a molecularly imprinted polymer that shows enzymatic activity

Biochemical Engineering Journal 14 (2003) 85–91

A molecularly imprinted polymer that shows enzymatic activity

Eiichi Toorisakaa, Kazuya Uezua,1, Masahiro Gotoa,∗, Shintaro Furusakib

a Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, Hakozaki, Fukuoka 812-8581, Japanb Department of Applied Life Science, Faculty of Engineering, Sojo University, Ikeda, Kumamoto 860-0082, Japan

Received 26 July 2002; accepted after revision 7 September 2002

Abstract

An enzyme-mimic polymer was prepared by the surface molecular imprinting technique. In the active site of the polymer, a substrateanalog was imprinted through the complex formation between a cobalt ion and alkyl imidazole that functions as the hydrolysis catalysis.The enzymatic performance of the imprinted polymer was evaluated by the hydrolysis reaction of an amino acid ester. Based on theMichaelis–Menten analysis,Vmax andKm were obtained. The imprinted polymer shows a high activity compared to that of the controlunimprinted polymer. The result was supported by a lowKm value of the imprinted polymer, indicating a high affinity to the target substrate.The enzyme-mimic polymer was found to possess a substrate specificity by using several substrates that have a different structure. Acomputational modeling also supports the structure of the imprinted sites formed on the polymer surface.© 2002 Elsevier Science B.V. All rights reserved.

Keywords:Surface molecular imprinting; Hydrolysis; Enzyme-mimic; W/O emulsions; Amino acid ester; Computational modeling

1. Introduction

Molecular recognition for a target substrate and a highcatalytic activity of enzymes in a biological system arevery interesting. These enzymatic functions are created bywell-organized three-dimensional structure. In native en-zymes, however, many difficulties in practical use exist intheir sensitive properties such as instability against hightemperature, organic solvents, and serious pH conditions,etc. To mimic the highly organized functions in enzymes isone of the most challenging themes. Thus to date, variouscomplicate organic compounds possessing enzyme-mimicfunctions have been developed to overcome the drawbacksin enzymes. However, a multi-step procedure is requiredfor the preparation of these organic host compounds andoften causes low yield.

A molecular imprinting technique[1–5] has been re-ported as a novel methodology to synthesize polymers witha function of biocatalysts[6–19]. This technique enables toconstruct specific recognition sites into highly cross-linkedpolymers, and it is conceptually easy to apply to a widevariety of target molecules. However, it still has some fun-damental drawbacks yet unresolved, i.e., inapplicabilityto water-soluble substances which are important in a bio-

∗ Corresponding author. Tel.:+81-92-642-3576; fax:+81-92-642-3575.E-mail address:[email protected] (M. Goto).

1 Present address: Kitakyushu City University, Fukuoka, Japan.

logical or biomedical field, and the slow reaction kineticsarising from the inner diffusion of imprint molecules towardthe catalytic sites, which are deeply formed in the polymermatrix.

Recently, to overcome these problems, we have proposedan advanced molecular imprinting technique, called “sur-face molecular imprinting technique”[20–26]. The generalidea of the surface molecular imprinting technique is illus-trated in Fig. 1. The molecularly imprinted polymer wasprepared by polymerizing water-in-oil (W/O) emulsionscontaining a functional host molecule, an imprint moleculeand a cross-linking monomer. After polymerization, theorientation of the functional host molecules can be fixed onthe polymer surface. We have successfully prepared highlyselective imprinted polymer for optical resolution of aminoacids[27–29].

In the present paper, we introduce a novel functionalhost molecule possessing a high interfacial activity andan imidazole group to catalyze the hydrolysis reaction ofsubstrates. Using the synthesized functional host molecule,an enzyme-mimic polymer has been prepared by imprint-ing a substrate analog (N-�-t-boc-l-histidine) through thecomplex formation between a cobalt ion and the imida-zole molecule. The catalytic properties of the molecularlyimprinted enzyme polymers are discussed on the hydrol-ysis reaction of an amino acid ester (N-t-boc-l-alaninep-nitrophenyl ester) by comparing the performance of sev-eral control polymers. The enzyme-mimic activity was also

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86 E. Toorisaka et al. / Biochemical Engineering Journal 14 (2003) 85–91

Fig. 1. Schematic illustration of surface molecular imprinting technique for the preparation of artificial enzyme polymers.

evaluated based on the Michaelis–Menten kinetics. Thespecificity of the imprinted polymers was also investigatedby using different substrates in which the shape and thelength of the side chain are different. Finally, we discuss thestructure of a catalytic site formed on the polymer surfacewith computational modeling.

