Biochemical Properties and Potential Applications of a Solvent-Stable Protease from the High-Yield Protease Producer <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PT121 |
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Authors: | Xiao-Yu Tang Bin Wu Han-Jie Ying Bing-Fang He |
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Institution: | (1) State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, 5 Xinmofan Road, Nanjing, 210009, China;(2) College of Pharmaceutical Engineering, Nanjing University of Technology, 5 Xinmofan Road, Nanjing, 210009, Jiangsu, China; |
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Abstract: | 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°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. |
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