1

Modification of Mesoporous Silicates for Immobilization of Enzymes

Darragh Gaffneya,b

, Jakki Cooneyb,c

and Edmond Magnera,b*

Department of Chemical and Environmental Sciencesa and Materials and Surface

Science Instituteb, University of Limerick, Limerick, Ireland.

Department of Life Sciencesc, Materials and Surface Science Institute

b, University of

Limerick, Limerick, Ireland.

* corresponding author, e-mail: edmond.magner@ul.ie

Contact details:

Darragh Gaffney, Department of Chemical and Environmental Sciences and Materials

and Surface Science Institute, University of Limerick, Limerick, Ireland.

Jakki Cooney, Department of Life Sciences, Materials and Surface Science Institute,

University of Limerick, Limerick, Ireland.

Edmond Magner, Department of Chemical and Environmental Sciences and Materials

and Surface Science Institute, University of Limerick, Limerick, Ireland.

Abstract

Mesoporous silicates (MPS) possess ordered pore structures with pore diameters

sufficiently large to accommodate a range of enzymes. The successful immobilization of

an enzyme on MPS usually requires modification of the surface of MPS to optimize the

interactions between the support and the enzyme. Recent developments on the

2

functionalization of MPS for the immobilization of enzymes are described in this

review.

Keywords: enzyme immobilization, mesoporous silicates, biocatalysis

Introduction

Immobilization of enzymes is used to improve the stability of the enzyme while at the

same time providing a means of recovering and reusing the enzyme with retention of its

catalytic function. A wide range of methods of immobilizing enzymes on a variety of

supports have been described [1]. The ideal support for immobilization of an enzyme

should have a high surface area with good mechanical and chemically stability, be

available at low cost and have minimal non-specific adsorption properties. MPS have

been widely used as supports for enzymes in biocatalytic applications [2-4]. A method

of preparation of MPS for use as low density silica was first described in 1969 [5]. A

range of highly ordered MPS with large surface areas and pore diameters were

subsequently described in 1991 [6]. Since the publication of this work there has been a

surge in interest in using these materials for a broad range of applications. In particular,

their ordered pore structure together with pore diameters which are comparable in size to

those of enzymes make them ideal candidates for the immobilization of enzymes [2-

4]. Transmission electron micrographs of three representative MPS materials depicting

the ordered porosity of the materials are shown in Fig. 1. The ordered porous structures

of MPS materials provide a sheltered or protected environment where reactions with

selected substrates can proceed.

3

Synthesis of MPS

The synthesis of an MPS material such as MCM-41 (Mobil Composition of Matter)

relies on the use of a surfactant template. The surfactant forms micelles and in the

presence of a silicate source and the appropriate solution conditions, the surfactant forms

liquid crystals which act as templates for the formation of MPS. The surfactant can then

be removed by heating or by extraction to produce the porous silicate. Many different

types of mesoporous materials have been synthesized. For example, Santa Barbara

Amorphous (SBA) materials which have thicker walls and higher thermal stability than

MCM-41 materials are prepared using non-ionic and block copolymer surfactants [7].

A range of synthetic procedures have been developed which enables control of the

pore diameter, structure, particle size, chemical composition and stability of MPS. As

the synthesis and characterisation of MPS materials has been extensively reviewed [7-9],

the focus of this review will be on the use of functionalized MPS for immobilization of

enzymes. Functionalised MPS can be prepared directly, post-synthesis and by the

addition of bridged silesquioxanes as the source of silica. MPS modified with functional

groups such as thiol, amine, chloro, carboxylic acid, N-(trimethoxysilylpropyl)ethylene

diamine triacetic acid (EDTA), oxirane rings, cyano, alkane and aromatic functional

groups together with a range of metal ions have been prepared [8-17].

Direct functionalization

Direct functionalization involves the co-condensation of siloxane and

organosilane precursors to produce a material containing a homogenous dispersion of

4

functional groups which protrude into the pore while also enabling access to the surface

silanol groups for adsorption [7-9]. An example of this method of functionalization is

the preparation of cyano modified MPS (CNS) [17]. A trypsin bioreactor based on CNS

has been used to digest proteins in clinical samples [18]. Confinement of trypsin and of

the proteins within the pores reduced the time required for sample digestion from 12

hours to 15 min.

