Extracellular acid protease from Rhizopus oryzae: Purification and characterization

Sushil Kumar, Neeru S. Sharma, Mukh R. Saharan, Randhir Singh

Research output: Contribution to journalArticlepeer-review

130 Scopus citations


Extracellular aspartate protease from Rhizopus oryzae was purified 91 times with 26% recovery using (NH4)2SO4 fractionation, ion-exchange and size-exclusion chromatographic techniques. The enzyme was found to be monomeric in nature having a molecular mass of 34 kDa. The enzyme acts optimally at 60°C with activation energy of 15.16 kcal/mol and was stable in the temperature range of 30-45°C. The purified enzyme is an acid protease with optimum pH of 5.5 and retained 96% of residual activity between pH 5.5 to 7.5. Ca2+ activation (250 times) and varying substrate concentration gave an hyperbolic response. The Lineweaver-Burk plot showed Km value of 5 mg/ml, when skim milk was used as substrate. The enzyme inhibition of 73 and 93% by pepstatin at 10 and 20 μM, respectively proved it to be an aspartate protease; however, the additional requirement of histidine residue for enzyme activity has been indicated by differential spectra of diethyl pyrocarbonate treated versus untreated enzyme.

Original languageEnglish (US)
Pages (from-to)1701-1705
Number of pages5
JournalProcess Biochemistry
Issue number5
StatePublished - Apr 2005
Externally publishedYes


  • Aspartate protease
  • Cheese
  • Microbial enzyme
  • Milk clotting activity
  • Rhizopus oryzae

ASJC Scopus subject areas

  • Bioengineering
  • Biochemistry
  • Applied Microbiology and Biotechnology


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