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

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. The 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. The 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. The enzyme was inhibited by 0.37 mM diisopropylfluorophosphate and by 0.013 mM p‐chloromercuribenzoate. The 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. 6‐Aminohexanoic‐acid‐oligomer hydrolase was classified as a new member of the linear amidases (EC 3.5.1.‐).

Characterization of the 6-aminohexanoate-dimer hydrolase from Pseudomonas sp. NK87

“…100 -however, inhibited approximately 80 YO of the initial activity, implying that a serine residue plays an important role in the catalytic function of the enzyme. Similar results were observed for the F-EII enzyme (Kinoshita et al, 1981) . Basic characteristics of the P-EII and F-EII enzymes are summarized in Table 3.…”

“…2). From these results, P-EII was estimated to be a homodimer enzyme like F-EII (Kinoshita et al, 1981). The purified P-EII enzyme had nearly equal activities against various oligomers of 6-aminohexanoate, from the dimer to the pentamer (AL2-AL5) ( Table 2).…”

“…is similar to that defined for the F-EII enzyme (Kinoshita et al, 1981). Initial rates of the enzyme reaction were measured at 30 "C by using various concentrations of AL2 (0.2-2.0mM).…”

“…NK87 , which can degrade 6-aminohexanoate-cyclic dimer (AC2), a by-product of the nylon-6 industry. In both strains, 6-aminohexanoate-cyclic-dimer hydrolase (EI, EC 3.5.2.12) (Kinoshita et al, 1977) and, 6-aminohexanoate-dimer hydrolase (EII, EC 3.5.1 .46) (Kinoshita et al, 1981) are responsible for the degradation of the cyclic dimer. In Flavobacterium sp.…”

High Yield Synthesis of 12-Aminolauric Acid by “Enzymatic Transcrystallization” of ω-Laurolactam Using ω-Laurolactam Hydrolase fromAcidovoraxsp. T31

“…KI72 has been studied as a model of how microorganisms evolved specific enzymes that degrade nylon oligomers. [18][19][20][21][22][23][24] Since Acd hydrolase has been found to be one of the nylon oligomers degrading enzymes encoded on plasmid pOAD2, it has been considered that the enzymes degrading nylon oligomers are active specifically toward 6-aminohexanoate-cyclicdimer. 14) There is a hypothesis for the birth of nylon oligomer degradation mechanisms that wild-type cells can be maintained in the starved condition for a long period, since the nylon oligomer has no detectible toxicity toward microorganisms.…”