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Biochemical Engineering Journal 48 (2009) 6–12

Contents lists available at ScienceDirect

Biochemical Engineering Journal

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nzyme immobilization on amphiphilic polymer particles having graftedolyionic polymer chains

asahiro Yasuda a,∗, Hibiki Nikaido a, Wilhelm R. Glomm b, Hiroyasu Ogino a,osaku Ishimi a, Haruo Ishikawa a

Department of Chemical Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, JapanDepartment of Chemical Engineering, Norwegian University of Science and Technology, Sem Sælands vei 4, Trondheim N-7491, Norway

r t i c l e i n f o

rticle history:eceived 13 April 2009eceived in revised form 22 June 2009ccepted 24 June 2009

a b s t r a c t

Previously, we have shown that amphiphilic polymer particles functionalized with both hydrophilicguanidino groups and hydrophobic acyl groups have been shown to immobilize a large amount of lipasewith the immobilized lipase retaining high transesterification activity in organic solvent. However, thestability of immobilized lipase in organic solvent was found to be insufficient. In the present study, thechemical environment surrounding the immobilized enzyme was made more hydrophilic in order to

eywords:nzyme immobilizationolyionic graft chainmphiphilic polymer particlenzymatic reaction in organic phase

enhance the stability of immobilized lipase in organic solvent. For this purpose, polyionic polymer chainscontaining amino group in addition to amphiphilic groups were introduced. Monodisperse acrylic poly-mer particles (∼10 �m) were synthesized by seed polymerization in the presence of pore forming agent,and subsequently modified with poly(allylamine) and various reagents to introduce amphiphilic func-tional groups. The half-life of Rhizopus delemar lipase immobilized on these macroporous amphiphilicpolymer particles in hexane was 2.46 times high compared to lipase immobilized on amphiphilic polymer

and s

particles with guanidino

. Introduction

Enzymes are usually not stable in the presence of organic sol-ents. Therefore, despite the advantages [1–3] of carrying outnzymatic reactions in organic solvents or aqueous solutions con-aining organic solvents (nonaqueous media), enzymatic reactionsre limited within their stable reaction conditions. In order tovercome this disadvantage, various modification methods haveeen employed, including modification of the enzyme with variousynthetic polymers, lipids, or surfactants, enzyme immobilizationia covalent attachment or adsorption on solid material, enzymeross-linking and encapsulation, and development of organic sol-ent tolerant enzyme via molecular cell biology ([3] and referencesherein). Since immobilized enzyme can be easily collected fromhe reaction mixture and thermal and organic solvent stabilitiesre superior to those of free enzymes, many studies have been

erformed for the development of immobilized enzyme for theeaction in nonaqueous media [4].

Yang et al. [5] studied immobilization of subtilisin and ther-olysin using acrylic polymers and reported that the stiff acrylic

∗ Corresponding author. Tel.: +81 072 254 9299; fax: +81 072 254 9911.E-mail address: yasuda@chemeng.osakafu-u.ac.jp (M. Yasuda).

369-703X/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.bej.2009.06.011

tearoyl groups.© 2009 Elsevier B.V. All rights reserved.

polymer was useful for recycling use in organic solvent. PEG modi-fication prevented not only autolysis but also conformation change.In our previous works, the transesterification activity of a freeze-dried lipase, which was prepared from Rhizopus chinensis lipasein the presence of a fatty acid methylester or tert-octylphenoxypolyethoxy ethanol (Triton X-100), was considerably higher thanwithout additives [6]. This result suggested that these additivesimproved the solubility and stability of lipase in nonaqueous media.In the presence of hydrophobic interface, lipases convert their con-formation from “lid” to “open structure” in which substrate canaccess their active site and internal hydrophobic micro-domain canadsorb on interface. Therefore, lipase immobilization on hydropho-bic support enhances lipase activity dramatically (hyperactivation)[7,8]. Consequently, immobilization of lipase on polymer particlefunctionalized with amphiphilic acyl groups can be effectively usedfor lipase transesterification in nonaqueous media. We have previ-ously synthesized amphiphilic acryl particles – functionalized withboth a hydrophilic amino group and a hydrophobic acyl group –which can be used in transesterification reactions in hexane [9].Using Rhizopus delemar lipase as a model enzyme, we showed that

the guanidino group (GA) performed best as the amino group ofthe amphiphilic group in the amphiphilic polymer particles, andthat the optimal number (Cn) of carbon atoms in the chain of theacyl group was 18. Furthermore, using monodisperse amphiphilicpolymer particles synthesized by the two-step seeding method,

Engineering Journal 48 (2009) 6–12 7

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Table 1Composition of seed polymerization.

