Introduction

Lignocellulosic material is of significant interest due to its potential capability to produce substitutions for petroleum-derived chemicals [39, 48]. Lignin, consisting of phenylpropane units connected by ether and C–C linkages, is a branched polymer of high complexity, and is mainly derived from three monolignols: the 4-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units, respectively [5, 33, 47]. The β-O-4 aryl ether linkages are the most abundant (accounting for 50–70%) followed by β-β, β-5, 5–5, and 5-O-4 linkages [45].

As part of an efficient biorefinery process, the pretreatment of lignocellulosic material is a key step to break down or loosen highly resistant lignin. Various physical, chemical, and biological approaches have been attempted for this pretreatment [19, 21, 27, 65]. Besides, fungi [23, 51, 66] and bacteria [61] have been studied for use in the degradation of lignin. Although some bacteria have been reported to depolymerize lignin, the enzymology of bacterial lignin degradation is poorly understood until recently. The dye-decolorizing peroxidase (DyP)-type enzymes (EC 1.11.1.19) have been studied for the ability to degrade lignin. The novel peroxidase DypB from the lignolytic soil bacterium Rhodococcus jostii RHA1 was demonstrated to catalyze the peroxide-dependent oxidation of divalent manganese [53]. Three DyPs were found in Pseudomonas fluorescens Pf-5, and Dyp1B enzyme showed oxidation activity for Mn(II) and Kraft lignin [40]. Different bacteria may employ different pathways of lignin degradation [3].

In this study, the ability of Pseudomonas sp. Q18 to degrade native lignocellulosic material and alkali lignin (AL) was investigated. Pseudomonas sp. Q18 was first isolated from rotten wood in Qinling Mountain, Shaanxi province China [63]. The degradation results were used to identify and characterize the DyP-type peroxidase from Pseudomonas sp. Q18.

Materials and methods

Microorganism, culture conditions, and chemicals

Pseudomonas sp. Q18 (Accession No. KX822686) was isolated from rotten wood in the forests of Qinling Mountain in Shaanxi province, China, as described in our previous article [63]. The phylogenetic tree of strain Q18 is shown in Fig. S1. Escherichia coli DH5α and BL21(DE3) (TransGen Biotech Co., Ltd) were used for gene cloning, and expression. The plasmid pET-28a(+) (Takara Bio, Otsu, Japan) was used as the expression vector. Wheat straw, corn stalk, and switchgrass were collected from a farmhouse in Xianyang City of Shaanxi province. All chemicals and biochemical reagents used in this work were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Biodegradation of native lignocellulosic material by strain Q18

Corn stalk, switchgrass, and wheat straw were used as the raw lignocellulosic material. The plant material was pulverized, screened by a 0.38 mm screen, and then dried at 60 °C for 48 h. Strain Q18 was grown in liquid LB medium until reaching an OD 600 of 1.0. Then, they were inoculated (10%, v/v) to the biodegradation medium: 2.5 g of NaNO3, 1.0 g of KH2PO4, 1.0 g of K2HPO4, 1.0 g of MgSO4, 1.0 g of NaCl, 0.5 g of CaCl2, 1.0 g of NH4Cl, and 3.0% (w/v) raw lignocellulosic material in 1000 mL of deionized water (pH 6.0). Strain Q18 was separately inoculated into the medium containing corn stalk, switchgrass, or wheat straw. The cultures were then incubated at 37 °C and 150 rpm for 15 days. Control samples were inoculated without bacteria.

The lignin content in the samples was determined after treatment. The total lignin content included the acid-insoluble lignin (AIL) and acid-soluble lignin (ASL) [36]. The ASL was determined by maximum UV absorbance of the supernatant, after the insoluble lignin was removed, at 205 nm using an extinction coefficient of 110 L g−1 cm−1 [1]. The formula was as follows:

$${\rm ASL\;content\,(\%)} = (d_{\text{f}} \times V \times A)/(110 \times W) \times 100,$$
(1)

where df is the dilution factor, V is the total volume of the supernatant, A is the UV absorbance, and W is the weight of the initial wood powder. The AIL was determined according to the standard Klason’s method [4, 24]. In a Soxhlet extractor, the samples were extracted with ethanol–benzene (1:1) for 8 h and then dried at 60 °C for 48 h. Next, 72% sulfuric acid was added and mixture was incubated at 15 °C for 2.5 h, before dilution of the sulfuric acid to 3% using deionized water. Subsequently, the samples were autoclaved at 121 °C for 30 min to remove the cellulose and hemicellulose in the residues and determine the lignin content. In this study, the AIL content was calculated by the following formula:

$${\rm AIL\; content \; (\%)} = {\rm Acid\; insoluble\; lignin \; (g)}/{\rm total\; biomass\; }({\text{g}})\; \times 100.$$
(2)

