microchannel enzyme reactors and their applications for processing

Microchannel enzyme reactors andtheir applications for processingMasaya Miyazaki and Hideaki Maeda

Nanotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST),

Tosu, Saga 841-0052, Japan

Review TRENDS in Biotechnology Vol.24 No.10

Microreaction technology is an interdisciplinary fieldcombining science and engineering. It has attractedthe attention of researchers from different fields forthe past few years, resulting in the development ofseveral microreactors. Enzymes are one of the catalystsused in microreactors: they are useful for substanceproduction in an environmentally friendly way and havehigh potential for analytical applications. However, fewenzymatic processes have been commercialized becauseof problems with stability and the cost and efficiency ofthe reactions. Thus, there have been demands for inno-vation in process engineering, particularly for enzymaticreactions, and microreaction devices can serve as effi-cient tools for the development of enzyme processes. Inthis review, we summarize the recent advances ofenzyme-immobilized microchannel reactors; fundamen-tal techniques for micro enzyme-reactor design andimportant applications of this multidisciplinary technol-ogy in chemical processing are also included in ourtopics.

IntroductionMicrochannel reaction systems are prepared by microfab-rication techniques (see Glossary) or the assembly andmodification of microcapillaries [1–3], and use reactionapparatus with small dimensions. Furthermore, thesesystems take advantage of micro- or nano-fluidics (seeGlossary) to enable the use of drastically reduced volumesof reactant solutions and they offer performance of highefficiency and repeatability. Therefore, micro-channelreaction systems are expected to be a new and promisingtechnology in the fields of chemistry, chemical engineeringand biotechnology [4–9]. They have several advantages forperforming chemical reactions compared with traditionaltechnologies; the key advantages are the rapid heatexchange and rapid mass transfer, which cannot beachieved by the conventional batch system. The streamsof solutions in a microfluidic systemmainly form a laminarflow, which differs from macro-scale systems with regardsto the strict control of reaction conditions and time. Inaddition, microchannel reaction systems provide large sur-face and interface areas, which are advantageous for manychemical processes such as extractions and catalytic reac-tions. Several chemical reaction devices demonstratepotential as applications [4–9]; moreover, many potential

Corresponding author: Maeda, H. ([email protected]).Available online 28 August 2006.

www.sciencedirect.com 0167-7799/$ – see front matter � 2006 Elsevier Ltd. All rights reserve

applications for miniaturized synthetic reactors requireonly small volumes of catalyst.

Enzymatic conversion has recently received attentionbecause of its environmentally friendly nature. Severalenzyme processes have been developed; however, improve-ment of the entire process is still required, to obtain thebenefit that can be derived from their use and for them tobe evaluated as common or standard technology [10,11].Reaction engineering might provide solutions to developan enzyme reaction process at the commercial level [12],and microreaction engineering is one example of suchtechnology. Micro enzyme-reactors have been constructedeither in the solution phase or by immobilizing enzymes.

Fundamental techniques for enzyme microreactorsContinuous-flow solution-phase reaction

Simplemicro enzyme-reactions have been performed usingsolution-phase methods. Continuous-flow microreactionwas performed on a chip-type microreactor fabricated ina PMMA (see Glossary) plate [13,14]. The reaction wasperformed by the simple loading of substrate and enzymesolutions into separate inlets using syringe pumps – suchreactions mainly rely on rapid mass transfer of the reac-tants. Trypsin-catalyzed hydrolysis of benzoyl-arginine-p-nitroanilide [13] and glycosidase-catalyzed hydrolysisreactions [14] were performed using this type of micro-reactor. In all cases, reaction yields improved betweenthreefold and fivefold in a microchannel system, whichdemonstrates the possibility of continuous-flow microreac-tion systems as a tool for further development of micro-reaction processes.

Stopped-flow reaction

In addition to continuous-flow microreactors, stopped-flowmode microreactors were also examined. Kitamori et al.reported stopped-flow microreactor devices using glassmicrochips with Y-shaped channel. The stopped-flow pro-cedure involves mobilization of reagents through thedevice for a designated period using an applied chemicaland/or physical field. The flow is subsequently paused nsecs duration) by the removal of the applied field before re-application of the field. Results from experiments using thestopped-flow mode reported an acceleration of a peroxi-dase-catalyzed reaction [15], attributed to an effectiveincrease in residence time within the device correspondingto the different kinetics associated with these reactions. Astopped-flow microreaction system with infrared (IR)heating was also developed [16]; this system enables

d. doi:10.1016/j.tibtech.2006.08.002

mailto:[email protected]

http://dx.doi.org/10.1016/j.tibtech.2006.08.002

Glossary

Microfluidics and nanofluidics: transporting and manipulating ml or nl amounts

of fluid through a microchannel.

