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Purification,characterization and application of alkaline protease enzymeproduced by Streptomyces rochei NRC24

Abdallah N.A1., Farid M.A2., Nayera A. M. Abdelwahed and Asmaa2 I. El Shazly

1- Microbiology Department, Faculty of Science, Ain-Shams University, Cairo, Egypt

2-      Chemistry of Natural and Microbial Products Dept., National Research Center,

12311,   Dokki, Cairo, Egypt.

ABSTRACT

             The crude extracellular alkaline protease from Streptomyces rochei NRC 24 was partially purified by fractional precipitation with ammonium sulphate yielded the highest specific activity of 365.5(U/mg of protein)  at 50% saturation and reached 13.18 fold purification of the culture filtrate. Gel filtration was carried out with columns of sephadex G-100 revealed the presence of two protein peaks. The first protein component covered by fractions 7-13 comprised the majority of alkaline protease activity. The molecular weight of partial purified enzyme was about 38 KDa. The maximum activity was found at 60oC, pH 8 after 10 min. incubation time and 10 mg/ml casein using enzyme concentration of 0.096 mg. The calculated Km value was found to be 2.43 mg/ml, while Vmax was 0.124 µg/ml/min. The enzyme was nearly stable for 60 min at temperature 40-50 oC. Maximum activity was also recorded with CaCl2, MnCl2.4H2O, MgCl2.6H2O, ZnSO4.7H2O, BaCl2.2H2O and H2O2 that caused an increase in the enzyme activity with different ratios. The enzymatic activity was strongly inhibited by EDTA, CuSO4.5H2O, HgCl2, FeCl3, and KMnO4 and (NH4)2S2O8.The enzyme was able to hydrolyze different keratin-containing wastes with different ratios. X-ray film decomposition was investigated where gelatin layer was completely stripped within 60 min at pH 8 and 37oC.

Key words: Streptomyces rochei NRC 24, alkaline protease, purification, characterization, applications