- Open Access
Dye-decolorizing peroxidases in Irpex lacteus combining the catalytic properties of heme peroxidases and laccase play important roles in ligninolytic system
© The Author(s) 2018
- Received: 3 September 2018
- Accepted: 26 October 2018
- Published: 8 November 2018
The white rot fungus Irpex lacteus exhibits a great potential in biopretreatment of lignocellulose as well as in biodegradation of xenobiotic compounds by extracellular ligninolytic enzymes. Among these enzymes, the possible involvement of dye-decolorizing peroxidase (DyP) in lignin degradation is not clear yet.
Based on the extracellular enzyme activities and secretome analysis, I. lacteus CD2 produced DyPs as the main ligninolytic enzymes when grown in Kirk’s medium supplemented with lignin. Further transcriptome analysis revealed that induced transcription of genes encoding DyPs was accompanied by the increased expression of transcripts for H2O2-generating enzymes such as alcohol oxidase, pyranose 2-oxidase, and glyoxal oxidases. Meanwhile, accumulation of transcripts for glycoside hydrolase and protease was observed, in agreement with abundant proteins. Moreover, the biochemical analysis of IlDyP2 and IlDyP1 confirmed that DyPs were able to catalyze the oxidation of typical peroxidases substrates ABTS, phenolic lignin compounds DMP, and guaiacol as well as non-phenolic lignin compound, veratryl alcohol. More importantly, IlDyP1 enhanced catalytic activity for veratryl alcohol oxidation in the presence of mediator 1-hydroxybenzotriazole, which was similar to the laccase/1-hydroxybenzotriazole system.
The results proved for the first time that DyPs depolymerized lignin individually, combining catalytic features of different peroxidases on the functional level. Therefore, DyPs may be considered an important part of ligninolytic system in wood-decaying fungi.
- Dye-decolorizing peroxidase
- White rot fungi
- Irpex lacteus
- Lignin biodegradation
Lignin is the second most abundant constituent of lignocellulosic biomass, amounting to 15–30% by weight or up to 40% by energy . The degradation of lignin represents a key step for carbon recycling in the land ecosystems, as well as a critical issue for cost-effective lignocellulosic biofuels and bio-based chemicals . However, due to the complex and random phenylpropanoic polymeric structure, lignin is highly recalcitrant toward chemical and biological degradations , resulting in lignocellulosic waste and environment pollution. White rot fungi, a large group of wood-decaying basidiomycetes, are able to completely decompose lignin into carbon dioxide and water by extracellular ligninolytic enzymes, which include an array of heme peroxidases and oxidases . Among them, the heme peroxidases, such as manganese peroxidase (MnP), versatile peroxidase (VP), lignin peroxidase (LiP), and laccase (Lac) have been considered to play important roles in lignin degradation [5–7].
Dye-decolorizing peroxidase (DyP) is a member of the novel heme peroxidase family (DyP-type peroxidase superfamily), showing no homology to classic fungal heme peroxidases including MnP, VP, and LiP [8, 9]. So far only eleven fungal DyPs have been purified and characterized . Compared with classic fungal heme peroxidases, the specific feature of all DyP is the ability to oxidize synthetic high redox potential dyes of the anthraquinone type . DyP can oxidize phenolic compounds, such as 2,6-dimethoxyphenol and guaiacol . Recently, there are a few reports about its catalytic ability to non-phenolic lignin model compound veratryl alcohol (VA) and Mn2+, which is attributed to high redox potential peroxidase LiP/VP and MnP, respectively [7, 10, 11]. These findings indicate that DyP might be an important part of ligninolytic system in white rot fungi, although biological roles of DyP are ambiguous in terms of different substrate specificities.
Irpex lacteus is a white rot fungus with a significant potential for various biotechnological applications such as bioremediation of organopollutants in water and soil environments and biopretreatment of lignocellulose [12, 13]. Its biotechnological applications were attributed to the extracellular ligninolytic enzymes, including MnP, LiP, laccase-like, and DyP [14–18]. Our preliminary work demonstrated I. lacteus CD2 could degrade all kinds of lignocellulose and dyes [13–15]. Genome analysis reveals that I. lacteus CD2 has seven mnp genes, two lip genes, and four dyp genes, without lac gene . Compared with MnP, the main ligninolytic enzyme of I. lacteus, DyP is scarcely known for catalytic properties and substrate specificities, especially in lignin degradation. Herein, the main ligninolytic enzymes of I. lacteus CD2 grown in lignin medium were determined, combining extracellular enzyme activities and secretome analysis. Furthermore, the mechanisms of lignin degradation by the main ligninolytic enzymes DyPs were elucidated using transcriptomics and biochemical analysis.
