synthesis of amphiphilic polymer particles by seed polymerization and their application for lipase...

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Synthesis of Amphiphilic Polymer Particles by SeedPolymerization and Their Application for LipaseImmobilization

Masahiro Yasuda,* 1 Miho Kobayashi,1 Takuya Kotani,1 Kouji Kawahara,1 Hibiki Nikaido,1 Atushi Ueda,2

Hiroyasu Ogino,1 Haruo Ishikawa1

1 Department of Chemical Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan2 Special Division Green Life Technology, National Institute of Advanced Industrial Science and Technology,

1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, JapanFax: +81-72-254-9911; E-mail: [email protected]

Keywords: amphiphiles; dispersions; enzymes; particle size distribution; polyamines;

IntroductionThere are several advantages in conducting enzymaticreactions in organic solvents or aqueous solutions con-taining organic solvents.[1–3] However, since enzymes areusually not stable in the presence of organic solvents,methods to overcome this disadvantage must be devel-oped.

To obtain enzymes which exhibit high activities inorganic solvents, several enzymes were modified with

lipids,[4, 5] amphiphilic compounds (surfactants),[6–8] andsynthetic polymers.[9] These modified enzymes exhibithigh activities in organic solvents compared with unmo-dified powder enzymes.[4–9]

From the viewpoint of industrial applications, immobi-lized enzymes are useful because they can easily berecovered from the reaction mixtures and used repeat-edly.[10] A majority of the immobilized enzymes exhibithigh activity and high stability in aqueous solutions.

Full Paper: To develop excellent immobilized enzymes,monodisperse amphiphilic polymer particles that haveboth hydrophilic guanidino groups and hydrophobic acylgroups were synthesized and characterized. The monodis-perse amphiphilic particles, which had an average diam-eter of 7.92 lm, had macropores with diameters rangingfrom 50 nm to 500 nm. The amount of Rhizopus delemarlipase immobilized on the monodisperse amphiphilicpolymer particles was 150–8700 times those of theimmobilized lipases prepared by previous investigatorsusing Dowex MWA-1, porous glass beads, and Sepharose4B. The specific transesterification activity of the immo-bilized lipase prepared with the monodisperse amphiphilicpolymer particles was 93.4 times that of the lyophilizedlipase.

Macromol. Chem. Phys. 2002, 203, No. 2 i WILEY-VCH Verlag GmbH, 69469 Weinheim 2002 1022-1352/2002/0201–0284$17.50+.50/0

Macromol. Chem. Phys. 2002, 203, 284–293

Hydrolytic activities of the immobilized lipases preparedwith the monodisperse GA-C18 particles. The immobilizedlipases were prepared at 4 8C under the different lipase con-centrations of 0.25–8.0 kg N m–3. The specific hydrolyticactivities of the immobilized lipases were calculated from theamount of lipase immobilized and the hydrolytic activities ofthe immobilized lipases.

Synthesis of Amphiphilic Polymer Particles by Seed Polymerization ... 285

However, there are a few immobilized enzymes whichexhibit high activity in organic solvents.[11]

To conduct enzymatic reactions in organic solvents,polydisperse amphiphilic polymer particles which haveboth hydrophilic amino groups and hydrophobic acylgroups were synthesized to form the support ofenzymes.[12] Furthermore, to decide which is the bestamphiphilic group, using Rhizopus delemar lipase as amodel enzyme, the transesterification activity of theimmobilized lipase was studied.[12] It was found that theamphiphilic polymer particles in which a guanidino groupand stearoyl group C18 were introduced as the aminogroup and the acyl group, respectively, were the best par-ticles as the support of lipase for use in enzymatic reac-tions in organic solvents.[12] However, the hydrolyticactivity of the immobilized lipase prepared with theamphiphilic polymer particles was equal to or lower thanthat of Pseudomonas cepacia lipase immobilized onpoly(propylene) support,[13] Candida rugosa lipase immo-bilized on polydisperse acrylic polymer particles,[14] orRhizopus delemar lipase on various supports.[15]

In the present work, to obtain the immobilized lipasewith a high activity, monodisperse amphiphilic polymerparticles were synthesized by the two-step seedingmethod.[16]

Experimental PartChemicals

Allyl methacrylate (AMA), 2,29-azobis (2,4-dimethylvalelo-nitrile) (ADVN), 2,29-azobis (2-methylpropionitrile) (AIBN),glycidyl methacrylate (GMA), hexanoyl chloride, myristoylchloride, stearoyl chloride, olive oil, poly(vinyl alcohol)(88% hydrolyzed, average molecular mass 22000) and sty-rene monomer were purchased from Wako Pure ChemicalsCo. Ltd. (Osaka, Japan). Acetone, 5,59-dithiobis (2-nitroben-zoic acid), guanidine carbonate, 1-hexadecanol, lauryl chlor-ide, methyl laurate, poly(vinylpyrrolidone) K-30 (PVP),sodium dodecyl sulfate (SDS) and molecular sieves (3A 1/16) were purchased from Nacalai Tesque (Kyoto, Japan). 3-dimercaptopropan-1-ol tributyroate was purchased fromSigma Chemical Co. Ltd. (St. Louis, MO). All the reagentswere of the highest grade and used without purification.

