PRODUCTION AND PARTIAL CHARACTERIZATION OF A MILK-CLOTTING PROTEINASE PRODUCED BY BACILLUS SUBTILIS SMDFS-2B MN715837 IN SUBMERGED CULTURES

Abstract

This study focused on the production and partial characterization of a milk-clotting protease produced by Bacillus subtilis SMDFS 2B in submerged cultures, under partially optimized conditions. The crude enzyme was recovered in the culture supernatant and concentrate was produced after cell removal and subsequent dialysis. Inhibition studies were conducted employing four distinct protease inhibitors: Pepstatin-A, Phenylmethane-sulphonyl-fluoride (PMSF), Ethylenediaminetetraacetic acid (EDTA), and iodoacetamide (IA). The effect of temperature, pH, metal ions and substrate concentration on milk-clotting activity were also evaluated. The thermal stability of the enzyme was determined by incubating the crude enzyme at a temperature value ranging from 35 ^oC to 60 ^oC. Similarly, pH stability was determined at pH values ranging between 4.5 and 8.0. The highest milk-clotting activity was observed at a temperature of 55 ^oC and pH 5.5. The crude enzyme preparation remained stable on incubation at 35 ^oC and 40 ^oC for 15 min and at pH 5.5. The enzyme also showed the lowest residual milk-clotting activity in the presence of EDTA (7.94%) and Pepstatin-A (26.71%). The addition of Mg²⁺ and Mn²⁺ significantly increased milk-clotting activity. The enzyme also showed an elevation in its apparent milk-clotting activity upon increasing the substrate (skim-milk) concentration. Thus, the milk-clotting protease produced by B. subtilis SMDFS 2B by submerged fermentation revealed some interesting milk-clotting characteristics. This may open the way for applications in the food and dairy industries.