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  • DOI: 10.1002/adsc.200606224

    Facile Preparation of an Enzyme-Immobilized Microreactor usinga Cross-Linking Enzyme Membrane on a Microchannel Surface

    Takeshi Honda,a Masaya Miyazaki,a,b,* Hiroyuki Nakamura,a

    and Hideaki Maedaa,b,*a Nanotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 807-1Shuku, Tosu, Saga 841-0052, JapanPhone: (+81)-942-81-3676, Fax: (+81)-942-81-3657; e-mail: m.miyazaki@aist.go.jp or maeda-h@aist.go.jp

    b Department of Molecular and Material Science, Interdisciplinary Graduate School of Engineering Sciences, KyushuUniversity, 61 Kasuga Koen, Kasuga, Fukuoka 816-8580, JapanPhone/Fax: (+81)-92-583-8891

    Received: May 19, 2006; Accepted: August 15, 2006

    Supporting information for this article is available on the WWW under http://asc.wiley-vch.de/home/.

    Abstract: The enzyme microreactor has considerablepotential for use in biotechnological syntheses andanalytical studies. Simplifying the procedure ofenzyme immobilization in a microreactor is attrac-tive, and it is achievable by utilizing enzyme immobi-lization techniques and taking advantage of the char-acteristics of microfluidics. We previously developeda facile and inexpensive preparation method for anenzyme-immobilized microreactor. The immobiliza-tion of enzymes can be achieved by the formation ofan enzyme-polymeric membrane on the inner wall ofthe microchannel through cross-linking polymeri-zation in a laminar flow. However, this method is un-suitable for use in conjunction with electronegativeenzymes. Therefore, a novel preparation methodusing poly-l-lysine [poly ACHTUNGTRENNUNG(Lys)] as a booster and anadjunct for the effective polymerization of electro-negative enzymes was developed in this study. Using

    aminoacylase as a model for an electronegativeenzyme, the reaction conditions for the enzyme-cross-linked aggregation were optimized. On thebasis of the determined conditions, an acylase-immo-bilized tubing microreactor was successfully preparedby cross-linking polymerization in a concentric lami-nar flow. The resulting microreactor showed a higherstability against heat and organic solvents comparedto those of the free enzyme. The developed methodusing poly ACHTUNGTRENNUNG(Lys) was applicable to various enzymeswith low isoelectric points, suggesting that this micro-reactor preparation utilizing a cross-linked enzymein a laminar flow could be expanded to microreac-tors in which a broad range of functional proteinsare employed.

    Keywords: cross-linked enzyme aggregation; enzymemicroreactor; immobilization; surface modification


    Microreactors are potentially powerful tools in thefields of chemistry and biotechnology.[1] The excellentperformance of microreaction systems is achieved bytaking advantage of microchannel system featuressuch as rapid heat and mass transfer, and their largersurface/interface area. These attractive features arefavorable for conducting catalytic reactions throughcatalysts that are immobilized on the reactor.[2] En-zymes are useful catalysts for the production of cer-tain types of chemicals and analysis, which are fre-quently applied to various reactors. There have beendemands for enzymatic microreaction devices includ-ing enzyme-immobilized microreactors in several

    fields, especially for high-throughput biotechnologicalsyntheses and bioanalyses.[3] In such microreactorpreparations, nanostructure fabrication and chemicalmodification of the microchannel are useful technolo-gies.[4] We have also developed a method for immobi-lizing enzymes on a glass-capillary surface using achemical modification containing sol-gel techniques.[5]

    However, these methods require high-level techni-ques and multi-step procedures, leading to low costperformance. More simple methods using carrierswith immobilized enzymes have also been proposed.The carriers were constructed by forming monolithsof a sol-gel and a polymer, and simply incorporatingcommercial beads and membranes in a microchan-nel.[6] These structures are often responsible for the

    Adv. Synth. Catal. 2006, 348, 2163 2171 E 2006 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim 2163


  • generation of high pressure, which is unfavorable formicrosynthesis and -analysis systems with multipleprocesses based on highly-controlled microfluids,[7]

    and are energy-intensive to operate. Therefore, in ourprevious study, we designed an enzyme-immobilizedmicroreactor which was simply and inexpensively pre-pared and does not require high pressure.[8] In our mi-croreactor process, a substrate membrane covers theinternal surface of the microchannel. The cylindricalmembrane is composed of the cross-linked polymer-ized enzyme product. The hollow structure of this re-actor prevents high pressure, compared to carrier-fill-ing microreactors.[6,8] In addition, this carrier-freemethod would be expected to have the advantage ofan absence of interactions between the enzyme andthe carrier material, which often leads to enzyme in-activation.[9] This bottom-up method is suitable forthe formation of miniature structures, and has recent-ly been utilized in various nanotechnological applica-tions such as enzyme-polymerized nanoparticles.[10]

