Purification and some properties of an alkaline protease from bacillus macerans

ABSTRACT

Partial purification of the crude alkaline protease produced by Bacillus macerans was carried out by fractional precipitation with acetone, ethyl alcohol or ammonium sulphate separately. The fraction obtained at 65% ammonium sulphate saturation was the most active showing 2.8-fold purification. Further purification was conducted by gel filtration on Sephadex G-100 and ion exchange chromatography of the most active protein peak on DEAE-cellulose. A pure alkaline protease enzyme was obtained [molecular weight 24000]. Maximum activity of the pure enzyme was obtained at a protein concentration of 25 micro g/reaction mixture, and optimum substrate concentration was 10 mg casein/reaction mixture. The amino acid composition of the pure enzyme was similar to that of other bacterial alkaline protease. The optimum temperature of the reaction was 50°C and the optimum pH value was 10.8. The enzyme was fairly stable to heat treatment and retained 56.5% of its activity after heating for 15 min at 60°. Ca [2+] ions slightly activated the enzyme, while Co[2+], Ba[2+], Fe[3+]and EDTA partially inhibited the enzyme activity. Cu[2+], Hg[2+]and Zn[2+]strongly inhibited the enzyme activity. Phenylmethylsulforyl fluoride [PMSF] inhibited the enzyme suggesting the presence of serine in the active site of the enzyme