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

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<ul><li><p>DOI: 10.1002/adsc.200606224</p><p>Facile Preparation of an Enzyme-Immobilized Microreactor usinga Cross-Linking Enzyme Membrane on a Microchannel Surface</p><p>Takeshi Honda,a Masaya Miyazaki,a,b,* Hiroyuki Nakamura,a</p><p>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</p><p>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</p><p>Received: May 19, 2006; Accepted: August 15, 2006</p><p>Supporting information for this article is available on the WWW under http://asc.wiley-vch.de/home/.</p><p>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</p><p>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.</p><p>Keywords: cross-linked enzyme aggregation; enzymemicroreactor; immobilization; surface modification</p><p>Introduction</p><p>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</p><p>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]</p><p>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</p><p>Adv. Synth. Catal. 2006, 348, 2163 2171 E 2006 Wiley-VCH Verlag GmbH&amp;Co. KGaA, Weinheim 2163</p><p>FULL PAPERS</p></li><li><p>generation of high pressure, which is unfavorable formicrosynthesis and -analysis systems with multipleprocesses based on highly-controlled microfluids,[7]</p><p>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]</p><p>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).</p><p>Results</p><p>The Effect of a Cationic Polymer on CLEAFormation of an Electronegative Protein</p><p>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</p><p>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-</p><p>Scheme 1. Reaction of enzyme amino groups and/or poly-ACHTUNGTRENNUNG(Lys) with aldehyde groups of the cross-linker.</p><p>2164 www.asc.wiley-vch.de E 2006 Wiley-VCH Verlag GmbH&amp;Co. KGaA, Weinheim Adv. Synth. Catal. 2006, 348, 2163 2171</p><p>FULL PAPERS Takeshi Honda et al.</p></li><li><p>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</p><p>various enzymes, we examined whether CLEAs wereformed using poly ACHTUNGTRENNUNG(Lys), using several industriallyuseful enzymes with distinct isoelectric points. Table 1shows the relationship between the isoelectric pointsof each enzyme and CLEA formation. Electropositiveenzymes, including chymotrypsin, cucumisin, papainand trypsin easily formed a CLEA without poly ACHTUNGTRENNUNG(Lys).In other enzymes, with low isoelectric points (pI</p></li><li><p>Figure 2.[15] A stable laminar flow is important for thesuccessful formation of a CLEA-based membrane onthe internal surface of a CEM-tube,[8] and appropriateflow rates should be selected for each solution. Weset the pumping rate of the cross-linker (correspond-ing to a central stream) to a value 12.5 times higherthan that of the acylase-poly ACHTUNGTRENNUNG(Lys) mixture (outerstream). The formation of a concentric laminar flowwas confirmed by confocal laser scanning microscopy(see Supporting Information, Figure S3). Calculatingfrom the two distinct pumping rates and microfluidicvolumes estimated from the confocal data, averagelinear velocities of the central and outer streams were187.6 mmmin1 and 6.4 mmmin1, respectively. In theCEM-tube, the residence time of the central streamwas much shorter (0.6 min) than that of the outerstream (39.1 min). According to the kinetics of CLEAformation in a batch (see Supporting Information,Figure S4), this residence time for the acylase-poly-ACHTUNGTRENNUNG(Lys) mixture solution was sufficient to form theCLEA.The resulting CEM was washed with distilled</p><p>water, dried and the enzyme-membrane observed.Figure 3 shows CCD images of the cylindricalenzyme-membrane (dry state) exposed from thePTFE tube and a sectional view of the CEM. The cy-</p><p>lindrical membrane was shrunk by drying, and thethickness of the obtained membrane was 3560 mm.When a suspension of CLEA prepared in a batchwisesystem was passed through a PTFE tube, no mem-brane was observed on the inner wall (data notshown). This result indicates that polymerization in amicrofluid enables the enzyme-polymer product toadopt a membrane structure on the inner wall of theCEM tube. The amount of acylase, estimated by aquantitative amino acid analysis as described previ-ously,[8] was 3.250.17 nmol per acylase-CEM.</p><p>Performance of the Acylase-CEM</p><p>The enzymatic performance of the obtained acylase-CEM was evaluated using the hydrolysis of acetyl-d,l-phenylalanine (Ac-d,l-Phe) at various conditions.The CEM was cut off at both ends to a length of12.2 cm, corresponding to 24 mL of inner volume. Thehydrolysis at 4 mM of substrate was complete at thisflow rate (1 mL min1) (Figure 4). The MichaelisMenten constant (Km) and maximal velocity (Vmax),calculated from a LineweaverBurke plot, were4.58 mM and 1.10 mMmin1, respectively. All prod-ucts showed high enantiopurities (99.30.8%) com-pared to those obtained in a batch process (99.20.4%). When treating 100 mM substrate at a flowrate of 4 mL min1, the highest amount of l-Phe pro-duction was 45 nmol per minute. Under similar reac-tion conditions, the Km and Vmax values for the freeacylase were estimated to be 2.33 mM and4.69 mMmin1, respectively. The Vmax value wassomewhat lower than that of the free acylase while ahigher Km value was observed for the immobilizedacylase. These results suggest that chemical modifica-tion with the cross-linker somewhat suppressed thehydrolytic ability of the acylase.When a PTFE tube was passed through the acy-</p><p>lase-polyACHTUNGTRENNUNG(Lys) solution without a cross-linker, no en-</p><p>Figure 2. Schematic illustration of the procedure used to prepare an acylase-CEM. The cross-linking polymerization was per-formed in a concentric laminar flow. A silica capillary was fitted to the outer diameter of the T-shape connector by attachingto a PTFE tube using heat-shrink tubing. The capillary was set in the connector located at the concentric position of theCEM tube. The cross-linker solution was supplied to the substrate PTFE tube through the silica capillary, corresponding to acentral stream in the concentric laminar flow. A solution of acylase-polyACHTUNGTRENNUNG(Lys) mixture was poured from the other inlet of theT-shape connector, and formed an outer stream of the laminar flow.</p><p>Figure 3. CCD images of a) cylindrical enzyme-membrane(dry state) exposed from the PTFE tube, which forms onthe inner wall of the tube and b) a sectional view of the ob-tained CEM.</p><p>2166 www.asc.wiley-vch.de E 2006 Wiley-VCH Verlag GmbH&amp;Co. KGaA, Weinheim Adv. Synth. Catal. 2006, 348, 2163 2171</p><p>FULL PAPERS Takeshi Honda et al.</p></li><li><p>zymatic activity was observed. This result indicatesthat the enzymatic activity of the acylase-CEM wasdue to the acylase immobilized in the CLEA-mem-brane, and not by non-specific adsorbed free acylaseand/or a water soluble acylase-poly ACHTUNGTRENNUNG(Lys) complex.It is very important for enzymes incorporated in a</p><p>microreaction system to have a high stability againstconditions such as pH, temperature, and solvent. ThepH activity profile of the free acylase and acylase-CEM was obtained by incubating the enzyme in50 mM phosphate buffer for pH 57.5, 50 mM Tris-HCl buffer for pH 89 at 40 8C for 30 min. The acy-lase in the CEM showed a maximum activity atpH 7.0...</p></li></ul>