Isolation of alkalophilic nylon-oligomer-degrading bacterium: Cloning and sequencing analysis of 6-aminohexanoate-oligomer hydrolase

We isolated alkalophilic bacterium, Agromyces sp. KY5R, which degrades 6-aminohexanoate (Ahx)-oligomer (by-product of nylon-6 manufacture, designated as nylon-oligomer), from activated sludge of sewage disposal plant. The strain KY5R exhibited good growth on LBplate containing insoluble Ahx-oligomer (pH10), and produced clear zone by hydrolysis of the oligomers. 6-Aminohexanoate-oligomer hydrolase from KY5R (A-EIII) had optimum pH at 7.5-8.0. This value was 0.5 point higher than the optimum pH of P-EIII (EIII encoded on plasmid pOAD2 in Arthrobacter sp. KI72). In addition, thermostability test revealed that the A-EIII was stable up to 60 °C, while the P-EIII was stable up to 50 °C under the same condition. Cloning and sequencing analysis demonstrated that the amino acid sequence of the two EIII enzymes differs only at 5 positions among 355 amino acid residues. These results suggest that at least one of the fi ve alterations affects the differences in the enzyme characteristics.

[1]  S. Negoro.  Biodegradation of Nylon and other Synthetic Polyamides , 2002 .

[2]  K. Komagata,et al.  Agromyces mediolanus sp. nov., nom. rev., comb. nov., a species for "Corynebacterium mediolanum" Mamoli 1939 and for some aniline-assimilating bacteria which contain 2,4-diaminobutyric acid in the cell wall peptidoglycan. , 1996, International journal of systematic bacteriology.

[3]  T. Yomo,et al.  A plasmid encoding enzymes for nylon oligomer degradation: nucleotide sequence and analysis of pOAD2. , 1995, Microbiology.

[4]  P. Hengen.  Methods and reagents. Cloning PCR products using T-vectors. , 1995, Trends in biochemical sciences.

[5]  I. Urabe,et al.  Nylon oligomer degradation gene, nylC, on plasmid pOAD2 from a Flavobacterium strain encodes endo-type 6-aminohexanoate oligomer hydrolase: purification and characterization of the nylC gene product , 1993, Applied and environmental microbiology.

[6]  I. Urabe,et al.  A new nylon oligomer degradation gene (nylC) on plasmid pOAD2 from a Flavobacterium sp , 1992, Journal of bacteriology.

[7]  I. Urabe,et al.  Amino acid alterations essential for increasing the catalytic activity of the nylon-oligomer-degradation enzyme of Flavobacterium sp. , 1991, European journal of biochemistry.

[8]  T. Holton,et al.  A simple and efficient method for direct cloning of PCR products using ddT-tailed vectors. , 1991, Nucleic acids research.

[9]  S. Takeshita,et al.  High-copy-number and low-copy-number plasmid vectors for lacZ alpha-complementation and chloramphenicol- or kanamycin-resistance selection. , 1987, Gene.

[10]  H. Okada,et al.  Purification and characterization of 6-aminohexanoic-acid-oligomer hydrolase of Flavobacterium sp. Ki72. , 1981, European journal of biochemistry.

[11]  V. Bisaria,et al.  6-Aminohexanoic acid cyclic dimer hydrolase. A new cyclic amide hydrolase produced by Achromobacter guttatus KI74. , 1977, European journal of biochemistry.