inhibitors in Mycobacterium smegmatis.

The fatty acid synthase type II (FAS-II) multienzyme system is the main target of drugs to inhibit mycolic acid synthesis in mycobacterium. Meromycolate extension acyl carrier protein (AcpM) serves as the carrier of fatty acyl chain shuttling among the individual FAS-II components during the progression of fatty acid elongation. In this paper, MSMEG_5634 in Mycobacterium smegmatis was determined to be a helix-grip structure protein with a deep hydrophobic pocket, preferring to form a complex with acyl-AcpM containing a fatty acyl chain at the C36-52 length, which is the medium product of FAS-II. MSMEG_5634 interacted with FAS-II components and presented relative accumulation at the cellular pole. By forming the MSMEG_5634/acyl-AcpM complex, which is free from FAS-II, MSMEG_5634 could transport acyl-AcpM away from FAS-II. Deletion of the MSMEG_5634 gene in M. smegmatis resulted in a mutant with decreased sensitivity to isoniazid and triclosan, two inhibitors of the FAS-II system. The isoniazid and triclosan sensitivity of this mutant could be restored by the ectopic expression of MSMEG_5634 or Rv0910, the MSMEG_5634 homologous protein in Mycobacterium tuberculosis H37Rv. These results suggest that MSMEG_5634 and its homologous proteins, forming a novel acyl-AcpM-binding protein family in mycobacterium, confer intrinsic sensitivity to FAS-II inhibitors.

Controllably crosslinked dual enzymes enabled by genetic-encoded non-standard amino acid for efficiently enantioselective hydrogenation.

In traditional method for preparing crosslinked enzymes aggregates using glutaraldehyde, random linkage is inevitable, which often destroys the enzyme active sites and severely decreases the activity. To address this issue, using genetic encode expanding, nonstandard amino acids (NSAAs) were inserted into enzyme proteins at the preselected sites for crosslinking. When aldehyde ketone reductase (AKR), alcohol dehydrogenase (ADH) and glucose dehydrogenase (GDH) were utilized as model enzymes, their mutants containing p-azido-L-phenylalanine were bio-orthogonally crosslinked with diyne to form crosslinked dual enzymes (CLDEs) acting as a cascade biological oxidation and reduction system. Then, the resultant self-purified CLDEs were characterized using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM), etc. In the asymmetric synthesis of (S)-1-(2,6-dichloro-3-fluorophenyl) ethanol using CLDEs, high product yield (76.08%), ee value (99.99%) and reuse stability were achieved. The yield and ee value were 12.05 times and 1.39 times higher than those using traditional crosslinked enzyme aggregates, respectively. Thus, controllable insertion NSAAs in number and location can engender reasonable linkage and metal-free self-purification for target enzyme proteins. This facile and sustainable method could be further expanded to other dual and multienzyme systems for cascade biocatalysis.

A membraneless starch/O2 biofuel cell based on bacterial surface regulable displayed sequential enzymes of glucoamylase and glucose dehydrogenase

Enzymatic biofuel cells (EBFCs) provide a new strategy to enable direct biomass-to-electricity conversion, posing considerable demand on sequential enzymes. However, artificial blend of multi-enzyme systems often suffer biocatalytic inefficiency due to the rambling mixture of catalytic units. In an attempt to construct a high-performance starch/O2 EBFC, herein we prepared a starch-oxidizing bioanode based on displaying a sequential enzyme system of glucoamylase (GA) and glucose dehydrogenase (GDH) on E.coli cell surfaces in a precise way using cohesin-dockerin interactions. The enzyme stoichiometry was optimized, with GA&GDH (3:1)-E.coli exhibiting the highest catalytic reaction rate. The bioanode employed polymerized methylene blue (polyMB) to collect electrons from the oxidation of NADH into NAD+, which jointly oxidized starch together with co-displayed GA and GDH. The bioanode was oxygen-insensitive, which can be combined with a laccase based biocathode, resulting in a membranless starch/O2 EBFC in a non-compartmentalized configuration. The optimal EBFC exhibited an open-circuit voltage (OCV) of 0.74 V, a maximum power density of 30.1 ± 2.8 μW cm−2, and good operational stability.

