Purification and characterization of a solvent stable aminopeptidase from Pseudomonas aeruginosa: Cloning and analysis of aminopeptidase gene conferring solvent stability

Aminopeptidase from a solvent tolerant strain Pseudomonas aeruginosa PseA was purified and studied for its biochemical and molecular characteristics. Ion-exchange chromatography resulted in 11.9-fold purification and 38% recovery of the 56 kDa enzyme. The enzyme was found to be stable over a pH rang...

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Bibliographic Details
Published in:Process biochemistry (1991) 2010-05, Vol.45 (5), p.757-764
Main Authors: Gaur, Ruchi, Grover, Tripti, Sharma, Rita, Kapoor, Sanjay, Khare, Sunil K.
Format: Article
Language:English
Subjects:
Aminopeptidase
Biochemistry
Enzymes
Genes
Ion-exchange chromatography
Metallopeptidase
Pseudomonas aeruginosa
Purification
Residues
Solvent tolerant
Solvents
Stability
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Summary:Aminopeptidase from a solvent tolerant strain Pseudomonas aeruginosa PseA was purified and studied for its biochemical and molecular characteristics. Ion-exchange chromatography resulted in 11.9-fold purification and 38% recovery of the 56 kDa enzyme. The enzyme was found to be stable over a pH range of 6.0–8.0 and appreciably thermostable up to 70 °C. PseA aminopeptidase exhibited K m of 3.02 mM and V max of 6.71 μmol/mg/min towards l-Leu- p-nitroanilide. Remarkable stability in both hydrophilic and hydrophobic solvents makes PseA aminopeptidase unique. Partial N-terminal sequence of enzyme showed exact match with probable aminopeptidase of P. aeruginosa PAO1, coded by gene pepB. Polymerase chain reaction amplified the 1611-bp open reading frame encoding a 57.51 kDa, 536 amino acid PseA PepB polypeptide. The deduced PseA PepB protein sequence contained a 24-residue signal peptide (2.57 kDa) followed by a 1.28 kDa propeptide and a mature product of 500 residues. Search for conserved domain in PseA aminopeptidase explored its place in zinc-metallopeptidase family. Primary sequence analysis showed the hydrophobic inclination of the protein; and the 3D structure modeling elucidated the presence of a high content of hydrophobic residues on its surface probably imparting solvent stability to it. The enzyme might find potential applications in non-aqueous enzymology due to its marked thermostability and striking solvent stability.
ISSN:1359-5113
1873-3298
DOI:10.1016/j.procbio.2010.01.017