Microfabrication: microstructures manufactured by methods used in the

microelectronics field such as micromachining, photolithography, molding

and embossing.

Monolith: porous material that can entrap molecules within its pore-like cage

and is usually used for the preparation of a porous catalyst.

PDMS: poly(dimethylsiloxane) (silicone).

PMMA: poly(methyl methacrylate) (acryl plastic).

PVDF: poly(vinyldenfluoride).

Hydrogel: a network of water-soluble polymer chains.

N,N-bis(carboxymethyl)glycine (NTA): binds nickel (II) ions and is usually

applied for identification of histidine tag of the engineered enzyme molecule.

His-tag: an amino acid motif in proteins that consists of at least six histidine

(His) residues, often introduced at the N- or C-terminus of the protein for

identification and affinity purification using Ni-NTA.

Parallel scale-out: production-scale plant constructed by parallel operation of

several reaction devices.

464 Review TRENDS in Biotechnology Vol.24 No.10

non-contact partial heating of the reaction solution. Aperoxidase-catalyzed reaction was performed in a cooledchip equipped with IR diode laser. The rate of the enzymereaction, which was initially inhibited owing to the coolingof the chip to lower the temperature, was increased by non-contact heating using the photothermal effect produced bya diode laser. Their findings suggest the possibility tocontrol nanoscale reactions and to synthesize, precisely,substances using photothermal stimulation.

Other solution-phase techniques

Amultiplex enzyme assay with several simultaneous enzy-matic reactions was performed in an electrophoretic micro-reaction device [17]: the resolving power of electrophoresisenables several enzyme assays to be analyzed at highspeed. Not only can the activities of individual enzymecatalysts be determined independently of other enzymesbut the effects of inhibitors can also be analyzed. Thisapproach enables high-throughput analysis on amicrochip.

A centrifugal microchip that uses a CD player-likeapparatus has also been described [18]. This microreactordoes not require the usual pump mechanism, rather cen-trifugal and capillary forces are used for pumping instead.This method was applied to an enzyme-linked immuno-sorbent assay (ELISA), with each step of the ELISA pro-cess carried out by controlling the rotation speed. Thismethod might be useful for the development of analyticalmicrobioreaction systems for multiple analyses of singlesamples.

Enzyme immobilization within microchannels

In the development of enzyme processes, the use ofimmobilized enzymes is preferable. Several methods areavailable to immobilize enzymes on supports in conven-tional reaction apparatus, and these techniques have alsobeen applied to immobilize enzymes within a microspace(Table 1).

In batchwise reactors, immobilization on beads ormono-liths (see Glossary) has been used for the separation andrecycling of enzymes. This approach has also been appliedto microreaction systems. Microreactors with enzymesimmobilized on glass beads have been prepared by fillingthe reaction chamber with the beads – such a device was

www.sciencedirect.com

used for the determination of xanthine usingchemiluminescent detection [19]. Crooks and co-workersdeveloped advanced analytical microreactors usingenzyme-immobilized-microbead mixing (Figure 1a) [20],and efficiently performed multistep enzyme reactionsusing glucose oxidase and horseradish peroxidase immo-bilized on polystyrene. Furthermore, the immobilization ofenzymes on nickel–nitrilotriacetic acid (Ni–NTA) agarosebeads has also been reported and was applied to immobi-lize bacterial P450 [21] – this immobilized enzyme is lessdenaturated because binding of the enzyme is achievedusing a His-tag (see Glossary). Magnetic beads were alsoused for enzyme immobilization within the microchannel:glucose oxidase was immobilized within a Teflon tubeusing a magnet [22] and was stable and active for morethan eight months.

Monolithic microreactors were prepared using severalmethods. A trypsin-immobilized microreactor was pre-pared in a microchannel by moulding a porous polymermonolith, prepared from 2-vinyl-4,4-dimethylazlactone,ethylene dimethacrylate and acrylamide or 2-hydroxyethylmethacrylate, with an enzyme, [23]: this microreactor wasused for mapping protein-digested fragments. Preparationof a microreactor by filling a silica monolith made fromtetraethoxysilane with an enzyme was also developed. Atrypsin-encapsulated monolith was fabricated in situ ontoa PMMA microchip to produce an integrated bioreactorthat can perform enzymatic digestion, electrophoreticseparation and detection in one chip [24]. Another exampleis a protease P-including monolith, which is prepared froma 1:4 mixture of tetramethoxysilane and methyltrimethox-ysilane and used to fill in a poly(ether ether ketone)(PEEK) microcapillary to produce a microreaction system[25]. Horseradish peroxidase was immobilized with3-aminopropylsilane using aluminium oxide as the solidsupport, and then placed within the microdevice [26]. Thismethod takes advantage of the porous nature of ceramicmicrostrut.