Component Amount (g)

Lauryl chloride 0.585Sodium dodecyl sulfate 0.22Acetone 1.17Distilled water 100.0Poly (vinyl alcohol) 2.0Seed particlesa 0.502,2′-Azobis (2,4-dimethyl valeronitrle) 0.05Glycidyl methacrylate 1.76Allyl methacrylate 4.10

M. Yasuda et al. / Biochemical

e showed that the surface area of the macro-pores (pore diam-ters of 50–500 nm) and the external surface area were importanto increase the amount of lipase immobilized [10,11]. In order toncrease the macro-pores with diameters of 50–500 nm, seed poly-

erization of acrylic monomers was performed in the presence of aonpolar inert solvent and/or a polar inert solvent. Immobilizationf lipases on these macroporous polymer particles yielded specificransesterification activities that were 74.8–85.9 times as high ashat of the lyophilized lipase. However, the stability of immobi-ized lipase in hexane was found to be suboptimal for industrialpplications of immobilized lipase [10,11].

As organic solvents interact with enzyme, the enzyme under-oes conformational changes from native to inactivate. Thus, inrder to reduce enzyme inactivation Abian et al. proposed thatigidification of enzymes via multipoint covalent immobilizationnd generation of hyper-hydrophilic microenvironment having aery open structure and fully surrounding every enzyme moleculeere effective for enzymes immobilized inside porous supports

12]. When penicillin G acylase and �-galactsidase were immo-ilized on porous support and modified with polyionic polymer,uch as dextran sulfate or polyethyleneimine, microenvironmentsround immobilized enzymes became hydrophilic, increasing thetability in organic solvent [12–18].

Therefore, introducing grafted polyionic polymer with chainengths was approaching the diameter of globular enzyme is anffective means to enhance the stability of immobilized lipasen porous acrylic polymer particles. Poly(allylamine) (PAA) is aydrophilic polymer having amino groups, with a random coilolecular size of approximately the same dimensions as the

nzyme. Furthermore, amino groups can be used not only forupport binding but also for hydrophobic group modification.he local density of amino group around immobilized lipase wasept high because of the large remaining amount of PAA nativemino groups. The objective of the present work is to synthesizend characterize amphiphilic polymer particle having grafted PAAhain and study the immobilization and organic solvent stabilityf lipase.

. Materials and methods

.1. Chemicals and enzyme

Allyl methacrylate (AMA), 2,2′-azobis (2,4-dimethylvalelo-itrile), 2,2′-azobis (2-methylpropionitrile), 1,4-butanediol digly-idyl ether, glycidyl methacrylate (GMA), stearoyl chloride, oliveil, poly(vinyl alcohol) (88% hydrolyzed, average molecular mass2,000) and styrene monomer were purchased from Wako Purehemicals Co. Ltd. (Osaka, Japan). Acetone, 5,5′-dithiobis (2-itrobenzoic acid) (DTNB), guanidine carbonate, 1-hexadecanol,

auryl chloride, methyl palmitate, poly(vinyl pyrrolidone) K-30,odium dodecyl sulfate and molecular sieves (3A 1/16) were pur-hased from Nacalai Tesque (Kyoto, Japan). 3-Dimercaptopropan--ol tributyroate was purchased from Sigma Chemical Co. Ltd. (St.ouis, MO, USA). Poly(allylamine) acetate (average molecular mass5,000, average degree of polymerization 260) was purchased fromittobo Co. Ltd. (Tokyo, Japan). Triolein was purchased from Kantoagaku (Tokyo, Japan). All the reagents were of the highest gradend used without purification.

A fine-grade lipase of R. delemar was purchased from Seikagakuougyo Co. (Tokyo, Japan) and used without purification.

.2. Seed polymerization

The seed copolymerization AMA and GMA was performed usingeed particles synthesized by the dispersion polymerization oftyrene in ethanol [19]. The composition of the reaction mixture

n-Decane 8.78

a Seed particle with a diameter of 2.21 �m was synthesized by the dispersionpolymerization of styrene [19].

was shown in Table 1. Reaction conditions were described in aprevious study [10,11].

