Facilitation of cascade biocatalysis by artificial multi-enzyme complexes——A review

Multi-enzyme complexes are the results of natural evolution to facilitate cascade biocatalysis.Through enzyme colocalization within a complex,the transfer efficiency of reaction intermediates between adjacent cascade enzymes can be promoted,resulting in enhanced overall reaction efficiency.Inspired by nature,a variety of approaches have been developed for the assembly of artificial multi-enzyme complexes with different spatial organizations,aiming at improving the catalytic efficiency of enzyme cascade.A recent trend of this research area is the creation of enzyme complexes with a controllable spatial organization which helps with the mechanistic studies and bears the potential to further increase metabolic productivity.In this review,we summarize versatile strategies for the assembly of artificial multi-enzyme complexes,followed by an inspection of the mechanistic studies of artificial multi-enzyme complexes for their enhancement of catalytic efficiency.Furthermore,we provide some highlighted in vivo,ex vivo,and in vitro examples that demonstrate the ability of artificial multi-enzyme complexes for enhancing the overall production efficiency of value-added compounds.Recent research progress has revealed the great biotechnological potential of artificial multi-enzyme complexes as a powerful tool for biomanufacturing.

Tandem Biocatalysis by CotA-TJ102@UIO-66-NH2 and Novozym 435 for Highly Selective Transformation of HMF into FDCA

2,5-Furandicarboxylic acid (FDCA) is a potential biorenewable chemical for applications including plastics, polyamides, drugs, etc. The selective biosynthesis of FDCA from 5-hydroxymethylfurfural (HMF) by a speci c enzyme poses a great challenge. In this study, we reported an e cient strategy to produce FDCA from HMF by the tandem biocatalysis of laccase (CotA-TJ102@UIO-66-NH 2 ) and Novozym 435. For the rst step, a nanoparticle metal organic framework was synthesized as a carrier to immobilize CotA-TJ102@UIO-66-NH 2 , which was assigned for the production of 5-formyl-2-furancarboxylic acid (FFCA) and featured an enzyme loading of 255.54 mg/g, speci c activity of 135.90 U/mg, and solid loading ratio of 99.65%. Under optimal conditions, an ideal FFCA yield of 98.5% was achieved, and the CotA-TJ102@UIO-66-NH2 pre- sented a high recycling capacity after 10 cycles. For the second step, Novozym 435 was applied for the further conversion of FFCA into FDCA, presenting a high FDCA yield of 95.5% under the optimized conditions. Novozym 435 also exhibited a high recyclability after eight cycles. As a result, the tandem biocatalysis strategy provided a 94.2% FDCA yield from HMF, indicating its excellence as a method for FDCA production.

Efficient acetoin production from pyruvate by engineered Halomonas bluephagenesis whole-cell biocatalysis

Acetoin is an important platform chemical,which has a wide range of applications in many industries.Halomonas bluephagenesis,a chassis for next generation of industrial biotechnology,has advantages of fast growth and high tolerance to organic acid salts and alkaline environment.Here,α-acetolactate synthase andα-acetolactate decarboxylase from Bacillus subtilis 168 were co-expressed in H.bluephagenesis to produce acetoin from pyruvate.After reaction condition optimization and further increase ofα-acetolactate decarboxylase expression,acetoin production and yield were significantly enhanced to 223.4 mmol·L^(-1) and 0.491 mol·mol^(-1) from 125.4 mmol·L^(-1) and 0.333 mol·mol^(-1),respectively.Finally,the highest titer of 974.3 mmol·L^(-1)(85.84 g·L^(-1))of acetoin was accumulated from 2143.4 mmol·L^(-1)(188.6 g·L^(-1))of pyruvic acid within 8 h in fed-batch bioconversion under optimal reaction conditions.Moreover,the reusability of the cell catalysis was also tested,and the result illustrated that the whole-cell catalysis obtained 433.3,440.2,379.0,442.8 and 339.4 mmol·L^(-1)(38.2,38.8,33.4,39.0 and 29.9 g·L^(-1))acetoin in five repeated cycles under the same conditions.This work therefore provided an efficient H.bluephagenesis whole-cell catalysis with a broad development prospect in biosynthesis of acetoin.

