RESUMO
Exploiting the vast diversity of soil samples, we have isolated three actinomycetes strains producing alkaline protease inhibitors API-I (242 U/ml). API-II (116 U/ml) and API-III (186 U/ml). The inhibitors exhibited different properties in their molecular nature and in their pH and temperature stabilities. API-I and API-II were high molecular weight (> 10 kD) proteinaceous inhibitors whereas API-III was a low molecular weight inhibitor (< 10 kD). API-I and API-II exhibited stability over a pH range of 5-12 whereas API-III displayed a wide pH stability from 2-12. API-I was stable at 60 degrees C with a half-life of 2 h but API-II showed a half-life of 1 h at 45 degrees C. API-III exhibited the least thermal stability with complete loss of activity at 37 degrees C after 1 h. The stability of API-I, II and III at 65, 55 and 45 degrees C, respectively, was enhanced by the addition of various additives. Glycine (I M) offered complete protection to the three APIs. Polyethylene glycol 8000 (10 mM) prevented the thermoinactivation of API-I. In the presence of glycerol and sorbitol (10%) increase in stability by 40 60% of API-I and API-II was obtained. API-I offered enhanced stability to the target alkaline protease at 50 degrees C by forming a reversible enzyme-inhibitor complex.