2.3. Introduction of functional group

The produced polymer particles (epoxy particles) functionalizedwith epoxy groups were reacted with an amino compound underbasic conditions. Into the 4-necked 300-mL glass flask, an aminocompound of which the molar amount was 1.5 times as high asthat of the epoxy group in the epoxy particles (1.92 × 10−3 mol (gparticle)−1), 0.6 g of sodium hydroxide and 50 g of distilled waterwere added, and the mixture was stirred at 300 rpm and 70 ◦Cfor 10 min. To this mixture, a suspension consisting of 5.0 g ofthe epoxy particles and 50 g of 1,4-dioxane was added, and thereaction mixture was stirred at 200 rpm and 70 ◦C for 24 h. Theamino-functionalized particles (amino particles) were collectedand washed with distilled water by the same method as describedabove. In this study, poly(allylamine) and guanidine carbonatewere reacted with the epoxy particle. The produced particles,functionalized with poly(allylamino) groups were labelled PA par-ticles, and the particles having guanidino groups were labelledGA particles.

PA particles were further reacted with 1,4-butanediol diglycidylether as shown in Scheme 1. The composition of the reaction mix-ture and the reaction conditions were almost the same as thosefor the synthesis of GA particles except the ratio of the amino par-ticles and 1,4-butanediol diglycidyl ether. 5.0 g of the PA particleswere reacted with 0.8 g of 1,4-butanediol diglycidyl ether. The pro-duced particles (PAE particle) were further reacted with guanidinecarbonate under the same condition at the synthesis of GA parti-cles (Scheme 1). The particles which have both poly(allylamino)group and guanidino groups were named as PAG particles. Here-after, GA, PA, and PAG particles are collectively referred to as aminoparticles.

The amino particles were reacted with stearoyl chlorides in1,4-dioxane. Stearoyl chlorides of which the molar amount was3 times that of the amino groups in the amino particles, 3.0 g ofthe amino particles, and 100 g of 1,4-dioxane were added in the4-necked 300-mL glass flask, and the reaction mixture was stirredat 200 rpm and 70 ◦C for 24 h. The particles (amphiphilic particles)were collected and washed with distilled water by the same methodas previously described [10,11]. The three produced amphiphilicparticles were labelled GA-C18, PA-C18, and PAG-C18 particles,respectively.

To use the amphiphilic particles for immobilizing lipase, unre-acted monomer and impurities must be completely removed. For

this purpose, the amphiphilic particles were packed in a glasscolumn (1.0 cm ID × 20 cm) and 200 mL of 0.05N HCl, 200 mL of dis-tilled water, 200 mL of methanol and 200 mL of 10 mM phosphatebuffer (pH 5.5) were flowed through the column at a flow rate of1.1 × 10−8 m3 s−1.

8 M. Yasuda et al. / Biochemical Engine

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seed particles for the seed polymerization of GMA and AMA. Theproduced monodisperse reactive polymer particles were function-alized with epoxy groups, and are hereafter referred to as epoxyparticles.

cheme 1. Reaction between the amino group in the particle and stearoyl chloride.

.4. Particle characterization

The particle diameter distribution of the aqueous suspensionas measured using a laser particle diameter analyzer MICROTRAC

RA (Leeds & Northrup, Sumneytown Pike, USA). Scanning electronicrographs (SEMs) were taken using a HITACHI S-2150 (Tokyo,

apan). The amount of the epoxy groups in the particles was mea-ured by the HCl-dioxane method [20]. The amount of the aminoroup of PAA was measured by titration and calculated from themount of nonreacted epoxy group. The pore area and pore vol-me of the polymer particles were measured with a Pascal 140nd 240 (Thermo Fisher, Waltham, MA, USA) automated mercuryorosimeter.

.5. Measurement of protein concentration and the hydrolyticctivity

The protein concentration was determined by the method ofradford [21]. The hydrolytic activities of lipase were assayed byhe BALB–DTNB method [22] which was described at the previousaper [10,11]. One unit (U) of activity was defined as the amount ofnzyme that liberated 1 �mol of SH groups (lipase) per min at 30 ◦C.

.6. Enzyme immobilization

10 mg of the polymer particles was added to 1 mL of 10 mMotassium phosphate buffer (pH 5.5) containing 0.50 or 3.78 mgf enzyme. Following incubation of the resulting suspension

◦

t 4 C for 24 h, the particles were collected by filtration. Thearticles collected on filter paper were washed with 10 mL of0 mM potassium phosphate buffer (pH 5.5), and the amount ofnzyme immobilized on the polymer particles was determinedy measuring the difference between the protein concentrations

ering Journal 48 (2009) 6–12

of the enzyme solution before adding polymer particles and ofthe filtrates.