A disadvantage associated with direct functionalization is that the structure and

order of the final material can be affected if the appropriate balance of surfactant,

silicate, acid/base and solvent is not maintained throughout the reaction process. In

addition, the functional group may become incorporated into the main body of the

material and not on the surface. In the synthesis of MCM-48 materials containing

phenyl, allyl, aminopropyl and mercaptopropyl functional groups [19], the desired cubic

mesophase was observed only when phenyltriethoxysilane (PTES) was used at a 10%

molar content. No discernible structural order was present with the other materials. The

direct synthesis of hybrid MPS materials has been described using two approaches [20].

Template substitution involving the partial substitution of tetraethoxy silane (TEOS) and

alkylamine surfactant with an organosilane was suitable for the synthesis of materials

where the organic moiety was comparable in size to the amine surfactant. In contrast, the

direct addition of organosilane, while maintaining the ratio of TEOS and amine

surfactant used, was useful for the incorporation of organic moieties which are smaller

than the amine surfactant.

5

Post-synthesis functionalization

Post-synthesis functionalization involves direct grafting of a suitable functional

group onto the surface of MPS and has been used to graft chloro, alkoxy, and silyl and

amine groups [8-15] onto the exposed surfaces of the mesoporous channels and external

surface. The advantages of post-synthesis functionalization include the fact that no

alteration of the structural ordering of the starting mesoporous material occurs and that

functionalization occurs only on the surface. Disadvantages of the method include a

decrease in the pore diameter, the requirement of extra steps in the synthesis procedure

and lower levels of modification than can be achieved by co-condensation.

Functionalization of the pore surfaces can alter the efficiency of covalent binding.

Comparison of the immobilization of penicillin G acylase (PGA) on SBA-15 materials

functionalised with different ratios of silanol:oxirane groups on the surface of MPS

demonstrated that the partially functionalised materials had a higher enzyme loading as

well as faster binding kinetics than the fully functionalised surfaces [21].

It is important that functional groups be distributed evenly throughout the

material. Post-synthesis grafting of vinyl groups on MPS was observed to occur

predominantly on the external surface and around the pore openings [22], due to the fact

that silylation of the external SiOH groups is kinetically favourable. Such

inhomogeneous distributions can arise from diffusional and steric factors. A two-step

approach to functionalising the internal surface of the channels has been described [23]

in which the external surface was first modified with Ph2SiCl2 followed by

functionalization of the internal surface with (CH3O)3Si(CH2)3NH2. An important point

6

to note when preparing materials using this approach is that the modified material

should be sufficiently stable to withstand the solvent extraction process required to

remove the surfactant.

Bridged silesquioxanes

Periodic mesoporous organosilicate (PMO) materials are prepared using a

bridged silesquioxane (RO)3Si-RI-Si(OR)3 as a precursor. PMOs may be synthesised

under acidic or basic conditions. PMOs were initially prepared using ionic surfactants

such as cetyltrimethylammonium bromide [24-26] to generate pores of 2-5 nm in

diameter. Larger pores, ranging from 6-20 nm in diameter, have been generated with

non-ionic triblock co-polymers such as P123 (EO20PO70EO20), F127 (EO106PO70EO106)

and B50-6600 (EO39PO47EO39) [27]. As an example of the stability of these materials,

PMO-SBA-15 synthesized from P123 in acidic media [28], was stable for 60 days when

placed in boiling water. In comparison SBA was stable for only 10 days under the same

conditions. This stability can be attributed to the thick pore walls, the highly

hydrophobic surface nature of the organic silicate network and the enhanced degree of

polymerization present in PMO-SBA-15.

The self-assembly of PMOs through surfactant templates requires a tailored

structure, which balances the competing elements of static coulombic interactions

between the surfactant head groups and the silica precursor, as well as the hydrophobic

interaction between surfactant head groups and hydrophobic parts of the silesquioxanes.