Analysis of field-emission scanning electron microscope (FE-SEM)

Shredded lignocellulosic material degraded by incubation with Pseudomonas sp. Q18 for 15 days were inspected with FE-SEM to visually study the decay processing [28]. To do this, 50 μL medium of the wheat straw sample was placed on a glass slide. The samples were immobilized by the addition of glutaraldehyde (2.5%) for 1 h. Next, a gradient of 30–90% ethanol was used to dehydrate the samples. After the above processing, the samples were coated with a layer of Au/Pd alloy using an ion sputter (HITACHI E-1045) [52]. Samples of AL degraded by strain Q18 for 7 days were collected and centrifuged at 8000×g for 10 min. The supernatant was dried in a vacuum freeze-drying apparatus, before imaging of these samples by FE-SEM (HITACHI S-4800). The controls were cultures lacking bacteria.

Gel-permeation chromatography (GPC) of degradation of alkali lignin

The biodegradation of AL was carried out in medium containing 2.5 g of NaNO3, 1.0 g of KH2PO4, 1.0 g of K2HPO4, 1.0 g of MgSO4, 1.0 g of NaCl, 0.5 g of CaCl2, 1.0 g of NH4Cl, and 3.0 g of alkali lignin in 1000 mL of deionized water (pH 6.0). The strain Q18 was cultured in LB medium until reaching an OD 600 value of 1.0. Then, the flasks containing 50 mL medium were inoculated with bacterial culture (10% v/v), and then, they were incubated at 37 °C, 150 rpm for 7 days. The sample lacking bacteria were analyzed as the control. The molecular weight distribution of AL was determined by GPC [58, 59]. The supernatant of each sample was dried in a vacuum freeze-drying apparatus. Then, a solution of AL in water (20 mg/mL, 5 mL) was loaded onto an Ultrahydrogel™ Linear 300 mm × 7.8 mmid column. The mobile phase was 0.1 mol/L NaNO3 solution with pH 10.7 and the flow rate was 0.5 mL/min. The sample was detected by UV–vis spectroscopy at 254 and 280 nm.

GC–MS analysis of degradation of AL

Samples to assay the amount of AL degraded by Pseudomonas sp. Q18 after 3 and 7 day incubation were prepared as described [7, 20, 25, 32, 37, 42,43,44, 57, 60, 69]. The cultures (50 mL) without (control) and with bacteria were centrifuged at 8000×g for 10 min. The pH of the supernatant was adjusted to 1–2 with the addition of concentrated HCl. After adding ethyl acetate (three volumes), the organic layer of the mixture was collected and dried. Then, the derivatization reaction occurred in the presence of trimethyl silyl [BSTFA (N, O-bis (trimethylsilyl) trifluoroacetamide and TMCS (trimethylchlorosilane)] (Sigma-Aldrich), after heating at 60 °C for 15 min.

The injection volume was set to 1 µL and the samples were applied and separated by GC–MS (GCMS-QP2010 Ultra). The flow rate of helium was 1 mL/min. The column was held at 50 °C for 5 min, increased to 300 °C at a rate of 10 °C/min, and then held for 5 min. The transfer line and ion source temperatures were maintained at 200 and 250 °C. Electron ionization (EI) mass spectra were recorded at 30-550 (m/z) at electron energy of 70 eV. Based on the retention times (RT), the compounds were identified by comparison to the NIST library.

Cloning and expression of pmDyP in Escherichia coli

Pseudomonas sp. Q18 was cultured in LB medium at 37 °C, 150 rpm for 24 h. The cells were obtained by centrifugation at 4 °C, 8000×g, and the genomic DNA was extracted using an Ezup column bacteria genomic DNA purification kit (Shanghai Sangon Biotech Co., Ltd.), from a total of 1 mL of the culture. The chromosomal DNA was used as a template for gene-specific primed PCR amplification. The gene-encoding pmDyP was amplified using primers, designed based on the gene sequence of the hypothetical DyP-type peroxidase of Pseudomonas sp. JY-Q (CP011525.1): pm-F: 5′-AATTCCATGGGCATGCCGTTCCAGCAAGGTCTGCTTGCC, and pm-R: 5′-AATGCTCGAGGGCCCGCAGCAAGGAGCTCAGAT (the restriction sites NcoI and XhoI were underlined). Then, the recombinant plasmid pET28a-pmDyP was transformed into E. coli DH5α. The successful cloning of the pmDyP gene was confirmed by sequencing by Shanghai Sangon Biotech Company (China).

