Covalent Immobilization of Organophosphorus Hydrolase oto Insoluble Bovine Collagen Fibers

Abstract

An organophosphorous hydrolase (OPH) was prepared and partially purified from Flavobacterium sp. The crude enzyme, with an activity of 1030U/g, was immobilized onto insoluble bovine collagen fibers (hide powder), instead of collagen membrane, through glutaraldehyde coupling. Optimal conditions of enzyme immobilization and properties of the immobilized enzyme preparation were investigated. Compared to glutaraldehyde tanned and chrome tanned hide powders, non-tanned hide powder showed higher immobilized enzyme activity. The optimal enzyme immobilization conditions are as follows: 10mg of enzyme was immobilized onto 500mg of hide powder in a 50mM phosphate buffer of pH 7.5 at 200C, and 20% glutaraldehyde offer (based on the hide powder weight) was used to couple the enzyme and collagen fibers. The enzyme activity yield was about 35% and the hide powder immobilized OPH has an enzyme activity of about 7U/g. The immobilized enzyme showed the same temperature and pH profiles as the free enzyme, and it performed at much higher pH and with better thermal stability. The Km value of the immobilized enzyme was a somewhat higher (0.388mM) than that of the free enzyme (0.215mM). The reusability test showed that about 85% activity was retained after 10 use cycles. After storing for eight months at 20oC, the residual activity of the immobilized enzyme preparation was 98%.