    The enzyme-polymerized membrane was based on across-linked enzyme aggregate (CLEA)[9] formedusing a cross-linker with aldehyde groups which reactwith amino groups of the enzyme (Scheme 1). There-fore, CLEAs could be not obtained for electronega-tive enzymes due to a lack of reactive amino groups(relatively lower number of lysine residues). In thepresent study, to demonstrate the applicability of theCLEA-based enzyme-microreactor (CEM) methodfor various enzymes, we developed a novel methodfor preparing a CEM that is applicable to electroneg-ative enzymes utilizing poly-l-lysine [poly ACHTUNGTRENNUNG(Lys)]. Thesuccessful preparation was found to realize an effi-cient and fast CLEA formation in electronegative en-zymes by using poly ACHTUNGTRENNUNG(Lys).


    The Effect of a Cationic Polymer on CLEAFormation of an Electronegative Protein

    The efficient formation of a CLEA is essential for thesuccessful preparation of a CEM. We first exploredan efficient method in a batchwise system. Glutaral-dehyde (GA) and paraformaldehyde (PA) were usedas cross-linkers, as previously described.[8] Whenmixing the cross-linker and enzyme, CLEAs for elec-tropositive enzymes such as chymotrypsin and trypsin,which contain a high number of lysine residues, arereadily formed. On the other hand, no CLEAs wereobserved in the case of electronegative enzymes. Therelatively low content of lysine residues in these en-zymes would cause a low degree of cross-linking,leading to inefficient aggregation. To achieve effectivecross-linking polymerization, we proposed the closepacking of electronegative enzyme molecules byenzyme-trapping using a cationic polymer matrixprior to the cross-linking reaction.[9] The polymermatrix, possessing multiple basic functional groups,and electronegative enzymes will be adsorbed by elec-trostatic interactions, and form an enzyme-enzymecomplex bridged by the matrix. Actually, poly ACHTUNGTRENNUNG(Lys)immobilized on a carrier is frequently utilized to cap-ture electronegative proteins.[11,12] In this study, weused poly ACHTUNGTRENNUNG(Lys) as coupling agent for a carrier-free ag-gregate of the electronegative enzyme.The effect of poly ACHTUNGTRENNUNG(Lys) on CLEA formation was

    compared between chymotrypsin and aminoacylase(hereafter referred to as acylase) as electropositiveand electronegative model enzymes, respectively. Theturbidity in the reaction mixture increases with in-creasing amounts of insoluble aggregates. Thus, theprogress of CLEA formation could be evaluated bythe turbidity of the solution, as estimated by measur-ing the absorbance at 630 nm. As shown in Figure 1,the cross-linker consisted of GA (0.25%, v/v) and PA(4%, v/v) agglutinated chymotrypsin, but not acylase.No turbidity was observed when concentrated acylase(5-fold; 100 mgmL1) and/or cross-linker (5-fold;1.25% GA and 20% PA) was used (data not shown).In contrast, the addition of the cross-linker into themixture of acylase and poly ACHTUNGTRENNUNG(Lys) (Mn=20 kDa) led toa remarkable increase in the turbidity of the reactionsolution. When mixing acylase and poly ACHTUNGTRENNUNG(Lys) withoutcross-linker, in which an acylase-poly ACHTUNGTRENNUNG(Lys) complexwould be formed as a result of electrostatic interac-tions, the solution contained negligible suspendedsolids (Figure 1). Poly ACHTUNGTRENNUNG(Lys) and the cross-linker mix-ture without acylase, in which the cross-linking reac-tion would occur between poly ACHTUNGTRENNUNG(Lys) molecules,showed no turbidity (Figure 1). These results can beattributed to the high hydrophilicity of poly ACHTUNGTRENNUNG(Lys)which enables each polymeric complex to prevent in-

    Scheme 1. Reaction of enzyme amino groups and/or poly-ACHTUNGTRENNUNG(Lys) with aldehyde groups of the cross-linker.

    2164 www.asc.wiley-vch.de E 2006 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim Adv. Synth. Catal. 2006, 348, 2163 2171

    FULL PAPERS Takeshi Honda et al.

  • solubilization. It is clear that the turbidity of the acy-lase-polyACHTUNGTRENNUNG(Lys)-cross-linker mixture is a result of theconcerted reaction of the three reagents. A reactionusing lysine instead of poly ACHTUNGTRENNUNG(Lys) showed no evidenceof CLEA formation, indicating the importance of thepolymeric structure of poly ACHTUNGTRENNUNG(Lys) for effective CLEAformation. The addition of lysine to a mixture of chy-motrypsin-cross-linker greatly reduced the turbidityof the reaction mixture. This deleterious effect mightbe due to competition between amino groups of theenzyme and lysine, which possess little cross-linkingability.In order to achieve broad applicability for CEM for

    various enzymes