Overall, preparation of the immobilized enzyme withpowdered material or a monolith is significantly easier;however, it is unfavourable in large-scale processingbecause they are susceptible to increasing pressure.

Immobilization of enzymes on microchannel surface

Methods for the immobilization of enzymes on amicrochannel surface have also been developed becausethey can have the advantage of the larger surface area ofmicroreaction systems without the increase in pressure.Physical immobilization is an easy way to immobilizemolecules. In microchannel systems, a biotin–avidinsystem was most frequently used to immobilize enzymes:the biotinylated polylysine was physically immobilizedon a glass surface to capture streptavidin-conjugatedalkakine phosphatase [27]. This microreactor wasapplied for rapid determination of enzyme kinetics.Biotinylated lipid-bilayer [28] and partial biotinylationby photo patterning on fibrinogen [29] were also used forimmobilization; however, these methods are not suitablefor long-term use because of their instability. In addition,these applications are limited to streptavidin-conjugatedenzymes.

Table 1. Typical techniques for preparation of enzyme immobilized microchannel reactor

Technique Media Immobilization method Immobilized

enzyme

Advantage Disadvantage Ref.

Particle

entrapment

Glass Cross-linking

(3-aminopropylsilane–

glutaraldehyde)

�Xanthine

oxidase

�Ease in preparation �Limited number of

enzymes are

applicable due to

denaturation

[19]

�Horseradish

peroxidase

�Enables multistep

reaction�Pressure gaining

Polystylene Biotin–Avidin

(avidin-coated

beads were

used)

�Horseradish

peroxidase

�Ease in preparation �Biotin-label

is required

[20]

�Glucose

oxidase

�Enables multistep

reaction �Pressure gaining

Agarose Complex formation

(Ni-NTA

and His-tag)

�Bacterial P450 �Ease in preparation �Higher pressure by

increasing flow rate

and particles can be

crushed

[21]

�Applicable for

engineered enzymes

Magnetic beads Cross-linking


glutaraldehyde)

�Glucose

oxidase

�Preparation is easy �Amount of enzyme

particle is limited

because of plugging

[22]

�Enzyme can be

immobilized on any

place using a magnet

Polymer

monolith

Entrapment

(2-vinyl-4,4-

dimethylazlactone,

ethylene

dimethacrylate,

2-hydroxyethyl

methacrylate, acrylamide)

�Trypsin �Stabilization of

enzyme structure

and activity

�Requirement of

skill in preparation

[23]

�Denaturation during

entrapment process

Silica

monolith

Entrapment within porous

silica

�Trypsin �Stabilization of

enzyme structure and

activity

�Requirement of

skill in preparation

[24,25]

�Protease P

�Compatibility in

organic solvent

�Denaturation

possible during

entrapment process

Aluminium

oxide

Cross-linking


glutaraldehyde)

�Horseradish

peroxidase

�Large surface area

due to porous nature

�Complicated

preparation

[26]

�Applicable for

heterogeneous

reactions

�Not applicable for

large-scale processing

Surface

modification

SiO2 surface Physical adsorption of

biotinylated poly-lysine

/biotin-avidin

�Alkakine

phosphatase

�Ease in preparation �Requirement for

avidin conjugation

[27]

�Possible occurrence

of detachment

PDMS

(O2 plasma

treated)

Physical adsorption of

lipid

bilayer/biotin-avidin

�Alkakine

phosphatase

�Enable

immobilization of

enzyme on plastic

surface

�Possible occurrence

of detachment

[28]

�Expensive reagents

�Requirement for

avidin- conjugation

PDMS Physical adsorption of

fibrinogen/Photochemical

reaction of

fluorescein-biotin

�Alkaline

phosphatase

�Enable partial

modification of

microchannel

�Special equipment

is required

[29]

Slilicon Cross-linking

(3-aminopropylsilane/

glutaraldehyde)

�Trypsin �Simple operation �Difficulty in

channel preparation

[30]

�Poor

reproducibility

Fused silica

(Sol-gel

modified)

Cross-linking


succinate)

�Cucumisin �Simple operation �Need several steps [31–34]

�Lipase �Immobilize �10 times

more enzymes than

single-layer

immobilization and

therefore performs

with higher reaction efficiency

�Reproducibility

strongly affected by

characteristics of

silica surface

�L-Lactic

dehydrogenase

�Several chemistries

are available (amide,

disulfide, His-tag)

PMMA –

modified with

butyl

methacrylate

and/or g-

methylacryloxy

propyltrimethoxysilicane]

Cross-linking

(Si-O bond between

modified surface and

silica monolyth)

�Trypsin �Stabilize enzyme

under denaturation

condition

�Complicated

preparation method

[35]

Review TRENDS in Biotechnology Vol.24 No.10 465


Table 1 (Continued )

Technique Media Immobilization method Immobilized

enzyme

Advantage Disadvantage Ref.

