Han Zhang,Yunpeng Bai,Ning Zhu,Jianhe Xu,*

1 State Key Laboratory of Bioreactor Engineering,East China University of Science and Technology,Shanghai 200237,China

2 College of Biotechnology and Pharmaceutical Engineering,Nanjing Tech University,Nanjing,211800,China

ABSTRACT 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.

Keywords:Microfluidic immobilized enzyme reactor Immobilization strategies Biocatalysis Bioconversion Bioanalysis

1.Introduction

The application of microfluidic technology in biocatalytic reactions has become a research hotspot in the field of biocatalysis and biotransformation.A variety of chemical reactions can be realized in microfluidic devices,including multi-enzyme catalysis,cascade reaction,rapid characterization and analysis of biotransformation processes,as well as the design and application of microfluidic biofuel cells [1–5].

Microflow is composed of the development of approaches and devices for the manipulation of bioprocess on a micron-or submicron scale [1,6,7].The decrease of operating scale promotes the investigation of particular microscale physics,providing crucial preponderances compared with normal-scale reactor system[8,9].Heat transfer and mass transfer can be radically enhanced on account of high specific surface area and short diffusion distance supplied by the microfluidic system,while the flow is laminar and highly ordered.In this way,fluid mixing becomes steerable and laminar flow regimes are adequately developed [3,7,8].

Enzymes are highly selective catalysts,with the major advantages of mild reaction conditions (atmospheric pressure and room temperatures),environmental friendliness and high catalytic efficiency.Currently,enzyme catalysis has greatly improved the efficiency of chemical reactions due to its unique catalytic properties and has become a green technology to synthesize fine chemicals [10–12].However,enzyme reactors (EMs) are limited by enzyme stability,underlining the necessity for enzymatic process engineering.

Microfluidic technology is one of the manner with great promise for such development.EMs can be scaled down as small as microfluidic devices or microcapillary devices,which can be fabricated on silicon wafers,glasses,polydimethylsiloxane(PDMS) and polymethylmethacrylate (PMMA) by photolithography,etching and mechanical processing methods for continuous-flow production[13–16].On account of miniaturization of EMs,the volumetric productivity is intensified.In the first place,it is caused by the high value of enzyme-to-substrate ratio which leads to a large local excess of biocatalyst molecules relative to substrate concentration and next on account of the large value of the surface-to-volume ratio [17,18].These factors are advantageous to reducing diffusion-related limitations by the reduction of the diffusion distance,which in turn strengthens the ability of the substrate molecules to reach the active sites of immobilized enzymes [1,4,6,19].For EMs,the substrate residence time in the microfluidic reactor can be readily adjusted by the flow rate.In addition,this method makes it possible to reduce the reagent consumption which is beneficial with respect to sample quantity needed and the concentration of the target product (bioanalysis and bioassay) [20,21].It makes the entire system more economical and hazardous substances also less risky (safer for some hazardous substances).That is why the microfluidic reactor can be adopted for the selection of mass transfer limiting reaction conditions and provides an effective way to evaluate the process economy before scale-up [22,23].

In this review,first,the main enzyme immobilization strategies will be briefly discussed,followed by summarizing the recent developments in the field of MIER for biocatalysis.It also discussed the special applications of microfluidic reactors to meet the needs of special reactions and to improve work efficiency.

2.Microfluidic Reactor with Immobilized Enzyme

2.1.A short overview

Miniaturization occupies a vital position in flow chemistry,and efficient MIERs prepared on this basis have become the main tools for developing novel and high-efficient continuous processes[4,7,24].Microtube(microcapillary)and chip devices are the main categories of the microfluidic reactor [25,26].The former directly takes advantage of the microchannel as the reaction space,which is commonly prepared using gas/liquid chromatography parts.In addition,microtube reactors can be easily scaled up by simply increasing the length of microtubules through the assembly of a bundle of microcapillaries [8,17].The chip reactor is composed of many microchannels connected in parallel,which usually have a high degree of integration and either an identity card or with external dimensions within several centimeters [27].The features enable the integration of multiple functions and processes in a single device,including sample treatment,mixing,reaction,parameter adjustment and product purification/detection [28,29].