Enzymes

A purified lipase was prepared from R. chinensis accordingto the method described in a previous paper.[17] A fine-gradelipase of R. delemar was purchased from Seikagaku KougyoCo. (Tokyo, Japan) and used without purification.

Dispersion Polymerization of Styrene

The seed particles used for polymerization were synthesizedby the dispersion polymerization of styrene in ethanol usinga glass batch reactor.[18] A clean and dry 300-mL separableflask was charged with 72.4 g of ethanol, 1.80 g of PVP, and0.57 g of 1-hexadecanol, and covered with a 4-necked separ-

able cover attached with a Dimroth condenser. The flask washeated to 708C with mild shaking for 20 min. 0.25 g ofAIBN was put into 25.0 g of styrene and the resulting solu-tion was quickly poured into the flask. The reaction was per-formed with agitation at 30 rpm for 24 h. The flask was thencooled in an iced-bath and the reaction mixture was trans-ferred to four 50-ml test tubes. The particles were washed byrepeating centrifugation and suspension in ethanol and dis-tilled water.

Seed Polymerization

0.65 g of the seed particles synthesized by dispersion poly-merization were dispersed in 7.7 g of distilled water contain-ing 0.005 g of SDS under ultrasonication for 15 min. 0.585 gof lauryl chloride was taken into 7.7 g of distilled water con-taining 0.015 g of SDS and the mixture was homogenized at10000 rpm for 10 min using a POLYTRON PT-MR3100homogenizer (KINEMATICA, Luzernerstrasse, Switzerland)attached to a 20-mm generator shaft. The homogenized solu-tion was sonicated using a UD-200 sonicator (TOMY,Tokyo, Japan). To this solution the dispersed seed particlesprepared above, 1.17 g acetone, and 0.2 g distilled waterwere added. The resulting solution was poured into the300-mL separable flask and then covered with the 4-neckedseparable cover. The seed particles were swollen with laurylchloride at 100 rpm and 308C for 24 h.

0.114 g of ADVN was taken into a mixture consisting of8.19 g of AMA and 3.21 g of GMA. The resulting monomermixture was mixed with 84.4 g of distilled water containing0.2 g of SDS and the mixture was homogenized at 10000rpm for 10 min using the POLYTRON PT-MR3100 homo-genizer. The mixture was added to the above suspension con-taining the seed particles swollen with lauryl chloride. Theseed particles were further swollen with the monomers withstirring at 100 rpm and 308C for 2 h. 2.0 g of poly(vinylalcohol) was put into 8.0 g of distilled water and the resultingsolution was quickly poured into the 300-mL separable flask.The flask was heated to 708C and the reaction was carriedout for 24 h with agitation at 100 rpm. The flask was thencooled in the iced-bath. The conversion was measured by agravimetric method. The produced polymer particles werecollected by filtering the reaction mixture using a funnelwith 5C filter paper (Toyo Roshi Kaisha Ltd., Tokyo). Theparticles were washed with methanol three times by repeatedfiltration and suspension. After washing with methanol, theparticles were similarly washed with distilled water threetimes.

Reaction between the Epoxy Groups in the Particles and anAmino Compound

The polymer particles (epoxy particles) that have epoxygroups were reacted with an amino compound under basicconditions. Into the 4-necked 300-mL glass flask, 5.0 g ofthe epoxy particles, an amino compound of which the molaramount was 1.5 times that of the epoxy group in the epoxyparticles, 0.6 g of sodium hydroxide and 50 g of distilledwater were taken and the mixture was stirred at 300 rpm and70 8C for 10 min. To this mixture, a suspension consisting of

286 M. Yasuda et al.

5.0 g of the epoxy particles and 50 g of 1,4-dioxane wasadded, and the reaction mixture was stirred at 200 rpm and708C for 24 h. The particles that had the amino group(amino particles) were collected and washed with distilledwater by the same method described above.

Reaction between the Amino Particles and Fatty AcidChloride

The amino particles were reacted with fatty acid chlorides in1,4-dioxane. One of the fatty acid chlorides of which themolar amount was 0.6–3 times that of the amino groups inthe amino particles, 3.0 g of the amino particles, and 100 gof 1,4-dioxane were taken into the 4-necked 300-mL glassflask, and the reaction mixture was stirred at 200 rpm and708C for 24 h. The particles (amphiphilic particles) werecollected and washed with distilled water by the samemethod described above.

To use the amphiphilic particles for immobilizing lipase,unreacted monomer and impurities must be completelyremoved. For this purpose, the amphiphilic particles werepacked in a glass column (1.0 cm ID610 cm) and 200 mL of0.05 n HCl, 200 mL of distilled water, 200 mL of methanol and200 mL of 10 mm phosphate buffer (pH 5.5) were flowedthrough the column at a flow rate of 3.33610–9 m3 N s–1.

