Three problems concerning the operation of enzyme reactors for the recycle of ATP were investigated. The potential of a simple enzyme thermistor system for monitoring reactor effluent was studied. Initial results from physical characterisations showed the interdependence of operating parameters on overall detector sensitivity. Subsequent results demonstrated the detection of urea using an immobilized urease column. Attempts to improve the flexibility of the system by replacing covalently bound enzymes with an adsorption system proved unsuccessful. Ultrafiltration reactors were considered the most suitable type for cofactor requiring systems. The effects of various operating parameters on the problem of concentration polarisation were studied. Results indicated that transmembrane pressure drop was not linearly related to protein concentration at the membrane, as determined by the 'Gel Polarisation' model. The degree of concentration polarisation was found to be a function of temperature, tangential flow and ionic strength of the solvent. The possibility of in situ removal of denatured enzymes was considered. It was found that with suitable adjustments to the operating conditions, proteases could be profitably used for this purpose. A simple two step coupling procedure was developed for binding ATP/ADP to dextran. In the preliminary step, dextran was activated using a bis oxirane in a reaction time of 45 minutes. Subsequent binding of ATP to activated dextran was found to be time dependent allowing a range of complex loading to be produced. The resultant complex was shown to be active with a range of enzymes. Essentially all cofactor groups were shown to be available to hexokinase at the highest loading produced. The effect of cofactor loading on the apparent kinetic constants obtained with hexokinase suggests that the cofactor groups are all equally available. The potential of this complex was demonstrated in a small scale ultrafiltration reactor with hexokinase and acetate kinase.
|Date of Award||1982|