Advances in engineered Bacillus subtilis biofilms and spores, and their applications in bioremediation, biocatalysis, and biomaterials

Bacillus subtilis is a commonly used commercial specie with broad applications in the fields of bioengineering and biotechnology.B.subtilis is capable of producing both biofilms and spores.Biofilms are matrix-encased multicellular communities that comprise various components including exopolysaccharides,proteins,extracellular DNA,and poly-γ-glutamic acid.These biofilms resist environmental conditions such as oxidative stress and hence have applications in bioremediation technologies.Furthermore,biofilms and spores can be engineered through biotechnological techniques for environmentally-friendly and safe production of bio-products such as enzymes.The ability to withstand with harsh conditions and producing spores makes Bacillus a suitable candidate for surface display technology.In recent years,the spores of such specie are widely used as it is generally regarded as safe to use.Advances in synthetic biology have enabled the reprogramming of biofilms to improve their functions and enhance the production of value-added products.Globally,there is increased interest in the production of engineered biosensors,biocatalysts,and biomaterials.The elastic modulus and gel properties of B.subtilis biofilms have been utilized to develop living materials.This review outlines the formation of B.subtilis biofilms and spores.Biotechnological engineering processes and their increasing application in bioremediation and biocatalysis,as well as the future directions of B.subtilis biofilm engineering,are discussed.Furthermore,the ability of B.subtilis biofilms and spores to fabricate functional living materials with self-regenerating,self-regulating and environmentally responsive characteristics has been summarized.This review aims to resume advances in biological engineering of B.subtilis biofilms and spores and their applications.

Cascade biocatalysis for production of enantiopure(S)-2-hydroxybutyric acid using recombinant Escherichia coli with a tunable multi-enzyme-coordinate expression system

Racemize 2-hydroxybutyric acid is usually synthesized by organic methods and needs additional deracemization to obtain optically pure enantiomers for industrial application.Here we present a cascade biocatalysis system in Escherichia coli BL21 which employed L-threonine deaminase(TD),NAD-dependent L-lactate dehydrogenase(LDH)and alcohol dehydrogenase(ADH)for producing optically pure(S)-2-hydroxybutyric acid((S)-2-HBA)from bulk chemical L-threonine.To solve the mismatch in the conversion rate and the consumption rate of intermediate 2-oxobutyric acid(2-OBA)formed in the multi-enzyme catalysis reaction,ribosome binding site regulation strategy was explored to control TD expression levels,achieving an eightfold alteration in the conversion rate of 2-OBA.With the optimized activity ratio of the three enzymes and using ADH for NADH regeneration,the recombinant strain ADH-r53 showed increased production of(S)-2-HBA with the highest titer of 129 g/L and molar yield of 93%within 24 h,which is approximately 1.65 times that of the highest yield reported so far.Moreover,(S)-2-HBA could easily be purified by distillation,making it have great potential for industrial application.Additionally,our results indicated that constructing a tunable multi-enzyme-coordinate expression system in single cell had great significance in biocatalysis of hydroxyl acids.

Multifunctional biocatalysis:An unusual imine reductase

A multifunctional biocatalyst EneIRED capable of catalyzing amine-activated conjugate alkene reduction and subsequent reductive amination was discovered.The enzyme realized the coupling ofα,β-unsaturated carbonyls with amines to efficiently synthesize a broad set of chiral amine diastereomers based on its unusual active site structure and catalytic mechanism.

