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Downstream processing, characterization, and structure–function relationship of solvent‐, detergent‐, psychro‐, thermo‐, alkalistable metalloprotease from metal‐, solvent‐tolerant psychrotrophic Pseudomonas putida SKG‐1 isolate
Author(s) -
Singh Santosh Kumar,
Singh Sanjay Kumar,
Tripathi Vinayak Ram,
Garg Satyendra Kumar,
Khare Sunil Kumar
Publication year - 2012
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1002/btpr.1654
Subject(s) - pmsf , chemistry , protease , metalloproteinase , pseudomonas putida , thermostability , zinc , chromatography , enzyme , casein , nuclear chemistry , biochemistry , organic chemistry
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. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013