Ikemoto, Hideki

Mesoporøse silicapartikler baseret på Santa Barbara Amorphous-15 (SBA-15), med varierende morfologi, struktur, porestørrelse og funktionelle grupper er blevet syntetiseret. To metalloenzymer og et fotosyntetisk membranproteinkompleks er blevet immobiliseret på partiklernes overflade eller i deres poresystem og de resulterende hybrider anvendt til videre studier.  Det ene metalloenzym, peberrodsperoxidase (HRP), blev immobiliseret ved physisorption på stavformet SBA-15. Den katalytiske aktivitet af det frie og immobiliserede enzym ved stuetemperatur blev først sammenlignet og detaljer i enzymkinetikken, den tilsyneladende Michaelis konstant (KM) og maksimale reaktionshastighed (Vmax) bestemt. Både termisk stabilitet og stabilitet over for to denatureringsreagenser, guanidiniumchlorid og urinstof (urea) for frie og immobilizerede enzymer blev dernæst sammenlignet. Termisk stabilitet af det immobiliserede enzym HRP er stærkt forbedret sammenlignet med det frie enzym Den katalytiske hastighed er nedsat, men Vmax er langt mere varmerobust end for det frie enzym. Enzymstabilitet over for kemisk denaturering afhænger af denatureringsreagensets kemiske natur og vekselvirkning med både enzym og nanoporevægge. Guanidiniumchlorid viste næsten ens nedsættelse af den katalytiske aktivitet affri og immobiliseret enzym. Til forskel herfra var immobiliseret HRP langt mere modstandsdygtigt over for urea end det frie enzym. Disse forskellige mønstre kan korreleres med vands forskellige hydrogenbindingsmønstre og proteinsolvatisering i silicananoporerne sammenlignet med den frie vandige opløsning.  Som sammenligning blev kobberenzymet galactose oxidase (GAOX) immobiliseret på SBA-15 med hexagonalt ordnet porestruktur ogpå cellulær “skum-struktureret (MCF)-type mesoporøs silica med kavitetslignende porestruktur. Enzymudslip kunne ikke detekteres efter physisorption af enzymet på SBA-15 med hexagonalt ordnede porer med en porediameter sammenlignelig med GAOX’s størrelse. Immobiliseret GAOX aktivitet var ca.30 % mindre end det frie enzyms. Det immobiliserede enzym viste ikke øget termisk stabilitet sammenlignet med det frie enzym, snarere tværtimod. GAOX immobilisering på MCF-type mesoporøs silica med kavitetslignende mesopore forbundet med snævrere åbninger (sammenlignelige med GAOX’s størrelse), kunne ikke opretholdes, men førte til betydeligt enzymudslip. Kovalent GAOX-binding kunne fastholde enzymet uden udslip.  Det lysabsorberende (Light harvesting) kompleks 2 (LH2) fra fotosyntetiske purpurbakterier blev endelig immobiliseret på SBA-15 med hexagonalt ordnede cylindriske porer og på MCF-type mesoporøs silica withuordnede kavitets-lignende mesoporer. LH2 kunne adsorberes på partikler med vide porer, men ikke på partikler med snævre porer. Efterfølgende fluorescensmikroskopi bekræftede, at LH2 adsorberes inde i de mesoporøse partikelporer. Hybriderne afLH2 og mesoporøs silica blev yderligere undersøgt med steady state fluorescens målinger fra stuetemperatur til kryogene temperaturer og desuden ved tidsopløst fluorescensspektroskopi ved stuetemperatur. Fluorescensspektre af LH2-silica hybriderne suspenderet i opløsning ved stuetemperatur var umiddelbart næsten identiske med spektrene for fri LH2 i opløsning. Fluorescensspektre af LH2-silica hybriderne på filmform fra stuetemperatur ned til 77 K kunne optages og sammenlignes med spektre for det frie LH2 ligeledes på filmform. Spektrene udviste temperaturafhængig båndbredde og maksimumforskydning De umiddelbare visuelle spektralforskelle mellem fri og immobiliseret LH2 var små, men efter numerisk spetralanalyse fandtes tydeligeforskelle i temperaturmønstrene for både båndbredder og fluorescensmaksima. Fluorescenslevetider gav andre interessante observationer. Fri LH2 og LH2 i uordnedekavitetslignende mesoporer viste monoexponentielt fluorescenshenfald. I modsætning hertil viste LH2 immobiliseret i hexagonalt ordnede cylindriskeporer biexponentielt fluorescenshenfald. Disse data er under yderligere behandling.Mesoporous silicas, based on Santa Barbara Amorphous-15 (SBA-15), with different morphology, structure, pore size and functional groups have been synthesized. Two metalloenzymes and a photosynthetic membrane protein were immobilized on or confined in the pores of the mesoporous silicas to prepare hybrid materials used for further study.  