Major extracellular proteins and ligninolytic enzymes of I. lacteus in lignin medium
Major extracellular proteins identified from the SDS-PAGE bands of culture supernatant of I. lacteus CD2 in lignin-Kirk’s medium
In addition to ligninolytic enzymes, some proteins involving in fungal growth, such as glycosidase hydrolases (including chitinases, glucanases, mannosidases and so on) and proteases were identified in the extracellular proteins. Chitinases, glucanases, and mannosidases are involved in hyphal cell wall biosynthesis . Proteases such as polyporopepsin, aspartic protease, and glutaminase are implicated in protein degradation and supplying nitrogen for fungal growth [22, 23]. Glycoside hydrolases are essential for cell wall synthesis and cell wall integrity, involving in protein maturation and transport, such as N-linked glycoproteins processing or carbohydrate structural degradation .
Comparative transcriptome analysis of I. lacteus grown in lignin and glucose medium
Ligninolytic enzyme system
Carbohydrate metabolism, nitrogen metabolism and related enzymes
In addition to oxidoreductases, genes encoding carbohydrate active enzymes (CAZYs) as well as other proteins were upregulated in lignin medium. 47 CAZYs genes of glycoside hydrolase, carbohydrate esterase (CE) or polysaccharide lyase (PL) families had significantly different transcript abundance in LIG3d relative to GLU3d (Additional file 2). The GHs mainly consisted of cellulose-degrading enzymes (GH6), hemicellulose-degrading enzymes (GH10/GH43/GH51), and pectin-degrading enzymes (GH28/GH78/GH88). These enzymes might be to hydrolyze a small amount of carbohydrate remained in lignin, and required for fungal growth. In particular, genes related to CAZYs including trehalase, chitinase, and mannosidase were upregulated. These enzymes might liberate carbon from major storage compound trehalose and be involved in fungal hyphal cell wall biosynthesis for growth [21, 31].
Numerous genes involved in mobilizing and recycling nitrogen were also expressed, including oligopeptide transporter, nucleoside transporter, acetamidase, amino acid permease, amine oxidase, arginase, aspartokinase, methionine synthase, nitrilase, and proteases (Additional file 3). In accordance with the abundance in the extracellular culture filtrates, polyporopepsins, and aspartic protease genes were early induced by lignin (LIG3d). Notably, polyporopepsin gene 0925.239 had the most expression level among extracellular proteases. In addition, acetamidases and amino acid permease genes were significantly differentially accumulated in lignin medium (LIG3d and LIG6d).
The above results suggested that DyPs were the main extracellular ligninolytic enzymes of I. lacteus in lignin medium, while in lignocellulose medium MnPs played key roles . Therefore, DyPs were purified from lignin medium for studying their characterization and catalytic properties.
Purification and characterization of DyPs from I. lacteus in lignin medium
Matching peptides with DyPs from I. lacteus after IlDyP2 and IlDyP1 trypsin digestion and PMF analysis
These differences in biochemical properties of DyP isoenzymes might be attributed to their divergent evolutionary origin. Furthermore, DyP isoenzymes had complementary effects on different pH values, the combination of IlDyP2 and IlDyP1 isoenzymes might result in wider pH range for efficient lignin degradation.
Kinetic constants for oxidation of ABTS, DMP, and guaiacol by IlDyP2 and IlDyP1 from I. lacteus
kcat/Km [s−1 M−1]
125.0 ± 1.4
396.2 ± 0.0
(3.2 ± 0.0) × 106
693.1 ± 8.5
356.3 ± 11.2
(5.1 ± 0.1) × 105
1631.7 ± 10.9
560.8 ± 19.3
(3.4 ± 0.2) × 105
892.7 ± 81.5
198.8 ± 12.6
(2.2 ± 0.1) × 105
229.1 ± 15.9
49.7 ± 0.9
(2.2 ± 0.1) × 105
592.1 ± 53.0
49.2 ± 1.5
(8.3 ± 0.5) × 104
Some proteins in DyP family have low amino acid sequence identity (lower than 15%), and significant differences in catalytic efficiency (kcat/Km) with a few orders of magnitude . Bacterial DyPs possess a lower oxidizing ability than fungal DyPs, oxidizing only less recalcitrant phenolic lignin model compounds and monophenolic substrates . However, Chen et al. found DyP from Thermomonospora curvata showed high catalytic efficiency with ABTS, close to that of fungal DyPs . Except for dye decolorization, DyP from Raoultella ornithinolytica OKOH-1 can directly decolorize melanin, and immobilization can improve its activity and stability . Whereas only a limited number of DyPs were purified and characterized. Further studies are still needed to assess the precise physiological roles and catalytic properties of DyPs, including fungal DyPs.
