• thermoalkaline protease;
  • solvent stable;
  • detergents;
  • secondary structure;
  • CD analysis


The purification and characterization of psychro-thermoalkalistable protease from psychrotrophic Pseudomonas putida isolate is being reported for the first time. A ∼53 kDa protease was purified 21.4-folds with 57.2% recovery by ultrafiltration and hydrophobic interaction chromatography. Kinetic analyses revealed the Km and Vmax to be 1.169 mg mL−1 and 0.833 mg mL−1 min−1, respectively. The kcat value of 3.05 × 102 s−1 indicated high affinity and catalytic efficiency toward casein. The protease was most active at pH 9.5 and 40°C, with 100% stability in pH and temperature range of 6.0–11.0 and 10–40°C, respectively. Presence of Zn2+ increased the thermostability of protease (at 70°C) by 433%. Ethylene diamine tetra acetic acid (EDTA) and 1,10-phenanthroline were inhibitory, whereas phenyl methyl sulfonyl fluoride (PMSF), p-chloro mercuric benzoate (PCMB), and β-mercaptoethanol were ineffective, revealing the enzyme to be a metalloprotease. Zinc, calcium, iron, nickel, and copper at 1 mM increased the enzyme activity (102–134%). Complete reversion of enzyme inhibition (caused by Ethylene diamine tetra acetic acid [EDTA]) by Zn2+ affirmed this enzyme as zinc-dependent metalloprotease. At 0.1% concentration, Triton X-100 and Tween 80 slightly increased, while SDS and H2O2 reduced the protease activity. In the presence of 0.1% commercial detergents, the enzyme was fairly stable (54–81%). In the presence of organic solvent, the protease was remarkably stable exhibiting 72–191% activities. In contrast, savinase exhibited good stability in the presence of hydrophilic solvents, while chymotrypsin showed elevated activities with benzene, toluene, and xylene only. Circular dichroism analysis revealed the protease as a β-rich protein, having large fraction (∼40%) of β-sheets. Presence of different environmental conditions altered the β-content, which accordingly affected the protease activity. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013