Biotechnology Letters22: 1953–1958, 2000.© 2000Kluwer Academic Publishers. Printed in the Netherlands.

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New immobilization method for enzyme stabilization involving amesoporous material and an organic/inorganic hybrid gel

Bo Li & Haruo Takahashi∗Toyota Central R&D Labs., Inc., 41-1, Yokomichi, Nagakute, Aichi 480-1192, Japan∗Author for correspondence (Fax: +81-561-63-6498; E-mail: e1092@mosk.tytlabs.co.jp)

Received 14 August 2000; Revisions requested 7 September 2000; Revisions received 5 October 2000; Accepted 6 October 2000

Key words:horseradish peroxidase, hybrid gel, immobilization, mesoporous materials

Abstract

Horseradish peroxidase (HRP) was immobilized in a mesoporous material (folded sheets mesoporous ma-terials, FSM-16) and then entrapped in organic/inorganic hybrid gel comprising various molar ratios ofdimethyldimethoxysilane (DMDMOS)/tetramethoxysilane (TMOS). When pore size of FSM-16 materials is muchlarger than the diameters of horseradish peroxidase (HRP), the residual enzymatic activity after thermal treatment(70◦C, 60 min) increased from 73 to 99%, and the oxidative conversion yield of 1,2-diaminobenzene in an organicsolvent increased from 59 to 79% after 4 h and the level of leakage of immobilized HRP decreased from 6 to 1.5%on washing by secondary hybrid gel entrapment comprising a molar ratio of DMDMOS/TMOS=1:3. When poresize of FSM-16 materials just matches the diameter of the enzyme, the conversion yield in an organic solvent andthe level of leakage of immobilized HRP did not change so much.

Introduction

In recent years, much attention has been focused onthe application of sol-gel techniques to the entrap-ment of proteins including enzymes (Kawakami &Yoshida 1995, Kawakami 1996, Shen & Tu 1999),microorganisms (Ellerbyet al. 1992, Gill & Balles-teros 1998), and cells for the development of novelbiosensors (Williams & Hupp 1998), and immobilizedbiocatalysts (Kawakamiet al. 1992, Gimon-Kinselet al.1998), etc. Sol-gel methodology, which has beendeveloped in the area of new materials science andtechnology, has now made it possible to entrap vari-ous biomolecules in inorganic silica matrices (Ellerbyet al. 1992). The advantages of using sol-gel materi-als for the mechanical entrapment of enzymes are thatthey permit the stabilization of the tertiary structuresof protein molecules because of the tight gel structureand the easy insertion of substituting groups into a sil-ica matrix, which may provide the entrapped enzymeswith beneficial microenvironments. Bio-catalysis inorganic media has been a major research subject inthe field of enzyme engineering. For example, per-

oxidases, which catalyze the oxidative reaction ofsubstrates, such as lignin or dioxin, have been studied(Bliukovskyet al.1994).

On the other hand, orderly mesoporous materialswith uniform pore diameters of 15–300 Å have beenreported (Becket al.1992, Cormaet al.1997, Inagakiet al. 1996, Zhaoet al. 1998). Because these porediameters approximate to the molecular diameters ofenzymes, their application as useful enzyme carriershas been suggested (Thomas 1994). We previouslyreported that the immobilization of horseradish per-oxidase (HRP) in mesoporous materials with suitablemesopore sizes gave the best stability (Takahashiet al.2000). In this work, we present a new method forthe stabilization of enzymes, the first step being theimmobilization of an enzyme in the pores of a meso-porous material such as folded sheets mesoporousmaterials (FSM-16), and the second step being the en-trapment of the enzyme molecules in the mesoporesusing an organic/inorganic hybrid gel. The mecha-nisms of the thermal stabilization and the increase inconversion yield in organic media with this method arealso discussed.