PDMS (O2 Plasma

treated)

Cross-linking

(Si-O-Ti or Si-O-Al bond

between titania or alumina

monolyth)

�Trypsin �Stabilize enzyme

under denaturation

condition

�Complicated

preparation method

[36]

Fused silica Cross-linking between

physically- immobilized

Silica particle


succinate)

�Lipase �Much larger surface

area (1.5 times

greater than sol-gel

modified surface) and

higher efficiency

�Complicated

preparation method

[38]

�Unstable with

physical force

PDMS Entrapment within

hydrogel

formed on surface

�Alkaline

phosphatase

�Fast reaction (90%

conversion at 10 min

reaction)

�Complicated

preparation method

[39]

�Urease

�Immobilization of

multiple enzyme

�Not applicable for

higher flow rate

Membrane PDMS/Glass Place PVDF

membrane which

adsorb enzymes

�Trypsin �Easy preparation �Less efficient [40]

�Possibility of

leakage at higher

flow rate

Glass Covalent cross-linking

with Nylon

membrane formed

at liquid-liquid interface

(glutaraldehyde)

�Horseradish

peroxidase

�Integration of

membrane permeation

and enzyme reaction

�Complicated

preparation method

[41]

�Preparation of

multiple membrane

�Unstable membrane

at higher flow rate

PTFE Enzyme-embedded

membrane

formation using

glutaraldehyde/

paraformaldehyde

�a-Chimotrypsin �Easy preparation �Membrane formation

dependent on the

number of amino

groups of enzyme

molecule

[42]

�Trypsin �Durable (>40days)

�Applicable in

organic solvents


The introduction of a functional group on themicrochannel surface was used for covalent cross-linking.A trypsin-immobilized microreactor was prepared bymodification with 3-aminopropylsilane and glutaralde-hyde, using the classical method [29]. Although thisimmobilization method is easy, fabrication of a complexedmicrostructure is required to obtain high performance. Ourgroup developed a modified sol-gel technique to formnanostructures on a silica microchannel surface(Figure 1b) [30] that modifies the microchannel surfacewith a polymerized copolymer of 3-aminopropylsilane and/or methylsilane. Using this method, increased surface areawas obtained, and at least ten times more enzymes can beimmobilized on these nanostructures by covalentcross-linking through amide-bond formation, disulfide orHis-tag, or by using a modifying succinate spacer,compared with single-layer immobilization [31,32]. Amicroreactor with immobilized cucumisin on thenanostructured surface could process substrate 15 timesfaster than the batchwise reaction [31].

Similar surface-modification methods incorporating thesol-gel technique were also developed. A PMMA surfacewas modified with a copolymer of butyl methacrylate and/or g-(methylacryloxy)propyltrimethoxysilicane and silica–sol-gel to immobilize enzymes; using this method, atrypsin-immobilized microreactor was developed. Inaddition, a trypsin-encapsulated titania (titanium dioxide)and alumina gel matrix was immobilized through the SiOHgroup formed on a PDMS (see Glossary) surface by plasmaoxidation [31]. Using this device, digestion times weresignificantly shortened (�2 seconds) and the applicationfor high-throughput protein identification was realized.


A particle-arrangement technique was also applied forenzyme immobilization. Silica nanoparticles were immo-bilized onto the surface using slow evaporation of theparticle suspension in a filled-in microchannel(Figure 1c) [32]. The resulting microchannel was subjectedto treatment with 3-aminopropyltriethoxysilane, andimmobilization of enzyme was achieved by covalentcross-linking through an amino group. Although physicalstability needs to be improved, a lipase-immobilized micro-reactor prepared by this method showed 1.5 times fasterkinetics than that of a microreactor obtained by sol-gelsurface modification [33]. This result showed goodcorrelation with the surface area: particle arrangementhas �1.5 times larger surface area and could immobilizemore enzymes.

