Biocatalytic hydrogel beads, which retain higher activity, expand, and contract with changes in pH, having biocompatibility, are developed. Composite spherical beads of chitosan having a diameter of 1-2 mm were prepared by ionic gelation using sodium tripolyphosphate (TPP). Above 3% TPP, the activity of the enzyme decreases. The mechanical strength of the chitosan-TPP beads was further improved by the addition of clay or cassava starch granules. The immobilization of protease (fungal, Aspergillus) was done with glutaraldehyde crosslinking. The chitosanstarch hydrogel beads showed significant increase in firmness and stiffness when compared with chitosan-clay beads. The swelling studies show that the particles expand at pH 1.2 and contract at pH 7.4. The activity retention of the immobilized protease was as high as 70% and exhibited a high pH and lower temperature optima than the free enzyme. Chitosan-starch hydrogel beads exhibited degradation peaks at about 90-110 degrees C in TGA analysis. The biocatalyst beads retained 85% of the original catalytic activity even after eight cycles of repeat use. The freeze-dried beads has good storage stability and can be used either as artificial bioreactor systems in detergent or in therapeutic formulations (c) 2007 Wiley Periodicals, Inc.