No abstract

Galactose oxidase (E.C. 1.1.3.9) was covalently immobilized to chemically modified porous silica particles by reaction of the native enzyme with pendant benzoyl azide groups on the carrier. The enzyme loading on the carrier was 100-150 units per milliliter. The immobilized enzyme was incorporated into a hardware assembly suitable for the determination of galactose or lactose concentrations in complex biological fluids. The prototype instrument as described is suitable for continuous, on-line monitoring or discrete sample analysis. Reaction conditions can be readily provided which maintain global first order kinetics within the reactor and strict linearity of the procedure over a wide range of sample concentrations. Auto-inactivation of the immobilized enzyme can be prevented by K3Fe(CN)6 and long-term reactor stability can be achieved by the periodic application of the reagent to the enzyme reactor in situ.

An analysis of the pore diffusion model involving a two-substrate enzymatic reaction is presented. The resulting equations have been applied to the case of galactose oxidase catalyzed oxidation of galactose when the enzyme is immobilized on porous glass particles. The physical constants of the system were obtained by theoretical predictions and the enzyme concentration in the porous medium was derived from the experimental results. The calculations were performed with the assumption that the kinetic parameters of the enzyme remain unchanged upon immobilization. The theoretically calculated effectiveness factors were compared with the experimental effectiveness factors determined from the batch kinetic experiments and were found to be in agreement. The results are presented as effectiveness factor plots graphed as functions of bulk galactose and oxygen concentrations. The model was extended in order to study the effect of external mass transfer coefficients and pore enzyme concentrations on the effectiveness factors.