2. Experimental

2.1. Preparation of imprinted polymers

A novel functional host molecule, oleyl imidazole(1C18IM), was synthesized from oleic acid chloride and his-tamine dihydrochloride. The emulsion stabilizerl-glutamicacid dioleylester ribitol (2C18�

9GE) was synthesized ac-cording to our previous work[30]. Divinylbenzene (DVB,Wako Pure Chemical Industries) was employed after treat-ment with silica gel to remove the inhibitor. Other reagentswere of commercially available grade.

An artificial enzyme polymer was prepared by the surfacemolecular imprinting technique utilizing W/O emulsions.A 20 cm3 solution of DVB, in which 1C18IM (150 mM)and 2C18�

9GE (30 mM) were dissolved, was mixed with10 cm3 toluene. A 15 cm3 aqueous solution containing100 mM Co(NO3)2 (pH = 7.0, buffered with 100 mol/m3

acetic acid–sodium acetate) and 100 mM N-�-t-boc-l-histidine was added to the toluene solution, and the mixturewas sonicated for 4 min to obtain W/O emulsions. After the

addition of the powder initiator (2,2′-azobis(2,4′-dimethyl-valeronitrile), 0.18 g, Wako Pure Chemical Industries), themixture was stirred at 55◦C for 3 h under a nitrogen at-mosphere. The obtained polymer was dried in vacuo andground into an appropriate size. The imprinted polymer waswashed with a 1 M hydrochloric acid solution to removecobalt ions and the imprint molecule N-�-t-boc-l-histidine,and then filtered off. This procedure was repeated severaltimes until the cobalt ions in the filtrate could not be de-tected. Finally, the N-�-t-boc-l-histidine-imprinted polymerwas dried in vacuo. The unimprinted polymer was similarlyprepared as a reference polymer without cobalt ions and theimprint molecule.

2.2. Catalytic activity measurement of imprintedpolymers

A hydrolysis reaction of an ester was performed in abiphasic system of isooctane and a phosphate buffer solu-tion. A 0.05 g of the polymer was placed in a sealed testtube (10 cm3 volume), to which a 5 cm3 isooctane solutioncontaining 1.3 × 10−6 mol N-t-boc-l-alaninep-nitrophenylester as a substrate and a 5 cm3 phosphate buffer (pH= 8.0)(functional host molecule/substrate= 7.5/1 (molar ratio))were added. The mixture was shaken in a thermostated wa-ter bath at 35◦C. The hydrolysis activity of the imprintedpolymer was determined by measuring the hydrolytic prod-uct (p-nitrophenol) produced in the phosphate buffer phase.The producedp-nitrophenol was detected at 400 nm with a

E. Toorisaka et al. / Biochemical Engineering Journal 14 (2003) 85–91 87

UV-Vis spectrophotometer (JASCO V-570). The degree ofself-hydrolysis of the substrate was also measured withoutthe functional host molecule under the same conditions. Ac-tivity measurement was conducted at least three times andthe data were plotted with the average values. The experi-mental errors were less than 7%.

2.3. Swelling ratio of imprinted polymers

The swelling ratio of the imprinted polymers was deter-mined by volumetric measurement. A 1.0 g sample of animprinted polymer was placed in a sealed Teflon tube andcentrifuged for 30 min at 5000 rpm. The volume of the poly-mers can be measured by the scale on the tube. The vol-ume of the imprinted polymer filled was measured asV1.Excess organic solvent was added as a swelling solvent,and was vigorously shaken with polymers to ensure com-plete mixing. After the mixture was centrifuged for 30 minat 5000 rpm again, it was left intact for several hours. Thevolume of the swelled imprinted polymers was remeasuredasV2. The swelling of the column volumes is the expansionof the imprinted polymers due to the swelling solvent. Thevolumetric swelling ratio,S, was defined by the followingequation:

S = V2 − V1

V1× 100

2.4. Computational calculation for energy minimumstructure of the cobalt ion complex

The molecular mechanic (MM) and molecular dynamic(MD) calculations were performed for the cobalt ion com-plex with a molecular modeling software, HyperChem Re-lease 5.1 (Hypercube, Canada). In the calculation, the forcefield parameter set of MM2 was employed. Before calculat-ing the optimized structure of the complex at the oil–waterinterface, two preliminary calculations were conducted (1)MM calculation to obtain the lowest energy structure of thecomplex in vacuum and (2) a toluene–water box to calcu-late the complex at the toluene–water interface. Firstly, thelowest energy structure of the complex in vacuum was cal-culated by the MM method. The toluene–water biphase box(30 Å×30 Å×60 Å) was built from two adjacent cubic boxes(30 Å×30 Å×30 Å) of pure toluene and water molecules setto be 158 and 932 by taking into account each molecular vol-ume, respectively. Finally, the complex optimized in vacuumwas placed at the toluene–water interface by replacing eleventoluene molecules that is almost equivalent to the complexvolume. The MD simulation was performed in the (N, V, T)ensemble after MM calculation in the biphase system, whichwas required to avoid a large strain energy raised when be-ing directly calculated by the MD method. The time stepwas adjusted to be 1 fs, and the temperature was controlledat 300 K by coupling to a thermal bath with a relaxation timeof 0.1 ps.