The ratio between surfactant and hybrid silesquioxanes is a key factor affecting their

synthesis. As a result the synthesis conditions need to be more strictly controlled than

7

for the synthesis of conventional MPS. The most significant advantage of this method of

functionalization is the incorporation of the functional group in the wall structure. As a

result, the functional groups do not protrude into the pore space and high loadings can be

obtained. The structure also maintains a periodic pore system that can be tailored to

specific needs through the adjustment of the organic spacer RI. A number of PMO’s

have been generated through this method, using bridging groups such as ethane,

methylene, saturated alkyl chains containing methylene groups, benzene, thiophene,

ferrocene [29-32]. The organic functionality of PMO materials can be tuned to suit the

application required. For example, Cornelius et al. [33] synthesized a PMO using 1,4-

bis((E)-2-(triethoxysilyl)vinyl) benzene to introduce two organic functionalities into the

pore wall structure, a vinyl group and an aromatic ring. These groups can be further

functionalised if required.

The enzyme chloroperoxdase has been successfully immobilized on an amine

based PMO. The optimal activity and stability of immobilized chloroperoxidase was

obtained using a PMO with 40% propylamine substitution. Stability studies using this

material showed no loss in activity over 20 cycles, compared to a 90% loss in activity

for chloroperoxidase immobilized on amino grafter PMO [34].

Metal functionalized MPS

Many metals including Ni, V, Fe, lanthanides, Ru, Cu, Zn, Co, Ti, Al, In and Sn

have been successfully incorporated into mesoporous materials. Direct grafting of

titanocene dichloride to the inner walls of MCM-41 [35] has been used to prepare

8

catalysts for the epoxidation of cyclohexane as well as for bulky cyclic alkenes. The

catalytic activity of MPS with Ti incorporated into the mesopore structure displayed low

catalytic activity as Ti was buried within the inner wall structure. Bein et al. [36]

synthesised a molybdenum containing structure through the reaction of a bimetallic tin-

molybdenum complex in the channels of MCM-41. Direct grafting of metal complexes

has been performed, generally through use of an oxo-bridge [22, 37]. This method

allows for the attachment of many metal oxides, with the optimal results obtained with

Sn, In, Ni and Co. However, the method has only been successful for mesoporous silica

monoliths and requires long processing times. Low catalytic activity is observed with

metals which are incorporated into the mesoporous wall structure during the synthesis

process. MPS prepared through a co-condensation method with a metal precursor retain

the accessibility of the metal and have shown good catalytic activity as stand-alone

catalysts [19]. However, the oxide moiety is not conducive to the coordination of

ligands, which is necessary for the attachment of proteins. The third option, using a

spacer ligand to post-synthetically tether the organometallic complex on the silicate

surface, has previously been employed with some success [20, 22, 38] This has been

demonstrated by the attachment of N-(trimethoxysilylpropyl) EDTA (ethylene diamine

triacetic acid) to the surface of the silicate and subsequent use as a tethering ligand for

nickel [42, 43]. The main advantage of this method is that the oxidation state of the

coordinated metal ion is retained.

9

Enzyme immobilization

Immobilization of enzymes generally occurs through one of the following

methods; non-covalent adsorption and deposition; covalent methods; cross-linking and

encapsulation in a polymeric gel or capsule. Immobilization of enzymes on MPS can

occur via adsorption or covalent binding with adsorption being the most widely used

method. The advantage of the latter is that it is a subtle approach and thus avoids

denaturation of the enzyme and minimises conformational changes in the enzyme. As

with any support, adsorption of enzymes on MPS, entails a range of electrostatic and

hydrophilic/hydrophobic interactions.

Immobilization of enzymes by adsorption

A systematic approach to the adsorption of enzymes on MPS was described by

Hudson et al. which entailed a detailed examination of the factors that can influence the

process of adsorption [2]. The factors include the relative sizes of the mesopore and the

enzyme, surface area, pore size distribution, mesopore volume, particle size, ionic

strength, isoelectric point and the surface characteristics of the enzyme and of the

mesoporous support. Using the enzyme subtilisin Carlsberg as an example (Fig. 2),

details of the electrostatic potentials and the degree of hydrophobicity/hydrophilicity of

the surface of the enzyme are essential requirements for the direct adsorption of an

enzyme on the surface of a material.