In the case of the measurement of the adsorption isotherms,lipase concentration in the phosphate buffer varied from 0.25 to8 mg cm−3 and temperature varied from 4 to 15 ◦C.

2.7. Measurement of the transesterification activity

The transesterification activities of lyophilized lipases and theimmobilized lipases prepared with various particles were mea-sured in hexane [6]. Water activity of hexane was attenuated at0.09 by using molecular sieve. The lyophilized lipases were pre-pared as follows: 10 mL of 10 mM potassium phosphate buffer (pH5.5) containing 37.8 mg of R. delemar lipase was well mixed with amagnetic stirrer and then lyophilized in a freeze-drying apparatusFD-5N (Tokyo Rikakikai Co., Tokyo, Japan). The immobilized lipasesprepared with 100 mg of various amphiphilic particles were alsolyophilized.

The transesterification activities were determined by measuringthe extent of the transesterification of olive oil and fatty acid methylesters. Water contained in hexane was removed using molecularsieves. Methyl laurate and methyl palmitate were used as fatty acidmethyl esters for activity measurements of the R. delemar lipase.One unit (U) of the transesterification activity was defined as theamount of enzyme that produced 1 �mol of methyl oleate per minat 37 ◦C.

2.8. Stabilities of immobilized enzymes

Thermostability of the immobilized lipases prepared withmonodisperse amphiphilic particles was measured using themethod described in a previous report [10,11]. To study the stabil-ity against hexane, the immobilized lipases prepared with 50 mg ofthe amphiphilic particles were lyophilized. The lyophilized samplesof the immobilized lipases were suspended in 3 mL of hexane andthe suspension was incubated at 37 ◦C for 72 h. The immobilizedlipases were then collected by filtration at various times and weredried to remove hexane. After the drying, the hydrolytic activity ofthe immobilized lipases was measured.

3. Results

3.1. Characterization of PA-C18, GA-C18 and PAG-C18 particles

Monodisperse seed particles (2.21 �m) were synthesized bydispersion polymerization of styrene, and subsequently used as

Fig. 1. SEM micrographs of particles: (a) GA-C18 particle and (b) PAG-C18 particle.

M. Yasuda et al. / Biochemical Engineering Journal 48 (2009) 6–12 9

Fig. 2. (a) Adsorption isotherm of lipase on the amphiphilic particles. 10.0 mg ofamphiphilic particles were added to 1.0 cm3 of 10 mM phosphate buffer contain-ing lipase (1.0–4.0 mg-lipase/cm3). The resulting mixture was incubated at 4 ◦Cfor 24 h, and then the amount of lipase immobilized was determined by mea-suring the concentration of lipase remaining in the supernatant. The lines in thisfigure were indicated theoretical line of Langmuir isotherm. (b) Langmuir plotfor lipase adsorption onto amphiphilic particles at 4 ◦C. C: lipase concentration[o(l

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particles have a smooth surface. However, there is no observabledifference between GA-C18 particles and PAG-C18 particles fromthe SEM micrographs.

The amount of amphiphilic group of particles affects the amountof lipase immobilized and the stability of immobilized lipase [11].

mg-lipase/cm3]. q: amount of lipase immobilized [mg-lipase/mg-particle]. 10.0 mgf particles were added to 1.0 cm3 of 10 mM phosphate buffer containing lipase1.0–4.0 mg-lipase/cm3). The resulting mixture was incubated for 24 h at 4 ◦C. Fol-owing incubation, the amount of immobilized lipase was measured.

Epoxy particles were further reacted with amino compoundnd stearoyl chloride, resulting in three kinds of functionalizedarticles, PA-C18, GA-C18 and PAG-C18. The diameters of GA-C18articles, PA-C18 particles and PAG-C18 particles were 9.32, 9.31nd 9.35 �m, respectively. Their standard deviation (SD) and coef-cient of variation (CD) were 0.44 �m and 4.76% (GA-C18 particles),.46 �m and 4.94% (PA-C18 particles), and 0.44 �m and 4.76% (PAG-18 particles), respectively. All particle diameters were almost

dentical, with a monodisperse distribution. The diameter of GA-18 particles was larger than that of GA-C18 particles described inhe previous paper (7.98 �m) [10]. This was because the amount ofhe seed particles on seed polymerization decreased. Therefore, thewelling amount of monomer with a particle was increased. Using

