title: Downstream processing, characterization, and structure-function relationship of solvent-, detergent-, psychro-, thermo-, alkalistable metalloprotease from metal-, solvent-tolerant psychrotrophic Pseudomonas putida SKG-1 isolate.
abstract: 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 K(m) and V(max) to be 1.169 mg mL(-1) and 0.833 mg mL(-1) min(-1) , respectively. The k(cat) value of 3.05 × 10(2) 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 Zn(2+) 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 Zn(2+) affirmed this enzyme as zinc-dependent metalloprotease. At 0.1% concentration, Triton X-100 and Tween 80 slightly increased, while SDS and H(2) O(2) 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.
authors.affiliation: Center of Excellence, Dept. of Microbiology, Dr. Ram Manohar Lohia Avadh University, Faizabad, Uttar Pradesh 224001, India.
authors.forename: Santosh Kumar
authors.initials: SK
authors.lastname: Singh
authors.forename: Sanjay Kumar
authors.initials: SK
authors.lastname: Singh
authors.forename: Vinayak Ram
authors.initials: VR
authors.lastname: Tripathi
authors.forename: Satyendra Kumar
authors.initials: SK
authors.lastname: Garg
authors.forename: Sunil Kumar
authors.initials: SK
authors.lastname: Khare
mesh_terms: D003902:Detergents / Q000494:pharmacology*; D004492:Edetic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D045726:Metalloproteases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification* / Q000378:metabolism; D019216:Metals, Heavy / Q000737:chemistry / Q000378:metabolism*; D010618:Phenanthrolines / Q000494:pharmacology; D055550:Protein Stability / Q000187:drug effects; D017433:Protein Structure, Secondary / Q000187:drug effects; D011549:Pseudomonas / Q000201:enzymology*; D012997:Solvents / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D013696:Temperature
chemical_list: D003902:Detergents; D004791:Enzyme Inhibitors; D019216:Metals, Heavy; D010618:Phenanthrolines; D012997:Solvents; D004492:Edetic Acid; D045726:Metalloproteases; C025205:1,10-phenanthroline