Multienzyme co-immobilization-based bioelectrode:Design of principles and bioelectrochemical applications

Enzyme cascade reactions play significant roles in bioelectrochemical processes because they permit more complex reactions. Co-immobilization of multienzyme on the electrode could help to facilitate substrate/intermediate transfer among different enzymes and electron transfer from enzyme active sites to the electrode with high stability and retrievability. Different co-immobilization strategies to construct multienzyme bioelectrodes have been widely reported, however, up to now, they have barely been reviewed. In this review, we focus on recent state-of-the-art techniques for constructing co-immobilized multienzyme electrodes including random and positional co-immobilization. Particular attention is given to strategies such as multienzyme complex and surface display. Cofactor co-immobilization on the electrode is also crucial for the enhancement of catalytic reaction and electron transfer, yet, few studies have been reported. The up-to-date advances in bioelectrochemical applications of multienzyme bioelectrodes are also presented. Finally, key challenges and future perspectives are discussed.

Toward modular construction of cell‐free multienzyme systems

The implementation of multiple enzymes for chemical production in a cell‐free scenario is an emerging field in biomanufacturing.It enables the redesign and reconstitution of new enzymatic routes for producing chemicals that may be hard to obtain from natural pathways.Although the construction of a cell‐free multienzyme system is highly flexible and adaptable,it is challenging to make all enzymatic reactions act in concert.Recently,modular construction has been conceptual‐ized as an effective way to harmonize diverse enzymatic reactions.In this review,we introduce the concept of a multienzyme module and exemplify representative modules found in Nature.We then categorize recent developments of synthetic multienzyme modules into main‐reaction modules and auxiliary modules according to their roles in reaction routes.We highlight four main‐reaction mod‐ules that can perform carbon metabolism,carbon assimilation,protein glycosylation and nonribo‐somal peptide synthesis,and exemplify auxiliary modules used for energy supply,protection and reinforcement for main reactions.The reactor‐level modularization of multienzyme catalysis is also discussed.

One-step synthesis of thermally stable artificial multienzyme cascade system for efficient enzymatic electrochemical detection

Recently,metal-organic framework(MOF)-based multienzyme systems integrating different functional natural enzymes and/or nanomaterial-basedartificial enzymes are attracting increasing attention due to their high catalytic efficiency and promising application in sensing.Simpleand controllable integration of enzymes or nanozymes within MOFs is crucial for achieving efficient cascade catalysis and high stability.Here,we report a facile electrochemical assisted biomimetic mineralization strategy to prepare an artificial multienzyme system for efficient electrochemicaldetection of biomolecules.By using the G0x@Cu-MOF/copper foam(G0x@Cu-MOF/CF)architecture as a proof of concept,efficientenzyme immobilization and cascade catalysis were achieved by in situ encapsulation of glucose oxidase(GOx)within MOFs layer grown onthree-dimensional(3D)porous conducting CF via a facile one-step electrochemical assisted biomimetic mineralization strategy.Due to thebio-electrocatalytic cascade reaction mechanism,this well-designed GOx@Cu-MOF modified electrode exhibited superior catalytic activityand thermal stability for glucose sensing.Notably,the activity of GOx@Cu-MOF/CF still remained at ca.80%after being incubated at 80℃.In sharp contrast,the activity of the unprotected electrode was reduced to the original 10%after the same treatment.The design strategypresented here may be useful in fabricating highly stable enzyme@MOF composites applied for efficient photothermal therapy and otherplatform under high temperature.

Multi-shell nanocomposites based multienzyme mimetics for efficient intracellular antioxidation

Oxidative stress is associated with many acute and chronic inflammatory diseases.Development of nanomaterial-based enzyme mimetics for reactive oxygen species(ROS)scavenging is challenging,but holds great promise for the treatment of inflammatory diseases.Herein,we report the highly ordered manganese dioxide encapsulated selenium-melanin(Se@Me@MnO_(2))nanozyme with high efficiency for intracellular antioxidation and anti-inflammation.The Se@Me@MnO_(2)nanozyme is sequentially fabricated through the radical polymerization and the in-situ oxidation-reduction.In vitro experimental results demonstrated that the Se@Me@MnO_(2) nanozyme exhibits multiple enzyme activities to scavenge ROS,including catalase(CAT),glutathione peroxidase(GPx)and superoxide dismutase(SOD).Mechanism researches illustrated that the Se core possesses GPx-like catalytic activity,the Me and the MnO_(2) possess both the SOD-like and the CAT-like activities.What’s more,due to the stable unpaired electrons existing in the nanozyme,the Se,Me and MnO_(2) provide synergistic and fast electron transfer effect to achieve the quickly scavenging of hydrogen peroxide,hydroxyl radical,and superoxide anion.Further in vivo experimental results showed that this biocompatible nanozyme exhibits cytoprotective effects by resisting ROS-mediated damage,thereby alleviating the inflammation.This multienzyme mimetics is believed to be an excellent ROS scavenger and have a good potential in clinical therapy for ROS-related diseases.