Microfluidic reactor can be made from a variety of materials such as silicon,glass,polymers and biodegradable papers etc.It’s worth noting that the choice of materials largely depends on the operating conditions(pressure and temperature,etc.),physical properties of fluids(pH,viscosity),cost,and capability of manufacturing [30–32].Microchannels can be prepared via disparate means,mainly including photolithography,machining,wet etching,and so on,which could give rise to various kinds of topographical structures in the microchannel [33].Due to its unique microstructure,the microfluidic reactor has excellent performance in mixing efficiency and heat exchange efficiency,amplification process,integration and continuity,reaction time control,and safety,which are all the key factors to improve the yield,selectivity and quality of the target product.

Enzymes have different forms in microfluidic reactors.In some reports,free enzymes are used [34,35].For example,Gruberet al.[34]demonstrated the first full conversion of (2S,3R)-2-amino-1,3,4-butanetriol by the participation of free transketolase and transaminase in a capillary microfluidic reactor.Similarly,Tuseket al.[35]carried out enzymatic reaction of phenolic compounds,catechol andl-DOPA via free laccase fromTrametes versicolorin an aqueous two-phase chip-type microfluidic reactor.

Due to the problems of enzyme stability and the limitation of reaction cost and efficiency,so far only a few enzymatic reaction processes has been commercialized.Compared to the free enzymes,the enzymes immobilized by varieties of ways not only improves device is an effective tool for strengthening the catalytic process [2,3,10,13],Table 1 shows the advantages and disadvantages of some immobilization methods [2,3].The microchannel immobilized enzyme technology can realize the separation of free enzymes from products,increasing the reusability of enzymes,and stabilize the enzyme activity.Therefore,it has great application potential for biosynthesis of high value-added products [1,4].

2.2.Physical adsorption method

The physical adsorption method mainly immobilizes the enzyme on the surface of the carrier through intermolecular interaction,which has the advantages of mild conditions,conducive to retaining enzyme activity,and easy to update the reactor.While,the stability of the reactor is poor.

By the significant hydrophobic interactions and/or π-π stacking between the graphene oxide backbone and the aromatic residues of L-lactate dehydrogenase,the MIER was fabricated,which was used for analyzing the pyruvate content of beer samples in the market [36].

In addition to the enzyme combination onto the inner surface of the microfluidic reactor via hydrophobic interactions,the immobilization of biocatalyst can be done by electrostatic interactions too.This place the isoelectric point of enzyme should be well understood to supplied an appropriate charge under the condition of specific pH which can also be optimized to the activity demand.For instance,fused silica capillary was first treated by NaOH to increase the negative charges of the inner surface,and then acetylcholinesterase (AChE) was adsorpted to the surface of silica capillary [37].

The stability could be improved via the introduction of layerby-layer assembly technology and accessional protective coating of the enzyme layer.In the work of Heet al.[38],the inner surface of the negatively charged fused silica capillary was covered with a layer of positively charged polyethyleneimine (PEI).And then,the glucose oxidase(GOD)solution was introduced into the microcapillary.For the sake of improving the stability of the system,after immobilization,the additional protective layer was created (chitosan and PEI layer).The good adsorption and biocompatibility of the multilayer film can not only maintain the activity of the enzyme,but also provide a good environment for enzyme catalysis.

Table 1 Immobilization methods:advantages and disadvantages

2.3.Covalent immobilization

The approach adopted for immobilization by covalent bonds of the enzymes is concerned with the selection of the carriers including the following functional groups:carbonyl,carboxyl,succinimide or amidogen.While both the schiff and carbodiimide chemistries are the most fundamental covalent immobilization manners [39,40].