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Materials and methods

Materials

Horseradish peroxidase (HRP) was purchased fromCalzyme Labs., Inc. FSM-16 materials with aver-age pore diameters of 51 Å (FSM-16/50) and 89 Å(FSM-16/90) were prepared from kanemite usinghexadecyltrimethyl ammonium chloride and 1,3,5-triisopropylbenzene (TIPB) in the molar ratio ofTIPB/surfactant= 3:1, and dococyltrimethyl am-monium chloride and TIPB in the molar ratio ofTIPB/surfactant= 4:1, respectively, according to themethod reported previously (Takahashiet al. 2000).An intense (100) diffraction peak and three otherpeaks (110, 200 and 210) of highly arranged ordersconnecting hexagonal structures were observed. Thenitrogen adsorption isotherms at 77 K were mea-sured with a Quantachrome Autosorb-1. The datawere analyzed by the BJH (Barrett–Joyner–Halenda)method using the Halsey equation for multiplayerthickness. The pore size distribution curve came fromthe analysis of the adsorption branch of the isotherm,and the pore volume was taken at the P/Po (relativepressure) where the isotherm sharply increased. Thespecific surface areas were calculated by the BET(Brunauer–Emmett–Teller) equation using the adsorp-tion data at the range from P/Po= 0.05 to P/Po justbelow the capillary condensation. Tetramethoxysi-lane (TMOS), methyltrimethoxysilane (MTrMOS),dimethyldimethoxysilane(DMDMOS), and trimethyl-methoxysilane (TrMMOS), as gel formation materials,were obtained from Tokyo Chemical Industries, Co.,Ltd. and Wako Pure Chemical Industries, Co., Ltd.

Enzyme immobilization in mesoporous materials

An immobilized enzyme was prepared according tothe following procedure. FSM-16 powder (250 mg)was added to 5 ml of a 10 mg HRP ml−1 water in acentrifuge tube. The mixture was stirred at 4◦C for16 h and then centrifuged at 20 000× g for 10 min at4◦C, the resulting pellet being washed with deionizedwater three times and then vacuum-dried.

Secondary gel entrapment of the FSM- immobilizedenzyme

Sol solutions were prepared by mixing various or-ganic silanes (MTrMOS, DMDMOS, or TrMMOS)and TMOS in certain molar ratios. The molar ratios of

organic silane to inorganic silane (TMOS) were 3:1,2:2, 1:3 and TMOS only, respectively.

As a typical example, 2.72 mmol DMDMOSand 8.16 mmol TMOS (DMDMOS:TMOS= 1:3,mol/mol) were dissolved in 0.4 ml of deionized water,and then 10µl 40 mM HCl was added with stirring toform a homogeneous sol at room temperature. Afterthe mixture had been cooled to 4◦C, 0.7 ml of 0.1 Mphosphate buffer (pH 7.5) was added and then 50 mgHRP/FSM-16 containing 9 mg HRP was mixed withthe sol solution. After 1 min stirring, the resultantmixture was left at room temperature over night. Thegels formed were vacuum-dried for 10 h after wash-ing three times with deionized water. Native HRP alsowas directly added to various sol solutions accordingto the above procedures. Each dried gel was washedwith deionized water with stirring for 1 h, and then theabsorbance at 405 nm was measured and the level ofleakage of the enzyme was calculated.

Thermal stability

The thermal stability of HRP immobilized in FSM-16(HRP/FSM-16) with various pore sizes or the gel-entrapped form was examined by means of phenolpolymerization (Klibanovet al. 1983). HRP/FSMpowder (5 mg) was added to 200µl of 50 mM sodiumacetate buffer (pH 4.0) in an Eppendorf tube, followedby heating at 70◦C for 30, 60, 90 or 120 min. Aftercentrifugal separation for 10 min, the precipitate waswashed twice with deionized water, and then 200µl of50 mM Tris-HCl buffer (pH 7.5), 4µl of an aqueoussolution of 5000 mg phenol l−1 and 1µl 30% (v/v)H2O2 were added to above the precipitate for the en-zymatic reaction at 37◦C for 30 min. After centrifugalseparation for 10 min, 50µl of the suspension wasadded to 50µl of 1% (w/w) potassium ferricyanidein 1 M glycine (pH 9.6) and 100µl 1% (w/w) 4-aminoantipyrine in 1 M glycine (pH 9.6). The residualactivity was determined spectroscopically by imme-diately measuring the absorbance of the reactant at490 nm.