Particle Characterization

The particle diameter distribution of the aqueous suspensionwas measured using a laser particle diameter analyzerMICROTRAC FRA (Leeds & Northrup, Sumneytown Pike,USA). Scanning electron micrographs (SEMs) were takenusing a HITACHI S-2150 instrument (Tokyo, Japan). Thepore area and pore volume of the polymer particles weremeasured using an automated mercury porosimeter Autopore9500 (Shimadzu, Kyoto, Japan). The amount of the epoxygroups in the particles was measured by the HCl-dioxanemethod.[19]

Measurement of the Protein Concentration and theHydrolytic Activity

The protein concentration was determined by the method ofBradford.[20] The hydrolytic activities of lipase were assayedby the BALB-DTNB method[21] and emulsificationmethod.[15]

BALB-DTNB Method

The BALB-DTNB method was employed for measuring thehydrolytic activities of the immobilized lipases and nativelipase dissolved in buffer solutions. In this method, 2,3-dimercaptopropan-1-ol tributyroate (BALB) was used as asubstrate and 5,59-dithiobis(2-nitrobenzoic acid) (DTNB)was used to measure the hydrolyzed BALB by colorimetry.One unit (U) of the hydrolytic activity was defined as theamount of enzyme that liberated 1 lmol of SH groups permin at 30 8C.

Emulsification Method

The emulsification method was used to measure the hydroly-tic activities of the immobilized lipases prepared with var-ious particles. This method was based on the measurementof fatty acids liberated from olive oil by the hydrolysis withlipase. One unit (U) of the hydrolytic activity was defined asthe amount of enzyme that liberated 1 lmol of a fatty acidper min at 37 8C.

Lipase Immobilization

10 mg of the polymer particles was taken into 1 ml of 10 mm

potassium phosphate buffer (pH 5.5) containing 0.50 mg ofR. chinensis lipase or 3.78 mg of R. delemar lipase. Theresulting suspension was incubated at 48C for 24 h. Then theparticles were collected from the suspension by filtration.The particles collected on filter paper were washed with 10mL of 10-mm potassium phosphate buffer (pH 5.5). Theamount of lipase immobilized on the polymer particles wasdetermined by measuring the difference between the proteinconcentrations of the enzyme solution before adding polymerparticles and of the filtrates.

In the case of the measurement of the adsorption iso-therms, the lipase concentration in the phosphate buffer var-ied from 0.25 kg N m–3 to 8 kg N m–3 and temperature variedfrom 48C to 158C.

Measurement of the Transesterification Activity

The transesterification activities of lyophilized lipases andthe immobilized lipases prepared with various particles weremeasured in hexane. The lyophilized lipases were preparedas follows: 10 ml of 10 mm potassium phosphate buffer (pH5.5) containing 5.0 mg of R. chinensis lipase or 37.8 mg ofR. delemar lipase was well mixed with a magnetic stirrer andthen lyophilized in a freeze-drying apparatus FD-5N (TokyoRikakikai Co., Tokyo, Japan). The immobilized lipases pre-pared with 100 mg of various amphiphilic particles werealso lyophilized.

The transesterification activities were determined by meas-uring the extent of the transesterification of olive oil andfatty acid methyl esters.[12] Water contained in hexane wasremoved using molecular sieves. Methyl laurate and methylpalmitate were used as fatty acid methyl esters for activitymeasurements of the R. chinensis lipase and the R. delemarlipase, respectively. One unit (U) of the transesterificationactivity was defined as the amount of enzyme that produced1 lmol of methyl oleate per min at 378C.

Stabilities of Immobilized Lipases

The thermostability and pH stability of the immobilizedlipases prepared with monodisperse amphiphilic particleswere measured using the method described in a previousreport.[12]

To study stability against hexane, the immobilized lipasesprepared with 50 mg of the amphiphilic particles were lyo-philized. The lyophilized samples of the immobilized lipaseswere suspended in 3 mL of hexane and the suspension wasincubated at 378C for 72 h. The immobilized lipases were


then collected by filtration at various times and were dried toremove hexane. After drying, the hydrolytic activities of theimmobilized lipases were measured.

Results and Discussion

Synthesis and Characterization of MonodisperseAmphiphilic Particles

Monodisperse seed particles were synthesized by the dis-persion polymerization of styrene. The diameter andcoefficient of variation of the synthesized particles were2.21 lm and 9.6%, respectively. The monodisperse poly-styrene seed particles were first swollen with lauryl chlor-ide and then with the acrylic monomers, GMA and AMA.The produced monodisperse reactive polymer particles(epoxy particles) had epoxy groups from GMA. The con-versions of monomer to polymer at 24 h were 95–98%.The average particle diameter of the synthesized mono-disperse epoxy particles was 7.80 lm and the coefficientof variation was 12.4%. The molar amount of the epoxygroups in the particles was 1.92 mol N (kg particle)–1. Thediameter of the monodisperse epoxy particles was about1/36 of that of the polydisperse epoxy particles synthe-sized in our previous paper.[12] However, the content ofthe epoxy groups in the monodisperse epoxy particleswas almost the same as that in the polydisperse epoxyparticles.

When the monodisperse epoxy particles were reactedwith guanidine carbonate to produce amine particles,about 38% of the epoxy groups in the epoxy particleswere reacted. This result was in agreement with the caseof the polydisperse epoxy particles.[12]

When the polydisperse amino particles were reactedwith a fatty acid chloride, one mole of the hydroxylgroups and one mole of the amino groups in the aminoparticles could react with about 3 mol of a fatty acidchloride.[12] In the present study, the amount of a fattyacid chloride reacted with the hydroxyl groups and guani-dino groups in the monodisperse amino particles wasalmost the same as in the case of the polydisperse aminoparticles.