Polymer coating is another option for enzymeimmobilization. Poly(ethylene glycol)-based hydrogel(see Glossary), which incorporated alkaline phosphatase,was prepared within microchannels by exposure to UVlight (Figure 1d). This method was also applied to immo-bilize urease and other enzymes on a microchannelsurface [34].

Membrane-formation

Enzymes can be immobilized on a membrane using aporous poly(vinylidene fluoride) membrane embeddedwithin the microchannel, whereby a miniaturized mem-brane reactor was prepared by absorption of enzymes ontothe membrane [35].

Hisamoto et al. reported that a nylonmembrane could beformed at the interface of two solutions formed in a micro-channel (Figure 1e). Peroxidase was immobilized on this

Figure 1. Images of immobilization technique for micro enzyme-reactor. Enzyme can be easily immobilized by trapping enzyme-immobilized polystylene beads within a

microchannel (a). Modified surfaces are also useful for enzyme immobilization. Surfaces obtained by the sol-gel technique (b), nanoparticle arrangement (c), and hydrogel

formation. Also, membranes formed within the microchannel can be used as support for enzyme immobilization. A nylon membrane formed at liquid–liquid interface (e) or

the membrane of a cross-linking enzyme-aggregate formed at microchannel surface (f) was used for immobilization. These images were reproduced, with permission, from

[20,31,37,39,41] and [42], respectively.


membrane, which was used as a chemicofunctionalmembrane [36]; however, immobilization of the membraneis technically difficult, and application of this method islimited because the nylon-membrane is unstable in organicsolvents.

We have developed a technique that forms anenzyme-immobilising membrane on the microchannelsurface [37]; this is a modification of CLEA (cross-linkedenzyme aggregate) formation, which is used in batchwiseorganic synthesis [38]. Simple loading of the enzymesolution and a mixture of glutaraldehyde and paraformal-dehyde into the microchannel forms a CLEAmembrane onthe microchannel wall (Figure 1f). The resultingmicroreactor can be used for prolonged periods (>40 days)and shows excellent stability against organic solvents.Taking into account these advantages, this method isconsidered ideal for the development of an enzymaticreactor tailored for specific applications.

Advanced applications of micro enzyme-reactors forprocessingNumerous analytical micro enzyme-reactors have beendeveloped that take advantage of the reduction in reaction


time and the minimal amount of reagents used inmicrochannel systems [39,40]. However, there havebeen only a few reports of continuous-flow enzymaticmicroreaction processes (Table 2).

Although the solution-phase reaction is not afavourable process due to the large volume of enzymesrequired, several important achievements have beenreported. Enzymatic oligosaccaride synthesis wasperformed using b-galactosidase in a continuous-flowmicroreactor [14,41]. The reaction was performed bythe separate loading, into inlets, of the enzyme inphosphate buffer solution and the substrate solution inacetonitrile, and was terminated by heating therecovered solution: the reaction in the microchannelwas approximately five times faster than that in thebatch reaction.

A biphasic continuous-flow microreaction was alsodevised. Goto and co-workers performed a dehalogenationreaction in a chip-type glass microreactor using laccase[42] by separately loading an aqueous solution of theenzyme, and the substrate solution in organic solvent.They performed detailed kinetic analysis, and concludedthat the reaction kinetics in of a biphasic stream in a

Table 2. Enzymatic processing performed in micro enzyme-reactor

Reaction Technique Enzyme Result Ref.

OH

HO

HHO H

HNH

H

O

HO

NO

O CH

OOH

H

HHO H

HOH

H

O

HO

NO

OOH

H

HHO H

HOH

HHO

OH

OHHO

H

H

HN

H

OH

OH

O

CH

OHO

H

HHOH HOH

HO

HO O

HOH

H

OH

H

HHN

H

HO

O

CH

OOH

H

HHO H

HOH

H

O

HO

O

HHO

HH

HNH

H

OH

OH

O

H C

Solution-phase

continuous-flow

�b-Glucosidase �5 times better

yield was obtained than that

of batchwise reaction

[14,

41,46]

�Isomers were not isolated

Cl

OH

O

O

Biphasic

solution-phase

continuous flow

reaction

�Laccase �Degradation of

p-chlorophenol occurred

mainly at the aqueous–organic

interface in the

microchannel.