Conversion yield in an organic solvent

For evaluation of the catalytic activity of HRP im-mobilized in mesoporous silica materials, the oxida-tive reaction of 1,2-diaminobenzene in toluene wasselected, tert-butylhydroperoxide being used as theoxidant (Kamiyaet al. 1997). A portion (20 ml) of50 mM 1,2-diaminobenzene in anhydrous toluene and5 ml of 1.1 M tert-butylhydroperoxide in decane were

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Fig. 1. Comparison of the entrapped ratios (closed symbols)and leakage ratios (open symbols) for HRP with various or-ganic/inorganic silane gels. ( , #) DMDMOS/TMOS, (�, �)TrMMOS/TMOS, and (N,4) MTrMOS/TMOS. The percentage ofordinate at the left indicated a ratio of gel-entrapped HRP amongtotal amount of HRP in the solution, and that at the right indicate aratio of leaked amount of HRP from the supports.

mixed in a triangular flask. The reaction was initiatedby adding a complex of solid immobilized HRP con-taining 0.5 mg protein with constant stirring at 37◦C.The conversion at 37◦C in 4 h via the oxidative reac-tion was determined spectroscopically by measuringthe absorbance of the supernatant at 470 nm. The cat-alytic activity of same weight of porous silica onlywas also measured as a background value. Changes inconversion yields after sample pretreatment in tolueneat 37◦C for 15 h were also determined with the aboveprocedures.

Results and discussion

HRP entrapment in an organic/inorganic hybrid gel

The effects of HRP entrapped in various or-ganic/inorganic hybrid gels are shown in Figure 1.By changing the ratio of organic silane [(n)methyl-(m)methoxysilane, nMmMOS] and monomer silane[tetramethoxysilane, TMOS], the morphology of thehybrid gel was changed.

The entrapment efficiency for HRP in the gel was75% (w/w) and the leakage on washing was about2% (w/w) with the monomer silane. The entrap-ment efficiency for HRP increased and the leakagedecreased with organic/inorganic hybrid gels. In thecase of nMmMOS:TMOS= 1:3 (mol/mol), the en-

trapment efficiencies increased to 88, 98 and 100%(w/w), and the leakage ratios decreased to 1.4, 0.2 and0.6% (w/w) for MTrMOS, DMDMOS and TrMMOS,respectively. In the case of nMmMOS:TMOS=2:2(mol/mol), the entrapment efficiencies were 93, 97 and83% (w/w), and the leakage ratios were 1.1, 2.0 and2.8% (w/w) for MTrMOS, DMDMOS and TrMMOS,respectively. If the proportion of organic silane wasincreased to nMmMOS:TMOS= 3:1 (mol/mol), theentrapment efficiency drastically decreased to lowerthan 65% (w/w) and the leakage ratio increased up to4% (w/w). When the HRP molecules were entrappedto the hybrid comprising DMDMOS:TMOS= 1:3 or2:2 (mol/mol), HRP showed the best stability, whichentrapment efficiencies were 99%, 98%, and leak-age ratios were 0.2%, 2%, respectively. Accordingto the above results, we used DMDMOS as the or-ganic silane component for further experiments. Thehydrophobicity of a gel will depend on the amount oforganic silane or chains of alkyl groups in the organicsilane. Many enzymes have a hydrophobic region, soa hydrophobic interaction may be important for thestabilization of enzymes. The organic silane content ina gel will be critical for the stabilization of enzymes.The hydrophobicity value of each enzyme is different,so the suitable organic silane content among a hybridgel will also be different for each enzyme.

