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/intermed...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.展开更多
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 chemica...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.展开更多
D-Allulose is a rare sugar with multiple physiological functions whose application has been restricted by low conversion rate,weak thermostability of the used enzymes,and complex production.In this study,a simplified ...D-Allulose is a rare sugar with multiple physiological functions whose application has been restricted by low conversion rate,weak thermostability of the used enzymes,and complex production.In this study,a simplified multienzyme cascade strategy was designed for biosynthesis of D-allulose from D-glucose,and a detailed investigation of the crucial enzyme allulose 6-phosphate phosphatase was conducted.Under optimized conditions,the yield of D-allulose reached 4.12±0.14 and 80.20±3.20 mmol/L using 10.0 and 200.0 mmol/L D-glucose as substrate,respectively.Furthermore,the influence of various factors on the cooling crystallization of D-allulose was investigated,and the crystallization process was optimized.Consequently,a crystallization recovery rate of 77.43%±2.50% and a purity of 98.70%±0.05% were achieved.Moreover,the obtained crystals of D-allulose were characterized by XRD,TGA-DSC,and optical microscopy.展开更多
Allitol is a valuable drug intermediate and a potential low-calorie sweetener in food industry.However,developing an efficient biotransformation system for large-scale production of allitol remains challenging.Here,a ...Allitol is a valuable drug intermediate and a potential low-calorie sweetener in food industry.However,developing an efficient biotransformation system for large-scale production of allitol remains challenging.Here,a customized multienzyme cascade immobilization system was designed for efficient biocatalytic synthesis of allitol from D-fructose.The system included three immobilized enzymes:amino resin(LX1000)-immobilized Dallulose 3-epimerase(SfDAE)from Sinorhizobium fredii,LX1000-immobilized ribitol dehydrogenase(KpRDH)from Klebsiella pneumoniae,and derived formate dehydrogenase(CbFDH)from Candida boidinii was directional immobilization on bacterial cellulose(BC)employ SpyCatcher/SpyTag modular.By employing a strategy of directional immobilization and SpyCatcher-functionalized BC,the poor cascade cooperativity in the rate-limiting enzyme(CbFDH)was successfully resolved.Under the optimum biotransformation conditions,this system was able to convert 500 g/L of D-fructose into 455 g/L of allitol with conversion yields as high as 90%.Importantly,they also had excellent stability,retaining 60%relative activity after 10 repeated cycles,and preserving 50.2%relative activity after 24 days of storage.Consequently,the design and application of this multienzyme cascade immobilization system successfully provides an effective strategy for the large-scale biosynthesis of allitol.展开更多
D-allulose,a low-calorie rare sugar,can be biosynthesized by D-allulose 3-epimerase(DAE).Here,DAE was covalently immobilized on polyethyleneimine-modified diatomite using two methods:a biopolymer coating approach(DAE@...D-allulose,a low-calorie rare sugar,can be biosynthesized by D-allulose 3-epimerase(DAE).Here,DAE was covalently immobilized on polyethyleneimine-modified diatomite using two methods:a biopolymer coating approach(DAE@PEI-GA@PDP)and a direct attachment method(DAE-GA@PEI@PDP).Both immobilized DAE enzymes exhibited enhanced thermal stability and broader pH tolerance compared to the free DAE.They also exhibited excellent reusability and maintained more than 70%of their activity after 10 cycles.Additionally,a multienzyme cascade system was developed to produce D-allulose from low-cost D-glucose using immobilized glucose isomerase(GI)and immobilized DAE enzymes.This system obtained 76 g/L D-allulose from 500 g/L Dglucose.Notably,the application was extended to fruit tea drinks,successfully transforming high-calorie sugars(D-glucose and D-fructose)into D-allulose.This research offers a promising immobilization strategy for DAE on silica-based materials and provides robust biocatalysts for creating functional fruit tea drinks aimed at diabetics and individuals with specific dietary needs.展开更多