Furthermore,the azlactone groups are highly reactive with the amino and thiol functionalities,and hence the enzyme molecules can be easily contacted as firsthand reaction;it’s worth noting that,the functionalization of carriers with azlactone group has no requirement for any activation procedures[41].The epoxy groups,in turn,can be readily hydrolyzed to diol form under acidic conditions and then transformed into imidazole carbamate (through reaction with 1,1′-carbonyldiimidazole) or aldehyde groups (by oxidation),while it is also possible to immobilize the enzyme directly in one-step method.A manner for screening tyrosinase inhibitors from traditional Chinese medicines was successfully produced by the usage of glutaraldehyde as cross-linker at the outlet of the capillary [42].

The active influence on the basis of stability is continually counteracted by the decrease in enzyme activity.One reason is the reduced accessibility to the enzyme’s active site by the substrate on account of conformational restrictions via contacting the enzyme to the carrier [43,44].For the sake of overcoming this,the conservation of the natural environment for a certain enzyme has been verified to be of great significance[45].This can be cometrue by chemical modification of the surface,including by increasing the hydrophilicity of the surface.Functionalization with poly(ethylene glycol) (PEG) for instance indicates an improvement in enzymatic activity [46].In addition,the preparation of MIERs by modifying the inner wall of capillary tubes is already widely used(Table 2).

Although the microfluidic reactor prepared by the covalent method has good stability,it can not be regenerated or replaced once it is prepared,and is limited to the performance of the carrier,no matter the activity or the catalytic efficiency of the immobilized enzyme,which needs to be further improved[62,63].In view of the shortcomings,the researches mainly conducted researches from either the preparation methods or the functions of the carrier and the reactor.Microspheres and nanoparticles have obvious surface and interface effects and small size effects.As carriers,they can increase the amount of enzyme loading and increase its catalytic efficiency,and at the same time,it is easy to functionalize and assemble [64–66].

Systems including nano-or micrometer-sized magnetic particulates (barium ferrite,cobalt,nickel,iron nickel alloy and so on),appropriately functionalized,have been generally used as carriers to bind protein,enzyme,and drug [67–69].Magnetic particulate not only offers great specific surface areas,but also can be recycled easily and responsibly by the usage of external magnetic field.By this manner,the recycling process is rendered gentle to the immobilized enzymes,which avoids the shear forces giving rise to centrifugation.Longitudinal magnetic field was used to arrange the trypsin (Try) magnetic particles in the microchannel of the PDMS chip.The high-density microspheres in the channel augment the reaction concentration of the immobilized Try,and the high shear flow generated when the substrate penetrates shortens the diffusion distance in the channel,making the catalytic efficiency 100 times that of the free enzyme solution[70].Similarly,a fused silica capillary was used as reaction chamber in each case,loaded withsuperparamagnetic silica microparticles with immobilized human flavin-containing monooxygenase 3 [71].

Table 2 Application of coating in the preparation of immobilized enzyme microfluidic reactor by covalent method

2.3.1.DNA-directed immobilization technology

The DNA-directed immobilization technology (DDI) was based on the development of DNA microarrays,and it realized the development of DNA chips through the orderly arrangement of DNA on the carrier surface.The interaction between the enzyme and the carrier can easily lead to changes in the spatial structure of the enzyme,resulting in the loss of enzyme activity[72].DDI can make full use of the base complementary pairing(A-T,C-G)of DNA molecules to specifically immobilize biological macromolecules under mild physiological conditions.It is worth noting that the shortchain double helix DNA molecule has strong mechanical rigidity and physical and chemical stability [73,74].Therefore,an enzyme microarray can be formed,and the active site of the enzyme can be fully exposed,which is beneficial to reduce the mass transfer resistance,and the contact ability of enzyme with substrate can be improved by fixing the enzyme on the surface of the carrier through DDI.

A new kind of Try microfluidic reactor was prepared on the basis of DDI technique used for a fused silica capillary modified with PAMAM.Compared with routine modified microtubule,the usage of DDI technique enabled higher loads of Try,signally enhancing the enzymatic efficiency [75].