HRP adsorption on mesoporous materials andsecondary entrapment in an organic/inorganic hybridgel

HRP was selectively adsorbed to mesoporous silicamaterials prepared with a cationic surfactant such asFSM-16 materials when the average mesopore sizewas larger than the diameters of enzyme (Takahashiet al. 2000). We synthesized FSM-16 with differentpore diameters, FSM-16/50 and FSM-16/90 (aver-age pore diameter 51 and 89 Å, BET surface area848 and 770 m2 g−1, total pore volume 0.84 and1.22 cm3 g−1, respectively). The FSM-16/90 mate-rial showed the larger amount of adsorption of HRP(180 mg g−1) than that for FSM-16/50 (130 mg g−1).HRP molecule is an elongated subject with the longand short axes of HRP molecule ca. 64 and ca. 37 Å,respectively. HRP molecule may be adsorbed to themesopore of FSM-16/90 from all directions but ad-sorbed to the mesopore of FSM-16/50 from limiteddirections. If pore diameter is much larger than thediameters of a enzyme such as FSM-16/90 material,a lot of free spaces would exist in the mesopore even

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Fig. 2. Comparison of the entrapped ratios (closed symbols) andleakage ratios (open symbols) for hybrid gel-entrapped HRP immo-bilized in FSM-16. ( ,#) Gel-entrapped HRP/FSM-16/50, (�,�)gel-entrapped HRP/FSM-16/90. The percentage of ordinate at theleft indicated a ratio of secondary gel-entrapped HRP among totalamount of HRP immobilized in FSM-16 materials, and that at theright indicate a ratio of leaked amount of HRP from the supports.

after enzyme loading, inducing environmental changein the pore easily. This may account for the lowerstability of HRP immobilized in FSM-16/90 than thatof FSM-16/50 in spite of larger amount of immo-bilized enzyme in FSM-16/90. We investigated thestabilization effects of a secondary gel-entrapment onFSM-16 immobilized HRP. The gel-entrapped ratiofor HRP immobilized in mesoporous material FSM-16/50, whose pore size is just matched to the diametersof HRP, and FSM-16/90, whose pore size is far largerthan the diameters of HRP, are shown in Figure 2.The gel entrapment ratios did not change in spite of achange in the organic/inorganic silane ratios, when theHRP molecules were adsorbed to FSM-16/50. HRPmolecules would form a stable complex with silanolresidues on the mesopore surface of FSM-16/50. Inthe case of HRP immobilized in FSM-16/90, the gelentrapment ratio decreased to 93% and the leakageratio drastically increased to 6% with the molar ra-tio of DMDMOS:TMOS= 3:1. When molar ratiosof DMDMOS:TMOS was 2:2 or 1:3, the entrappedenzyme showed the best entrapped ratio (more than98%) and the lowest leakage ratio (less than 1.5%).When the organic silane content was very high, for ex-ample nMmMOS:TMOS= 4:1 (mol/mol), a gel wasnot formed.

Table 1. Residual activity of immobilized HRP afterthermal treatment for 60 min at 70◦C.

Sample Residual activity (%)

Native HRP/ HRP/

HRP FSM-16/50 FSM-1690

Non-gelated 22 81 73

Gelate 60 98 99

Thermal stability of the entrapped enzyme

HRP entrapped in a hybrid gel or immobilized in amesoporous silica material was treated at 70◦C ina buffer solution, the residual activities being sum-marized in Table 1. In the case of HRP being di-rectly entrapped in a hybrid gel (DMDMOS:TMOS= 1:3, mol/mol), the residual activity was about 60%after heat-treatment for 60 min, while native HRPwas inactivated to lower than 22%. HRP immobi-lized in FSM-16/50 exhibited high residual activity(81% of the initial activity), and the residual activityof HRP immobilized in FSM-16/90 was 73%. Gel-entrapped HRP after immobilization in FSM-16/50or FSM-16/90 showed the highest residual activity(residual activity more than 98%). In this case, theorganic/inorganicsilane would permeate into the poresof the mesoporous silica materials and form a stablehybrid gel in the spaces between the enzyme mole-cules and the pore walls, so the stereo-structure of theenzyme may be kept and stabilized.

