A thermo-stable lysine aminopeptidase from Pseudomonas aeruginosa: Isolation, purification, characterization, and sequence analysis.
Pseudomonas aeruginosa NJ-814, isolated from garden soil, produced an extracellular aminopeptidase that was purified using ammonium sulfate precipitation and ion exchange chromatography. The purity was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the Mr value of the enzyme was estimated to be 55 kDa. The purified enzyme shows maximum activity at pH 9.0 and 80 °C. It exhibits high thermo-stability. Half of the activity can remain after incubation at 80 °C for 119 min. It is stable within pH range of 7.5-10.5. It is strongly activated by Co(2+) and inhibited by Fe(2+) , Cu(2+) , Ni(2+) , Zn(2+) , and ethylene diamine tetraacetic acid (EDTA). The specificity of the enzyme was investigated. Within several aminoacyl-p-nitroanilines (AA-pNA), Lys-pNA is proven to be the optimal substrate. The Michaelis-Menten constant (Km ) of the enzyme for Lys-pNA and Leu-pNA were 2.32 and 9.41 mM, respectively. Peptide map fingerprinting shows that the sequence of the enzyme is highly similar to aminopeptidase Y from P. aeruginosa 18A. It can be speculated that this enzyme is a Zn(2+) -dependent enzyme and contains two zinc ions in its active site.
Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China.
Yan Tao
YT
Wu
Nan Di
ND
Zhou
Zhe Min
ZM
Zhou
Xin Xing
XX
Gao
Ya Ping
YP
Tian
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D012988:Soil Microbiology; D013379:Substrate Specificity; D013696:Temperature
D008670:Metals; D000626:Aminopeptidases
Aminopeptidase; Characterization; Pseudomonas aeruginosa NJ-814; Purification; Sequence analysis