Conjugation of Candida rugosa lipase with hydrophobic polymer improves esterification activity of vitamin E in nonaqueous solvent

We described a novel polymer-lipase conjugate for high-efficient esterification of vitamin E using vitamin E and succinic anhydride as the substrates in nonaqueous media.In this work,the monomer,N-isopropylacrylamide(NIPAM),was grafted onto Candida rugosa lipase(CRL)to synthesize poly(NIPAM)(pNIPAM)-CRL conjugate by atom transfer radical polymerization via the initiator coupled on the surface of CRL.The result showed that the catalytic efficiencies of pNIPAM-CRL conjugates(19.5-30.3 L·s^(-1)·mmol^(-1))were at least 7 times higher than that of free CRL(2.36 L·s^(-1)·mmol^(-1))in DMSO.It was attributed to a significant increase in Kcat of the conjugates in nonaqueous media.The synthesis catalyzed by pNIPAM-CRL co njugates was influenced by the length and density of the grafted polymer,water content,solvent polarity and molar ratio of the substrates.In the optimal synthesis,the reaction time was shortened at least 7 times,and yields of vitamin E succinate by pNIPAM-g-CRL and free CRL were obtained to be 75.4%and 6.6%at 55℃after the reaction for 1.5 h.The result argued that conjugation with pNIPAM induced conformational change of the lid on CRL based on hydrophobic interaction,thus providing a higher possibility of catalysis-favorable conformation on CRL in nonaqueous media.Moreover,pNIPAM conjugation improved the thermal stability of CRL greatly,and the stability improved further with an increase of chain length of pNIPAM.At the optimal reaction conditions(55℃and 1.5 h),pNIPAM-g-CRL also exhibited good reusability in the enzymatic synthesis of vitamin E succinate and kept~70%of its catalytic activity after ten consecutive cycles.The research demonstrated that pNIPAM-g-CRL was a more competitive biocatalyst in the enzymatic synthesis of vitamin E succinate and exhibited good application potential under harsh industrial conditions.

Creation of cytochrome P450 catalysis depending on a non-natural cofactor for fatty acid hydroxylation

Cytochrome P450 enzymes catalyze diverse oxidative transformations at the expense of reduced nicotinamide adenine dinucleotide phosphate(NADPH),however,their applications remain limited largely because NADPH is cost-prohibitive for biocatalysis at scale yet tightly regulated in host cells.A highly challenging task for P450 catalysis has been to develop an alternative and biocompatible electrondonating system.Here we engineered P450 BM3 to favor reduced nicotinamide cytosine dinucleotide(NCDH)and created non-natural cofactor-dependent P450 catalysis.Two outstanding mutants were identified with over 640-fold NCDH preference improvement and good catalytic efficiencies of over15,000 M^(-1)s^(-1)for the oxidation of the fatty acid probe 12-(para-nitrophenoxy)-dodecanoate.Molecular docking analysis indicated that these mutants bear a compacted cofactor entrance.Upon fusing with an NCD-dependent formate dehydrogenase,fused proteins functioned as NCDH-specific P450catalysts by using formate as the electron donor.Importantly,these mutants and fusions catalyzed NCDH-dependent hydroxylation of fatty acids with similar chain length preference to those by natural P450 BM3 in the presence of NADPH and also similar regioselectivity for subterminal hydroxylation of lauric acid.As P450 BM3 and its variants are catalytically powerful to take diverse substrates and convey different reaction paths,our results offer an exciting opportunity to devise advanced cell factories that convey oxidative biocatalysis with an orthogonal reducing power supply system.

