Stability studies of immobilized enzyme stir rods

Alcohol dehydrogenase has been covalently attached to the surfaces of nylon stir rods. Several rod types have been evaluated in terms of their mixing efficiency and enzyme loading. Fluorometric monitoring of the rate of conversion of NAD to NADH serves as a measure of the reaction rate under varying conditions. The rate of reaction of the enzyme stir rods has been evaluated in terms of RPM, buffer concentration, NAD reagent concentration, and pH. The rate of reaction is seen to reach a plateau at higher stir rates, indicating a lack of diffusional hindrances. The reaction rate also begins to level off at phosphate buffer concentration of 0.1M to 0.15M. Saturating conditions are reached at an NAD concentration of 2.5mM. The optimum pH is found to be 9.0. The Stability of the covalent bond between the enzyme and the nylon has been assessed by comparing the bond strength to the energies of various disruptive forces to which the enzyme is exposed. Centrifugal, drag, and shear forces are shown to be insufficient to cause rupture of the bond. The stability to the immobilized enzyme preparation has been investigated under varying conditions of immobilization and use. No effect on activity loss was found for rotation rate or for continuous versus intermittent use. It was found that enhanced stability occurred for hydrolytic cleavage of the nylon, using nitric acid, as compared to nonhydrolytic cleavage. Hydrolytic cleavage also led to some degree of adsorption of the enzyme to the surface of the nylon. Thus, the possibility of increased stability to multipoint attachment of the enzyme is discussed. Possible cause of activity loss are discussed, as well as the extension of the enzyme stir rod to use in scale model reactor studies.