Diabetic wound repair is a global challenge due to bacterial infection and the typical microenvironments of hyperglycemia,high pH value,hypoxia,persistent inflammation and insufficient angiogenesis.Herein,a glucoseact...Diabetic wound repair is a global challenge due to bacterial infection and the typical microenvironments of hyperglycemia,high pH value,hypoxia,persistent inflammation and insufficient angiogenesis.Herein,a glucoseactivated nitric oxide(NO)releasing and microenvironment regulation hydrogel dressing(COH-GB)has been developed for efficiently promoting infectious diabetic wound healing.Specifically,an artificial multienzyme nanoflower(GB nanoflower)composed of glucose oxidase(GOx)and hemoglobin(Hb)was synthesized and incorporated into the hydrogel formed by carboxymethyl chitosan(CMCS),oxidized sodium alginate(OSA)and hydroxyurea(HU).In which,the hyperglycemia-triggered cascade reaction could consume glucose,while providing hydrogen peroxide(H_(2)O_(2))for the generation of NO by HU in the presence of Hb with peroxidasemimicking activity.The COH-GB hydrogel with essential wound dressing characteristics could remodel the microenvironment of diabetic wounds by reducing local glucose and pH levels,alleviating hypoxia,and scav-enging excessive ROS;and releasing NO under different blood glucose levels for antibacterial or angiogenesis.Importantly,the prepared COH-GB hydrogel significantly accelerated the MRSA-infected diabetic wound healing by effectively reducing wound infection,inhibiting inflammation,and promoting collagen deposition,angio-genesis and the migration and differentiation of fibroblasts and keratinocytes.Notably,the formation of GB nanoflowers could enhance the activity and stability of enzymes,and improve the cascade reaction efficiency,thereby inducing a more efficient performance in microenvironment regulation,antibacterial and angiogenesis,as well as wound healing.Hence,the prepared artificial multienzyme nanoflower composite hydrogel provides an efficient and secure dressing for promoting infectious diabetic wound healing via glucose-activated NO releasing and microenvironment regulation.展开更多
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 cataly...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.展开更多
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 ...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.展开更多
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 gly...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.展开更多
Hydrogenating carbon dioxide to formate using formate dehydrogenase (FDH) is a sustainable approach for CO_(2) mitigation. Herein, we developed a biocatalytic system with cofactor regeneration by immobilizing multiple...Hydrogenating carbon dioxide to formate using formate dehydrogenase (FDH) is a sustainable approach for CO_(2) mitigation. Herein, we developed a biocatalytic system with cofactor regeneration by immobilizing multiple enzymes, namely FDH, carbonic anhydrase (CA), and glutamate dehydrogenase (GDH), on a hydrophobic surface modified MOF, SA-HKUST-1. The adsorption kinetics of the multiple enzymes on the SA-HKUST-1 surface were described using pseudo second-order model, while the equilibrium followed Freundlich isotherm. Formate production by the enzymes immobilized on SA-HKUST-1 was 3.75 times higher than that achieved by free enzymes and 8.4 times higher than that of FDH immobilized alone on SA-HKUST-1. The hydrophobic interaction between the enzymes and the support altered the secondary structure of enzymes, and the immobilized enzymes retained 94% of their activity after four reuse cycles. This study provides novel insights into the combined effect of hydrophobic support and multiple enzymes on the catalytic efficiency and stability of FDH. These findings can provide a basis for developing a highly stable biocatalytic system with cofactor regeneration for continuous hydrogenation of CO_(2) to formate at the industrial level.展开更多
D-Allulose is one of the most common rare sugars with low calorie and is extensively applied in the food,cosmetic,and pharmaceutical fields.Here,a novel in vitro multienzyme cascade route(MCAS)was designed to produce ...D-Allulose is one of the most common rare sugars with low calorie and is extensively applied in the food,cosmetic,and pharmaceutical fields.Here,a novel in vitro multienzyme cascade route(MCAS)was designed to produce D-allulose from sucrose.The MCAS route comprised three modules(sucrose phosphorolysis,phosphorylation-dephosphorylation,and ATP regeneration),including five enzymes(sucrose phosphorylase,fructokinase,D-allulose 6-phosphate 3-epimerase,D-allulose 6-phosphatephosphatase,and polyphosphate kinase).As a rate-limiting enzyme,D-allulose 6-phosphate 3-epimerase from Pantoea sp.was semi-rationally engineered,and variant Y143F led to 88.8%yield of D-allulose from 10 mM sucrose using one-pot strategy.ATP consumption was reduced to 5%of the sucrose concentration after introducing ATP-regeneration system.At elevated sucrose concentration of 100 mM by two-step strategy,a D-allulose yield of 70.4%was achieved with a space-time yield of 1.06 g L^(-1)⋅h^(-1).This study provides an efficient and cost-effective approach for producing D-allulose from sucrose.展开更多
Objective:To investigate the multienzyine complex formation of human malaria parasite Plasmodium falciparum[P.falciparum)orotate phosphoribosyltransferase(OPRT)and orotidine5'-monophosphate decarboxylase(OMPDC),th...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.展开更多