Revealing the Intrinsic Peroxidase-Like Catalytic Mechanism of Heterogeneous Single-Atom Co-MoS2

The single-atom nanozyme is a new concept and has tremendous prospects to become a next-generation nanozyme.However,few studies have been carried out to elucidate the intrinsic mechanisms for both the single atoms and the supports in single-atom nanozymes.Herein,the heterogeneous single-atom Co-MoS2(SA Co-MoS2)is demonstrated to have excellent potential as a high-performance peroxidase mimic.Because of the well-defined structure of SA Co-MoS2,its peroxidase-like mechanism is extensively interpreted through experimental and theoretical studies.Due to the different adsorption energies of substrates on different parts of SA Co-MoS2 in the peroxidase-like reaction,SA Co favors electron transfer mechanisms,while MoS2 relies on Fenton-like reactions.The different catalytic pathways provide an intrinsic understanding of the remarkable performance of SA Co-MoS2.The present study not only develops a new kind of single-atom catalyst(SAC)as an elegant platform for understanding the enzyme-like activities of heterogeneous nanomaterials but also facilitates the novel application of SACs in biocatalysis.

An enzyme-loaded reactor using metal-organic framework-templated polydopamine microcapsule

Ultrathin polydopamine microcapsules with hierarchical structure and porosity were prepared for the immobilization of multienzymes using metal-organic framework(MOF) as the template.The multienzyme/MOF composite was first prepared using a "one-pot" co-precipitation approach via the coordination and self-assembly of zinc ions and 2-methylimidazole in the presence of enzymes.The obtained nanoparticles were then coated with polydopamine thin layer through the self-polymerization of dopamine under alkaline condition.The polydopamine microcapsules with an ultrathin shell thickness of ~48 nm were finally generated by removing the MOF template at acidic condition.Three enzymes were encapsulated in PDA microcapsules including carbonic anhydrase(CA),formate dehydrogenase(FateDH),and glutamate dehydrogenase(GDH).FateDH that catalyzed the main reaction of CO_(2) reduction to formic acid retained 94.7% activity of equivalent free FateDH.Compared with free multienzymes,the immobilized ones embedded in PDA microcapsules exhibited 4.5-times higher of formate production and high catalytic efficiency with a co-factor-based formate yield of 342%.

Zwitterionic polymer-coated porous poly(vinyl acetate–divinyl benzene)microsphere: A new support for enhanced performance of immobilized lipase

Enzyme immobilization has attracted great attention for improving the performance of enzymes in industrial applications.This work was designed to create a new support for Candida rugosa lipase(CRL)immobilization.A porous poly(vinyl acetate–divinyl benzene)microsphere coated by a zwitterionic polymer,poly(maleic anhydride-alt-1-octadecene)and N,N-dimethylethylenediamine derivative,was developed for CRL immobilization via hydrophobic binding.The catalytic activity,reaction kinetics,stabilities and reusability of the immobilized CRL were investigated.It demonstrated the success of the zwitterionic polymer coating and subsequent CRL immobilization on the porous microsphere.The immobilized lipase(p2-MS-CRL)reached27.6 mg·g^-1 dry carrier and displayed a specific activity 1.5 times higher than free CRL.The increase of Vmax and decrease of Kmwere also observed,indicating the improvement of catalytic activity and enzyme-substrate affinity of the immobilized lipase.Besides,p2-MS-CRL exhibited significantly enhanced thermal stability and pH tolerance.The improved performance was considered due to the interfacial activation regulated by the hydrophobic interaction and stabilization effect arisen by the zwitterionic polymer coating.This study has thus proved the advantages of the zwitterionic polymer-coated porous carrier for lipase immobilization and its potential for further development in various enzyme immobilizations.

Preparation of cross-linked enzyme aggregates of nitrile hydratase ES-NHT-118 from E.coli by macromolecular cross-linking agent

Cross-linked enzyme aggregates(CLEAs) of nitrile hydratase(NHase) ES-NHT-118 from Escherichia coli were prepared by using ammonium sulfate as precipitating agent followed by cross-linking with dextran polyaldehyde for the first time. In this process, egg white was added as protein feeder for facilitating the formation of CLEAs. The optimal conditions of the immobilization process were determined. Michaelis constants(Km) of free NHase and NHase CLEAs were also determined. The NHase CLEAs exhibited increased stability at varied pH and temperature conditions compared to its free counterpart. When exposed to high concentrations of acrylamide, NHase CLEAs also exhibited effective catalytic activity.