A significant disadvantage of the use of adsorption to immobilize an enzyme is

the weak forces of interaction that ensue, with leaching of the enzyme from the support

being a major problem. In addition, the immobilization process is not a targeted one with

10

immobilization possible at a range of sites on the surface of the enzyme, some which

may enable the catalytic function of the enzyme, some of which may completely remove

any catalytic activity.

Immobilization of enzymes by covalent methods

Covalent immobilization of enzymes on MPS entails linking the enzyme to the

support, generally using a linking agent such as glutaraldehyde [1]. This approach, as

with any crosslinking approach, eliminates the possibility of leaching of the enzyme and

can confer increased stability on the immobilized enzyme. For example, α-chymotrypsin

supported on various mesoporous silicates functionalised with trimethoxysilylproponal

displayed a half-life greater than 1000 fold of that of the native enzyme in aqueous and

organic media [41]. However, the conditions employed and/or possible conformation

changes to the enzyme can affect the catalytic activity due to the rather indiscriminate

means of cross linking the enzyme and MPS.

Ideally, immobilization of enzymes should occur in a targeted manner with the

enzyme anchored at a point that does not affect either the conformation or activity of the

enzyme. In particular diffusion of the substrate and product to and from the active site

should not be hindered. A disadvantage of using MPS as enzyme supports lies in the

indiscriminate means of binding of the enzyme. A targeted method of attaching an

enzyme, which also minimises non-specific binding of the enzyme, can overcome this

disadvantage. Tailoring of the surface functional groups of MPS and of a protein can

enable anchoring of the protein so that binding occurs in a specific manner, thus

11

ensuring that the enzyme is not only irreversibly bound but also in an orientation which

is substrate accessible. Such an approach has been described recently by anchoring an

enzyme to MPS via a histidine tag which can coordinate to metal ions on the surface of

MPS [42, 43].

Immobilisation of enzymes by metal affinity methods

This use of nickel modified materials is in widespread use in protein recombinant

purification [24]. The adsorption of His-tagged proteins onto Ni(II) or Co(II)

functionalised surfaces has been demonstrated previously [25, 26, 44]. Cho et al.

reported a post-synthetic preparation of a mesoporous silicate containing finely

dispersed Ni particles through the use of N-(trimethoxysilylpropyl)ethylene diamine

triacetic acid (EDTA) as a tethering ligand [45]. Bruehwiler et al. have described the

covalent attachment of Ni [46], through a Ni-O-Si group, to the surface of MCM-41 by

ion exchange from aqueous solution.

A wide range of polysiloxane-immobilised ligands have been previously used for

the coordination of metal ions [47-49]. These generally consist of thiols, amines and N-

/S-containing ligands. However, such ligands may not have the stability required in

aqueous solution. The use of cyclic polyamines can alleviate this factor. Cyclam

(1,4,8,11-tetraazacyclotetradecane) is a widely used 14-membered tetra-amine

macrocycle. The synthesis of silica supports covalently functionalised with cyclam

derivatives has been described [50-61]. Cyclams can bind to a range of transition metals,

including nickel, often achieving very high thermodynamic and kinetic stability in terms

of metal ion dissociation which has lead to applications in catalysis [62] and

12

medicine[63,64]. Ni-cyclam complexes have been extensively studied [65], in particular

the tetra-N-substituted complexes [66, 67]. Mono-N-substituted cyclam they exist in a

trans-III (S, S, R, R) (square-planar) configuration with two accessible coordination

sites above and below the plane of the ring [68], sites which can facilitate attachment of

a protein to the surface of the silicate through a His6-tag on the protein (Scheme 1).

