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8. General Conclusions and Further Work

8.1 General Conclusions

The synthesis and characterisation of a range of mesoporous materials based on SBA-15 have been described. The materials are thick-walled and stable to heat treatment in aqueous conditions. The material was templated by surfactant, which resulted in a hexagonal structure (p6mm) with a narrow pore size distribution. The surfactant was removed, either by calcination or extraction by solvent. The surface of a calcined material is fully condensed, and this surface is hydrolysed in aqueous conditions without disruption of the structure, to generate a surface comprised largely of silanol groups.

The incorporation of organic functional groups was demonstrated by co- condensation and by post-synthesis grafting. These functional groups can alter the morphology of the structure when incorporated by co-condensation, and a change from hexagonal to cubic symmetry was observed with increasing propyl thiol concentration. A wide range of functional groups were incorporated, although incorporation of basic amine groups resulted in a poorly ordered material at high concentration. The pore size of the material was increased with the addition of trimethylbenzene during synthesis, which also changed the morphology of the material to a foam-like structure.

The immobilisation of CALB within the mesoporous materials synthesised was rapid, and a high uptake of enzyme was possible, with a maximum loading of 450 mg g-1 measured. The uptake of the enzyme was poor when the surfactant template was not extracted, indicating that the enzyme is located within the pore system of the support. This was supported by nitrogen adsorption experiments on immobilised CALB samples, with lower adsorption of nitrogen as the enzyme content was increased. However, the leaching of enzyme at a pH removed from the isoelectric point of the protein was a significant problem with unfunctionalised systems. This leaching was significantly reduced by the incorporation of sulfur containing groups on the surface of the material, which stabilise the enzyme via van der Waal's interactions. The uptake of enzyme by thiol functionalised materials is described by a two step model, in which the enzyme is quickly adsorbed by the support and then diffuses through the pore system at a slower rate. Once the enzyme was

adsorbed within thiol functionalised materials, it was not readily removed, with very low levels of leaching measured after washing with pH 8 phosphate buffer. CALB was also immobilised successfully on expanded pore supports, with rapid uptake at high levels of enzyme incorporation.

The rate of reaction in aqueous conditions for the hydrolysis of tributyrin was complicated by the leaching of enzyme from unfunctionalised supports that bind the enzyme poorly. The reusability with these supports was poor, with very low activity after only five reuses. For the supports which bind CALB strongly, including the thiol functionalised materials, little difference in hydrolysis activity was observed when the sample was washed with pH 8 buffer. The thiol supported CALB samples showed good reusability over successive reactions. CALB supported within thiol functionalised supports showed higher stability toward thermal degradation than unfunctionalised supports, and all supported CALB samples showed a large improvement over the free enzyme. This is due to strong binding to the surface of the support hindering unfolding of the enzyme at high temperature.

The enantioselective acylation of (R)-1-phenylethanol was catalysed by immobilised CALB in organic solvents, with much higher activity measured for the mesoporous supported enzyme than for a powder dispersion of free enzyme. The activity of the immobilised enzyme was higher with calcined supports, which may be due to the incorporation of ethoxide groups on the surface of the material during extraction, resulting in faster substrate diffusion. The incorporation of organic functional groups has a direct effect on the rate of the reaction, with hydrophobic organic groups increasing the initial rate, due to improved substrate diffusion. There is no leaching of enzyme observed from the mesoporous supports, with good reusability properties of the immobilised enzyme.

The encapsulation of CALB within mesoporous silica was also demonstrated, although retaining the activity of the enzyme was challenging. Although the initial rate of reaction was reduced relative to adsorbed CALB, the reusability properties were excellent, with no activity loss observed after five uses when the enzyme was encapsulated by 3- mercaptopropyltriethoxysilane.

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8.2 Further Work

There is considerable scope for further study and modification of the experiments previously described. The modification of the support material morphology is possible, with the properties of larger pore supports holding considerable interest, as larger pore supports not only have potentially better substrate diffusion properties, they can also potentially accommodate larger enzymes, such as cytochrome P450 or glycosidases.

As good reusability and low enzyme leaching from the thiol functionalised supports was demonstrated, the nature of binding for enzymes that have unbridged cysteine residues on the surface of the protein can be studied, and the potential formation of disulfide bonds can be investigated. This could be compared with established methods for covalent attachment, although attempts at binding CALB via glutaraldehyde and other covalent binding methods (not reported) were unsuccessful.

The preliminary studies on mesoporous systems for DKR could be explored further, as immobilised CALB within mesoporous support shows good activity and excellent selectivity for the kinetic resolution of secondary alcohols. This would require a study of racemisation catalysts, and the ruthenium catalysts described in section 7.1.1 may provide good targets for immobilisation studies.

The encapsulation methods described above are very promising methods for maintaining the advantages of adsorption systems, with very little enzyme leaching in aqueous conditions. Investigation with a smaller substrate may elucidate the reason for the activity loss during encapsulation. The development of alternative encapsulation methods, and a detailed study of the kinetics and stability of the encapsulated enzyme would be an interesting study. The adaptation of these methods to larger support materials and alternative enzymes could lead to simple methods of immobilisation that would produce highly robust heterogeneous catalysts. One potential route to encapsulation of an enzyme without deactivation would be to first immobilise the enzyme on a thiol functionalised support. The surface of the catalyst could then be reacted with a bulky compound incorporating a free thiol group, to encapsulate the enzyme via disulfide formation between the support and the bulky thiol.