Fig. 3. Hydrolytic activities of immobilized lipases prepared with amphiphilic par-ticles. Lipases were immobilized on amphiphilic particles (10.0 mg) at 4 ◦C withvarious lipase concentrations (1.0–4.0 mg-lipase/cm3).

mercury porosimetry, the pore area and pore volume distributionsof the three sets of particles were measured. The produced parti-cles were found to exhibit macropores with diameters in the rangefrom 50 to 500 nm. Total pore volume (1.34 × 10−6 m3/g-particle),the distributions of pore volume and area were almost the sameas those of the GA-C18 particles described previously [11]. Fig. 1shows SEM micrographs (8000×) of the GA-C18 particles and PAG-C18 particles. Using the inert solvent during seed polymerization,the particles display a rough surface whereas the polystyrene seed

Fig. 4. Specific hydrolytic activities of immobilized lipases prepared withamphiphilic particles. Lipases were immobilized on amphiphilic particles (10.0 mg)at 4 ◦C with various lipase concentrations (1.0–4.0 mg-lipase/cm3). Specifichydrolytic activities of immobilized lipases were calculated by the amount of lipaseimmobilized and hydrolytic activities of immobilized lipases.

10 M. Yasuda et al. / Biochemical Engineering Journal 48 (2009) 6–12

Table 2Amount of reacted epoxy group with amino compounds.

Particle Amount of epoxy group reacted withamino compound (mol/g-particle)

Amount of amino group introducedinto the particle (mol/g-particle)

Surface density ofamino groupa (mol/m2)

GA 4.74 × 10−4 9.48 × 10−4 3.30 × 10−4

PA 2.91 × 10−4 7.57 × 10−2 2.64 × 10−2

PAG 1.14 × 10−47.55 × 10−2 (amino group)

2.63 × 10−21.14 × 10−4 (guanidino group)

a Surface densities of amino group were calculated using surface area (2.87 m2/g-particle) of macro-pore (50–1000 nm).

Table 3Comparison of hydrolytic activity of immobilized lipases prepared with various particles.

Particle used for lipase immobilization Amount of lipase immobilized(mg/mg–particle)a

Hydrolytic activity(U/mg-particle)a

Specific hydrolytic activity(U/mg–lipase)

Epoxy particle 3.28 × 10−2 5.83 × 10−2 1.78GA-C18 particle 1.05 × 10−1 1.69 × 10−1 1.61PA-C18 particle 9.86 × 10−2 9.08 × 10−2 0.92P

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AG-C18 particle 5.88 × 10−2

a Lipases were immobilized on amphiphilic particles (10 mg) at 4 ◦C with 3.7 mgupernatant.

herefore, the amount of remaining epoxy group after reaction withmino compound and the amount of introduced epoxy group ofAE particles was measured. Table 2 shows the amount of aminoroup in the PA-C18, GA-C18 and PAG-C18 particles. In the reac-ion between guanidine carbonate and the epoxy group of thepoxy particle, about 24.5% of the epoxy group was reacted whereaseacted epoxy group was 15.2% in the case of the reaction betweenoly(allylamine) and the epoxy group of the particles. However,oly(allylamine) has about 260 amino groups in one moleculehereas guanidine has two amino groups. Therefore, PA parti-

les have 7.57 × 10−2 mol/g-particle of amino group (NH2) andAG particles have 7.55 × 10−2 mol/g-particle of amino group and.14 × 10−4 mol/g-particle of guanidino group. Furthermore, PA and

AG particles have many intermolecular amino groups in graftedolyionic chain and they were congested. It was expected thatongested amphiphilic groups may protect enzyme from organicolvent.

ig. 5. Heat stability of R. delemar lipase. 10.0 mg of particles were added to 1.0 cm3 of0 mM phosphate buffer containing 3.7 mg of lipase. The resulting mixture was incu-ated for 15 min at various temperatures, and then remaining hydrolytic activitiesf lipase were measured.