Enzymes immobilized in wood-derived cellulose scaffold for constructing a novel modular bioreactor

Modular bioreactors can provide a flexible platform for constructing complex multi-step pathways, which may be a solution for maximizing reactions and overcoming the complexity of multi-enzyme systems. Here, we selected wood-derived cellulose scaffold as a support for enzyme immobilization and constructed the modular bioreactor. Cellulose scaffold was prepared after removing lignin from wood, followed by citric acid functionalization and the addition of glutaraldehyde finally allowed the cross-linking of enzymes. Three enzymes, horseradish peroxidase (HRP), glucose oxidase (GOD), and catalase (CAT), were separately immobilized, resulting in the immobilized enzyme amount to over 40 mg/g. The introduction of carboxyl groups from citric acid facilitated the rapid enzyme adsorption on the support surface and immobilized enzymes possess ∼65% expressed activity. Modular bioreactors were constructed by using the immobilized enzymes. With the immobilized HRP module, reactor showed desired catalytic performance with the phenol degradation rate of > 90%. Also, a pH regulation can occur in the bioreactors for preserving enzyme activities and neutralizing acid products. In the GOD/CAT modular bioreactor, the cascade reaction with adjusting pH values can achieve a 95% yield of sodium gluconate and exhibit a favorable reusability of 5 operation cycles.

A Nanopore Sensing Assay Resolves Cascade Reactions in a Multienzyme System

AbstractEnzymatic cascade reactions are widely used to synthesize complex molecules from simple precursors. The major underlying mechanism of cascade reactions is substrate channelling, where intermediates of different enzymatic steps are not in equilibrium with the bulk solution. Here, we report a nanopore sensing assay that allows accurate quantification of all the reaction intermediates and the product of an artificial three‐enzyme system. A DNA–peptide complex is used as the initial substrate which undergoes sequential enzymatic cleavages in solution. All the temporal changes of the intermediates and product can be obtained through nanopore translocation recordings. Furthermore, we find that in a confined environment such as liposome, substrate channelling occurs between two sets of the three enzymes. Our results demonstrate a novel and powerful approach to determine and quantify substrate channelling effects, which is potentially useful for designing and evaluating multienzyme systems.

A Nanopore Sensing Assay Resolves Cascade Reactions in a Multienzyme System.

Enzymatic cascade reactions are widely used to synthesize complex molecules from simple precursors. The major underlying mechanism of cascade reactions is substrate channelling, where intermediates of different enzymatic steps are not in equilibrium with the bulk solution. Here, we report a nanopore sensing assay that allows accurate quantification of all the reaction intermediates and the product of an artificial three-enzyme system. A DNA-peptide complex is used as the initial substrate which undergoes sequential enzymatic cleavages in solution. All the temporal changes of the intermediates and product can be obtained through nanopore translocation recordings. Furthermore, we find that in a confined environment such as liposome, substrate channelling occurs between two sets of the three enzymes. Our results demonstrate a novel and powerful approach to determine and quantify substrate channelling effects, which is potentially useful for designing and evaluating multienzyme systems.

On paper synthesis of multifunctional CeO2 nanoparticles@Fe-MOF composite as a multi-enzyme cascade platform for multiplex colorimetric detection of glucose, fructose, sucrose, and maltose

This study reports an economical and portable point-of-care (POC) monitoring device based on artificial multi-enzyme cascade systems for multiple detection purposes. The device was made up of a disposable three dimensional microfluidic paper-based analytical device (3D μPAD) with multiple detection zones and a smartphone readout. On-paper synthesis of a multifunctional mimetic composite, based on the CeO2 nanoparticles embedded in the amino-functionalized Fe metal-organic frameworks (CeO2@NH2-MIL-88B(Fe)), for cascade reactions was the main achievement of this work. The 3D μPAD was applied for simultaneous quantification of glucose, fructose, sucrose and maltose, and the detection process consisted of the enzymatic reaction of each sugar by anchored enzymes on the metal-organic frameworks (MOF) and successive oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB). Utilizing the new artificial mimicking system improved the color development uniformity and resulted in a reliable detection tool, with excellent detection limits in the range of 20–280 μM. It was directly applied to analyze the sugars levels of human total blood, urine, semen, honey and juice samples with the relative errors of less than 7.7% compared with the HPLC method. The cost-effective and easy-to-use μPAD has a great potential to be used in either medical diagnostics or the food industry. Also, it can be considered as a competitive POC method for patients in disadvantaged communities or emergencies.

Cry3Aa*SpyCatcher Fusion Crystals Produced in Bacteria as Scaffolds for Multienzyme Coimmobilization.