From the results of the reaction of the epoxy particleswith the guanidine carbonate and the reaction of theamino particles with a fatty acid chloride, it was con-cluded that the preparation method of the epoxy particleshad no effect on the amount of the amphiphilic groups inthe amphiphilic particles.

The amino particles in which the guanidino group wasintroduced were named as GA particles. Using the num-ber n of carbon atoms in the chain of fatty acid chloride,the particles formed by the reaction with the GA particleswere denoted as GA-Cn particles.

Figure 1 shows SEM images of the synthesized seedparticles, monodisperse epoxy particles, monodisperseGA particles and monodisperse GA-C18 particles. Using

the monodisperse polystyrene seed particles as the start-ing particles, the monodisperse amphiphilic particlescould be synthesized. The average particle diameter, stan-dard deviation of the average particle diameter, and thecoefficient of variation of the average particle diameterare summarized in Table 1. The average particle diameterof the epoxy particles was 3.5 times that of the seed parti-cles. Although the coefficient of variation had slightlyincreased during the three reactions, monodisperseamphiphilic particles with an average particle diameter of7.92 lm were obtained.

The distributions of the pore area and the pore volumeof the amphiphilic particles affect the amount of theenzyme immobilized and the mass transfer of substrates.Therefore, the distributions of the pore area and the porevolume of the monodisperse GA-C18 particles and thepolydisperse GA-C18 particles which were synthesized inthe previous paper[12] were measured. The distributions ofthe pore volume of the monodisperse GA-C18 particlesand the polydisperse GA-C18 particles are shown in Fig-ure 2. The data contain the void volume between the par-ticles in addition to the pore volume. Judging from theaverage particle diameter, the pores whose diameter wasgreater than 1.0 lm were interstices. Therefore, the poreswhose diameter was smaller than 1.0 lm were taken intoconsideration in the case of the monodisperse GA-C18particles. In the case of the polydisperse GA-C18 parti-cles, the pores whose diameters were smaller than 40 lm

Figure 1. SEM micrographs of (a) seed particles, (b) epoxyparticles, (c) GA particles and (d) amphiphilic particles.

Table 1. Particle diameters, standard deviations and copeffi-cients of variation of various particles.

Particles Particlediameter

lm

Standarddeviation

lm

Coefficientof variation

%

Polystyrene 2.21 0.21 9.6Epoxy 7.80 0.97 12.4GA 8.78 2.02 23.0Amphiphilic (GA-C18) 7.92 1.45 18.3


were taken into consideration. In both the GA-C18 parti-cles, meso pores with diameters ranging from 3 nm to 20nm were found and the volume of the polydisperse GA-C18 particles was slightly greater than that of the mono-disperse GA-C18 particles. However, macro pores withdiameters ranging from 50 nm to 500 nm were foundonly in the monodisperse GA-C18 particles. The totalpore volumes in the monodisperse GA-C18 particles andthe polydisperse GA-C18 particles were 1.86610–4

m3 N (kg particle)–1 and 8.16610–5 m3 N (kg particle)–1,respectively. The total pore volume of the monodisperseGA-C18 particles was about 2.3 times that of the polydis-perse GA-C18 particles. This large pore volume in themonodisperse GA-C18 particles can be explained as fol-lows: in the seed polymerization, lauryl chloride wasused to increase the amount of monomer swollen in theseed particles because lauryl chloride is a poor solvent forthe produced polymer in polymerization, which resultedin the formation of macro pores with diameters rangingfrom 50 nm to 500 nm.

Bulk density, true density, and porosity of the monodis-perse GA-C18 particles were 983 (kg particle) N m–3,1200 (kg particle) N m–3 and 18.3%, respectively. Bulkdensity, true density, and porosity of the polydisperseGA-C18 particles were 986 (kg particle) N m–3, 1 030 (kgparticle) N m–3 and 4.57%, respectively.

Figure 3 compares the distribution of the pore area inthe monodisperse GA-C18 particles with that in the poly-disperse GA-C18 particles. The total pore areas (internalsurface area) of the monodisperse and polydisperse GA-C18 particles were 2.456103 m2 N (kg particle)–1 and2.806103 m2 N (kg particle)–1, respectively. Although thetotal pore volume in the monodisperse GA-C18 particleswas larger than that in the polydisperse GA-C18 particles,the internal surface area in the monodisperse GA-C18particles was smaller than that in the polydisperse GA-C18 particles. This was because the volume of meso

pores with diameters of 3 nm to 20 nm in the polydisperseGA-C18 particles was slightly larger than that in themonodisperse GA-C18 particles. The external surfaceareas of the monodisperse and polydisperse GA-C18 par-ticles were 7.716102 m2 N (kg particle)–1 and 2.17610m2 N (kg particle)–1, respectively. The specific surfaceareas, which included the external surface area, of themonodisperse and polydisperse GA-C18 particles were3.226103 m2 N (kg particle)–1 and 2.82610 m2 N (kg par-ticle)–1, respectively. The specific surface area of themonodisperse GA-C18 particles was 1.14 times that ofthe polydisperse GA-C18 particles.