Nicotiana tabacum is one of the representative high-value crops with distinctive aromatic flavors,holding sig-nificant economic importance and research value worldwide.Carotenoid cleavage products are known to be majo...Nicotiana tabacum is one of the representative high-value crops with distinctive aromatic flavors,holding sig-nificant economic importance and research value worldwide.Carotenoid cleavage products are known to be major contributors to plant flavor,but their expression patterns and underlying mechanisms in N.tabacum remain ambiguous.In this study,21 carotenoid cleavage dioxygenase(CCD)family members were identified in Nicotiana tabacum through genomic analysis and 3 novel members from the CCD1,CCD4,and CCD7 subfamilies were heterologously expressed.Functional assays revealed remarkable thermal tolerance,with NtCCD4 exhib-iting an optimal activity temperature of 50℃.All enzymes efficiently catalyzedβ-carotene cleavage,producingβ-ionone,β-cyclocitral,and other related compounds.Cleavage sites were inferred through targeted site muta-tions.By combining three enzymes in several complex preparations,distinct substrate conversion patterns emerged,with different enzymes dominating and altering the quantity and composition of aroma compounds.In post-roasted tobacco and tea leaves,treatment with a multienzyme system increased total aroma intensity(~2.5-5-fold),boosting sweet and roasted aromas in tobacco and grassy and floral notes in tea.This study re-ported the catalytic patterns of enzymes from three CCD subfamilies,and confirmed their positive contribution to enhancing plant aroma quality.They also paved the way for exogenous regulation of flavor synthesis.展开更多
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...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.展开更多
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 desig...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.展开更多
基金supported by the National Natural Science Foundation of China(21878324,21706273)the CAS Pioneer Hundred Talent Program(Type C,reference#2016-081)。
文摘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.
文摘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.
基金supported by the National Key Research and Development Program of China(2021YFE0101800)the S&T Support Program of Jiangsu Province(BE2022324)the College Students'Innovative Entrepreneurial Training Plan Program of Jiangnan University.
文摘D-Allulose is a rare sugar with multiple physiological functions whose application has been restricted by low conversion rate,weak thermostability of the used enzymes,and complex production.In this study,a simplified multienzyme cascade strategy was designed for biosynthesis of D-allulose from D-glucose,and a detailed investigation of the crucial enzyme allulose 6-phosphate phosphatase was conducted.Under optimized conditions,the yield of D-allulose reached 4.12±0.14 and 80.20±3.20 mmol/L using 10.0 and 200.0 mmol/L D-glucose as substrate,respectively.Furthermore,the influence of various factors on the cooling crystallization of D-allulose was investigated,and the crystallization process was optimized.Consequently,a crystallization recovery rate of 77.43%±2.50% and a purity of 98.70%±0.05% were achieved.Moreover,the obtained crystals of D-allulose were characterized by XRD,TGA-DSC,and optical microscopy.
基金supported by National Key Research and Develop-ment Program of China(2022YFC2104901)National Natural Science Foundation of China(32372279).
文摘Allitol is a valuable drug intermediate and a potential low-calorie sweetener in food industry.However,developing an efficient biotransformation system for large-scale production of allitol remains challenging.Here,a customized multienzyme cascade immobilization system was designed for efficient biocatalytic synthesis of allitol from D-fructose.The system included three immobilized enzymes:amino resin(LX1000)-immobilized Dallulose 3-epimerase(SfDAE)from Sinorhizobium fredii,LX1000-immobilized ribitol dehydrogenase(KpRDH)from Klebsiella pneumoniae,and derived formate dehydrogenase(CbFDH)from Candida boidinii was directional immobilization on bacterial cellulose(BC)employ SpyCatcher/SpyTag modular.By employing a strategy of directional immobilization and SpyCatcher-functionalized BC,the poor cascade cooperativity in the rate-limiting enzyme(CbFDH)was successfully resolved.Under the optimum biotransformation conditions,this system was able to convert 500 g/L of D-fructose into 455 g/L of allitol with conversion yields as high as 90%.Importantly,they also had excellent stability,retaining 60%relative activity after 10 repeated cycles,and preserving 50.2%relative activity after 24 days of storage.Consequently,the design and application of this multienzyme cascade immobilization system successfully provides an effective strategy for the large-scale biosynthesis of allitol.