In addition,DDI technique is also widely used in multienzyme cascade microfluidic reactor.By designing DNA base sequence,Vonget al.[76].modified the capillary wall by using two mutually unpaired ssDNA,and then the complex of DNA-GOD and DNAHorseradish peroxidase (HRP) paired with ssDNA complementary were immobilized in the capillary through DDI,and the double enzyme MIER was prepared,which showed the prepared capillary double-enzyme MIER has good enzymatic activity and outstanding substrate affinity.Utilizing the advantages of DDI that can control the prepared enzyme array,by adjusting the ratio of two ssDNA,the ratio of GOD and HRP can be controlled in the capillary,and then the construction of a high-efficiency cascade reaction system can be realized.

2.3.2.Click chemistry

Click chemistry strategies which were proposed by KB Sharpless,the winner of the Nobel Prize in Chemistry,were also employed for constructing microfluidic reactor.

These unique advantages make click chemistry widely used in the fields of drug development and chemical biology.Both of alkyne-azide and thiol-ene are the most representative reactions in click chemistry [77,78].By in situ thermal copolymerisation of glycidyl methacrylate and ethylene dimethacrylate,Celebiet al.[79]prepared common carrier and the process of azide functionality was followed.The enzyme,α-chymotrypsin which had attached of alkyne functionality,was covalently attached onto the monolith via triazole ring formation by click-chemistry.

Compared with the way of alkyne-azide,the manner of thiolene-based is more suitable for the preparation of enzyme microfluidic reactor.The “thiol-ene”reaction is one of the click chemical reactions,in which a thiol radical produced by an addition of radical source to an alkene double bond.The“thiol-ene”click reaction can be performed quickly under mild conditions,be insensitive to water and air in the medium,and avoid protease denaturation.Hence,the enzyme immobilized by thiol-ene chemistry is simpler and more convenient than former approaches due to thiol groups of the enzyme[80–82].Fanet al.[83]prepared a novel MIER based on trimethylolpropane trimethacrylate(TRIM)organic monolith by“thiol-ene”click reaction.There were residual double bonds on the surface of TRIM monolith,in which click reaction can be performed with the free thiol groups of Try at room temperature to bind the Try on the monolithic column without additionally introducing an active functional group.Similarly,the microtubule-based and SiO2chip-based Try-MIER on the surface of poly(trimethylolpropane trimethacrylate) monolith were prepared in the work of Weiet al.[84].

2.3.3.Coordination collateral

The immobilization of biomacromolecule onto the carrier by covalent bonding is irreversible,which limits the regeneration of the prepared microfluidic reactor as the activity of the immobilized biomacromolecule is impaired.There are a great deal of differences between the metal-ion-chelated immobilization of enzyme and common methods.By this way,the enzyme is bound to the carrier via Lewis acid-base interaction with divalent cation chelators such as iminodiacetic acid [85],which is chemically bound onto the matrix.Therefore,the enzyme can be taken down via ethylenediaminetetraacetic acid elution for regeneration of the carrier.

A metal-ion chelate immobilized enzyme reactor supported on organic–inorganic hybrid silica monolith was prepared for speedy hydrolysis of protein.The chip-carrier wasin situprepared in a fused silica capillary by the polycondensation between tetraethoxysilane hydrolytic sol and iminodiacetic acid conjugated glycidoxypropyltrimethoxysilane.Followingtheactivation treatment by Cu2+,Try was bound onto the chip-carrier by metal chelation [86].Liet al.[87]utilized a substitutable and regeneratable MIER with metal-ion-chelated adsorption of Try on magnetic silica microsphere.The renewability of the made MIER and great reproducibility of MIER before and after regeneration were verified too.

2.4.Embedding method

The embedding method is to encapsulate or embed the enzyme in the pore size or three-dimensional network of the carrier through physical action,and the enzyme does not directly chemically react with the carrier.The embedding method has strong versatility and large enzyme adsorption capacity,which can take into account enzyme activity and reactor stability,and can also be doped with specific functional particles.It is a widely used reactor preparation method.The key to preparing a MIER by the embedding method is the carrier material,which is commonly a variety of porous or net-shaped carriers,such as silica gel sol–gel,porous particles,porous membranes,and so on [88–92].Silica gel sol–gel embedding is a commonly used carrier in the embedding method because of its mild conditions and easy maintenance of enzyme performance.Janget al.[93]used tetramethoxysiloxane as a precursor to mix with phosphoprotease,and prepared an integrated monolithic column by sol–gel reaction during injection.The method has mild conditions,and the activity and stability of the enzyme embedded in the gel matrix network are significantly improved.

