Functionalization of porous solids plays an important role in many areas, including heterogeneous catalysis and enzyme immobilization. In this study, large-pore ordered mesoporous SBA-15 molecular sieves were synthesized with tetraethyl orthosilicate (TEOS) in the presence of the non-ionic triblock co-polymer Pluronic P123 under acidic conditions. These materials were grafted with 3 aminopropyltrimethoxysilane (ATS), 3-glycidoxypropyltrimethoxysilane (GTS) and with 3 aminopropyltrimethoxysilane and glutaraldehyde (GA-ATS) in order to provide covalent anchoring points for enzymes. The samples were characterized by nitrogen adsorption, powder X-ray diffraction, solid-state NMR spectroscopy, elemental analysis, diffuse reflectance fourier transform infrared spectroscopy and diffuse reflectance UV/Vis spectroscopy. The obtained grafted materials were then used for the immobilization of chloroperoxidase (CPO) and glucose oxidase (GOx) and the resulting biocatalysts were tested in the oxidation of indole. It is found that enzymes anchored to the mesoporous host by the organic moieties can be stored for weeks without losing their activity. Furthermore, the covalently linked enzymes are shown to be less prone to leaching than the physically adsorbed enzymes, as tested in a fixed-bed reactor under continuous operation conditions.
References
[1]
Hartmann, M; Jung, D. Biocatalysis with enzymes immobilized on mesoporous hosts: the status quo and future trends. J. Mater. Chem?2010, 20, 844–857.
[2]
Ariga, K; Hill, JP; Lee, MV; Vinu, A; Charvet, R; Acharya, S. Challenges and breakthroughs in recent research on self-assembly. Sci Technol Adv Mater?2008, 9, 014109:1–014109:96.
[3]
Cabral, JMS; Kennedy, JF. Thermostability of Enzymes; Gupta, MN, Ed.; Springer: Berlin, Germany, 1993; pp. 163–179.
[4]
Ren, L; He, J; Zhang, S; Evans, DG; Duan, X. Immobilization of penicillin G acylase in layered double hydroxides pillared by glutamate ions. J. Mol. Catal. B: Enzym?2002, 18, 3–11.
[5]
Pan, J-L; Syu, M-J. A thermal study on the use of immobilized penicillin G acylase in the formation of 7-amino-3-deacetoxy cephalosporanic acid from cephalosporin G. J. Chem. Technol?2004, 79, 1050–1056.
[6]
Lü, Y; Lu, G; Wang, Y; Guo, Y; Guo, Y; Zhang, Z; Wang, Y; Liu, X. Functionalization of cubic Ia3d mesoporous silica for immobilization of penicillin G acylase. Adv. Funct. Mater?2007, 17, 2160–2166.
[7]
Shah, P; Sridevi, N; Prabhune, A; Ramaswamy, V. Structural features of Penicillin acylase adsorption on APTES functionalized SBA-15. Microporous Mesoporous Mater?2008, 116, 157–165.
[8]
Soares, CMF; Santana, MHA; Zanin, GM; de Castro, HF. Covalent coupling method for lipase immobilization on controlled pore silica in the presence of nonenzymatic proteins. Biotechnol. Prog?2003, 19, 803–807.
[9]
Godjevargova, T; Nenkova, R; Dimora, N. Covalent Immobilization of Glucose Oxidase onto New Modified Acrylonitrile Copolymer/Silica Gel Hybrid Supports. Macromol. Biosci?2005, 5, 760–766.
Kima, MI; Hama, HO; Ohb, S-D; Park, HG; Changa, HN; Choi, S-H. Immobilization of Mucor javanicus lipase on effectively functionalized silica nanoparticles. J. Mol. Catal. B: Enzym?2006, 39, 62–68.
[12]
Szymanska, K; Bryjak, J; Mrowiec-Bialon, J; Jarzebski, AB. Application and properties of siliceous mesostructured cellular foams as enzymes carriers to obtain efficient biocatalysts. Microporous Mesoporous Mater?2007, 99, 167–175.
[13]
Zhang, X; Guan, RF; Wu, DQ; Chan, KY. Enzyme immobilization on amino-functionalized mesostructured cellular foam surfaces, characterization and catalytic properties. J. Mol. Catal. B: Enzym?2005, 33, 43–50.