The specific adsorption of a His6 tagged protein on a Ni-cyclam functionalised

MPS was examined using a protease inhibitor (Spi) from the human pathogen

Streptococcus pyogenes as a model system. The His6-tagged form of Spi has a molecular

mass of 14.7 kDa an isoelectric point of 5.4 and does not possess a high surface charge,

ensuring that electrostatic interactions with the carrier will be minimized. Hydrophobic

interactions between the protein surface and the silicate surface were prominent, leading

to unspecific protein-silicate binding. In the presence of PEG400 these non-specific

interactions were suppressed enabling specific binding of His6-tagged Spi onto SBA-15-

Ni-cyclam. Based on this approach, histidine-tagged alanine racemase and histidine-

tagged Candida antarctica lipase B have been immobilised onto MPS [55].

Conclusions

MPS have been used extensively for the immobilization of enzymes. The

successful immobilization of enzymes requires the preparation of MPS with functional

groups that are tailored to provide a support that is compatible with providing a stable

means of sequestering the enzyme with retention of catalytic activity. The surface of

MPS can be functionalized directly, post-synthesis or via the use of bridged

13

silesquioxanes. Such modifications do not preclude non-specific adsorption. The

combination of using a tether on MPS such as Ni2+

cyclams has significant potential as

this approach enables specific binding of any enzyme which contains a His6 tag. Any

non-specific binding that does occur can be prevented by the addition of sodium chloride

and polyethylene glycol.

Acknowledgments

This work was funded by Science Foundation Ireland (RFP06/CHP001) and by the HEA

PRTLI4 programme, INSPIRE. Dr. Sarah Hudson is acknowledged for providing the

data in Fig. 1.

14

References

[1] L.Q. Ciao, Carrier-bound immobilized enzymes (Wiley-VCH, Weinheim, 2005).

[2] S. Hudson, J.C. Cooney and E. Magner, Ang. Chem. Int. Ed. 47 (2008) 8582.

[3] H.H.P. Yiu and P.A.Wright, J. Mater. Chem. 15 (2005) 3690.

[4] M. Hartman and D. Jung, Chem. Mater. 20 (2010) 844.

[5] V. Chiola, E. Ritsko and C.D. Vanderpool, US Patent No. 3556725 (1971).

[6] C.T. Kresge, M.E. Leonowicz, W.J. Roth, J.C. Vartuli and J.S. Beck, Nature 359

(1992) 710.

[7] Y. Wan and D. Zhao, Chem. Rev. 107 (2007) 2821.

[8] F. Hoffman, M. Cornelius, J. Morell and M. Froba, Angew. Chem. Int. Ed. 45 (2006)

3216.

[9] B. Platschek, A. Keilbach and T. Bein, Adv Mater 23 (2011) 2395.

[10] X. Feng, G.E. Fryxell, L.Q. Wang, A.Y. Kim, J. Liu and K.M. Kemner, Science

276 (1997) 923.

[11] H.H.P. Yiu, P.A. Wright, N.P. Botting, J. Mol. Cat. B Enzym. 15 (2001) 81.

[12] Y.S. Cho, J.C. Park, B. Lee, Y. Kim and J. Yi, Cat. Lett. 81 (2002) 89.

[13] R.J.P. Corriu, E. Lancelle-Beltran, A. Mehdi, C. Reye, S. Brandes and R. Guilard,

J. Mater. Chem. 12 (2002)1355.

[14] M. Alvaro, A. Corma, D. Das, V. Fornes and H. Garcia, Chem. Commun. (2004)

956.

[15] C.H. Lei, M.M. Valenta, K.P. Saripalli and E.J. Ackerman, J. Environ. Qual. 36

(2007) 233.

[16] J. Deere, E. Magner, J.G. Wall and B.K. Hodnett, Biotechnol. Prog. 19 (2003)

1238.

[17] J. Deere, E. Magner, J.G. Wall and B.K. Hodnett, J. Phys. Chem. B 106 (2003)

7340.

[18] L. Qiao L, Y. Liu, S. Hudson, P. Yang, E. Magner, B. Liu, Chem. Eur. J. 14 (2008)

151.

