Purification and characterization of a novel peroxidase from Geotrichum candidum dec 1 involved in decolorization of dyes

Abstract

A peroxidase (DyP) involved in the decolorization of dyes and produced by the fungus strain Geotrichum candidum Dec 1 was purified. DyP, a glycoprotein, is glycosylated with N-acetylglucosamine and mannose (17%) and has a molecular mass of 60 kDa and an isoelectric point (pI) of 3.8. The absorption spectrum of DyP exhibited a Soret band at 406 nm corresponding to a hemoprotein, and its Na2S2O4-reduced form revealed a peak at 556 nm that indicates the presence of a protoheme as its prosthetic group. Nine of the 21 types of dyes that were decolorized by Dec 1 cells were decolorized by DyP; in particular, anthraquinone dyes were highly decolorized. DyP also oxidized 2,6-dimethoxyphenol and guaiacol but not veratryl alcohol. The optimal temperature for DyP activity was 30 degrees C, and DyP activity was stable even after incubation at 50 degrees C for 11 h.

FIG. 1

Butyl Toyopearl chromatography of a peroxidase produced by G. candidum Dec 1. Symbols: ●, peroxidase activity; ○, absorbance at 280 nm.

FIG. 2

(a) SDS-polyacrylamide gel electrophoresis of DyP after each purification step. Lanes: 1, standard molecular mass markers; 2, sample after YM 10 ultrafiltration (amount of protein loaded, 10 μg); 3, sample after Super Q chromatography (amount of protein loaded, 10 μg); 4, purified DyP after Butyl Toyopearl chromatography (amount of protein loaded, 6 μg); 5, mixed DyPs after Butyl Toyopearl chromatography. (b) Isoelectric focusing of purified DyP and mixed DyPs obtained from Butyl Toyopearl chromatography. Lanes: 1, mixed DyPs after Butyl Toyopearl chromatography (amount of protein loaded, 5 μg); 2, purified DyP after Butyl Toyopearl chromatography (amount of protein loaded, 10 μg); 3, standard pI marker.

FIG. 3

Spectral characteristics of purified DyP, DyP oxidized by H₂O₂, and DyP reduced by dithionite. The inset shows the peak at 556 nm indicative of reduced DyP in the form of a heme-pyridine complex.

FIG. 4

(a) Optimal decolorization temperature of DyP. (b) Thermostability of DyP and HRP; enzyme activity at 30°C after treatment with DyP at 40°C (——), DyP at 50°C (●——●), DyP at 60°C (▪——▪), HRP at 40°C (- - -), and HRP at 60°C (▪- - -▪).

FIG. 5

H₂O₂ inhibition of DyP activity when RB5 and its simplified model compound, AQ-2′, were used as substrates. Symbols: □, degradation activity of 0.6 nM DyP for RB5; ●, degradation activity of 2.8 nM DyP for AQ-2′.