[42,47]

�Diffusion of the substrate

( p-chlorophenol) is the

rate-limiting step

�A simple theoretical model

for the degradation in the

microchannel was proposed

OHH C

O

O

OHH C OH

OH

FAD

NADH

NAD+

FADH2e-

Solution-phase

reaction using

electrochemical

microreactor

�Formate

dehydrogenase

�Regenerate coenzymes within

a single reactor

[43,48]

�Lactate

dehydrogenase

�Regeneration of NADH was as

high as 31%

OO

OCH

CHH C

CH

O

OH

CH

NCH

CH

OO

OCH

CHH C

CH

O

OH

CH

NCH

CH

HO

OO

OCH

CHH C

CH

O

OH

CH

NCH

CHHO

Ni-NTA agarose bead

immobilization

�PikC hydroxylase

(Bacterial P450)

�>90% conversion was

obtained at

70nm/min

[44]

H COH

O

O

H COH

OH

O

Surface

modification by

sol-gel

technique/Ni-NTA

immobilization

�L-Lactic

dehydrogenase

�Crude enzyme can be used for

immobilization

[32]

�Reversible immobilization

was achieved by EDTA treatment

�Reaction was completed

within 15 min

O OO

O

H C O OHO

Surface

modification of

silica capillary

by sol-gel technique/

immobilized by

amide bond formation

using succinate linker

�Lipase �1.5 times better yield was

obtained than with the

batchwise reaction

[34]

HO O OHOH OH

H C

O OH

H C

OO

O

OH

OH

OHEntrapment of

Novozym 435TM

within microchannel

�Novozym-435TM �Much less of the reactant was

required compared with the

batchwise test

[49]

OHO

O

O

CH

OO

O

H C

Silica monolith

entrapped within

microchannels (w 200 mm

or 1 mm � 10 cm length)

�Protease P �Conversion within

microreactor was higher

than that in batchwise

reaction at higher flow rates

[25]




microchannel depends on the diffusion of the substrate intothe aqueous phase.

A more complicated continuous-flow-type enzymemicroreaction system was also developed. Regenerationof a coenzyme is the most difficult point in an enzyme-catalyzed process; however, regeneration of NADH wasperformed in a Y-shaped microreactor containing an elec-trode within the microchannel [43]. Although the experi-ment is primitive, this result demonstrates that acontinuous-flow microreactor is one of the promisingdevices for the development of efficient enzyme reactionsystems.

Applications of enzyme-immobilized microreactors forprocessing were also presented, including hydroxylation ofmacrolides in a microreactor [21]: PikC hydroxylase wasimmobilized onto Ni-NTA agarose beads and then filledinto the microchannel. This microreactor was used forhydroxylation to produce methymycin and neomethylmy-cin, and >90% conversion was achieved at a flow rate of70 nl/min – such high efficiency might have resulted fromshorter residence time, which is preferable for enzymeswith inherent stability. This result opens the door for theapplication of micro bioreactors for enzymatic synthesis ofbioactive natural products.

Esterification hydrolysis reactions are an importantprocess in industry that have also been performed in amicrochannel system. Lipase-immobilized microreactorswere prepared using ceramicmicroreactor and glassmicro-capillaries [44], wherein hydrolysis of the ester was con-ducted. Both microreactors showed 1.5 times better yieldthan the batchwise reaction using the same volume andenzyme ratios. This could have resulted from an increase incontact due to the larger surface area of microchannelsystems. A microreaction using immobilized Novozym-435TM (http://www.novozymes.com) was also reported,where esterification of diglycerol with lauric acid wasperformed using a monolytic microreactor tethering pro-tease P for the bioconversion process [45]. Transesterifica-tion of (S)-(–)-glycidol and vinyl n-butyrate was alsoperformed using this microreaction device [24] but theconversion depended on the amount of immobilizedenzymes. So far, few enzymes have been applied to micro-reaction process development.

Concluding remarksMicrochannel devices can be useful in imitating biologicalreaction apparatus, such as cellular surface and vascularsystem, by providing the advantages of reduced space andlaminar flow compared with conventional reaction appa-ratus. The quest for microreaction technologies will lead tobetter process intensification and efficient analytical meth-ods. Increasingly, new findings are being achieved inmicrofluidics Further investigation could provide novelmechanisms not observed in conventional systems, andbetter understanding of the fluidics in microchannelsmight enable new reaction pathways not possible withconventional systems.

The strong advantages offered by microreaction devicesare useful, particularly in the development of microreac-tion systems for commercial purposes. Once a microreactoris optimized, it can be easily introduced into an industrial-


scale plant. Parallel scale-out (see Glossary) enablesextension of reaction conditions optimized in a singlereactor, and eliminates scale-up problems arising fromconventional processes. Parallel operation of the samemicroreaction provides high-throughput operation ofdifferent reagents at a single operation and serves as anexcellent tool for combinatorial processing. Althoughseveral problems, such as connection, parallel control offluid, monitoring and reaction conditions, are commonchallenges, the benefits offered by microreaction technol-ogy accumulate with the development of enzyme-reactiondevices.