The resulting plasmid with the pmDyP gene under the control of the T7lac promoter was transformed into the host expression strains E. coli BL21(DE3). Expression of PmDyP was induced by addition of IPTG (0.2 mM) when the culture had reached an optical density of about 0.6 at 600 nm. The cells were collected by centrifugation (6000×g for 10 min at 4 °C) and resuspended in 2 mL of 20 mM Tris–HCl buffer (pH 8.0) containing 5 mM NaCl, followed by ultrasonication for 20 min using a SCIENTZ-IID ultrasonic homogenizer (Ningbo Scientz Biotechnology Polytron Technologies Inc., Zhejiang province, China). The resulting cell lysates were centrifuged at 8000×g for 30 min at 4 °C. SDS-PAGE was then performed to analyze the supernatant and the insoluble fraction of the sonicated whole cell lysate [10, 63].

Purification and refolding of PmDyP from inclusion bodies

The precipitate was dissolved in extraction buffer (20 mM Tris–HCl, 150 mM NaCl, 20 mM imidazole with 8 M urea, pH 8.0) overnight at 4 °C. After centrifugation, the supernatant was loaded onto an Ni–NTA His Bind resin column (Novagen, Germany) and the protein of PmDyP was eluted by elution buffer (20 mM Tris–HCl, 150 mM NaCl, 8 M urea, with 250 mM imidazole, pH 8.0). The eluted protein was detected by SDS-PAGE. The eluate containing PmDyP was diluted with extraction buffer to a final protein concentration of 0.5 mg/mL. The samples were dialyzed to refold in renaturation buffer (20 mM Tris–HCl, 150 mM NaCl, 0.5 mM GSH (Glutathione reduced), 0.05 mM GSSG (l-glutathione oxidized), 10 mM glycine, containing different concentrations of urea) at 4 °C with stirring at 120 rpm, and the renaturation buffer was changed every 8 h with decreasing urea concentration. The protein concentration was determined by BCA method with bovine serum albumin as the standard [8, 67].

Bioinformatic and phylogenetic analysis of PmDyP

The plasmid pET28a-pmDyP was sequenced, and the databases of PeroxiBase and NCBI were used to explore the protein homology and conserved motifs of the deduced amino acid sequence of PmDyP obtained from Pseudomonas sp. Q18. The phylogenetic tree analysis of the reported DyP-type A–D protein was constructed using the MEGA 5.0 and iTOL based on the algorithm of neighbor-joining (NJ) [2, 49]. The sequence for the signal peptide, transmembrane regions, and the hydrophobicity of the protein were identified and analyzed by SignalP3.0, TMHMM, TMPRED at the ExPASy server, and ExPASy-ProtScale [15, 34]. Sequence alignments of the reported primary sequences of DyPs were performed using ClustalW Multalign [56, 71]. The deduced tertiary structure of PmDyP was predicted using I-TASSER (https://zhanglab.ccmb.med.umich.edu/I-TASSER/) [64].

Steady-state kinetic assays

All assays were determined at 25 °C in 100 mM phosphate buffer (pH 5.0) using a microplate reader (Victor X3, PerkinElmer, USA). Enzyme activity was determined at different concentrations of the following substrates. The oxidation of ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)), at concentrations 0.05–10 mM, was performed with 1 mM hydrogen peroxide, with monitoring at 420 nm (ε420 = 36,000 M−1 cm−1). The oxidation of guaiacol, at concentrations 0.05–5 mM, was studied with 1 mM hydrogen peroxide, with monitoring at 470 nm (ε470 = 26,600 M−1 cm−1). The oxidation of 2,6-DMP (2,6-Dimethylphenol), at concentrations 0.05–5 mM, was performed with 1 mM hydrogen peroxide, with monitoring at 468 nm (ε468 = 49,600 M−1 cm−1). The oxidation of MnCl2, at concentrations 0.05–25 mM, was performed with 1 mM hydrogen peroxide, with monitoring at 238 nm (ε468 = 6500 M−1 cm−1). Steady-state kinetic parameters were measured by non-linear curve fitting to the obtained enzyme activities (using Graphpad Prism 5 software) and were fitting to the Michaelis–Menten equation [6, 40, 56].

