Purification and Characterization of 6-Aminohexanoic-Acid-Oligomer Hydrolase of Flavobacterium sp. KI72

Authors


Abstract

6-Aminohexanoic-acid-oligomer hydrolase of Flavobacterium sp. KI72 was purified to homogeneity by column chromatography three times, and by preparative polyacrylamide gel electrophoresis twice. The purified enzyme had the following characteristics.

  • 1The molecular weight was estimated to be 84000 by Sephadex G-200 molecular-sieve chromatography. The enzyme consisted of two homologous subunits of 42000, judged from sodium dodecylsulfate/polyacrylamide gel electrophoresis.
  • 2The optimum pH for activity was between 8 and 9, the optimum temperature was 40° C for a 1-h reaction. The Michaelis-Menten constants and turnover numbers for the 6-aminohexanoic acid dimer and trimer were 5.9 mM and 2.4 s−1, and 6.2 mM and 2.0 s−1 respectively.
  • 3The enzyme was inhibited by 0.37 mM diisopropylfluorophosphate and by 0.013 mM p-chloromercuribenzoate.
  • 4The enzyme was active on 6-aminohexanoic acid oligomers from dimer to hexamer and icosamer but not on hectamer, and the activity decreased with the increase of the polymerization number of the ougomer. The oligomers were hydrolyzed so as to remove the 6-aminohexanoic acid residue successively from the amino terminus. The enzyme could not hydrolyze other linear amides, cyclic amides, dipeptides, tripeptides or casein.
  • 56-Aminohexanoic-acid-oligomer hydrolase was classified as a new member of the linear amidases (EC 3.5.1.-).
Enzyme
 

6-Aminohexanoic-acid-oligomer hydrolase (EC 3.5.1.-)

Ancillary