The Effect of the Amount of Amphiphilic Group in theAmphiphilic Particles on the Amount of LipaseImmobilized

In the previous paper, it was found that the kind of aminogroup and the number of carbon atoms in the chain of theacyl group of the polydisperse amphiphilic particlesaffected the amount of lipase immobilized and the stabi-lity and activity of the immobilized lipase.[12]

In the present work, the effect of the amount of theamphiphilic groups in the amphiphilic particles on theamount of lipase immobilized was studied. The monodis-perse GA-C18 particles in which the amounts of the gua-nidino group and the stearoyl group were varied weresynthesized. To express the amount of functional groups,the ratio (amphiphilic ratio) of the introduced amount ofthe acyl groups to that of the guanidino groups wasdefined.

Two types of monodisperse GA-C18 particles weresynthesized, in which the amounts of the guanidino groupwere 3.52610–1 mol N (kg particle)–1 (A-1 particles) and7.30610–1 mol N (kg particle)–1 (A-2 particles), both of

Figure 2. Distribution of the pore volume.

Figure 3. Distribution of the pore area.


which had an amphiphilic ratio of 3.0. The amount of R.delemar lipase immobilized on the A-1 and A-2 particleswas 2.26610–2 kg N (kg particle)–1 and 8.15610–2

kg N (kg particle)–1, respectively. Although the amount ofthe guanidino group in the A-2 particles were about 2times those of the A-1 particles, the amount of lipaseimmobilized on the A-2 particles was about 3.6 times thaton the A-1 particles. This result suggested that theamount of the stearoyl group of the A-2 particles, whichwas about 6 times those of the A-1 particles, affected theamount of lipase immobilized.

To study the effect of the amount of the acyl groups ofthe amphiphilic particles on the amount of lipase immobi-lized, monodisperse GA-C18 particles were synthesizedof which the amount of the guanidino group was7.30610–1 mol N (kg particle)–1, and the amphiphilic ratioranged from 0 to 3.0. Figure 4 shows the effect of theamphiphilic ratio of the amphiphilic particles on theamount of lipase immobilized. The amount (1.66610–2

kg N (kg particle)–1) of lipase immobilized on the epoxyparticles was taken as 1.0. The amount of lipase immobi-lized increased with increase in the amphiphilic ratio ofthe GA-C18 particles. In the previous study,[12] it wasfound that the amino groups in the amphiphilic particlesaffected the amount of lipase immobilized. In addition tothe importance of the amino groups in the amphiphilicparticles, the results shown in Figure 4 and those obtainedusing the A-1 and A-2 particles indicated that the acylgroups in the amphiphilic particles play a very importantrole in the lipase immobilization. Since the amount oflipase immobilized on the GA-C18 particles of which the

amount of the guanidino group was 7.30610–1 mol N (kgparticle)–1 and the amphiphilic ratio was 3.0 was highest,hereafter this particle was used for immobilizing R. dele-mar lipase.

Effect of Lipase Concentration on the Amount ofLipase Immobilized

The activity of the immobilized enzyme is affected by theamount of enzyme immobilized.[22] Therefore, the effectof the lipase concentration in the buffer solution on theamount of lipase immobilized was studied. Figure 5shows the adsorption isotherms of R. delemar lipase onthe monodisperse GA-C18 particles measured at 4, 10,and 158C. CE is the lipase concentration in the phosphatebuffer (pH 5.5) and q the amount of lipase immobilizedon the GA-C18 particles. The experimental data obtainedat a constant temperature could be correlated well with aline given by the following Langmuir equation (Langmuiradsorption isotherm):

q ¼ qmKCE

1þ KCEð1Þ

where qm is the saturated or maximum amount of adsorp-tion and K the Langmuir equilibrium constant. As can beseen in Figure 5, the adsorption isotherm of R. delemarlipase on the GA-C18 particles obeys the Langmuir equa-tion. The values of qm at 48C, 108C and 158C were1.14610–1 kg N (kg particle)–1, 1.03610–1 kg N (kg parti-cle)–1, and 5.50610–2 kg N (kg particle)–1, respectively.

Figure 4. The effect of the amphiphilic ratio of the GA-C18particles on the amount of lipase immobilized. To 1 mL of 10mm phosphate buffer (pH 5.5) containing 3.78 kg N m–3 lipase,10 mg of GA-C18 particles was added. The resulting mixturewas incubated at 4 8C for 24 h, and then the amount of lipaseimmobilized was determined by measuring the protein concen-tration of the lipase remaining in the supernatant.

Figure 5. Adsorption isotherm of R. delemar lipase on the GA-C18 particles. To 1 mL of 10 mm phosphate buffer (pH 5.5) con-taining 0.25–8.0 kg N m–3 lipase, 10 mg of GA-C18 particles wasadded. The resulting mixture was incubated at 4–15 8C for 24 h,and then the amount of lipase immobilized was determined bymeasuring the concentration of the lipase remaining in thesupernatant.