Fig. 2. Surface structure of molecular imprinted polymer.

3. Results and discussion

3.1. Synthesis of artificial enzyme polymers

A highly cross-linked polymer was prepared by im-printing the substrate analog using W/O emulsions. Theimprinted polymer was obtained at more than 90% yields.After polymerization, the bulk polymer was ground into par-ticles, which volume-averaged diameters were ca. 35�m.Fig. 2 shows a typical view of the imprinted polymerprepared from W/O emulsions by scanning electron mi-croscopy (SEM). Substantial traces of aqueous phases in theemulsions are observed in the polymer. The catalytic sitesfor the substrate are conceptually formed on the surfaces ofthe inner cavities in the polymer.

3.2. Catalytic characteristics of enzyme polymers

The catalytic property of the enzyme-mimic polymer(imprinted polymer) prepared by the surface molecular im-printing technique is shown inFig. 3. The catalytic activityof the imprinted polymer is found to be high comparedto that of the unimprinted polymer and the isooctane so-lution containing the same amount of the functional hostmolecule. The substrate N-t-boc-l-alanine p-nitrophenylester was not self-hydrolyzed under the present experi-mental conditions. The unimprinted polymer exhibited alittle higher catalytic activity than that of the functionalhost solution. Since the solubility of the functional hostmolecule in the organic phase is not high enough, the unim-printed polymer that has well-dispersed host moleculesshows a better performance. These results suggest thatthe imprinting technique is effective for the enhance-ment of binding ability of substrates, and complemen-tary recognition sites are constructed by the imprintingtreatment.


Fig. 3. Comparison of enzymatic systems on the hydrolysis reaction:(�) imprinted polymer, (�) unimprinted polymer, (�) organic solutioncontaining the same amount of the functional host molecule and (×) selfhydrolysis of the substrate.

3.3. Effect of the structure of functional host molecules

The molecular imprinted technique provides optimumcavities to recognize a target substrate on the polymer sur-face. If the reaction sites can be formed on the polymersurface, the reaction rate would be improved. To create therecognition sites on the polymer surface, the functional hostmolecule should have an aqueous–organic interfacial activ-ity. In the previous study, we synthesized oleyl imidazoleas the novel functional molecule, which has a high interfa-cial activity and an effective functional group imidazole tocatalyze the hydrolysis reaction[24].

Fig. 4shows the catalytic activity of each imprinted poly-mer prepared by using oleyl imidazole or 4-vinyl pyridineas the functional molecule. The catalytic activity of the im-printed polymer with oleyl imidazole was much higher than

Fig. 4. Effect of functional molecular structure on the hydrolysis reaction.

Fig. 5. Effect of reaction solvents on enzymatic activity.

that of 4-vinyl pyridine. Since 4-vinyl pyridine possesseslittle interfacial activity, recognition sites should be formedin the polymer matrix. This slow reaction rate indicates thatthe substrates are catalyzed after inner diffusion. On thecontrary, the oleyl imidazole possessing a high interfacialactivity can be fixed on the polymer surface. Thus, the re-action rate of substrates is enhanced. In our previous study,we reported that the interfacial adsorption constant of thefunctional host molecule is required to be above 40 mM forobtaining a high imprinted effect[22]. In this study, the in-terfacial adsorption constant of oleyl imidazole was mea-sured to be 47.99 mM that is high enough on the basis ofthe previous standard.

3.4. Effect of reaction solvents

Native enzymes often lost their activity when contactingwith an organic solvent. While, since the artificial enzymepolymer can maintain the activity even in organic solvents,it can catalyze the reaction of oil–soluble substrates. It isstill unclear what characteristics of organic solvents affectcatalytic activity. In the present study, we investigated a re-lationship between the swelling ratio of the enzyme poly-mer in organic solvents and catalytic activity (Fig. 5). Asa result, in aliphatic organic solvents such as isooctane andn-tetradecane the imprinted polymer provided higher cat-alytic activity than that in aromatic solvents. Since the im-printed polymer prepared with DVB strongly interacts witharomatic solvents, the polymer tends to swell. The catalyticactivity of the swelled polymer is found to be relatively lowdue to the deformation of the catalytic active sites, and thefunctional host molecules may be removed.