Qiet al.[94]developed an enzyme array biosensor by using hydrogel as the embedding carrier and doping with functional quantum dots (QDs).They added 2-hydroxy-2-methyl-1-phenylacetone photoinitiator,CdSe/ZnS QDs and tyrosinase to polyethylene glycol diacrylate (PEG-DA) to form a gel precursor solution.Drop it on the glass substrate modified with 3-(trichlorosilane) propyl acrylate and cover it with a lightshielding film with a microarray structure.The exposed part will generate free radicals under ultraviolet light to initiate gelation,and finally a hydrogel embedded tyrosine kinase microarray was constructed on the surface of the substrate.The embedding method can effectively maintain the enzyme structure and biochemical function,and which has good versatility,can simultaneously embed different types of enzymes,and can be developed into a high-throughput biological enzyme sensor.

3.Specialty Applications

As mentioned above,MIER is provided with numerous preponderance in performing chemical reactions over traditional technologies.In order to improve the working efficiency of the microfluidic reactor and meet the needs of some special reactions,the following auxiliary methods are used,including microparticles,ionic liquid,ultrasound,microwave,etc.[95–98].In addition,some special forms of microfluidic reactors have also been developed and applied.

3.1.Passive mixing microfluidic reactor

As reagent flows in routine microchannel at low Reynolds numbers (Re),well-proportioned mixture turns into difficult.Hence,varieties of active or passive mixed approaches have been applied to produce turbulence to make the length of microchannel minimum requested for accomplishing homogenization.Passive mixture is put into practiceviathe microchannel with specifical morphology or preparing certain pattern or feature in microchannel [99–101].Usual methods utilize T,Y or ψ connections,hydrodynamic focusing,and chaotic advection,so as to obtain different flow and to generate turbulence or fluid folding to minimize the channel length required for attaining homogeneity patterns in a microchannel [1,4,6,9](Fig.1).

By a fluorescence approach,Ringborget al.[102]characterized mixture in microchannel with slanted grooves as a function of distance,and this was compared to the mixture behaviour got in microchannel which had no grooves.Possibly due to higher local velocities over the electrode surfaces in the chaotic mixers giving rise to thinner diffusion layer at this surface and ameliorative reagent transmission and enhanced currents.In comparison with unstructured microchannel,higher oxidation or reduction current was found for one with slanted or herringbone grooves.They had also applied the microfluidic reactor for carrying out protein PEGylation (Fig.2).The staggered herringbone grooves embedded within the microchannels leaded to chaotic advection within the Stokes regime (i.e.,Re<1).It gave rise to rapid and steerable mixture which was fit for performing protein PEGylation.

A series of groove-typed channel microfluidic reactor with surface geometry patterns were first established for effective immobilization of naringinase,which was used to produce isoquercitrin from rutin via hydrolysis.Efficient transfer of heat and mass were attained in the grooved MIERs with surface geometry patterns[103].

3.2.Pressurized microfluidic reactor

Fig.2.PDMS-based microfluidic reactor with silicon tubing inlet and outlet ports.