Hydrolytic Activity of Lipase Immobilized on theMonodisperse GA-C18 Particles

Figure 6 shows the effect of the amount of R. delemarlipase immobilized on the monodisperse GA-C18 parti-cles on the hydrolytic activity. The hydrolytic activity ofthe immobilized lipase increased with increase in theamount of lipase immobilized. On the other hand, thespecific hydrolytic activity of the immobilized lipasedecreased with an increase in the amount of lipase immo-bilized. The specific hydrolytic activity of R. delemarlipase in the 10 mm phosphate buffer (pH 5.5) was3.206106 U N kg–1. The specific hydrolytic activities ofthe immobilized lipases prepared with the monodisperseGA-C18 particles were lower than that of free nativelipase. These results may suggest that the internal diffu-sional limitations affect the activities of the immobilizedlipases prepared with the monodisperse GA-C18 parti-cles. However, according to Lima et al.,[22] lipase wasmainly located on the external surface of the support andthe internal diffusional limitations were not importantwhen Mucor miehei lipase was immobilized on a porousanion exchange resin having meso pores with diametersof 4–9 nm. The monodisperse GA-C18 particles have notonly meso pores but also macro pores with diameters of50 nm to 500 nm, as described above.

To study quantitatively the effect of the diffusional lim-itations of the substrate on the hydrolytic activity of theimmobilized lipase, the hydrolysis reactions of BALBwere conducted using the immobilized lipases preparedwith monodisperse GA-C18 particles with diameters of

5.86 lm (particle 1) and 7.92 lm (particle 2). The amountsof lipase immobilized were 7.01610–2 kg N (kg particle)–1

(particle 1) and 7.08610–2 kg N (kg particle)–1 (particle 2)and the hydrolytic activities of the immobilized lipaseswere 1.286105 U N (kg particle)–1 (particle 1) and1.166105 U N (kg particle)–1 (particle 2) at 378C and 30rpm. When the stirring speeds of the reaction mixturechanged from 30 rpm to 200 rpm, the hydrolytic activitiesof the immobilized lipases were almost unchanged. Thisresult indicated that the external diffusional limitations ofthe substrate were negligible under these conditions.

Next, we studied the effect of internal diffusion limita-tions on the hydrolytic activity. The hydrolysis reactionof BALB catalyzed by lipase can be regarded to be firstorder with respect to the BALB concentration.[21] There-fore, the rate of the hydrolysis reaction catalyzed by animmobilized lipase is given by:[23–25]

ÿrA ¼ gk1CAS ð2Þ

when k1 is the rate constant of the hydrolysis reaction andCAS the BALB concentration at the external surface. g inEquation (2) is the catalytic effectiveness factor for aspherical catalyst and is given by:

g ¼ 1b

1tanh ð3bÞ ÿ

13b

� �ð3Þ

b is a dimensionless parameter (Thiele modulus) and isdefined for a spherical catalyst in which the reactionobeys irreversible first-order kinetics as:

b ¼ dp

3

ffiffiffiffiffiffiffiffiffiffiffik1qp

DeA

sð4Þ

where dp is the particle diameter, DeA the effective diffu-sivity of BALB in the particle and qp the density of thesupport particles.

The rates of the hydrolysis reaction (the hydrolyticactivities: 1U = 1.67610–8 mol N s–1) mentioned abovewere analyzed by the trial and error method using Equa-tion (2)–(4). The catalytic effectiveness factors of theimmobilized lipases prepared with the GA-C18 particleswere g1 = 0.860 and g2 = 0.779, respectively. The sub-scripts 1 and 2 refer to the parameter of the particle 1 andthat of the particle 2, respectively. These results indicatedthat the internal diffusional limitations of the substrate inthe particles affected the hydrolytic activity of the immo-bilized lipases. Therefore, the decrease in the specificactivity of the immobilized lipases with increase in theamount of lipase immobilized was partly explained bythe internal diffusional limitations of the substrate in theparticles.

For the above analysis, the rate constant k1 and theeffective diffusivity DeA of the BALB in the particleswere estimated to be 1.38610–3 m3 N (kg particle)–1 N s–1

Figure 6. Hydrolytic activities of the immobilized lipases pre-pared with the monodisperse GA-C18 particles. The immobi-lized lipases were prepared at 4 8C under the different lipaseconcentrations of 0.25–8.0 kg N m–3. The specific hydrolyticactivities of the immobilized lipases were calculated from theamount of lipase immobilized and the hydrolytic activities ofthe immobilized lipases.


and 1.77610–11 m2 N s–1, respectively. The rate constantof the hydrolysis reaction in bulk phase (2.96610–2 m3

N kg–1 N s–1) was about 1.5 times that of the immobilizedlipase (1.97610–2 m3 N kg–1 N s–1; this value was obtainedby dividing k1 by the amount of lipase immobilized onparticle 1). This may be because some amount of theimmobilized lipase could not contact with substrate, orthe specific activity of lipase immobilized was decreased.The effective diffusivity DeA of the BALB in the particleswas estimated by the parallel pore model (DeA = (e/s)DA).[25, 26] When the diffusivity (DA = 5.15610–10

m2 N s–1) of BALB in water estimated by the Wilke-Changmethod[27] in which the molar volume of BALB was esti-mated by the Le Bass additive volume table,[28] and thetortuosity (s = 6.0) and the porosity (e = 0.183) wereused, the effective diffusivity of BALB in the particleswas estimated to be 1.57610–11 m2 N s–1, the value beingalmost the same as that estimated above (1.77610–11

m2 N s–1). These results suggested that the above analysisof the effect of internal diffusion limitations on the hydro-lytic activity was reasonable.