[14]
Tortajada, M; Ramon, D; Beltran, D; Amoros, P. Hierarchical bimodal porous silicas and organosilicas for enzyme immobilization. J. Mater. Chem?2005, 15, 3859–3868.
[15]
Pandya, PH; Jasra, RV; Newwalkar, BL; Bhatt, PN. Studies on the activity and stability of immobilized α-amylase in ordered mesoporous silicas. Microporous Mesoporous Mater?2005, 77, 67–77.
[16]
Salis, A; Meloni, D; Ligas, S; Monduzzi, M; Solinas, V; Dumitriu, E. Physical and chemical adsorption of mucor javanicus lipase on SBA-15 mesoporous silica. Synthesis, structural characterization, and activity performance. Langmuir?2005, 21, 5511–5516.
[17]
Lei, C; Shin, Y; Liu, J; Ackerman, EJ. Synergetic effects of nanoporous support and urea on enzyme activity. Nano Lett?2007, 7, 1050–1053.
[18]
Yiu, HHP; Wright, PA; Botting, NP. Enzyme immobilisation using SBA-15 mesoporous molecular sieves with functionalised surfaces. J. Mol. Catal. B: Enzym?2001, 15, 81–92.
[19]
Lei, C; Shin, Y; Liu, J; Ackerman, EJ. Entrapping Enzyme in a Functionalized Nanoporous Support. J. Am. Chem. Soc?2002, 124, 11242–11243.
Wang, P; Dai, S; Waezsada, SD; Tsao, AY; Davison, BH. Enzyme stabilization by covalent binding in nanoporous sol-gel glass for nonaqueous biocatalysis. Biotech. Bioeng?2001, 74, 249–255.
[22]
Schlossbauer, A; Schaffert, D; Kecht, J; Wagner, E; Bein, T. Click chemistry for high-density biofunctionalization of mesoporous silica. J. Am. Chem. Soc?2008, 130, 12558–12559.
[23]
Oha, C; Lee, J-H; Lee, Y-G; Lee, Y-H; Kimb, J-W; Kangb, H-H; Oha, S-G. New approach to the immobilization of glucose oxidase on non-porous silica microspheres functionalized by (3-aminopropyl)trimethoxysilane (APTMS). Colloid. Surface. B: Biointerfaces?2006, 53, 225–232.
[24]
Jung, D; Streb, C; Hartmann, M. Oxidation of indole using chloroperoxidase and glucose oxidase immobilized on SBA-15 as tandem biocatalyst. Microporous Mesoporous Mater?2008, 113, 523–529.
[25]
Aburto, J; Ayala, M; Bustos-Jaimes, I; Montiel, C; Terres, E; Dominguez, JM; Torres, E. Stability and catalytic properties of chloroperoxidase immobilized on SBA-16 mesoporous materials. Microporous Mesoporous Mater?2005, 83, 193–200.
[26]
Montiel, C; Terres, E; Dominguez, J-M; Aburto, J. Immobilization of chloroperoxidase on silica-based materials for 4,6-dimethyl dibenzothiophene oxidation. J. Mol. Catal. B: Enzym?2007, 48, 90–98.
[27]
Hudson, S; Cooney, J; Hodnett, BK; Magner, E. Chloroperoxidase on periodic mesoporous organosilanes: Immobilization and reuse. Chem. Mater?2007, 19, 2049–2055.
[28]
Subramanian, A; Kennel, SJ; Oden, PI; Jacobson, KB; Woodward, J; Doktycz, MJ. Comparison of techniques for enzyme immobilization on silicon supports. Enzyme Microb. Technol?1999, 24, 26–34.
[29]
Impens, NREN; van der Voort, P; Vansant, EF. Silylation of micro-, meso- and non-porous oxides: A review. Microporous Mesoporous Mater?1999, 28, 217–232.
[30]
Chong, ASM; Zhao, XS. Functionalization of SBA-15 with APTES and Characterization of Functionalized Materials. J. Phys. Chem. B?2003, 107, 12650–12657.
[31]
Yiu, HHP; Wright, PA. Enzymes supported on ordered mesoporous solids: a special case of an inorganic–organic hybrid. J Mater Chem?2005, 15, 3690–3699.
