6-Aminohexanoate oligomer hydrolases from the alkalophilic bacteria Agromyces sp. strain KY5R and Kocuria sp. strain KY2

Abstract

Alkalophilic, nylon oligomer-degrading strains, Agromyces sp. and Kocuria sp., were isolated from the wastewater of a nylon-6 factory and from activated sludge from a sewage disposal plant. The 6-aminohexanoate oligomer hydrolases (NylC) from the alkalophilic strains had 95.8 to 98.6% similarity to the enzyme in neutrophilic Arthrobacter sp. but had superior thermostability, activity under alkaline conditions, and affinity for nylon-related substrates, which would be advantageous for biotechnological applications.

FIG. 1.

Mode of degradation of nylon oligomers by the Ahx cyclic-dimer hydrolase (NylA), the Ahx dimer hydrolase (NylB), and the Ahx endo-type-oligomer hydrolase (NylC).

FIG. 2.

TLC of various nylon oligomers and reaction products by nylon oligomer hydrolases. (A and B) Cell extracts (KI72, KY1, KY2, KY31, KY32, KY4, KY5R, and KY5S) were incubated for 3 h with 2 mg/ml of the Ahx cyclic oligomers (degree of polymerization, >3) (see panel C) at 30°C (A) or at 60°C (B) in 20 mM phosphate buffer containing 10% glycerol, pH 7.3 (buffer A). After a 1-μl aliquot was spotted, reaction products were developed and detected by spraying them with ninhydrin (12). Ahx, authentic Ahx; Ald, the authentic Ahx linear dimer; Aco, the Ahx cyclic oligomers. (C) The Ahx cyclic dimer and Aco (degree of polymerization, >3) were fractionated from the NOM by an enzyme reaction using purified NylA_p2 or NylC_p2. Samples at each preparation stage were analyzed by TLC (slots 1 to 10). Slot 1, unwashed NOM; slot 2, NOM-W (NOM washed with hot water on filter paper); slot 3, NOM-W digested with NylC_p2; slot 4, the purified Ahx cyclic dimer (used for NylA assay); slot 5, the Ahx cyclic dimer (sample 4) digested with NylA_p2; slot 6, the Ahx cyclic dimer (sample 4) digested with NylC_p2; slot 7, NOM-W digested with NylA_p2; slot 8, purified Aco (used for the NylC assay); slot 9, Aco (sample 8) digested with NylA_p2; slot 10, Aco (sample 8) digested with NylC_p2. (D) NylC purified from E. coli clones (0.1 mg/ml each) was incubated with a 10 mM concentration of the Ahx cyclic dimer, 10 mM Ald, and 2 mg/ml Aco at pH 7.3 at 30°C for 4 h, and reaction products were detected by TLC as described above. Slot 1, NylC_p2 plus the Ahx cyclic dimer; slot 2, NylC_A plus the Ahx cyclic dimer; slot 3, NylC_K plus the Ahx cyclic dimer; slot 4, NylC_p2 plus Ald; slot 5, NylC_A plus Ald; slot 6, NylC_K plus Ald; slot 7, NylC_p2 plus Aco; slot 8, NylC_A plus Aco; slot 9, NylC_K plus Aco.

FIG. 3.

Comparison of amino acid sequences and characterization of NylC_p2, NylC_K, and NylC_A. Amino acid residues are shown in the one-letter code. Fifteen residues differed between NylC_p2 and NylC_K. The five darkly shaded residues in both NylC_K and NylC_A were altered. Ten residues were unique to NylC_K (lightly shaded). The NylC enzymes were purified and characterizations were compared. For quantitative NylC assay, the enzyme reactions were performed at 30°C using 1 mg/ml of the Ahx cyclic oligomer as a substrate in buffer A (see the text) (standard assay conditions), and an increase in the amino group was assayed using trinitrobenzene sulfonic acid (4, 6, 7). For the kinetic studies, NylC activity was assayed under standard assay conditions, except that various concentrations of the Ahx cyclic oligomer were used. Kinetic parameters (k_cat and K_m values) were evaluated by directly fitting the Michaelis-Menten equation to the data using the program GraphPad Prism, version 5 (Graphpad, San Diego, CA). Since the Ahx cyclic-oligomer substrate used for the enzyme assay includes Ahx cyclic oligomers with different degrees of polymerization, K_m values are expressed in mg/ml. Assuming that the Ahx cyclic oligomer is composed of the homogeneous Ahx cyclic tetramer (M_r = 448), the Ahx cyclic oligomer (1 mg/ml) corresponds to a 2.2 mM concentration of the Ahx cyclic tetramer. k_cat values are expressed as turnover numbers per α- or β-subunit (M_r = 36,000). For determination of thermostability, each NylC enzyme (1 mg/ml) was incubated at 30 to 75°C (5°C intervals) in buffer A for 30 min and residual activity was measured under standard assay conditions. Even after the heat treatment at the temperatures shown in the figure or lower temperatures, more than 90% of the enzyme activities were retained. To determine the optimum pH, the enzyme reaction was performed under standard assay conditions, except that pH was changed using 100 mM phosphate buffer (pHs 6.0, 6.5, 7.0, and 7.5), 100 mM barbital-HCl buffer (pHs 7.5, 8.0, 8.5, 9.0, and 9.5), or 100 mM carbonate buffer (pHs 9.5, 10.0, and 10.5). The pH range that exhibited more than 80% of the maximum activities are shown.