Biochemical properties and potential applications of a solvent-stable protease from the high-yield protease producer Pseudomonas aeruginosa PT121.
An organic solvent-stable protease from Pseudomonas aeruginosa PT121 was purified in a single step with 55% recovery by hydrophobic interaction chromatography on a Phenyl Sepharose High Performance matrix. The purified protease was homogenous on SDS-PAGE and had an estimated molecular mass of 33 kDa. The optimal pH and temperature conditions for enzyme activity were 8.0 and 60 degrees C, respectively. The enzyme was classified as a metalloprotease based on its strong inhibition by EDTA and 1,10-phenanthroline and exhibited good stability across a broad pH range (6.0-11.0). The protease was quite stable in the presence of various water-miscible organic solvents. This is a unique property of the protease which makes it an ideal choice for application in aqueous-organic phase organic synthesis including peptides synthesis. The synthetic activity of the protease was tested using N-carbobenzoxy-L-asparagine (Z-Asp) and L-phenylalaninamide (Phe-NH2) as substrate in the presence of various water-miscible organic solvents for aspartame precursor synthesis. The highest yield was obtained in the presence of 50% DMSO (91%). The synthesis rate in the presence of DMSO was also much higher than the rates in the other tested organic solvents, and the initial rates of Z-Asp-Phe-NH2 synthesis in mixtures of various water-miscible organic solvents, with the exception of ethanol, correlated with the yields of Z-Asp-Phe-NH2. Furthermore, the PT121 protease was able to use various carboxyl components (Z-AA) and Phe-NH2 as substrates to catalyze the syntheses of the dipeptides, indicating that this protease has a broad specificity for carboxylic acid residue.
Tang
Xiao-Yu
XY
State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, 5 Xinmofan Road, Nanjing 210009, China.
Wu
Bin
B
Ying
Han-Jie
HJ
He
Bing-Fang
BF
D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004151:Dipeptides / Q000138:chemical synthesis; D004795:Enzyme Stability; D045726:Metalloproteases / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D012997:Solvents / Q000494:pharmacology; D013379:Substrate Specificity; D013501:Surface-Active Agents / Q000494:pharmacology; D013696:Temperature
D004151:Dipeptides; C548974:N-carbobenzoxy-asparagyl-phenylalaninamide; D012997:Solvents; D013501:Surface-Active Agents; D045726:Metalloproteases; D004121:Dimethyl Sulfoxide