Culture of yeast cells immobilized by alginate-chitosan microcapsules in aqueous-organic solvent biphasic system

Immobilization biocatalysis is a potential technology to improve the activity and stability of biocatalysts in nonaqueous systems for efficient industrial production.Alginate-chitosan(AC)microcapsules were prepared as immobilization carriers by emulsifi cation-internal gelation and complexation reaction,and their contribution on facilitating the growth and metabolism of yeast cells were testifi ed successfully in culture medium-solvent biphasic systems.The cell growth in AC microcapsules is superior to that in alginate beads,and the cells in both immobilization carriers maintain much higher activity than free cells,which demonstrates AC microcapsules can confer yeast cells the ability to resist the adverse effect of solvent.Moreover,the performance of AC microcapsules in biphasic systems could be improved by adjusting the formation of outer polyelectrolyte complex(PEC)membrane to promote the cell growth and metabolic ability under the balance of resisting solvent toxicity and permitting substrate diffusion.Therefore,these findings are quite valuable for applying AC microcapsules as novel immobilization carriers to realize the biotransformation of value-added products in aqueous-solvent biphasic systems.

Characterization ofanovel deep-seamicrobial esterase EstC 10 and its use in the generation o f(R)-methyl 2-chloropropionate

A novel esterase Est C10 from B acillus sp. CX01 isolated from the deep sea of the Western Pacific Ocean and the functionalities of Est C10 was characterized. At present, the reports about the kinetic resolution of racemic methyl 2-chloropropionate were quite rare. So we developed deep-sea microbial esterase Est C10 as a novel biocatalyst in the kinetic resolution of racemic methyl 2-chloropropionate and generate( R)-methyl 2-chloropropionate with high enantiomeric excess(>99%) after the optimization of process parameters such as p H, temperature, organic co-solvents, surfactants, substrate concentration and reaction time. Notably, the optimal substrate concentration(80 mmol/L) of esterase Est C10 was higher than the kinetic resolution of another esterase, Est12-7(50 mmol/L). The novel microbial esterase Est C10 identified from the deep sea was a promising green biocatalyst in the generation of( R)-methyl 2-chloropropionate as well of many other valuable chiral chemicals in industry.

Microfluidic reactor with immobilized enzyme-from construction to applications:A review

Microfluidic,as the systems for using microchannel(micron-or sub-micron scale)to process or manipulate microflow,is being widely applied in enzyme biotechnology and biocatalysis.Microfluidic immobilized enzyme reactor(MIER)is a tool with great value for the study of catalytic property and optimal reaction parameter in a flourishing and highly producing manner.In view of its advantages in efficiency,economy,and addressable recognition especially,MIER occupies an important position in the investigation of life science,including molecular biology,bioanalysis and biosensing,biocatalysis etc.Immobilization of enzymes can generally improve their stability,and upon most occasions,the immobilized enzyme is endowed with recyclability.In this review,the enzyme immobilization techniques applied in MIER will be discussed,followed by summarizing the novel developments in the field of MIER for biocatalysis,bioconversion and bioanalysis.The preponderances and deficiencies of the current state-of-the-art preparation ways of MIER are peculiarly discussed.In addition,the prospects of its future study are outlined.

Single‑Atom Cobalt‑Based Electrochemical Biomimetic Uric Acid Sensor with Wide Linear Range and Ultralow Detection Limit