Irpex lacteus CD2 grown in lignin medium secreted DyPs as the main extracellular ligninolytic enzymes. Transcriptomics analysis revealed that DyPs- and H2O2-generating enzymes including AOX, POX, and GLOX were coordinately expressed for efficient lignin degradation. Moreover, IlDyP2 and IlDyP1 could catalyze the oxidation of typical peroxidases substrates ABTS, phenolic lignin compounds DMP, and guaiacol as well as non-phenolic lignin compound VA. IlDyP1 could enhance the oxidation of non-phenolic lignin compound VA in the presence of mediator 1-HBT, the same as with Lac. These results proved for the first time that DyPs might depolymerize lignin when lacking classic heme peroxidases such as MnP, LiP, and Lac. DyPs could display different catalytic features of different peroxidases to different substrates, combining the catalytic properties of classic heme peroxidases and Lac. Therefore, DyPs may form an important constituent of the ligninolytic system in wood-decaying fungi.
Alkali lignin, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,6-dimethylphenol (DMP), guaiacol, reactive black 5 (RB5), veratryl alcohol (VA), and 1-hydroxybenzotriazole (1-HBT) were purchased from Sigma-Aldrich (St. Louis, MO). Reactive blue 19 (RB19) was purchased from Sinopharm Chemical Reagent Company (Beijing, China).
Strain and culture conditions
Irpex lacteus CD2  was maintained at 4 °C on potato dextrose agar plate. The inoculum was precultured in potato dextrose broth for 7 days at 28 °C, then 10% (v/v) inoculum was transferred into the modified Kirk’s medium, and shaken at 150 rpm. The Kirk’s medium contained: alkali lignin (or glucose) as the sole carbon source, 10 g/L; ammonium tartrate, 0.2 g/L; KH2PO4, 2 g/L; MgSO4·7H2O, 0.71 g/L; CaCl2, 0.1 g/L; and 70 mL trace element solution. The trace element solution contains NaCl, 1 g/L; CoCl2·6H2O, 0.184 g/L; FeSO4·7H2O, 0.1 g/L; ZnSO4·7H2O, 0.1 g/L; CuSO4, 0.1 g/L; H3BO3, 0.01 g/L; Na2MoO4·2H2O, 0.01 g/L; KAl(SO4)2·12H2O, 0.01 g/L; and nitrilotriacetic acid, 1.5 g/L.
Total ligninolytic enzyme activities were measured by monitoring the oxidation of ABTS (ε420 = 36,000 M−1 cm−1) at 420 nm, in 50 mM sodium tartrate buffer (pH 4.0) containing 1 mM ABTS, 1 mM Mn2+, and 0.1 mM H2O2. Manganese-independent peroxidase activity was also determined by ABTS oxidation in the absence of Mn2+. DyP activity was assayed by the decolorization of an anthraquinone dye RB19 (ε595 = 10,000 M−1 cm−1) at 595 nm. The reaction was performed in the same buffer containing 125 μM RB19 and 0.1 mM H2O2. One unit of enzyme activity was defined as the amount of enzyme that oxidized 1 μmol of ABTS or RB19 per minute at 25 °C.
The extracellular enzymes of I. lacteus CD2 grown in alkali lignin at different periods of time were collected and concentrated by 80% ammonium sulfate . The concentrated proteins were separated by one-dimensional SDS-PAGE, and the main bands on the third day were excised from the gel, digested with trypsin, and identified by nano LC–MS/MS. The peptides were separated in a reverse-phase C18 column, 0.18 mm × 100 mm, 5 μm particle size (Thermo). The mobile phases were A (water) and B (acetonitrile) containing 0.1% formic acid . The flow rate was maintained at 300 nL/min. The phase B gradient was started at 3%, followed by a linear gradient to 8% in 1 min, 8–40% in 5 min, 40–85% in 1 min, and held there for 1 min. All MS/MS spectra were searched using PEAKS software against I. lacteus CD2 protein database using the following criteria : enzyme trypsin; fixed modification of cysteine (+ 57.02 Da); and variable modification of methionine (+ 15.99 Da).
