A novel one-step method was developed for entrapment of proteolytic enzymes, namely carboxypeptidase B and trypsin, in polymer composite gel based on poly(N-vinyl caprolactam). Native proteases and enzymes previously stabilized by covalent attachment to poly(vinylpyrrolidone-co-acrolein) were immobilized in gels. After immobilization, about 90% and 75% of original trypsin and CPB activities, respectively, were retained. The immobilized enzymes were active within a wide pH range. The optimum temperature of the entrapped enzymes was approximately 25°C higher than that of the soluble enzymes. The entrapped enzymes were successfully used to obtain human insulin from recombinant proinsulin. © 1994 Science & Technology Letters.