[32]
Burkett, SL; Sims, SD; Mann, S. Synthesis of hybrid inorganic–organic mesoporous silica by co-condensation of siloxane and organosiloxane precursors. Chem Commun?1996, 1367–1368.
[33]
Macquarrie, JD. Direct preparation of organically modified MCM-type materials. Preparation and characterisation of aminopropyl–MCM and 2-cyanoethyl–MCM. Chem. Commun?1996, 16, 1961–1962.
[34]
Chong, MAS; Zhao, XS; Kustedjo, AT; Qiao, SZ. Functionalization of large-pore mesoporous silicas with organosilanes by direct synthesis. Microporous Mesoporous Mater?2004, 72, 33–42.
[35]
Faber, K. Biotransformations in Organic Chemistry, 5th ed ed.; Springer Verlag: Berlin-Heidelberg, Germany, 2004.
[36]
Felix, G; Descorps, V. Stereochemical resolution of racemates, in HPLC, using a chiral stationary phase based upon immobilized α-chymotrypsin. I. Structural chiral separations. Chromatographia?1999, 49, 595–605.
[37]
Ispas, C; Sokolov, I; Andreescu, S. Enzyme-functionalized mesoporous silica for bioanalytical applications. Anal. Bioanal. Chem?2009, 393, 543–554.
[38]
Mason, RD; Detar, CC; Weetall, HH. Protease covalently coupled to porous glass: Preparation and characterization. Biotechnol. Bioeng?1975, 17, 1019–1027.
[39]
Hartmann, M; Streb, C. Selective oxidation of indole by chloroperoxidase immobilized on the mesoporous molecular sieve SBA-15. J. Porous Mater?2006, 13, 347–352.
[40]
Morris, DR; Hager, LP. Chloroperoxidase: I. Isolation and properties of the crystalline glycoprotein. J. Biol. Chem?1966, 241, 1763–1768.
[41]
Shaw, PD; Hager, LP. Biological Chlorination: VI. Chloroperoxidase: A component of the β-ketoadipate chlorinase system. J. Biol. Chem?1961, 236, 1626–1630.
[42]
Barret, EP; Joyner, LG; Halenda, PP. The Determination of pore volume and area distributions in porous substances. I. Computations from nitrogen isotherms. J. Am. Chem. Soc?1951, 73, 373–380.
[43]
Zhao, DY; Huo, QS; Feng, JL; Chmelka, BF; Stucky, GD. Nonionic Triblock and Star Diblock Copolymer and Oligomeric Surfactant Syntheses of Highly Ordered, Hydrothermally Stable, Mesoporous Silica Structures. J. Am. Chem. Soc?1998, 120, 6024–6036.
[44]
Zhao, DY; Feng, JL; Huo, QS; Melosh, N; Fredrickson, GH; Chmelka, BF; Stucky, GD. Triblock Copolymer Syntheses of Mesoporous Silica with Periodic 50 to 300 Angstrom Pores. Science?1998, 279, 548–552.
[45]
Sing, KSW; Everett, DH; Haul, RAW; Moscow, L; Pierotti, RA; Rouquérol, J; Siemieniewska, T. Reporting physisorption data for gas/solid systems with special reference to the determination of surface area and porosity. Pure Appl. Chem?1985, 57, 603–619.
[46]
Saam, WF; Cole, MW. Excitations and thermodynamics for liquid-helium films. Phys. Rev. B?1975, 11, 1086–1105.
[47]
Jaroniec, CP; Gilpin, RK; Jaroniec, M. Comparative studies of chromatographic properties of silica-based amide-bonded phases under hydro–organic conditions. J. Chromatogr. A?1998, 797, 103–110.
[48]
Chiang, CH; Ishida, H; Koenig, JL. The structure of γ-aminopropyltriethoxysilane on glass surfaces. J. Colloid Interface Sci?1980, 74, 396–404.
[49]
Huang, X; Wang, J; Liu, X; Cong, R. A new type of chemically bonded phase for reversedphase HPLC. Anal. Sci?2003, 19, 1391–1394.
[50]
Hesse, M; Meier, H; Zeeh, B. Spektroskopische Methoden in der organischen Chemie, 5th ed ed.; Georg Thieme Verlag: New York, NY, USA, 1995.