Expression and characteristics of manganese peroxidase from Ganoderma lucidum in Pichia pastoris and its application in the degradation of four dyes and phenol

Abstract

Background: Manganese peroxidase (MnP) of white rot basidiomycetes, an extracellular heme enzyme, is part of a peroxidase superfamily that is capable of degrading the different phenolic compounds. Ganoderma, a white rot basidiomycete widely distributed worldwide, could secrete lignin-modifying enzymes (LME), including laccase (Lac), lignin peroxidases (LiP) and MnP.

Results: After the selection of a G. lucidum strain from five Ganoderma strains, the 1092 bp full-length cDNA of the MnP gene, designated as G. lucidum MnP (GluMnP1), was cloned from the selected strain. We subsequently constructed an eukaryotic expression vector, pAO815:: GlMnP, and transferred it into Pichia pastoris SMD116. Recombinant GluMnP1 (rGluMnP1) was with a yield of 126 mg/L and a molecular weight of approximately 37.72 kDa and a specific enzyme activity of 524.61 U/L. The rGluMnP1 could be capable of the decolorization of four types of dyes and the degradation of phenol. Phenol and its principal degradation products including hydroquinone, pyrocatechol, resorcinol, benzoquinone, were detected successfully in the experiments.

Conclusions: The rGluMnP1 could be effectively expressed in Pichia pastoris and with a higher oxidation activity. We infer that, in the initial stages of the reaction, the catechol-mediated cycle should be the principal route of enzymatic degradation of phenol and its oxidation products. This study highlights the potential industrial applications associated with the production of MnP by genetic engineering methods, and the application of industrial wastewater treatment.

Keywords: Degradation; Ganoderma lucidum; Manganese peroxidase; Phenolic compound; Yeast expression system.

Fig. 1

Decolorization of O-methoxyphenol with five G. lucidum strains. G. lucidm 00679, 50044, 50817, 51562 and 00680 was cultured on PDA medium for 7 d, and then was taken photographs. a displayed on the front of the petri dish, and (b) displayed the reverse side of the petri dish

Fig. 2

Diameters of colored red-brown circled with G. lucidum 00679 by N-limited and N-rich cultures at 3 μM and 200 μM Mn²⁺

Fig. 3

Electrophoresis of PCR amplification of GluMnP1 from pAO815::GluMnP1 Lane M: DNA marker DL 10000; lane NC: negative control; lane PC: positive control; Lane 1–13: selected transformants. The hollow arrow showed the DNA bands of AOX gene from yeast (about 2200 bp). The solid arrow showed the DNA bands of the GluMnP1 gene plus a part of vector sequences (about 1300 bp)

Fig. 4

SDS-PAGE (a) and western blot analysis (b) of positive clones of recombinant Ganoderma MnP after 2 days of induction (a), lane M: protein marker; lanes 1–13: recombinant plasmid pAO815::MnP; Lane NC: negative control. Arrows showed the expressed bands. b, lane M: protein marker; lanes 1–13: re-pAO815-MnP; lane NC: negative control; lane PC: positive control. An arrow indicated the target band

Fig. 5

Time course and visual effect of decolorization of four dyes by the crude enzymatic solution. A1, B1, C1 and D1 show the in vitro decolorization rate of four dyes over time by consumption of crude rGluMnP1, which included Drimaren Blue CL-BR (A1), Drimaren Yellow X-8GN (B1), Drimaren Red K-4Bl (C1) and Disperse Navy Blue HGL (D1). The black line in each image shows the decolorization rate after treatment by rGluMnP1; the red line shows the results for the negative control. A2, B2, C2 and D2 showed the visual decolorization effect of four dyes by control (untransformed yeast) and crude rGluMnP1 solutions (transformed yeast). (a) and (b) show the visual decolorization effects before/after treated by the untransformed yeast, (c) and (d) show the visual decolorization effects before/after treated by the yeast transformants. Yeasts were broken to prepare the crude enzyme solutions. The reactions were carried out in a 2 mL EP tube

Fig. 6

HPLC chromatography of phenol and the main degradation products. HPLC analysis of the phenol (retention time = 16.37 ± 0.1 min) and its main degradation products of hydroquinone (retention time = 4.96 ± 0.1 min), pyrocatechol (retention time = 8.74 ± 0.1 min). The aqueous solutions of phenol were treated by 5% (Fig. 6a, upper spectrum), 10% (Fig. 6b, middle spectrum) and 15% (Fig. 6c, lower spectrum) rGlMnP1 enzyme solutions, respectively

Fig. 7

Pictorial scheme of the enzymatic degradation route of phenol. ① Hydroxylation of benzene formed the dihydroxybenzene and quinones. ② Dihydroxybenzene and quinones dehydrogenated and opened loop to form carboxylic acids. ③ Carboxylic acids mineralized to carbon dioxide and water. Dashed frame represented the compounds that were detected in this experiment