1.44 × 10−1 2.45

/cm3. Amount of lipase immobilized was measured by the lipase concentration of

3.2. Lipase immobilization on PA-C18, GA-C18 and PAG-C18particles

The amount of lipase immobilized and the specific hydrolyticactivities of the immobilized lipases prepared with GA-C18, PA-C18, and PAG-C18 particles were studied, with lipase concentrationbeing varied from 0.8 to 4.0 mg-lipase/cm3. Fig. 2a shows an adsorp-tion isotherm of lipase on the amphiphilic particles. As can beclearly seen from Fig. 2a, the PA-C18 particles immobilize the high-est amount of lipase of the particles tested in this work. Fig. 2bshows Langmuir plots for lipase adsorption onto amphiphilic parti-cles at 4 ◦C. C is the lipase concentration in the phosphate buffer (pH5.5) and q the amount of lipase immobilized on the particles. Theexperimental data obtained at a constant temperature could be cor-related well with a line given by the Langmuir equation [11]. As canbe seen in this figure, the adsorption isotherm of R. delemar lipaseon the three amphiphilic particles obeys the Langmuir equation.The values of the maximum amount of adsorption, qm of GA-C18, PA-C18, and PAG-C18 particles were 2.41 × 10−1, 1.33 × 10−1,and 8.83 × 10−2 mg-lipase/mg-particle, respectively. From fittingthe data to the Langmuir equation, qm of the GA-C18 particleswas highest. This was because enzyme diffusion in particle poreswas inhibited with the introduction of bulky PAA group, withthe immobilized lipase being restricted to the outer surface andneighborhood of particle macro-pores. Fig. 3 shows the effect ofthe amount of R. delemar lipase immobilized on the amphiphilicparticles on the hydrolytic activity. The hydrolytic activity of theimmobilized lipase increased with an increase in the amount oflipase immobilized. However, the specific hydrolytic activity ofthe immobilized lipase decreased with an increase in the amount

of lipase immobilized as shown in Fig. 4. The specific hydrolyticactivity of the immobilized lipase prepared with PAG-C18 particleswas highest of all tested in this study while the PA-C18 parti-cles were found to immobilize the highest amount of lipase. Theintroduction of bulky PAA groups restricts immobilized lipase to

Table 4Half-lives of various immobilized lipases.

Particle used for lipaseimmobilization

Half-life of immobilizedlipase in hexane (h)

Epoxy particles 27.8GA-C18 particles 67.0PA-C18 particles 133PAG-C18 particles 165

M. Yasuda et al. / Biochemical Engineering Journal 48 (2009) 6–12 11

Table 5Comparison of transesterification activities of immobilized lipases and lyophilized lipase.

Lipase Amount of lipase immobilized(mg/mg-particle)

Transesterification activity(U/mg-particle)a

Specific transesterificationactivity (U/mg-lipase)

Lyophilized lipase 0.0440Lipase immobilized on GA-C18 particles 1.05 × 10−1 9.17 × 10−2 0.873Lipase immobilized on PA-C18 particles 9.86 × 10−2 5.85 × 10−2 0.593L 6.82 × 10−2 1.16

ith the amphiphilic particles and 5.0 mg of the lyophilized lipase, the transesterificationr rpm and 37 ◦C for 5 h.

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ipase immobilized on PAG-C18 particles 5.88 × 10−2

a Using 100 mg each of the lyophilized samples of immobilized lipases prepared weactions between triorein and methyl palmitate were carried out in hexane at 500

he outer surface and the vicinity of particle macro-pores. There-ore, the effect of substrate diffusion on hydrolytic activity was

inimalized. Moreover, as shown in Table 2, amino group densi-ies of PAG-C18 particles and PA-C18 particles were about 80-folds high as that of GA-C18 particles. Thus, the nano-environmentf immobilized lipase using PAG-C18 particles and PA-C18 par-icles is more hydrophilic, with a ratio of hydrophobic stearoylroup to hydrophilic guanidino group of 3 [9]. This hydrophilicano-enviroment would affect the promotion of mass transfer ofubstrate in the pores, hence the enhancement of hydrolytic activityf immobilized lipase. Since the specific activity of lipase immo-ilized on PAG-C18 particles was higher than that observed forA-C18 particles, both the amino group of poly(allylamine) anduanidino groups were necessary for lipase immobilization andptimal expression of its enzymatic activity.

Table 3 compares the amount of lipase immobilized, theydrolytic activities, and the specific hydrolytic activities. The spe-ific hydrolytic activity of R. delemar lipase in the 10 mM phosphateuffer (pH 5.5) was found to be 3.20 U/mg-lipase. The specificydrolytic activities of the immobilized lipases prepared with themphiphilic particles were lower than that of free native lipase forll the particles studied here. This can be attributed to the inter-al diffusional limitation affecting the activity of the immobilized

ipases as has been reported earlier [10].