[19] S.R. Hall, C.E. Fowler, S. Mann and B. Lebeau, Chem. Commun. (1999) 201.

[20] L. Mercier and T.J. Pinnavaia, Chem. Mater. 12 (2000) 188.

[21] X.S. Zhao, X.Y. Bao, W. Guo, and F.Y. Lee, Materials Today 9 (2006) 32.

[22] M.H. Lim and A. Stein, Chem. Mater. 11 (1999) 3285.

[23] D.S. Shephard, W. Zhou W, T. Maschmeyer, J.M. Matters, C.L. Roper, S. Parsons,

B.F.G. Johnson and M.J. Duer, Angew. Chem. Int. Ed. 37 (1998) 2719.

[24] T. Asefa, M.J. MacLachlan, N. Coombs and G.A. Ozin, Nature 402 (1999) 867.

[25] T. Asefa, M.J. MacLachlan, H. Grondey and N. Coombs, Angew. Chem. Int. Ed. 39

(2000) 1808.

[26] B.J. Melde, C.F. Blanford and A. Stein, Chem. Mater. 11 (1999) 3302.

[27] A. Saya and M. Jaroniec, Recent developments in the synthesis and chemistry of

periodic mesoporous organosilicas (Elsevier, Amsterdam, The Netherlands, 2002).

[28] W. Guo, X. Li and X.S. Zhao, Micro. Meso. Mat. 93 (2006) 285.

[29] F. Hoffmann, M. Cornelius, J. Morell and M. Fröba, Angew. Chem. Int. Ed. 45

(2006) 3216.

[30] X.Y. Bao, X.S. Zhao, X. Li, P.A. Chia and J. Li , J Phys. Chem. B 108 (2004) 4684.

15

[31] J. Hunks and G.A. Ozin, J. Mater. Chem. 15 (2005) 3716.

[32] A. Sayari and S. Hamoudi, Chem. Mater.13 (2001) 3151.

[33] M. Cornelius, F. Hoffmann and M. Froba, Chem. Mater. 17 (2005) 6674.

[34] S. Hudson, J.C. Cooney, B.K. Hodnett and E. Magner, Chem. Mat. 19 (2008)

2049.

[35] T. Maschmeyer, F. Rey, G. Sankar and J.M. Thomas, Nature 378 (1995) 159.

[36] C. Huber, K. Moller and T. Bein, Chem. Commun. (1994) 2619.

[37] H. Yang, Y. Lu, F. Gao, Q. Shi, Y. Yan, G.Q.Zhang, S.H. Xie, B. Tu and D.Y.

Zhao, Adv. Funct. Mater. 15 (2005) 1377.

[38] Y.C. Liang and R. Anwander, Dalton Trans. 15 (2006) 1909.

[39] C.P. Jaroniec, M. Kruk, M. Jaroniec and A. Sayari, J. Phys. Chem. B 102 (1998)

5503.

[40] D.S. Shephard, W. Zhou, T. Maschmeyer, J.M. Matters, Angew. Chem. Int. Ed. 37

(1998) 2719.

[41] P. Wang, S. Dai, S.D. Waezsada, A.Y. Tsao and B.H. Davison, Biotech. Bioeng. 74

(2001) 249.

[42] K.E. Cassimjee, T. Martin, C. Branneby and P. Berglund P, Biotech. Bioeng. 99

(2008) 712.

[43] D.A. Gaffney, S. O’Neill, M.C. O’Loughlin, U. Hanefeld, J.C. Cooney and E.

Magner, Chem. Commun. 46 (2010) 1124.

[44] R.M. Tindwa, D.K. Ellis, G.J. Peng and A. Clearfield, J. Chem. Soc. Faraday Trans.

81 (1985) 545.

[45] Y.S. Cho, J.C. Park, B. Lee and Y. Kim, Cat. Lett. 81 (2002) 89.

[46] D. Bruhwiler and H. Frei, J. Phys. Chem. B 107 (2003) 8547.

[47] M. Cornelius, F. Hoffmann and M. Froba, Chem. Mater. 17 (2005) 6674.

