Purification and characterization of a solvent stable protease from Pseudomonas aeruginosa PseA.
A solvent tolerant Pseudomonas aeruginosa PseA strain was isolated from soil. It secreted a novel alkaline protease, which was stable and active in the presence of range of organic solvents, thus potentially useful for catalysis in non-aqueous media. The protease was purified 11.6-fold with 60% recovery by combination of ion exchange and hydrophobic interaction chromatography using Q-Sepharose and Phenyl Sepharose 6 Fast Flow matrix, respectively. The apparent molecular mass based on the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was estimated to be 35,000 Da. The enzyme was stable in the pH range of 6.0-9.0, the optimum being 8.0. The Km and Vmax towards caseinolytic activity were found to be 2.7 mg/ml and 3 micromol/min, respectively. The protease was most active at 60 degrees C and characterized as a metalloprotease because of its sensitivity to EDTA and 1,10-phenanthroline. It was tested positive for elastase activity towards elastin-orcein, thus appears to be an elastase, which is known as pseudolysin in other strains of P. aeruginosa. The protease withstands range of detergents, surfactants and solvents. It is stable and active in all the solvents having log P above 3.2, at least up to 72 h. These two properties make it an ideal choice for applications in detergent formulations and enzymatic peptide synthesis.
Gupta
Anshu
A
Chemistry Department, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110016, India.
Roy
Ipsita
I
Khare
S K
SK
Gupta
M N
MN
D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D002413:Cations, Divalent / Q000494:pharmacology; D002852:Chromatography, Ion Exchange; D003510:Cyclohexanes / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification*; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D045726:Metalloproteases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D012988:Soil Microbiology; D012997:Solvents / Q000494:pharmacology
D001426:Bacterial Proteins; D002413:Cations, Divalent; D003510:Cyclohexanes; D012997:Solvents; D010450:Endopeptidases; D045726:Metalloproteases; C087822:alkaline protease