Protein containing porous polymers for biocatalytic applications
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
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