Enzyme Immobilization Using Chitosan-Xanthan Complexes (including Aspergillus sojae protease type XIX)

The immobilization of enzymes is a technique extensively studied since the late 1960s, and the knowledge accumulated on enzyme immobilization studies has grown steadily since then. Hydrogels are a particular category of support material that can be used for convenient immobilization. The preparation of hydrogels can be achieved by a variety of methods: reticulation of linear polymers, grafting of synthetic polymers onto naturally occurring macromolecules, chelation of polycations, and complexation between polyanions and polycations. In this chapter, we describe novel hydrogels prepared from naturally occurring polymers, namely chitosan and xanthan. Immobilization and Assay of Protease 1. Protease type XIX (E.C. from Aspergillus sojae, 0.4 U/mg (Sigma). 2. Hemoglobin powder (Sigma). 3. 0.25M phosphate buffer, pH 7.2. 4. 0.05M acetate buffer, pH 5 3. 5. 0.5Macetate buffer, pH 5.3. 6. Trichloroacettc acid, 5 wt% m water. 7. Substrate: Prepare 2% hemoglobin by dissolvmg (wtth gentle stirring) 5 g of hemoglobm in a solution of urea (80 g urea/80 mL water). Heat the mixture at 37°C for 60 mm, then add 50 mL of 0.25Mphosphate buffer, pH 7.2. Adjust the pH of solution to 7.2, then dilute the solution to 250 mL with water.

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