Gene cloning, overexpression and biochemical characterization of the peptide amidase from Stenotrophomonas maltophilia
- 1 May 2002
- journal article
- research article
- Published by Springer Nature in Applied Microbiology and Biotechnology
- Vol. 58 (6) , 772-780
- https://doi.org/10.1007/s00253-002-0943-6
Abstract
The peptide amidase (Pam) from the gram-negative bacterium Stenotrophomonas maltophilia catalyzes predominantly the hydrolysis of the C-terminal amide bond in peptide amides. Its gene (pam) was isolated by Southern hybridization using a DNA probe derived from the known N-terminal amino acid sequence. Pam is a member of the amidase signature family and was identified as a periplasmic protein by an N-terminal signal peptide found in the gene. The processed protein consists of 503 amino acids with a molecular mass of 53.5 kDa. The recombinant enzyme with a C-terminal His6 tag has a monomeric structure and its isoelectric point is 6.3. The dipeptide amide L-Ala-L-Phe-NH2 is hydrolyzed in the absence of cofactors to L-Ala-L-Phe-OH and ammonia with Vmax=194 U/mg and Km Ki95% pure enzyme was obtained, which corresponds to a total activity of 416,000 units.Keywords
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