As described here, few enzymes have been applied tomicroreaction process development, and not many patentsdescribing the construction of micro enzyme-reactors arepublished: indications that the field is still in its initialstage. Efforts directed at the development, optimizationand application of micro enzyme-reactors will open a newera for biochemical processing.

References1 Ehrfeld, W. et al. (2000) Microreactors-New Technology for Modern

Chemistry. Wiley-VCH2 Ziaie, B. et al. (2004) Hard and soft micromachining for bioMEMS:

review of techniques and examples of applications in microfluidics anddrug delivery. Adv. Drug Deliv. Rev. 56, 145–172

3 Szekely, L. and Guttman, A. (2005) New advances in microchipfabrication for electro-chromatography. Electrophoresis 26, 4590–4604

4 Hessel, V. et al. (2004) Chemical Micro Process Engineering. Wiley-VCH

5 Watts, P. and Haswell, S.J. (2005) The application of micro reactors fororganic synthesis. Chem. Soc. Rev. 34, 235–246

6 Chovan, T. and Guttman, A. (2002) Microfabricated devices inbiotechnology and biochemical processing. Trends Biotechnol. 20,116–122

7 Anderson, H. and van den Berg, A. (2003) Microfluidic devices forcellomics: a review. Sens. Actuators B 92, 315–325

8 Cullen, C.J. et al. (2004) Microfluidic systems for high-throughput andcombinatorial chemistry. Curr. Opin. Drug Discov. Devel. 7, 798–806

9 Wang, H. and Holladay, J.D. (2005) Microreactor Technology andProcess Intensification, ACS Symp. Ser., 914, American ChemicalSociety

10 Schoemaker, H.E. et al. (2003) Dispelling the myths – biocatalysis inindustrial synthesis. Science 299, 1694–1697

11 Garcia-Junceda et al. (2004) Enzymes in the synthesis of bioactivecompounds: the prodigious decades.Bioorg. Med. Chem. 12, 1817–1834

12 Schmid, A. et al. (2001) Industrial biocatalysis today and tomorrow.Nature 409, 258–268

13 Miyazaki, M. et al. (2001) Improved yield of enzyme reaction inmicrochannel reactor. Chem. Lett. 30, 442–443

14 Kanno, K. et al. (2002) Rapid enzymatic transglycosylation andoligosaccharide synthesis in a microchip reactor. Lab Chip 2, 15–18

15 Tanaka, Y. et al. (2001) Acceleration of enzymatic reaction in amicrochip. Anal. Sci. 17, 809–810

16 Tanaka, Y. et al. (2000) Non-contact photochemical control of enzymereactions on amicrochip by using a compact diode laser. J. Chromatogr.A 894, 45–51

17 Xue, Q. et al. (2001) Multiplexed enzyme assays in capillaryelectrophoretic single-use microfluidic devices. Electrophoresis 22,4000–4007

18 Lai, S. et al. (2004) Design of a compact disk-like microfluidic platformfor enzyme-linked immunosorbent assay. Anal. Chem. 76, 1832–1837

19 Richter, T. et al. (2002) Bi-enzymatic and capillary electrophoreticanalysis of non-fluorescent compounds in microfluidic devicesdetermination of xanthine. Sens. Actuators B 81, 369–376

20 Seong, G.H. and Crooks, R.M. (2002) Efficient mixing and reactionswithin microfluidic channels using microbead-supported catalysts. J.Am. Chem. Soc. 124, 13360–13361

http://www.novozymes.com/


21 Srinivasan, A. et al. (2004) Bacterial P450-catalyzed polyketidehydroxylation on a microfluidic platform. Biotechnol. Bioeng. 88,528–535

22 Nomura, A. et al. (2004) Preparation, characterization, and applicationof an enzyme-immobilized magnetic microreactor for flow-injectionanalysis. Anal. Chem. 76, 5498–5502

23 Sakai-Kato, K. et al. (2004) An enzyme-immobilization method forintegration for biofunctions on a microchip using a water-solubleamphiphilic phospholipid polymer having a reaction group. LabChip 4, 4–6

24 Sakai-Kato, K. et al. (2003) Creation of an on-chip enzyme reactor byencapsulating trypsin in sol-gel on a plastic microchip. Anal. Chem. 75,388–393

25 Kawakakami, K. et al. (2005) Development and characterization of asilica monolith immobilized enzyme micro-bioreactor. Ind. Eng. Chem.Res. 44, 236–240

26 Heule, M. et al. (2003) A miniaturized enzyme reactor based onhierarchically shaped porous ceramic microstruts. Adv. Mater. 15,1191–1194

