Collection of Czechoslovak Chemical Communications - digital archive 

Abstract

A rapid and relatively simple procedure is described for kinetic measurement of low activities of enzymes hydrolyzing phenol conjugates. Phenol generated by the enzyme reactions in the pH-optimum range 5.0-9.5 is continuously monitored by an oxygen membrane electrode of the Clark type. The surface of the electrode exposed to the solution is coated by a layer of chemically insolubilized polyphenol oxidase. The current response of the electrode, indicating the uptake of oxygen - diffusing from the solution together with phenol into the enzyme membrane - is proportional to the enzyme concentration (according to the kind of hydrolase in the range from 0.2 to at least 2-9 ncat). The dependences of the initial rate of enzyme reaction on enzyme and substrate concentration and the K_m- and V_max-values at optimal pH were measured with alkaline phosphatase, emulsin (β-glucosidase) and β-glucuronidase. The kinetic measurements showed that sweet almond emulsin contains two enzymes differing in their affinity toward phenyl-β-D-galactopyranoside.