Prowess to combine reactivity with selectivity in O2dependent reactions carried out under mild reaction conditions makes enzymes interesting candidates for use as oxidation catalysts in process chemistry applications.While enzymatic catalysis takesplace in water system and affording O2to such system is up against lots of proverbial restrictions [104–106].As a matter of fact,the dominating factors of enzymatic reaction efficiency (product concentration,space–time yield,catalyst turnover) all rest with,and are often gravely restricted by,how effectually O2is became gainable within the liquid phase.Furthermore,it is foremost that the microfluidic reactor design and the preparation of the enzyme used(e.g.,immobilized enzyme,whole cell) both are brought in good accordance with the demands of O2supply to the continuous bioreaction envisaged.The oxygen transfer coefficient (kLa) value of up to 30 min-1was reported for segmented gas–liquid flow in microchannel [105].A falling-film microfluidic reactor performed in continuous countercurrent gas–liquid flow showed akLaof 450 min-1.Based on the above reasons,Bolivar et al.[105]showed that continuous flow microfluidic reactor technology provides a all-sided approach (Fig.3).It does so by expanding the process window to the medium pressure range (here:34 bar,1 bar=105Pa) and enables enzymatic reaction to be carried out in a simplex liquid phase at enhanced concentration of the dissolved O2(here:43 mmol·L-1).Reactions of GOD and D-amino acid oxidase were performed in such a system for the sake of demonstrating that pressor microfluidic reactor shows a vigorous engineering means inimitably apt to surmount limitations immanent to the certain O2-dependent reaction.The usage of soluble enzymes in liquid flow,the reaction rate improvement (up to 6-fold) caused by the influence of enhanced O2concentration on the oxidase kinetics was exhibited.

Fig.1.Basic structure unit of microfluidic reactor systems together with microchannels and disparate inlet shapes.

Fig.3.The flowchart of the high pressure reactor performed with soluble enzymes is shown.The system comprised the reactor coil,a mass flow controller for gas delivery,two pumps controlling liquid inflow,two flow-through pressure sensors at the inlet and the outlet of the reactor unit,and a backpressure regulator.The reactor components were made of stainless steel.Observation windows made from Teflon tubes were included as indicated (1 bar=105 Pa).

In a similar way,a novel method was presented for the collection of such kinetic data using a pressurized tube-in-tube microfluidic system (Fig.4),operated in the low-dispersed flow regime to generate time-series data,with minimal material consumption[107].

3.3.Slug flow microfluidic reactor

Large interfacial surface areas can be got by heterogeneous intake,via bubbling,stirring,etc.which is especially significant in heterogeneous reactions.But soluble enzymes are usually unstable under these conditions,possibly owing to the mechanical stress leading to irreversible inactivation of the biocatalyst,slug flow reactor occupies a special position in solving such problem[108,109].

In the course of an oxidation reaction,dissolved O2is consumed rapidly and diffusion of O2into the reaction medium can easily become overall rate-limiting.The O2diffusion rate into the reaction medium directly correlates with the interfacial area between aqueous medium and the gas phase.High mass-transfer coefficients are generally the consequence of small vortices induced by the segmented flow regime.This flow pattern guarantees an enhanced contact between the two phases and provides a uniform gas concentration in the liquid segment [109].Based on this,alcohol oxidase-catalysed oxidation of alcohols to aldehydes has been greatly improved [110].

Immobilized enzymes have been widely applied in the synthesis of biodiesel,for example,by the (trans)esterification of waste cooking oils and ethanol [111].The immobilization of enzymes has the advantages of increased catalyst stability,ease of reuse,and lower downstream processing costs as no additional catalyst separation is needed.However,immobilized enzymes may be less active than free enzymes,and the immobilization procedure can be expensive and time-consuming.In contrast,by performing the free lipase-catalyzed (trans)esterification reactions in a biphasic aqueous-organic system with the enzyme in the aqueous phase and oil and biodiesel product in the organic phase(in the presence of a solvent),the lipase can be easily separated and reused.Moreover,such biphasic systems promote the enzyme performance of certain lipases by interfacial activation,where active sites are generated on the aqueous-organic interface by the induced lidopening of the enzyme [112].

In Hommes’s work,the enzymatic esterification of oleic acid and 1-butanol to butyl oleate was performed in an aqueousorganic system in capillary microfluidic reactor under slug flow.The freeRhizomucor mieheilipase in the aqueous phase was used as a catalyst andn-heptane as the organic solvent.A close to 100%yield of butyl oleate could be achieved in the microfluidic reactor made of polytetrafluoroethylene within 30 min residence time at 30 °C [113].