Enzymatic activities in organic reaction immobilizedenzyme

Figure 3 shows the conversion yields of 1,2-diaminobenzene through the oxidative reaction cat-alyzed by native HRP, or FSM-16 immobilized HRPentrapped in an organic/inorganic hybrid gel. The en-zyme content of each immobilized enzyme used forthe reaction was the same, 0.5 mg HRP, for comparingthe activities of the enzymes. Native HRP was quicklyinactivated in an organic solvent. The HRP entrappedin an organic/inorganic hybrid gel exhibited relativelyhigh conversion yield. In the case of HRP immobi-lized in FSM-16/50, the oxidative conversion yieldhad slightly decreased from 67% to 56% after gel-entrapment (4 h). In the case of HRP immobilized inFSM-16/90, conversion yield increased from 59% to79% after gel entrapment (4 h). The conversion yieldof the same weight of enzyme free hybrid gel was

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Table 2. Change of conversion yield (%) of 1,2-diaminobenzene with HRP immobilized by various methods.Samples were pretreated in toluence for 15 h at 37◦C.

Sample Native Before gelation After gelationb

HRP HRP/ HRP/ HRPc HRP HRP/ HRP/

FSM-16/50a FSM-16/90 TMOS FSM-16/50 FSM-16/90

Non-pretreatment 10 67 65 66 66 63 78

Pretreatment 2 64 50 28 50 61 70

aThe immobilized HRP in FSM-16.bSecondary organic/inorganic hybrid gel (DMDMOS:TMOS=1:3, mol/mol) entrapment of the FSM-16 immobi-lized enzyme.cGelated with only TMOS.

Fig. 3. Conversion yields of 1,2-diaminobenzene by various formsof immobilized HRP (0.5 mg each) in an organic solvent.( ) HRP/FSM90/gel, (H) HRP/FSM50, (�) HRP/FSM90, (�)HRP/gel, (N) HRP/FSM50/gel, (#) native HRP, (�) hybrid gel.

also measured as the background value. The hybridgel showed a lower conversion yield than native HRP.

The various forms of immobilized HRP were im-mersed in toluene for 15 h, the conversion yields ofbefore and after the pretreatment in organic solvent be-ing shown in Table 2. The oxidative conversion yieldwith native HRP was 10%, but pretreated one wasonly 2%. The conversion yield of HRP entrapped inthe inorganic gel comprising only TMOS markedlydecreased by pretreatment (from 66% to 28%), whilethat in hybrid gel with the molar ratio of DMD-MOS:TMOS= 1:3 remained 50% conversion yield.When HRP immobilized in FSM-16/50 are secondaryentrapped in a hybrid gel, the conversion yield afterpretreatment was 61%. When HRP immobilized inFSM-16/90 was secondary entrapped in a hybrid gel,

the conversion yield after pretreatment was 70% con-version. HRP immobilized in FSM-16/90 showed ahigher conversion yield than that in FSM-16/50 on gelentrapping. In the case of the pore diameter of FSM-16being far larger than the diameters of enzyme mole-cule, a lot of free spaces would exist in the pores ofthe mesoporous silica materials after enzyme loading.This would allow free access of the substrate to theactive site of FSM-16/90 immobilized HRP even aftergelation and make out a high conversion yield.

Conclusion

We investigated the stabilities of immobilized HRPafter hybrid gel entrapment, it being found thatthe thermal stability and catalytic activity of gelatedHRP/FSM-16/90 in organic solvent greatly improved,although those of entrapped HRP/FSM-16/50 did notchange so much. Using other enzyme such as trypsinor subtilisin, the same results were obtained. A largeamount of HRP molecules would be adsorbed in themesopores of FSM-16/90 having a large free spacein the mesopore and further stabilized by the hybridgel. When HRP molecules were adsorbed by a FSM-16/50 material having mesopores that just matched thediameter of HRP, HRP molecules would be stronglysupported by the mesopores, so the stability was notimproved so much by the entrapment in the hybrid gel.

Our new enzyme stabilization method involvingmesoporous materials and organic/inorganic hybridgels will be useful for the development of novelbiosensors or biocatalysts, especially in the field ofgreen chemistry.

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