World Journal of Microbiology and Biotechnology

, Volume 13, Issue 5, pp 573–578

Purification and characterization of a milk clotting protease from Rhizomucor miehei

Authors

  • S. Preetha
    • Department of BiotechnologyBharathiar University
  • R. Boopathy
    • Department of BiotechnologyBharathiar University
Article

DOI: 10.1023/A:1018525711573

Cite this article as:
Preetha, S. & Boopathy, R. World Journal of Microbiology and Biotechnology (1997) 13: 573. doi:10.1023/A:1018525711573

Abstract

Benzamidine, an inhibitor of serine proteases, was used as an affinity ligand for the purification of aspartyl protease from culture filtrate of Rhizomucor miehei. The two step purification protocol (ion-exchange and affinity chromatography) resulted in a homogenous enzyme preparation with seven-fold purification and a final recovery of 22%. The purified enzyme was free of brown pigmentation, a factor inherently associated with the enzyme; it was stable and active at acidic pH (optimum pH 4.1 for proteolytic activity and 5.6 for milk clotting activity). The significant positive characteristic of the enzyme is its comparatively lower thermostability; the enzyme was comparable to calf rennet in its properties of thermostability, milk-clotting to proteolytic activity ratio and sensitivity to CaCl2. Limited protease digestion of the purified enzyme with proteinase K yielded a 20kDa fragment as shown by SDS–PAGE. Native gel electrophoresis of the digest showed an additional peak of activity corresponding to the 20kDa fragment on SDS–PAGE, this fragment retained both milk-clotting and proteolytic activities. It was also inhibited by pepstatin A and hence it is presumed that this fragment contained the active site of the enzyme.

Milk clotting proteasepurificationRhizomucor miehei

Copyright information

© Chapman and Hall 1997