基金supported by the National Key R&D Program of China[grant number 2022YFC2104904]Major Science and Technology Innovation Projects in Dezhou City,Shandong Province[grant number DZJBGS-2022-0010-2]the Shandong Province Key R&D Program[grant number 2022CXPT018].
文摘D-allulose,a low-calorie rare sugar,can be biosynthesized by D-allulose 3-epimerase(DAE).Here,DAE was covalently immobilized on polyethyleneimine-modified diatomite using two methods:a biopolymer coating approach(DAE@PEI-GA@PDP)and a direct attachment method(DAE-GA@PEI@PDP).Both immobilized DAE enzymes exhibited enhanced thermal stability and broader pH tolerance compared to the free DAE.They also exhibited excellent reusability and maintained more than 70%of their activity after 10 cycles.Additionally,a multienzyme cascade system was developed to produce D-allulose from low-cost D-glucose using immobilized glucose isomerase(GI)and immobilized DAE enzymes.This system obtained 76 g/L D-allulose from 500 g/L Dglucose.Notably,the application was extended to fruit tea drinks,successfully transforming high-calorie sugars(D-glucose and D-fructose)into D-allulose.This research offers a promising immobilization strategy for DAE on silica-based materials and provides robust biocatalysts for creating functional fruit tea drinks aimed at diabetics and individuals with specific dietary needs.
基金funded by the National Natural Science Foundation of China(82103893,82073606 and 82373630)。
文摘Diabetic wound repair is a global challenge due to bacterial infection and the typical microenvironments of hyperglycemia,high pH value,hypoxia,persistent inflammation and insufficient angiogenesis.Herein,a glucoseactivated nitric oxide(NO)releasing and microenvironment regulation hydrogel dressing(COH-GB)has been developed for efficiently promoting infectious diabetic wound healing.Specifically,an artificial multienzyme nanoflower(GB nanoflower)composed of glucose oxidase(GOx)and hemoglobin(Hb)was synthesized and incorporated into the hydrogel formed by carboxymethyl chitosan(CMCS),oxidized sodium alginate(OSA)and hydroxyurea(HU).In which,the hyperglycemia-triggered cascade reaction could consume glucose,while providing hydrogen peroxide(H_(2)O_(2))for the generation of NO by HU in the presence of Hb with peroxidasemimicking activity.The COH-GB hydrogel with essential wound dressing characteristics could remodel the microenvironment of diabetic wounds by reducing local glucose and pH levels,alleviating hypoxia,and scav-enging excessive ROS;and releasing NO under different blood glucose levels for antibacterial or angiogenesis.Importantly,the prepared COH-GB hydrogel significantly accelerated the MRSA-infected diabetic wound healing by effectively reducing wound infection,inhibiting inflammation,and promoting collagen deposition,angio-genesis and the migration and differentiation of fibroblasts and keratinocytes.Notably,the formation of GB nanoflowers could enhance the activity and stability of enzymes,and improve the cascade reaction efficiency,thereby inducing a more efficient performance in microenvironment regulation,antibacterial and angiogenesis,as well as wound healing.Hence,the prepared artificial multienzyme nanoflower composite hydrogel provides an efficient and secure dressing for promoting infectious diabetic wound healing via glucose-activated NO releasing and microenvironment regulation.
基金This work was supported by the National Key Research and Development Program of China(Nos.2017YFA0206500 and 2017YFA0206801)the National Basic Research Program of China(No.2015CB932301)the National Natural Science Foundation of China(Nos.21671163,21721001,and J1310024).
文摘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.
基金supported by the Innovation Zone Project(No.18-163-12-ZT-003-077-01)Health Major Project(Nos.BWS17J028 and AWS16J018)National Natural Science Foundation of China(Nos.81872835,21621003,and 21563010).
文摘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.
基金financial support from the National Natural Science Foundation of China(31972032)the National Natural Science Foundation of China(31730067)+1 种基金the Independent Innovation Project of Jiangsu Province(CX(21)3039)the Science Foundation of Jiangsu Province(BK20190586).
文摘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.
基金support from the College of Graduate Studies (fund number 31N438)UAEU and Zayed Center of Health Sciences (grant number 12R077) to carry out this study.
