Determination of intrinsic properties of immobilized enzymes : 1. Kinetic studies on sepharose-staphylococcal nuclease in the absence of diffusional limitations.
Staphylococcal nuclease has been insolubilized, directly through its amino groups, on CNBr-activated Sepharose 2B. For kinetic studies, a small substrate (thymidine 5'-(p-nitrophenyl phosphate) 3'-phosphate) has been used to measure the hydrolytic activity. With this system the absence of diffusional limitation has been proven. Eadie-Hofstee analysis of the data has been employed to determine the intrinsic kinetic constants of the insolubilized enzyme. Thek cat-pH andK M-pH profiles and the activation energies are similar for the soluble and for the insolubilized nuclease. At the same time conditions are established in which a stirred batch reactor containing particles of insolubilized nuclease behaves as an open system.
Instituto de Catálisis y Petroleoqúimica, Consejo Superior de Investigaciones Cientificas, Madrid, Spain.
J M
JM
Guisán
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FV
Melo
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A
Ballesteros