Purification and characterization of a nylon-degrading enzyme

Abstract

A nylon-degrading enzyme found in the extracellular medium of a ligninolytic culture of the white rot fungus strain IZU-154 was purified by ion-exchange chromatography, gel filtration chromatography, and hydrophobic chromatography. The characteristics of the purified protein (i.e., molecular weight, absorption spectrum, and requirements for 2,6-dimethoxyphenol oxidation) were identical to those of manganese peroxidase, which was previously characterized as a key enzyme in the ligninolytic systems of many white rot fungi, and this result led us to conclude that nylon degradation is catalyzed by manganese peroxidase. However, the reaction mechanism for nylon degradation differed significantly from the reaction mechanism reported for manganese peroxidase. The nylon-degrading activity did not depend on exogenous H2O2 but nevertheless was inhibited by catalase, and superoxide dismutase inhibited the nylon-degrading activity strongly. These features are identical to those of the peroxidase-oxidase reaction catalyzed by horseradish peroxidase. In addition, alpha-hydroxy acids which are known to accelerate the manganese peroxidase reaction inhibited the nylon-degrading activity strongly. Degradation of nylon-6 fiber was also investigated. Drastic and regular erosion in the nylon surface was observed, suggesting that nylon is degraded to soluble oligomers and that nylon is degraded selectively.

FIG. 1

Morphological disintegration of nylon-66 membrane. (A) Nylon-66 membrane (1 mg) and 5 μl of 200 mM MnSO₄ were added to 1 ml of fivefold-concentrated culture fluid on day 6. The preparation was incubated for 2 days at 30°C. (B) Results obtained when the concentrated culture fluid was used after it was boiled for 5 min.

FIG. 2

Hydrophobic chromatography of a partially purified nylon-degrading enzyme preparation from 6-day-old cultures of IZU-154. Dotted line, absorbance at 270 nm; solid line, absorbance at 405 nm; dashed line, (NH₄)₂SO₄ gradient.

FIG. 3

Electrophoretic analysis of purified enzyme. (A) SDS-PAGE analysis. Lane M contained molecular weight markers. (B) IEF gel electrophoresis analysis. The arrows indicate the pI values of markers.

FIG. 4

Absorption spectrum of purified enzyme. Protein was dissolved in 20 mM sodium acetate (pH 4.5), and the spectrum was determined at room temperature.

FIG. 5

pH profiles of peroxidase activity and nylon-degrading activity. Symbols: ○, 2,6-DMP oxidation; •, weight average molecular weight of nylon-66 membrane. Peroxidase activity was assayed by using 2,6-DMP in a reaction mixture containing 20 mM acetate and 50 mM lactate.

FIG. 6

Scanning electron micrographs of nylon-6 fiber. (A) Unincubated control fiber. (B) Fiber after incubation for 1 day with enzyme. (C) Fiber after 2 days of incubation with enzyme. (D) Fiber after 4 days of incubation with enzyme.