The production of Cry3Aa enzyme fusion crystals in Bacillus thuringiensis provides a direct method to immobilize individual enzymes and thereby improve their stability and recyclability. Nevertheless, many reactions require multiple enzymes to produce a desired product; thus a general strategy was developed to extend our Cry3Aa technology to multienzyme coimmobilization. Here, we report the direct production of particles comprising a modified Cry3Aa (Cry3Aa*) fused to SpyCatcher002 (Cry3Aa*SpyCat2) for coimmobilization of model enzymes MenF, MenD, and MenH associated with the biosynthesis of menaquinone. The resultant coimmobilized particles showed improved reaction rates compared to free enzymes presumably due to the higher local enzyme substrate concentrations and enhanced enzyme coupling made possible by colocalization. Furthermore, coimmobilization of these enzymes on Cry3Aa*SpyCat2 led to increased thermal stability and recyclability of the overall multienzyme system. These characteristics together with its overall simplicity of production highlight the benefits of Cry3Aa*SpyCat2 crystals as a platform for enzyme coimmobilization.

Application of Nucleic Acid Frameworks in the Construction of Nanostructures and Cascade Biocatalysts: Recent Progress and Perspective

Nucleic acids underlie the storage and retrieval of genetic information literally in all living organisms, and also provide us excellent materials for making artificial nanostructures and scaffolds for constructing multi-enzyme systems with outstanding performance in catalyzing various cascade reactions, due to their highly diverse and yet controllable structures, which are well determined by their sequences. The introduction of unnatural moieties into nucleic acids dramatically increased the diversity of sequences, structures, and properties of the nucleic acids, which undoubtedly expanded the toolbox for making nanomaterials and scaffolds of multi-enzyme systems. In this article, we first introduce the molecular structures and properties of nucleic acids and their unnatural derivatives. Then we summarized representative artificial nanomaterials made of nucleic acids, as well as their properties, functions, and application. We next review recent progress on constructing multi-enzyme systems with nucleic acid structures as scaffolds for cascade biocatalyst. Finally, we discuss the future direction of applying nucleic acid frameworks in the construction of nanomaterials and multi-enzyme molecular machines, with the potential contribution that unnatural nucleic acids may make to this field highlighted.

Compartmentalized Immobilization of Multi-enzyme Systems.

The methods of compartmentalized immobilization in multi-enzyme systems containing inorganic complexes and organic scaffolds (i.e. nucleic acid (RNA and DNA), protein and lipid) have been thoroughly investigated. Compartmentalization mostly focuses on dividing individual enzyme(s) into specific location or orientation of the enzymes cooperating in cascade reaction. Organic scaffolds are preferred because of their capability for simultaneous synthesis in biological systems. Besides, the most required methods of horseradish peroxidase (HRP) and glucose oxidase (GOD) enzymes including enzyme activity measurement, enzyme immobilization, removal, and re-hybridization, and enzyme attaching have been provided because they have been extensively applied in multi-enzyme systems. Organic scaffolds have a wide range and properties. Therefore, two methods including dockerin-cohesin linker and nucleotides interaction have been demonstrated for immobilization of enzyme on protein and DNA scaffold, respectively.

A Four-enzyme Nanoassembly Consisting of Hydrolases and Oxidoreductases for Multi-step Cascade Reactions.

Cascade reactions catalyzed by multi-enzymatic systems have attracted enormous scientific interest over the last decade. They are an emerging technology that significantly expands the applicability of biocatalysts in several biotechnological processes, such as the synthesis of high value-added products. Immobilization of enzymes on a solid carrier is a commonly used strategy to improve the stability and reuse of multiple enzyme systems. Magnetic nanoparticles have been applied as promising nanocarriers for either the immobilization of one enzyme or the co-immobilization of multiple enzymes. In this chapter, we describe the preparation of magnetic iron oxide nanoparticles γ-Fe2O3 modified with 3-(aminopropyl)-triethoxysilane (APTES), for the simultaneous covalent co-immobilization of oxidoreductases and hydrolytic enzymes, such as cellulase, β-glucosidase (bgl), glucose oxidase (GOx), and horseradish peroxidase (HRP). Several spectroscopic techniques that are used to characterize the structure and the catalytic performance of such systems are also described.

DNA Nanoscaffolds for Multienzyme Systems Assembly.

Multienzyme reactions play an important role in cellular metabolic functions. The assembly of a metabolon is often observed, in which the position and the orientation of composite enzymes are optimized to facilitate the substrate transport. The recent progress of DNA nanotechnology is promising to organize the assembly of bimolecular complexes with precise controlled geometric patterns at nanoscale, such as enzyme cascades assembly, biomimetic substrate channeling, and compartmentalization. Here, we present detailed protocols of using DNA nanoscaffolds to assemble a multienzyme system with control over spatial interactions and arrangements of individual components. The protocols include the preparation and purification of DNA nanostructures, the bioconjugation of DNA with proteins and cofactors, the chromatography purification of DNA-conjugated biomolecules, the characterization of assemblies by routine gel electrophoresis and advanced AFM imaging, as well as the activity evaluation of multienzyme assemblies.