文摘Hydrogenating carbon dioxide to formate using formate dehydrogenase (FDH) is a sustainable approach for CO_(2) mitigation. Herein, we developed a biocatalytic system with cofactor regeneration by immobilizing multiple enzymes, namely FDH, carbonic anhydrase (CA), and glutamate dehydrogenase (GDH), on a hydrophobic surface modified MOF, SA-HKUST-1. The adsorption kinetics of the multiple enzymes on the SA-HKUST-1 surface were described using pseudo second-order model, while the equilibrium followed Freundlich isotherm. Formate production by the enzymes immobilized on SA-HKUST-1 was 3.75 times higher than that achieved by free enzymes and 8.4 times higher than that of FDH immobilized alone on SA-HKUST-1. The hydrophobic interaction between the enzymes and the support altered the secondary structure of enzymes, and the immobilized enzymes retained 94% of their activity after four reuse cycles. This study provides novel insights into the combined effect of hydrophobic support and multiple enzymes on the catalytic efficiency and stability of FDH. These findings can provide a basis for developing a highly stable biocatalytic system with cofactor regeneration for continuous hydrogenation of CO_(2) to formate at the industrial level.
基金supported by the National Key Research and Development Program of China(2021YFC2102700)the National Natural Science Foundation of China(31871738,22077054)+1 种基金the National First-Class Discipline Program of Light Industry Technology and Engineering(LITE2018-07)the Program of Introducing Talents of Discipline to Universities(111-2-06).
文摘D-Allulose is one of the most common rare sugars with low calorie and is extensively applied in the food,cosmetic,and pharmaceutical fields.Here,a novel in vitro multienzyme cascade route(MCAS)was designed to produce D-allulose from sucrose.The MCAS route comprised three modules(sucrose phosphorolysis,phosphorylation-dephosphorylation,and ATP regeneration),including five enzymes(sucrose phosphorylase,fructokinase,D-allulose 6-phosphate 3-epimerase,D-allulose 6-phosphatephosphatase,and polyphosphate kinase).As a rate-limiting enzyme,D-allulose 6-phosphate 3-epimerase from Pantoea sp.was semi-rationally engineered,and variant Y143F led to 88.8%yield of D-allulose from 10 mM sucrose using one-pot strategy.ATP consumption was reduced to 5%of the sucrose concentration after introducing ATP-regeneration system.At elevated sucrose concentration of 100 mM by two-step strategy,a D-allulose yield of 70.4%was achieved with a space-time yield of 1.06 g L^(-1)⋅h^(-1).This study provides an efficient and cost-effective approach for producing D-allulose from sucrose.
基金supported in part by Faculty of Graduate School(to W.L)Faculty of Medicine(contract no. RAH/54(1) to J.K.),Chulalongkorn University
文摘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.
基金supported by the Key Science and Technology Program of Hunan Provincial Tobacco Corporation(HN2023KJ04,HN2024KJ01,HN2025KJ01)support provided by the China Postdoctoral Science Foundation Program(2025T015HN,2025M782868).
文摘Nicotiana tabacum is one of the representative high-value crops with distinctive aromatic flavors,holding sig-nificant economic importance and research value worldwide.Carotenoid cleavage products are known to be major contributors to plant flavor,but their expression patterns and underlying mechanisms in N.tabacum remain ambiguous.In this study,21 carotenoid cleavage dioxygenase(CCD)family members were identified in Nicotiana tabacum through genomic analysis and 3 novel members from the CCD1,CCD4,and CCD7 subfamilies were heterologously expressed.Functional assays revealed remarkable thermal tolerance,with NtCCD4 exhib-iting an optimal activity temperature of 50℃.All enzymes efficiently catalyzedβ-carotene cleavage,producingβ-ionone,β-cyclocitral,and other related compounds.Cleavage sites were inferred through targeted site muta-tions.By combining three enzymes in several complex preparations,distinct substrate conversion patterns emerged,with different enzymes dominating and altering the quantity and composition of aroma compounds.In post-roasted tobacco and tea leaves,treatment with a multienzyme system increased total aroma intensity(~2.5-5-fold),boosting sweet and roasted aromas in tobacco and grassy and floral notes in tea.This study re-ported the catalytic patterns of enzymes from three CCD subfamilies,and confirmed their positive contribution to enhancing plant aroma quality.They also paved the way for exogenous regulation of flavor synthesis.
基金financial supports from the National Natural Science Foundation of China(31961133004,21977013,21903045)the National Key R&D Program of China(2018YFA0902200)+1 种基金China Post-doctoral Science Foundation(2019M661842)the Fundamental Research Funds for the Cornell University(PT1917,buctrc201,30920021122)。
文摘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.
基金supported by the National Natural Science Foundation of China(51773155 and 51873156)the National Key Research and Development Program(2016YFA0100800 and 2018YFC1803100)。
文摘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.
