Differential stabilities of alkaline protease inhibitors from actinomycetes: effect of various additives on thermostability

Abstract

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 °C with a half-life of 2 h but API-II showed a half-life of 1 h at 45 °C. API-III exhibited the least thermal stability with complete loss of activity at 37 °C after 1 h. The stability of API-I, II and III at 65, 55 and 45 °C, respectively, was enhanced by the addition of various additives. Glycine (1 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 °C by forming a reversible enzyme–inhibitor complex.