Several continuous flow systems utilising reactors containing soluble enzymes have been designed and built. Various reactor configurations have been examined using the urease catalysed hydrolysis of urea as a model. Preliminary experiments showed that the attainment of a steady state within a continuous stirred tank reactor was dependent on a number of environmental conditions. Loss of enzyme activity was partially prevented by using EDTA in the inlet feed. However enzyme activity was also partially lost in a thin channel reactor by shear forces and adsorption of the enzyme onto the membrane surface. Studies on the enzyme suggest that the shear effect is reversable. Analysis of the residence time distributions in a thin channel reactor showed that under certain controlled conditions this reactor could be considered as an ideally-mixed vessel. Kinetic parameters obtained using this assumption agreed well with those obtained from steady state kinetics. The effect of flow rate on conversion and the minimising of substrate inhibition also indicated a similarity between this reactor and an ideally-mixed vessel. The possibility of using glycerol kinase within a thin channel reactor to produce sn-glycerol 3-phosphate was investigated. Results indicated that reactor operation at the pH optimum of the enzyme did not produce the greatest enzyme stability. Carbamate kinase v/as successfully used within the reactor to reconvert the ADP produced by glycerol kinase back to ATP, thus increasing the efficiency of cofactor utilisation. To further increase the efficiency of ATP usage the cofactor was successfully immobilized to a soluble high molecular weight dextran by a new facile method so that ATP would be retained within the reactor. The immobilized cofactor acted as a substrate for glycerol kinase and several other kinases. However it was not active with carbamate kinase obtained from Streptococcus faecalis is R, and thus could not be used profitably within the reactor.
|Date of Award||1977|