Effect of the Surface Area of the Monodisperse GA-C18 Particles on the Amount of Lipase Immobilized

When the polydisperse GA-C18 particles were used forimmobilizing R. delemar lipase, the amount of lipaseimmobilized was 8.90610–4 kg N (kg particle)–1.[12] Asdescribed above, the amount of lipase immobilized on themonodisperse GA-C18 particle, of which the amount ofthe amphiphilic groups was almost the same as the poly-disperse GA-C18 particles, was 8.15610–2 kg N (kg parti-cle)–1. The amount of lipase immobilized on the monodis-perse GA-C18 particles was about 92 times that of thepolydisperse GA-C18 particles. As described in the sec-tion regarding particle characterization, the specific sur-face area of the monodisperse GA-C18 particles was 1.14times that of the polydisperse GA-C18 particles. If lipasewas adsorbed only on the external surface of the supportas described by Lima et al.,[22] the amount of lipase immo-bilized was proportional to the external surface area.However, the external surface area of the monodisperseGA-C18 particles was 36 times that of the polydisperseGA-C18 particles. Therefore, the surface of the macropores with diameters of 50 nm to 500 nm and the externalsurface were responsible for the increase in the amount ofthe lipase immobilized.

Comparison of the Hydrolytic Activity of theImmobilized Lipase Prepared with the MonodisperseGA-C18 Particles with Those of Immobilized LipasePrepared with Preexisting Supports

Table 2 compares the hydrolytic activity of the immobi-lized lipase prepared with the monodisperse GA-C18 par-

ticles with those of the immobilized lipases prepared withpolydisperse GA-C18 particles,[12] Dowex MWA-1, por-ous glass beads, and Sepharose 4B.[15] The latter threeimmobilized R. delemar lipases were believed to exhibitthe highest hydrolytic activities.[14] Since three hydrolyticactivities were measured by the emulsification method,[15]

the hydrolytic activity of the immobilized lipase preparedwith the monodisperse GA-C18 particles were also mea-sured by the emulsification method. The hydrolytic activ-ity of the immobilized lipase prepared with the monodis-perse GA-C18 particles was 150–8700 times higher thanthose prepared with the others. The high hydrolytic activ-ity of the immobilized lipase prepared with the monodis-perse GA-C18 particles was probably attributed to thefact that the monodisperse GA-C18 particles could immo-bilize a large amount of lipase.

Stabilities and Transesterification Activity of theImmobilized Lipase Prepared with the MonodisperseGA-C18 Particles

The thermostability and pH stabilities of the immobilizedR. delemar lipase prepared with the monodisperse GA-C18particles were enhanced similarly to the results observed inthe previous report.[12] The half-lives of the activities of theimmobilized R. delemar lipases in hexane prepared withthe GA-C18 and epoxy particles were 68.5 h and 12.5 h,respectively. The lipase immobilized on the GA-C18 parti-cles exhibited much higher stability in hexane comparedwith the lipase immobilized on the epoxy particles.

Using 100 mg of lyophilized sample of the immobi-lized R. delemar lipase prepared with the GA-C18 parti-cles and 5 mg of lyophilized lipase, the transesterificationreactions between olive oil and methyl palmitate werecarried out in hexane. The specific hydrolytic activities ofthe lyophilized lipase and the immobilized lipase andtheir specific transesterification activities are comparedin Table 3. Although the specific hydrolytic activity ofthe immobilized lipase was about 0.58 times that of thelyophilized lipase, the specific transesterification activityof the former lipase was 93.4 times higher than that of thelatter lipase. The very high transesterification activity ofthe immobilized lipase was attributed to the fact that it

Table 2. Comparison of the hydrolytic activities of immobi-lized lipases prepared with various supports.

R. delemar lipase immobilized on: Hydrolyticactivity

U N ðkg particleÞÿ1

Monodisperse GA-C18 particles 6.126105

Polydisperse GA-C18 particles 5.246103

Dowex MWA-1a) 7.006103

Porous glass beadsa) 1.106103

Sepharose 4Ba) 4.006103

a)Reported in ref.[15]


disperses well in n-hexane whereas the lyophilized lipasedoes not.

Immobilization of R. chinensis Lipase on theMonodisperse Amphiphilic Particles

The GA-C18 particles in which a guanidino group and astearoyl group were introduced were the best amphiphilicparticles as the support of R. delemar lipase for use in thetransesterification reactions in hexane.[12] To studywhether this amphiphilic group was suitable for immobi-lizing other lipases, the effect of the hydrophilic aminopart and the hydrophobic acyl part of the amphiphilicgroups on the amount of R. chinensis lipase immobilized(the specific hydrolytic activity was 6.596106 U N kg–1)was studied. The GA-C14 particles in which guanidinogroups and myristoyl groups were introduced were thebest particles as the support of R. chinensis lipase. Theamount of lipase immobilized on the GA-C14 particleswas 2.74610–2 kg N (kg particle)–1. The half-lives of theimmobilized lipases prepared with the epoxy particlesand the monodisperse GA-C14 particles in hexane were18.5 h and 99.2 h, respectively. The transesterificationactivity and specific transesterification activity of theimmobilized lipase prepared with the monodisperse GA-C14 particles were 1.046105 U N (kg particle)–1 and3.816106 U N kg–1 in hexane, respectively. The specifictransesterification activity of the immobilized lipase pre-pared with the monodisperse GA-C14 particles was 81times higher than that of the lyophilized R. chinensislipase (4.706104 U N kg–1). Although the best acyl groupwas different depending on lipase, the monodisperseamphiphilic particles that had the hydrophilic guanidinogroup and hydrophobic acyl group were useful as the sup-port of lipase for use in the transesterification reactions inorganic solvents.

