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Title: Protein containing porous polymers for biocatalytic applications
Author: Long, James.
Awarding Body: Liverpool John Moores University
Current Institution: Liverpool John Moores University
Date of Award: 2005
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Over the last twenty years there has been a great deal of research conducted into the use of enzymes as catalysts for reactions conducted in non-aqueous media. Not only has it been shown that enzymes can function effectively in organic media, but also that the range of reactions that they can catalyse has been vastly increased. The major disadvantages associated with non-aqueous enzymatic catalysis are relatively slow reaction rates compared to traditional aqueous catalysis, poor enzyme stability towards polar organic solvents and protein agglomeration, which leads to reduced efficiency, making recovery and reuse of the enzyme very difficult. Immobilisation of the enzyme on a suitable support material has been shown to be an effective method in overcoming these problems. This study examined several different methods for immobilising a-chymotrypsin on novel support materials. The catalytic activities of the preparations were assayed by following the transesteriflcation reaction between N-acetyl-L-tyrosine ethyl ester (A TEE) and propan-l-ol by high performance liquid chromatography (HPLC). Immobilised enzyme activities were compared to those obtained for simple unsupported lyophilised preparations of a-chymotrypsin. Uniform porous poly(acrylamide) beads were loaded with various quantities of enzyme via an adsorption procedure. Catalytic activity was measured over a wide range of thermodynamic water activities and was found to be similar to the lyophilised preparations. However, the immobilised enzyme was shown to be more resistant to changes in pH and temperature, and could easily be recovered from the reaction mixture and reused. Design of experiment methodology was employed to optimise support matrices constructed from six component materials. The enzyme was immobilised via a noncovalent entrapment method. The best composites prepared displayed a fifty-fold increase in catalytic activity and a three-fold increase in mechanical strength relative to the equivalent a-chymotrypsin controls. These materials could be reused more than ten times whilst still retaining useful catalytic activity. Porous poly(acrylamide) monoliths containing entrapped a-chymotrypsin were synthesised using a novel carbon dioxide high internal phase emulsion templating technique. The effects of enzyme loading, carbon dioxide pressure and monomer to crosslinker ratio were examined. The corresponding enzyme activity of all the emulsion templated materials was shown to be higher than for the unsupported lyophilised preparations, with the best materials exhibiting a ten-fold increase in activity. Multipoint covalent enzyme immobilisation was also studied. The structure of the enzyme was first modified so as to include a polymerisable functionality. This modified enzyme was then dissolved in organic solvents via the formation of ion-pairs with various anionic surfactants. It was shown that the enzyme remained in solution when transferred from organic solvents to a mixture of monomers. The dense gas porogen R134a was then added to the solution of enzyme in monomer, prior to the initiation of a polymerisation reaction. The resulting crosslinked enzyme-containing polymers were shown to possess useful catalytic activity
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available