Fig.4.Experimental setup of the Tube-in-Tube Reactor.The three syringe pumps on the left deliver a liquid solution to the inner membrane tube.Two mass flow controllers are used to vary the gas composition,supplied to the outer tube.The gas is wetted and heated before entering the reactor,and was fed through an outer tube.A pressure regulator and a manometer were located at both ends of the two tubes to control the pressure,as well as to ensure an equal or higher pressure on the liquid side of the membrane (1 bar=105 Pa).

Fig.5.(A) Scheme of HbpA-catalyzed 2-hydroxybiphenyl to 3-phenylcatechol;(B) Schematic representation of a TiTR with an aqueous-organic two liquid segmented flow.

3.4.Microfluidic reactor based on two-parallel-plates

One solution,differs from the methods mentioned above,to the inadequate supply of O2for O2-dependent reaction is a bubble-free aeration system by membrane.Its feasibility has been verified for cyclohexane monooxygenase and laccase in a common batch equipment[114,115].A weakness of this method was the unsatisfactory membrane surface area to liquid volume ratio,which restricted the transformation of O2and thus the general reaction rate.An analogical technology has later been used for the preparation of a tube-in-tube reactor(TiTR),where the membrane surface area to volume ratio would be maximized.In comparison to slug flow microfluidic reactor,the gaseous substrate can be supplied with higher efficiency,furnishing an invariable gas concentration within the whole reaction process.

A report indicated the preparation of 3-phenylcatechol with the usage of a continuous segmented flow TiTR (Fig.5).2-Hydroxybiphenyl 3-monooxygenase(HbpA)was used as a catalyst for the hydroxylation reaction,which was dependent on the substrate 2-hydroxybiphenyl,NADH,and oxygen.The oxygen transfer rate by the membrane of the TiTR was determined to be 24 μmol·min-1·ml-1emphasizing the potential of the TiTR as hopeful technology for realizing gas-dependent enzymatic reactions [114].

Different from Tomaszewskiet al.,Zhouet al.[115]prepared a novel microfluidic aqueous two-phase system with immobilized enzyme under parallel-laminar flow condition was constructed to prepare a new high-quality mulberry red pigment for resource utilization.After optimization,the purity of cyanidin-3-O-glucoside reached the maximum of 88.78 % within 8.59 s,which was 68 %higher than the original.Zhanget al.[116]used diatomites to immobilize lipase in the polyethylene glycol phase of the aqueous two-phase system,showing the highest enzyme activity for purification.

4.Summary and Outlook

This review indicates the abreast of the times applications of microfluidic immobilized enzyme reactor.Instrumented microstructured reactor is promising tool for enzymatic reaction engineering at each scale of the biocatalytic process development,with the characteristics of high reaction yield and turnover efficiency.The major challenges that remain for microfluidic enzymatic processes are the integration of various components for different unit operations including reaction,analysis,extraction,separation,and concentration.Can’t be ignored is the difficulties for integrating cascaded microfluidic reactors under different reaction conditions.The different microfluidic reactors,especially those with enzymeinhibiting reactants/products,need careful and effective matches among various microfluidic reactor conditions,for instance by regulating the flow rate of the reactor or by assembling a variety of equipments,such as thermo-and pH-stat devices,to optimize the various microfluidic reactors to achieve the best reaction performance.Many superiorities have been verified with the usage of microfluidic reactor in enzymatic reactions,which strongly overcomes its own limitations in enzymatic production.

The application of microfluidic reactor in the field of enzymatic transformations has been gaining momentum to accelerate process development in an economically effective method.The productivities of enzymatic microfluidic reactors in the presented cases are significantly higher than the classical batch reactors that are applied currently in industry.But,the examples of such microdevices used in large bioproduction scales are still scarce,probably because the widespread implementation of these microdevices for enzyme screening and high-throughput reaction optimization will be highly dependent on the levels of automation and integration with analytics.As demonstrated by the developing trend in the fields related to its application,it can be expected that miniaturization within the enzyme-catalyzed transformations will become increasingly relevant and widespread.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.