Gene cloning, overexpression and biochemical characterization of the peptide amidase from Stenotrophomonas maltophilia

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 terminal amino acid sequence....

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Bibliographic Details
Published in:Applied microbiology and biotechnology 2002-05, Vol.58 (6), p.772-780
Main Authors: Neumann, S, Kula, M.R
Format: Article
Language:English
Subjects:
amidase
Amides
Amidohydrolases - chemistry
Amidohydrolases - genetics
Amidohydrolases - isolation & purification
Amidohydrolases - metabolism
Amino Acid Sequence
Amino acids
ammonia
Bacteria
Base Sequence
Biological and medical sciences
Biology of microorganisms of confirmed or potential industrial interest
Biotechnology
Chromatography, Gel
Cloning
Cloning, Molecular
DNA
DNA Probes
E coli
Electrophoresis, Polyacrylamide Gel
Enzyme Inhibitors - pharmacology
Enzymes
Escherichia coli
Fundamental and applied biological sciences. Psychology
gene overexpression
genes
Gram-negative bacteria
hydrolysis
isoelectric point
Miscellaneous
Mission oriented research
molecular cloning
Molecular Sequence Data
molecular weight
Peptides
Proteins
Sequence Homology, Amino Acid
signal peptide
Southern blotting
Stenotrophomonas maltophilia
Stenotrophomonas maltophilia - enzymology
Stenotrophomonas maltophilia - genetics
Substrate Specificity
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Description
Summary: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 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 V(max)=194 U/mg and K(m)
ISSN:0175-7598
1432-0614
DOI:10.1007/s00253-002-0943-6