Uric acid(UA)detection is essential in diagnosis of arthritis,preeclampsia,renal disorder,and cardiovascular diseases,but it is very challenging to realize the required wide detection range and low detection limit.We present here a single-atom catalyst consisting of Co(Ⅱ)atoms coordinated by an average of 3.4 N atoms on an N-doped graphene matrix(A-Co-NG)to build an electrochemical biomimetic sensor for UA detection.The A-Co-NG sensor achieves a wide detection range over 0.4-41,950μM and an extremely low detection limit of 33.3±0.024 nM,which are much better than previously reported sensors based on various nanostructured materials.Besides,the A-Co-NG sensor also demonstrates its accurate serum diagnosis for UA for its practical application.Combination of experimental and theoretical calculation discovers that the catalytic process of the A-Co-NG toward UA starts from the oxidation of Co species to form a Co^3+-OH-UA*,followed by the generation of Co^3+-OH+^*UA_H,eventually leading to N-H bond dissociation for the formation of oxidized UA molecule and reduction of oxidized Co^3+to Co^2+for the regenerated A-Co-NG.This work provides a promising material to realize UA detection with wide detection range and low detection limit to meet the practical diagnosis requirements,and the proposed sensing mechanism sheds light on fundamental insights for guiding exploration of other biosensing processes.

Recent advance of chemoenzymatic catalysis for the synthesis of chemicals: Scope and challenge

Chemoenzymatic catalysis can give full play to the advantages of versatile reactivity of chemocatalysis and excellent chemo-,regio-,and stereoselectivities of biocatalysis.These chemoenzymatic methods can not only save resource,cost,and operating time but also reduce the number of reaction steps,and avoid separating unstable intermediates,leading to the generation of more products under greener circumstances and thereby playing an indispensable role in the fields of medicine,materials and fine chemicals.Although incompatible challenges between chemocatalyst and biocatalyst remain,strategies such as biphasic system,artificial metalloenzymes,immobilization or supramolecular host,and protein engineering have been designed to overcome these issues.In this review,chemoenzymatic catalysis according to different chemocatalysis types was classifiably described,and in particular,the classic dynamic kinetic resolutions(DKR)and cofactor regeneration were summarized.Finally,the bottlenecks and development of chemoenzymatic catalysis were summarized,and future development was prospected.

Enzymatic oxidation of aqueous pentachlorophenol

The influence of the nonionic surfactant Tween 80 on pentachlorophenol （PCP） oxidation catalyzed by horseradish peroxidase was studied. The surfactant was tested at concentrations below and above its critical micelle concentration （CMC）. Enhancement of PCP removal was observed at sub-CMCs. The presence of Tween 80 in the reaction mixture reduced enzyme inactivation which occurred through a combination of free radical attack and sorption by precipitated products. A simple first-order model was able to simulate time profiles for enzyme inactivation in the presence or absence of Tween 80. At supra-CMCs, the surfactant caused noticeable reductions in PCP removal, presumably through micelle partitioning of PCP which precluded the hydrophobic PCP molecule from interacting with the enzyme.

Tailoring polymeric composite gel beads-encapsulated microorganism for efficient degradation of phenolic compounds

Phenol and its derivatives are highly toxic pollutants in industrial wastewater for the ecological environments,so there is essential attention to develop effective means of removing these harmful substances from water.In this work,the microorganism was immobilized into polymeric composite gel beads prepared by the effective recombination of natural abundant chitosan(CS)and industrial polyvinyl alcohol(PVA)for treating phenolic compounds.The degradation rate of 99.5%can be achieved to treat 100 mg·L^(1)of phenol at 30℃using the fresh resultant immobilized microorganism,where only 21.1%degradation rate was obtained by the free microorganism under the identical conditions.The recycling experiments of repeated 90 times to treat 100 mg·L^(1)of phenol displayed that the degradation rate of phenol was stable to 99%with the appearance of beads unchanged significantly,indicating the immobilized microorganism possessed excellent operating stability.Moreover,while the phenol derivatives of 100 mg·L^(1)were treated catalytically including pmethylphenol,catechol,and oaminophenol for 24 h by the immobilized microorganism,the degradation rates were all above 95%.The immobilized microorganism into PVACS polymeric composite with excellent operating stability and degradation activity would provide a feasible solution for treating phenolic compounds in water in industrial applications.