Purification and properties of an aminopeptidase from a protamine-degrading marine bacterium.
A protamine-degrading marine bacterium was isolated from marine soil and identified as Aeromonas salmonicida subsp. based on its taxonomical characteristics. An alanine-specific aminopeptidase, called aminopeptidase K, from an extract of the strain was purified and characterized. The aminopeptidase K was purified about 80-fold by fractionation with ammonium sulfate and column chromatography on QA-52 cellulose, Phenyl Superose and Superose 12. The purified enzyme is composed of 6 subunits of 86 kDa with a molecular mass of 520 kDa according to gel filtration and SDS-PAGE. The N-terminal sequence of the enzyme was H.Gly-Gln-Gln-Pro-Gln-Ile-Lys-Try-Tyr-His-Asp-Tyr-Asp-Ala-Pro-Asp-Tyr-Ty r- Ile-Thr-. It is inhibited by monoiodoacetate, N-ethylmaleimide, and puromycin. The Michaelis constant (K(m)) and the maximal rate of hydrolysis (Vmax) were, respectively, 0.28 mM and 49.4 mumol/min/mg for the L-Ala-beta-naphthylamide substrate. The optimum pH and optimum temperature were 6.5 and 45 degrees C, respectively. The purified enzyme was highly specific to L-Ala-beta-naphthylamide.
Obata
H
H
Department of Biotechnology, Faculty of Engineering, Kansai University, Osaka, Japan.
Sugiyama
A
A
Kawahara
H
H
Muramatsu
T
T
D000333:Aeromonas / Q000378:metabolism*; D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis / Q000494:pharmacology; D000626:Aminopeptidases / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D004795:Enzyme Stability; D005285:Fermentation; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011479:Protamines / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D017421:Sequence Analysis; D013379:Substrate Specificity; D013696:Temperature
D000596:Amino Acids; D011479:Protamines; D011480:Protease Inhibitors; D000626:Aminopeptidases; C070939:aminopeptidase K; D000409:Alanine