Preparation of high-quality resistant dextrin through pyrodextrin by a multienzyme complex

As a soluble food raw material with a low calorie content,resistant dextrin (RD) has broad application prospects in the food industry.Branching enzymes (BEs),as a key enzyme for RD preparation,can break the α-1,4 glycosidic bonds of donor chains and reconstruct the cleaved chains to acceptor chains through the α-1,6 glycosidic bonds.BEs with high transglucosidic activity toward amylopectin and short-chain substrates are urgently needed to increase the quality of RD.Herein,BE derived from Thermuobifida fusca (TfBE) was mined and characterized.The optimal temperature and pH of the TfBE were 40 ℃ and 6.5,respectively.A total of 1500 U/g substrate TfBE reacted with 20% (w/v) pyrodextrin for 12 h,the ratio of α-1,4 to α-1,6 glycosidic bonds was changed from 3.52:1 to 2.33:1,and the content of enzyme-resistant components notably increased from 44.0% to 53.8%.Furthermore,to make full use of receptor chains and small molecular sugars in the reaction system,a multienzyme complex of TfBE with T.fusca α-cyclodextrin glucosyltransferase (TfCGTase),TfBE with TfCGTase and Aspergillus nidulans α-glucosidase (AnGS) was used to further increase the enzyme resistance of RD from 44.0% to 65.3% and 70.6%,respectively.The developed multienzyme complex method could effectively contribute to improving the production quality and efficiency of RD preparation.

A unique insertion of low complexity amino acid sequence underlies protein-protein interaction in human malaria parasite orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase

Objective:To investigate the multienzyine complex formation of human malaria parasite Plasmodium falciparum[P.falciparum)orotate phosphoribosyltransferase(OPRT)and orotidine5'-monophosphate decarboxylase(OMPDC),the fifth and sixth enzyme of the de novo pyrimidine biosynthetic palhway.Previously,we have clearly established that the two enzymes in the malaria parasite exist physically as a heterotetrameric(OPRT)_2(OMPDG)_2 complex containing two subunits each of OPRT and OMPDC.and that the complex have catalytic kinetic advantages over the monofunetional enzyme.Methods:Both enzymes were cloned and expressed as recombinant proteins.The protein-protein interaction in the enzyme complex was identified using bifunctionul chemical cross-linker,liquid chromatography-mass spectrometric analysis and homology modeling,Results:The unique insertions of low complexity region at the a 2 and a 5 helices of the parasite OMPDC,characterized by single amino acid repeat sequence which was not found in homologous proteins from other organisms,was located on the OPRT-OMPDC interface.The structural models for the protein-prolein interaction of the helerotetrameric(OPRT)_2(OMPDC)_2multienzyme complex were proposed.Conclusions:Based on the proteomic data and structural modeling,it is surmised that the human malaria parasite low complexity region is responsible for the OPRT-OMPDC interaction.The structural complex of the parasite enzymes,thus,represents an efficient functional kinetic advantage,which in line with co-localization principles of evolutional origin,and allosteric control in protein-protein-interactions.

Boosted activity by engineering the enzyme microenvironment in cascade reaction: A molecular understanding

Engineering of enzyme microenvironment can surprisingly boost the apparent activity.However,the underlying regulation mechanism is not well-studied at a molecular level so far.Here,we present a modulation of two model enzymes of cytochrome c(Cty C)and D-amino acid oxidase(DAAO)with opposite pH-activity profiles using ionic polymers.The operational pH of poly(acrylic acid)modified Cyt C and polyallylamine modified DAAO was extended to 3-7 and 2-10 where the enzyme activity was larger than that at their optimum pH of 4.5 and 8.5 by 106%and 28%,respectively.The cascade reaction catalyzed by two modified enzymes reveals a 1.37-fold enhancement in catalytic efficiency compared with their native counterparts.The enzyme activity boosting is understood by performing the UV-vis/CD spectroscopy and molecular dynamics simulations in the atomistic level.The increased activity is ascribed to the favorable microenvironment in support of preserving enzyme native structures nearby cofactor under external perturbations.

Construction of self-assembled nanogel as mulitenzyme mimics for bioresponsive tandem-catalysis imaging

The self-assembled phospholipid-or cytosolassociated multienzyme complexes constitute necessary components of the foundation of life.As a proof of concept,metalcoordinated supramolecular nanogels (MCSGs) have been designed,with the self-assembly of di-lysine coordinated iron(Fe(Lys)_(2))-functionalized peptide gelators on the interface by an in situ amidation-induced protonation process.The monoatomic and highly dispersed active centers of Fe(Lys)_(2) offered the nanogel mimics with excellent reaction rates due to the high density and nano compartmental structure similar to the natural matrix-associated multienzyme complex.SiO_(2)@MCSGs show both superoxide dismutase (SOD) activity and peroxidase (POD) activity,and the higher activities compared with the activity of free Fe(Lys);molecules can be detected.After loading the substrate 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate)(ABTS),SiO_(2)@MCSGs;can responsively convert O^(-)_(2) in the tumor microenvironment into H_(2)O_(2) intermediates and then tandem catalyze the oxidization of ABTS for contrast photoacoustic (PA) imaging of tumor by the SOD-POD mimic activity,showing their great potential as the efficient enzymatic agents for pathological theranostics.