A new one-step procedure for entrapping proteases into a polymeric composite calcium alginate-poly(N-vinyl caprolactam) hydrogel was developed that provided 75-90% retention of the activity of entrapped enzymes compared to soluble ones. Properties of entrapped carboxypeptidase B, trypsin, and thrombin were investigated. The immobilized enzymes were active within a wide pH range. The temperature optima of entrapped trypsin and carboxypeptidase B were approx 25°C higher than that of the soluble enzymes, and the resistance to heating was also increased. The effects of various polar and nonpolar organic solvents on the entrapped proteases were investigated. The immobilized enzymes retained their activity within a wide concentration range (up to 90%) of organic solvents. Gel-entrapped trypsin and carboxypeptidase (CPB) were successfully used for obtaining human insulin from recombinant proinsulin. The developed stabilization method can be used to catalyze various reactions proceeding within wide pH and temperature ranges. © Copyright 1997 by Humana Press Inc. All rights of any nature whatsoever reserved.