Irpex lacteus CD2 was grown in the modified Kirk’s medium containing lignin or glucose as carbon source. The total RNA was extracted from mycelia on days 3 and 6 using the TRIZOL reagent (Invitrogen, Waltham, MA) according to the manufacturer’s instructions. The total RNA was sent to Annoroad Genomics (Beijing, China) for sample preparation and sequencing. All samples were in duplicate. The cDNA was synthesized and prepared for sequencing using the Illumina mRNA-Seq Sample Prep Kit (San Diego, CA). The samples were run on independent lanes, and paired-end sequences of 150 bp were obtained at 4 Gb clean data for each sample using the Illumina Hiseq 2500. The raw reads were trimmed and filtered using Trimmomatic software to remove adapters and low-quality bases . Then clean reads were assembled into transcripts using TopHat and Cufflinks with the I. lacteus CD2 genome as a Ref. [14, 44]. All sequences of transcripts were extracted from reference sequence using gffread from cufflinks pipeline. The gene expression levels were conducted using the fragments per kilobase of exon per million fragments (FPKM) mapped method , and read counts were analyzed for differential expression using DESeq with a q value < 0.05 .
Purification and characterization of IlDyPs
The liquid cultures of I. lacteus CD2 grown in alkali lignin for 3 days were collected and concentrated by 80% ammonium sulfate at 4 °C. 20 mM sodium acetate buffer (pH 5.0) was used to dissolve the pellets and dialyzed using 30-kDa cutoff membrane. Then IlDyPs were purified using a HiTrap Q HP anion exchange column (GE Health, Fairfield, CT) pre-equilibrated with the same acetate buffer. The IlDyPs were eluted with a linear gradient of 0–1.0 M NaCl, and fractions containing active enzymes were pooled after SDS-PAGE. Meanwhile, the bands were excised and identified by peptide mass fingerprinting.
To determine the optimal pH, 50 mM sodium tartrate buffers with pH ranging from 2.0 to 7.0 were used for all substrates including ABTS, DMP, guaiacol, VA, RB19, and RB5 at 25 °C. The maximum activities of IlDyP2 and IlDyP1 were considered to be 100%. For catalysis properties, the reactions were performed in optimal pH at 25 °C using 50–4000 μM substrates by monitoring corresponding oxidation products. The nonlinear least square fitting method was used to calculate the Km, kcat, and kcat/Km parameters of IlDyP2 and IlDyP1 using the GraphPad Prism 5 software.
1-HBT was used as the mediator in evaluating the abilities of IlDyP1 and IlDyP2 to oxidize the non-phenolic lignin compound VA. The oxidation of VA was performed in 50 mM sodium tartrate buffer (pH 4.0 or pH 3.0) containing 1 mM VA, 1 mM 1-HBT, 0.1 mM H2O2, and 0.5 U/mL IlDyP1 or IlDyP2, without 1-HBT or DyP as corresponding control. The reaction proceeded at 30 °C. After 24 h, the reaction products were analyzed by HPLC using a reverse-phase C18 column, 4.6 mm × 250 mm, 5-m particle size (Waters XTerra). The isocratic elution condition was performed with 55% methanol containing 0.1% formic acid at a flow rate of 1 mL/min. The elution peaks were monitored at 310 nm.
XZ, XS, and BY conceived and designed the experiments. XQ performed the experiments. XQ, HL, and FM analyzed the data. XQ, FM, and XS wrote the manuscript. All authors read and approved the final manuscript.
We are grateful to Dr. Rui Ma for her suggestion of the manuscript content and Mr. Zhaohui Zhang for his help in HPLC analysis.
The authors declare that they have no competing interests.
Availability of supporting data
All data supporting the conclusions of this article are included within the manuscript and additional files.
Consent for publication
All authors provide their consent for publication of this manuscript in Biotechnology for Biofuels.
Ethics approval and consent to participate
This research was supported by the National Natural Science Foundation of China (31570577, 31672458), the National Key Research and Development Program of China (2016YFD0501409-02), the National Science Fund for Distinguished Young Scholars of China (31225026), the China Modern Agriculture Research System (CARS-42), and the Elite Youth Program of Chinese Academy of Agricultural Sciences.
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