ConclusionsTo develop an excellent support for lipase immobiliza-tion, the monodisperse amphiphilic particles that had

both the hydrophilic guanidino groups and hydrophobicacyl groups were synthesized and characterized. Mono-disperse amphiphilic particles with an average particlediameter of 7.92 lm were obtained and the synthesizedmonodisperse amphiphilic particles had macro pores withdiameters ranging from 50 nm to 500 nm.

The amount of the amphiphilic groups of the GA-C18particles affected the amount of R. delemar lipase immo-bilized. The adsorption of R. delemar lipase on the GA-C18 particles obeyed the Langmuir adsorption isotherm.Although the hydrolytic activity of the immobilizedlipase increased with increase in the amount of lipaseimmobilized, the apparent specific hydrolytic activity ofthe immobilized lipase decreased with increase in theamount of lipase immobilized. Estimation of the catalyticeffectiveness factor of the immobilized lipase showedthat the internal diffusional limitations of the substrate inthe particles affected the hydrolytic activity of the immo-bilized lipase and resulted in the decrease in the apparentspecific activity of the immobilized lipase.

The hydrolytic activity of the immobilized lipase pre-pared with the monodisperse GA-C18 particles was 150–8700 times higher than those of the immobilized lipasesprepared with Dowex MWA-1, porous glass beads, andSepharose 4B. The stability and the transesterificationactivity of lipase were enhanced by immobilizing lipaseon the monodisperse GA-C18 particles. The specifictransesterification activity of the immobilized lipase pre-pared with the GA-C18 particles was 93.4 times higherthan that of the lyophilized lipase.

When R. chinensis lipase was immobilized on theamphiphilic particles, the GA-C14 particles in which gua-nidino groups and myristoyl groups were introduced werethe best particles, whereas the GA-C18 particles were thebest particles as the support of R. delemar lipase. How-ever, from the results obtained using R. delemar lipaseand R. chinensis lipase, it was found that the monodis-perse amphiphilic particles which have hydrophilic gua-nidino groups and hydrophobic acyl groups were usefulas the support of lipase for the transesterification reac-tions in organic solvents.

In the present work, the surface area of the macro poreswith diameters of 50 nm to 500 nm and the external sur-face area were important to increase the amount of lipaseimmobilized. From an industrial viewpoint, it is neces-sary to develop immobilized lipases of which the amountof lipase immobilized is higher. This is expected to beaccomplished by producing porous particles.

NomenclatureCA substrate concentration [mol N m–3]CAS substrate concentration at the particle surface

[mol N m–3]CE lipase concentration in phosphate buffer [kg N m–3]

Table 3. Comparison of the specific activities if immobilizedlipase and native lipase.

HydrolyticactivityU N kgÿ1

TransesterificationactivityaÞ

U N kgÿ1

Immobilized R. delemar lipaseprepared with GA-C18 particles

1.186106 4.116106

Lyophilized R. delemar lipase 3.206106 4.406104

a) Using a freeze-dried sample (100 mg) of the immobilizedlipase (8.15610–2 kg N (kg particle)–1) prepared with theGA-C18 particles and 5 mg of lyophilized native lipase, thetransesterification reactions between olive oil and methyl pal-mitate were carried out in hexane at 500 rpm and 37 8C for5 h.


DA diffusivity of substrate in water [m2 N s–1]DeA effective diffusivity of substrate in particles [m2 N s–1]dp particle diameter [lm]K Langmuir equilibrium constant [–]k1 rate constant of hydrolysis reaction [m3 N (kg parti-

cle)–1 N s–1]q amount of lipase adsorbed on the particle [kg N (kg

particle)–1]qm saturated or maximum amount of lipase adsorbed

[kg N (kg particle)–1]–rA rate of hydrolysis reaction [mol N (kg particle)–1 N s–1]e porosity of spherical catalyst [–]b Thiele modulus [–]g catalytic effectiveness factor [–]qp bulk density of support particles [(kg particle) N m–3]s tortuosity [–]

Acknowledgement: This work was supported in part by a Pro-posal-Based Immediate-Effect R & D Promotion Program fromthe New Energy and Industrial Technology Development Orga-nization of Japan (NEDO, project ID 98Z36-013-1) and Grant-in-Aids for Scientific Research from the Ministry of Education,Science, Sports and Culture of Japan (No. 07750889, 08750896,11555209, 12750682).

Received: June 7, 2001Revised: September 3, 2001

Accepted: September 5, 2001

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