[48] J. Aguado, J.M. Arsuaga, A. Arencibia, M. Lindo and V. Gascón, J. Haz. Mat. 163

(2009) 213.

[49] A. Walcarius and C. Delacôte, Anal. Chim. Acta 547 (2005) 3.

[50] R.J.P. Corriu, A. Mehdi, C. Reye and C. Thieuleux, Chem. Commun. (2003) 1564.

[51] F. Barbette, F. Rascalou, H. Chollet, J.L. Babouhot, F. Denat and R. Guilard, Anal.

Chim. Acta 502 (2004) 179.

[52] G. Dubois, R. Tripier, S. Brandès, F. Denat and R. Guilard. J. Mat. Chem. 12

(2002) 2255.

[53] G. Dubois, R.J.P. Corriu, C. Reyé, S. Brandès, F. Denat and R. Guilard, Chem

Commun. (1999) 2283.

[54] G. Dubois, C. Reyé, R.J.P. Corriu, S. Brandès, F. Denat and R. Guilard, Ang.

Chem. Int. Ed. 40 (2001) 1087.

[55] D.A.Gaffney, Tailored adsorption of enzymes onto mesoporous silcates (PhD

thesis, University of Limerick, 2010).

[56] R.J.P. Corriu, A. Mehdi, C. Reye, C. Thieuleux, A. Frenkel and A. Gibaud. New J.

Chem. 27 (2003) 905.

[57] A. Kassiba, M. Makowska-Janusik, J. Alauzun, W. Kafrouni, A. Mehdi, C. Reye,

R.J. Corriu and A. Gibaud, J. Phys. Chem. Sol. 67 (2006) 875.

[58] R. Mouawia, A. Mehdi, C. Reyé and R. Corriu, New J. Chem. 30 (2006)1077.

16

[59] R.J.P. Corriu, E. Lancelle-Beltran, A. Mehdi, C. Reye, S. Brandes and R. Guilard,

J. Mat. Chem. 12 (2002) 1355.

[60] M. Etienne, S. Goubert-Renaudin, Y. Rousselin, C. Marichal, F. Denat F, B.

Lebeau and A. Walcarius, Langmuir 25 (2009) 3137.

[61] S. Goubert-Renaudin, M. Etienne, S. Brandes, M. Meyer, F. Denat, B. Lebeau and

A. Walcarius, Langmuir 25 (2009) 9804.

[62] M. Beley, J.P. Collin, R. Ruppert and J.P. Sauvage, J. Am. Chem. Soc. 108 (1986)

7461.

[63] R. Delgado, V. Felix, L.M.P. Lima and D.W. Price, Dalton Trans. 26 (2007) 2734.

[64] X. Liang and P.J. Sadler, Chem. Soc. Rev. 33 (2004) 246.

[65] T.H. Kaden Helv. Chim. Acta 53 (1970) 617.

[66] M. Meyer, V. Dahaoui-Gindrey, C. Lecomte and R. Guilard, Coord. Chem. Rev.

178 (1998) 1313.

[67] C. Bucher, E. Duval, J.M. Barbe, J.N. Verpeaux, C. Amatore, R. Guilard, L. Le

Pape, J.M. Latour, S. Dahaoui and C. Lecomte, Inor. Chem. 40 (2001) 5722.

[68] T.M. Hunter, I.W. McNae, X. Liang, J. Bella, S. Parsons, M.D. Walkinshaw and

P.J. Sadler, Proc. Nat. Acad. Sci. 102 (2005) 2288.

17

Fig 1 Transmission electron micrographs of (A) a periodic mesoporous organic silicate,

(B) MCM-41 and (C) SBA-15

A

B

C

18

Fig 2 Structure of subtilisin (A) as ribbon diagram, (B) in CPK format displaying

hydrophobic (yellow) and hydrophilic (blue) residues and (C) with Poisson-Boltzmann

electrostatic potentials at pH 7.0

A C B A C B

19

Scheme 1 Surface modification of SBA-15 with Ni-cyclam and subsequent attachment

of a His6-tagged protein