The purification and characterization of psychro-thermoalkalistable protease from psychrotrophic Pseudomonas putida isolate is being reported for the first time. A approximate to 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 mL1 and 0.833 mg mL1 min1, respectively. The kcat value of 3.05 x 102 s1 indicated high affinity and catalytic efficiency toward casein. The protease was most active at pH 9.5 and 40 degrees C, with 100% stability in pH and temperature range of 6.011.0 and 1040 degrees C, respectively. Presence of Zn2+ increased the thermostability of protease (at 70 degrees 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 -mercaptoethanol were ineffective, revealing the enzyme to be a metalloprotease. Zinc, calcium, iron, nickel, and copper at 1 mM increased the enzyme activity (102134%). 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 (5481%). In the presence of organic solvent, the protease was remarkably stable exhibiting 72191% 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 -rich protein, having large fraction (approximate to 40%) of -sheets. Presence of different environmental conditions altered the -content, which accordingly affected the protease activity. (c) 2012 American Institute of Chemical Engineers Biotechnol. Prog. 29;99-108, 2013