Porcine pepsin was immobilized by chemical aggregation using glutaraldehyde as a bifunctional crosslinking agent. The immobilized pepsin followed Michaelis-Menten kinetics (K, = 5.3 x 10-5M) and the yield of immobilization was 91%. The activation energy of the immobilized preparation was 90,613 cal/mol as compared to 67,532 cal/mol for native pepsin. Using acid-denatured hemoglobin and N-acetyl phenyl-alanyl-3, 54iiodotyrosine (APDT) as substrates, the activities shown by the immobilized pepsin were, respectively, 67 and 79% that of the soluble pepsin. The immobiized pepsin showed marked stabilization against pH, temperature, urea, and guanidine hydrochloride. The activity of the immobilized preparation in the presence of urea was greater when hemoglobin was used as the substrate than when APDT was used as substrate. Storage of the preparation under refrigerated conditions for 160 days showed 58% retention in enzyme activity. The immobilized pepsin can be removed from the reaction mixture volume easily, retaining nearly 100"/0 of its activity even after being used in seven consecutive ass a y s .
INTRODUCTlON
Immobilized enzymes have great potential in the chemical and food industries due to their high stability and reusability.'.' Pepsin, known to undergo rapid inactivation above pH 6, has been fixed to a large number of inert support^,^ including glutaraldehyde-activated amino-ethyl cellulose9 are amino-hexyl Sepharose." All these methods of immobilization are time consuming, uneconomical, and lead to increase in reaction mixture volume. This article describes the preparation and properties of a fine suspension of insoluble pepsin using glutaraldehyde as the crosslinking agent.