.3. Heat stability and hexane stability of immobilized lipase

The thermostabilities of the immobilized R. delemar lipaserepared with the PAG-C18 particles and GA-C18 particles wereompared with native lipase. As shown in Fig. 5, immobilizationesulted in enhanced thermostabilities for both lipases at a highemperature. The thermostability of immobilized lipase preparedith the PAG-C18 particles was higher than that prepared with theA-C18 particles due to the PAG-C18 particle having high aminoroup content and congested amphiphilic groups.

The half-lives of the hydrolytic activities of the immobilizedipases in hexane prepared with the three amphiphilic particles andpoxy particles are shown in Table 4. The stability of the immo-ilized lipase prepared with the PAG-C18 particles in hexane wasound to be much higher in hexane compared with the lipase immo-ilized on the other particles.

.4. Transesterification activity and repeated use of themmobilized lipase

Using 100 mg of lyophilized sample of the immobilized lipaserepared with the three amphiphilic particles and 5 mg of

yophilized lipase, the transesterification reactions between oliveil and methyl palmitate were carried out in hexane. The specificydrolytic activities of the lyophilized lipase and the immobilized

ipase and their specific transesterification activities are comparedn Table 5. In the case of PAG-C18 particles, while the specificydrolytic activity of the immobilized lipase was about 0.58 timeshat of the lyophilized lipase, the specific transesterification activ-ty of the former lipase was 26.3 times higher than that of the latter

was used for the transesterification reaction (500 rpm, 37 ◦C, and 5 h). Followingreaction, the immobilized lipases were recovered from the reaction mixture by fil-tration and washed with hexane. These immobilized lipases were repeatedly usedfor the next cycle of the transesterification reaction.

lipase. The very high transesterification activity of the immobilizedlipase was attributed to the fact that it disperses well in hexanewhereas the lyophilized lipase does not.

The immobilized lipases must be usable repeatedly or for longperiods of time in order to be suited for industrial applications.Each of the immobilized lipases (100 mg) prepared with the GA-C18 particles and the PAG-C18 particles were used repeatedly forthe transesterification reactions in hexane. After each reaction for5 h, the immobilized lipases were recovered by filtration with afunnel with JGWP01300 filter paper (Nihon Millipore Ltd., Tokyo,Japan) and were washed with hexane and used for the next reac-tions. Fig. 6 shows the effect of the number of repeated uses ofthe immobilized lipases on the remaining transesterification activ-ity. For the immobilized lipase prepared with the GA-C18 particles,the transesterification activity decreased slightly faster with thenumber of cycles compared with that prepared with the PAG-C18particles. After seven cycles (the total reaction time was 35 h), thetransesterification activities of the immobilized lipases preparedwith the PAG-C18 particles and the GA-C18 particles were 83% and70%, respectively, of the initial transesterification activities.

4. Conclusions

To enhance the stability of immobilized enzyme in organicsolvent, new amphiphilic polymer particles functionalized withgrafted polyionic polymer chains were synthesized. The amountof amino group of PAG-C18 particles was 662 times as high asthat of GA-C18 particles. The reaction between epoxy particle and

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oly(allylamine) was restricted to the external particle surface dueo steric hindrance. Thus, the amphiphilic group of PAG-C18 existsolely in the vicinity of the external surface, yielding a much higherurface density of amino group compared to that of GA-C18 par-icles. The specific hydrolytic activity of the immobilized lipaserepared with PAG-C18 particles was highest of all tested in thistudy while the amount of lipase immobilized on the GA-C18 par-icles was highest. This was because the high amount of amphiphilicroup localized at particle surface increased the amount of lipasemmobilized and enhanced the stability of immobilized lipase.herefore, the half-life of Rhizopus delemar lipase immobilized onhe macroporous amphiphilic polymer particles in hexane was 2.46imes those of the immobilized lipases prepared by GA-C18 parti-les.

cknowledgment

This work was supported in part by a Proposal-Basedmmediate-Effect R & D Promotion Program from the New Energynd Industrial Technology Development Organization of JapanNEDO, project ID 98Z36-013-1) and Grant-in-Aids for Scientificesearch from the Ministry of Education, Science, Sports and Cul-ure of Japan (No. 14750617 and 18560734).

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