3.5. Kinetic analysis of imprinted polymers

The hydrolytic activities of the imprinted polymer andthe unimprinted polymer were evaluated based on the


Table 1Kinetic parameters obtained for the hydrolysis reaction

Km (mM) Vmax (mM/min) Vmax/Km

Imprinted polymer 1.63 6.25× 10−2 3.83× 10−2

Unimprinted polymer 2.05 3.43× 10−2 1.67× 10−2

Michaelis–Menten kinetics. The analysis results are sum-marized in Table 1. The values ofVmax and Km wereobtained from the Lineweaver–Burk plots (Fig. 6). TheKmvalue represents the affinity of the polymer to the substrateand a low value indicates high affinity. TheKm value ofthe imprinted polymer is found to be lower than that of theunimprinted polymer. This result means that the imprintedpolymer having substrate recognition sites on the polymersurface is easy to incorporate the substrate. Also, theVmaxvalue of the imprinted polymer was higher than that of theunimprinted polymer. The catalytic sites of the imprintedpolymers were prepared through the complex formation be-tween a cobalt ion and the functional host molecules. Thus,the functional host molecules are orientated for catalyzingthe target molecule, and the good arrangement of the func-tional molecules improves the reactivity for the substrate.

3.6. Substrate specificity of imprinted polymers

We used four different substrates to investigate the sub-strate specificity of the imprinted polymers. Relative reactiv-ities in Fig. 7 were obtained from the following definition:(the initial rate catalyzed by the imprinted polymer)/(the ini-tial rate catalyzed by the unimprinted polymer). The highestactivity was observed for the substrate I whose structure ismost similar to that of the imprinted molecule. The secondactivity was observed for the substrates II and III. Thesereactivities were lower than that of the substrate I becausethese side chains are more bulky than that of the imprintedmolecule. The substrate IV provided the lowest reaction rate

Fig. 6. Kinetic analysis for the hydrolysis reaction by imprinted polymerand unimprinted polymer: (�) imprinted polymer and (�) unimprintedpolymer.

Fig. 7. Specific activity of imprinted polymers for various substrates:(I) N-t-boc-l-alanine p-nitrophenyl ester; (II) N-t-boc-l phenylalaninep-nitrophenyl ester; (III) N-t-boc-l-leucine p-nitrophenyl ester; (IV)p-nitrophenyl acetate.

in all the substrates because the shape and the size are quitedifferent from the imprinted molecule. Based on these fact,it was found that the enzyme-mimic polymer recognized thestructure of the imprinted molecule and selectively catalyzedthe substrate that has a similar structure of the imprintedmolecule.

3.7. Effect of reaction inhibitor on the hydrolysis reaction

To confirm that the complementary recognition sites forthe substrate is formed in the imprinted polymer, the imprintguest molecule was added to the reaction medium (Fig. 8).

Fig. 8. Effect of inhibitor concentration on the hydrolysis reaction byimprinted polymers.


Fig. 9. The optimized structure for the active site of the imprinted polymer. The lowest energy structure of the complex: (a) in vacuum before MDcalculation; (b) at the oil–water interface after MD calculation (1 ps).

The imprint molecule is thought to exist as an inhibitor inthe aqueous solution of the biphasic system. If an excessmolecular analog exists in the reaction medium, we consid-ered that the hydrolysis reaction should be reduced. As theresults, the hydrolysis reaction of the ester was inhibited byincreasing the inhibitor concentration. Since the imprintedpolymer memorizes the shape and the size of the imprintmolecule, the imprint molecule is easy to incorporate therecognition site. This result suggests that the complemen-tary recognition sites of the cobalt complex are formed onthe polymer surface.

3.8. Molecular dynamic calculation

We calculated the lowest energy structure of the complexin the toluene–water box by the MD method, and the snapshot at 1 ps is shown inFig. 9. In the complex as shownin Fig. 9, the imprint molecule forms the complex withthree functional host molecules and a cobalt ion. From theoverview of the lowest energy structure at the interface, wefound that the alkyl chain of the functional host moleculetakes a bulky structure in the toluene–water box. This resultsuggests that the functional host molecule is hard to be re-moved from the toluene–water interface. Also, it is shownthat the bulky boc group of the imprint molecule locatesalong the toluene–water interface. The imprint molecule canbe removed by washing with an acidic solution, and thecomplementary space to the substrate will be memorized onthe polymer surface.

4. Conclusion

An enzyme-mimic polymer was prepared by the surfacemolecular imprinting technique. The imprinted polymerexhibited higher catalytic activity towards an amino acid

ester than the unimprinted control polymer. The catalyticsite constructed on the polymer surface selectivity recog-nized the substrate and catalyzed the hydrolysis reaction.Furthermore, computational modeling clearly showed theformation between the imprint molecule and the functionalhost molecules at the toluene–water interface. The surfacemolecular imprinted polymer can be easily prepared andpossesses excellent chemical and physical stability. Thesurface molecular imprinting technique will be a useful toolfor the preparation of an enzyme-mimic polymer.

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a molecularly imprinted polymer that shows enzymatic activity

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