27 Gleason, N.J. and Carbeck, J.D. (2004) Measurement of enzymekinetics using microscale steady-state kinetic analysis. Langmuir20, 6374–6381

28 Mao, H. et al. (2002) Design and characterization of immobilizedenzymes in microfluidic systems. Anal. Chem. 74, 379–385

29 Holden, M.A. et al. (2004) Patterning enzymes inside microfluidicchannels via photoattachment chemistry. Anal. Chem. 76, 1838–1843

30 Ekstrom, S. et al. (2000) Integrated microanalytical technologyenabling rapid and automated protein identification. Anal. Chem.72, 286–293

31 Miyazaki, M. et al. (2003) Simple method for preparation ofnanostructure on microchannel surface and its usage for enzyme-immobilization. Chem. Commun. 5, 648–649

32 Miyazaki, M. et al. (2004) Preparation of functionalized nanostructureson microchannel surface and their use for enzyme microreactors.Chem. Eng. J. 101, 277–284

33 Miyazaki, M. et al. (2005) Efficient immobilization of enzymes onmicrochannel surface through His-tag and application formicroreactor. Protein Pept. Lett. 12, 207–210

34 Kaneno, J. et al. (2003) A simple method for surface modification ofmicrochannels. N. J. Chem. 27, 1765–1768

Elsevier.com – linking scientists

Designed for scientists’ information needs, Elsevie

customer-focused navigation and an intuitive arch

greater prod

As a world-leading publisher of scientific, technical

linking researchers and professionals to the best t

deepest coverage in a range of media types to

breakthroughs in research and discovery, and t

Elsevier. Building insightswww.elsev


35 Qu, H. et al. (2004) Stable microstructured network for proteinpatterning on a plastic microfluidic channel: strategy andcharacterization of on-chip enzyme micrreactor. Anal. Chem. 76,6426–6433

36 Wu, H. et al. (2004) Titania and alumina sol-gel-derived microfluidicenzymatic-reactors for peptide mapping: design, characterization, andperformance. J. Proteome Res. 3, 1201–1209

37 Wang, H. et al. (2002) Continuous particle self-arrangement in a longmicro-capillary. Adv. Mater. 14, 1662–1666

38 Nakamura, H. et al. (2004) A simple method of self-assembled nano-particles deposition on the micro-capillary inner walls and the reactorapplication for photo-catalytic and enzyme reactions. Chem. Eng. J.101, 261–268

39 Koh, W-G. and Pishko, M. (2005) Immobilization of multi-enzymemicroreactors inside microfluidic devices. Sens. Actuators B 106,335–342

40 Gao, J. et al. (2001) Integrated microfluidic system enabling proteindigestion, peptide separation, and protein identification. Anal. Chem.73, 2648–2655

41 Hisamoto, H. et al. (2003) Chemico-functional membrane forintegrated chemical processes on a microchip. Anal. Chem. 75,350–354

42 Honda, T. et al. (2005) Immobilization of enzymes on microchannelsurface through cross-linking polymerization. Chem. Commun. 5062–5064

43 Cao, L. et al. (2003) Immobilized enzymes: carrier-bound or carrier-free? Curr. Opin. Biotechnol. 14, 387–394

44 Krenkova, J. and Foret, F. (2004) Immobilized microfluidic enzymaticreactors. Electrophoresis 25, 3550–3563

45 Urban, P.L. et al. (2006) Enzymatic microreactors in chemical analysisand kinetic studies. Biotechnol. Adv. 24, 42–57

46 Kanno, K. et al. (2002) Enhanced enzymatic reactions in amicrochannel reactor. Aust. J. Chem. 55, 687–690

47 Maruyama, T. et al. (2003) Enzymatic degradation of p-chlorophenolin a two-phase flow microchannel system. Lab Chip 3, 308–312

48 Yoon, S.K. et al. (2005) Laminar-flow-based electrochemicalmicroreactor for efficient regeneration of nicotinamide cofactors forbiocatalysis. J. Am. Chem. Soc. 127, 10466–10467

49 Garcia, E. et al. (2000) Use of microreactors in biotransformationprocesses: study of the synthesis of diglycerol monolaurate ester.Proc. IMRET 4, 5–9

to new research and thinking

r.com is powered by the latest technology with

itecture for an improved user experience and

uctivity.

and health information, Elsevier is dedicated to

hinking in their fields. We offer the widest and

enhance cross-pollination of information,

he sharing and preservation of knowledge.

. Breaking boundaries.ier.com

microchannel enzyme reactors and their applications for processing

Documents