One metalloenzyme, horseradish peroxidase(HRP), was immobilized on rod-shaped SBA-15 by physical adsorption. The catalytic activity of free and immobilized enzyme was first compared at room temperature. Details of the enzyme kinetics including the apparent Michaelis constant (KM) and maximum rate (Vmax) were determined. Both thermal stability and the stability toward the denaturing agents guanidinium chloride and urea, of free and immobilized enzymes were compared next. The thermal stability of the immobilized enzyme is significantly improved in comparison with free HRP. The catalytic kinetics is slowed down notably, but Vmax is much more robust to heat than for the free enzyme. The stability resistance of the enzyme toward the denaturing agents depends on the chemical nature of the denaturing agentsand interactions between enzyme and silica nanopore walls. Guanidinium chloride showed similar attenuation of the catalytic activity of immobilized and free enzyme. In contrast, immobilized HRP was much more resistant to urea than the free enzyme. The different behavior of free and immobilized enzyme is most likely due to different hydrogen bonding of water and increased hydration strength of the protein inside the nanopores.  A copper-containing enzyme, galactose oxidase (GAOX), was immobilized on SBA-15 with a hexagonally ordered pore structure, or on mesocellular foam (MCF)-type mesoporous silica with a cage-like pore structure. Physical adsorption of this enzyme on the SBA-15 with a pore diameter comparable to the size of GAOX keeps enzyme stably immobilized with no enzyme leaching detected. Catalysis by immobilized GAOX was ca. 30 % less efficient than for the free enzyme. Immobilized enzyme did not show improved thermal stability compared to free GAOX.When GAOX was immobilized on the MCFv type mesoporous silica with cage-like mesopores interconnected by smaller openings (the opening comparable to the size of GAOX), significant leaching of enzyme was observed. Covalent attachment of GAOX was found toprevent leaching of the enzyme.  Light harvesting complex 2 (LH2) from purple photosynthetic bacteria was immobilized on SBA-15 with hexagonally ordered cylindrical pores or on MCF-type mesoporous silica with disordered cage-like mesopores. To identify the location of LH2, spherical particles with small or large pores were tested for the adsorption of LH2. LH2 was adsorbed to the particles with large pores, while little LH2 was adsorbed to the particles with small pores. Subsequent observation with fluorescence microscopy confirmed that LH2 is adsorbed in the pores of mesoporous silica. The conjugates of LH2 and mesoporous silica were studiedby steady state fluorescence measurements from ambient to cryogenic temperatures and also by time resolved fluorescence spectroscopy at room temperature. Fluorescence spectra of the LH2-silica conjugates suspended in solution measured at room temperature were found to bealmost the same as those of free LH2. Fluorescence spectra of the LH2-silica conjugates in a film form were measured from room temperature down to 77 K and compared with the spectraof LH2 alone in a film form. Temperature dependent bandwidth change and peak position shift were observed. The immediate difference between the samples was small, but systematic differences in the temperature-dependent patterns of the spectral band widths and band maximum positions were found by detailed bandshape analysis. Lifetime measurements brought other interesting results. Free LH2 and LH2 confined in disordered cage-like mesopores showed monoexponential fluorescence decay. In contrast, LH2 confined in hexagonally ordered cylindrical pores showed biexponential fluorescence decay. The data are under further scrutiny for proper interpretation.</p

Online Research Database In Technology

Catalytic Activity and Photophysical Properties of Biomolecules Immobilized on Mesoporous Silica

http://orbit.dtu.dk/ws/files/74274346/Hideki_Ikemoto..PDF

Catalytic Activity and Photophysical Properties of Biomolecules Immobilized on Mesoporous Silica

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