Statistical analysis

All experiments were performed in triplicate, and the results were expressed as mean ± standard deviation. Data analysis was performed by ANOVA and Duncan test using SPSS 16.0. In all analysis, p < 0.05 was considered significant.

Results and discussion

Biodegradation of native lignocellulosic material by strain Q18

The degradation results of the raw lignocellulosic material are shown in Fig. 1. The measured AIL amounts of native corn stalk, wheat straw, and switchgrass were (21.61 ± 0.57) %, (22.72 ± 0.49) %, and (18.95 ± 0.52) %, respectively. The ASL content of the three kinds of plants was less than 4%. After treatment by Pseudomonas sp. Q18, the amount of lignin remaining decreased. Most ASL was degraded, which was agreed with a previous report [12]. The amount of the remaining material compared with the total lignin was lower for switchgrass (14.60 ± 0.79) % than it was for corn stalk and wheat straw. The AIL content was also the lowest, which indicated that the Pseudomonas sp. Q18 exhibited the best degradation capability on switchgrass. The diversity of plant biomass types results in differences in composition and structure among these feedstocks, which plays an important role in process of the plant biomass conversion. Plant biomass consists of lignin, carbohydrate polymers, pectins, proteins, and other components [13]. Grass lignin is composed of all G, S, and H sub-units which differs from the composition present in hardwoods (G and S) and softwoods (G and H) [30, 50]. The S/G ratio can influence structural features, such as the amount of β-O-4 linkages, methoxyl content, or the degree of condensation [50]. Compared with wheat straw and corn stalk, the treatment of switchgrass by Pseudomonas sp. Q18 showed the highest weight loss of dry biomass, almost 25%. The degradation results of the lignin in native lignocellulosic material indicated that Pseudomonas sp. Q18 had high potential to break down lignin [11, 26]. Compared with Rhizobia sp. YS-1r, Pseudomonas sp. Q18 had great potential capacity for raw lignin degradation. Rhizobia sp. YS-1r was reported to degrade 15% of AIL in switchgrass [22], much less than the 25% observed here. However, the activity of Pseudomonas sp. Q18 was lower (about 20%) than the reported activity of the fungi, such as Ganoderma applanatum BEOFB 411 with a maximum rate of wheat straw lignin degradation of 35% during cultivation for 14 days [14]. The morphological changed in wheat straw after treatment by strain Q18 were observed by FE-SEM. There were obvious changes before and after degradation. The control wheat straw seemed smooth and plump on the surface. After 15 day microbial treatment, the structure was disrupted and many small fragments were attached to the surface.

Fig. 1
figure 1

Changes of total lignin content and loss weight after pretreatment by Pseudomonas sp. Q18 for 15 days. In addition, the images of degraded wheat straw treated by strain Q18 by FE-SEM. Control samples were inoculated without bacteria. AIL acid-insoluble lignin, ASL acid-soluble lignin, LW loss weight of dry native straw. Total lignin (%) = AIL content (%) + ASL (%). All experiments were performed in triplicate, and the results were expressed as mean ± standard deviation

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Degradation of AL by strain Q18

GPC was used to measure the molecular weight distribution of AL before and after the bacterial degradation by Pseudomonas sp. Q18. The number-average (Mn), weight-average (Mw) molecular weights, and polydispersity (Mw/Mn) are shown in Fig. 2 and Table 1. The AL was used, as a bacteria-free control, and showed a molecular weight (Mw) range from 53 to 68,599 Da. The Mw of the control sample indicated that the AL included both high and low Mw lignin fractions. The fraction with fragment of Mw greater than 2000 Da in size was about 20%. After 7 days of Pseudomonas sp. Q18 treatment, the highest Mw was decreased to 48772 Da, and only 10% of fragments had the Mw more than 1500 Da. This result indicated that the molecular weight tended to be low values after treatment by bacteria, which was coincidence with the lignin content after treatment. The GPC results indicated that the AL particles with high Mw were depolymerized into smaller ones after treatment. In addition, the analysis of untreated and treated AL by FE-SEM showed that the control sample maintained irregular spherical shapes with porous internal structure, and the surface was mostly smooth, although some breakage might have occurred during processing. The diameter of these AL particles was greater than 100 μm. However, the smooth surface of the lignin was completely eroded after microbial treatment. The Cupriavidus basilensis B-8 also could break down the kraft lignin from 15.1 to 1.65 kDa for 7 days [52]. For these processes, the degraded lignin fraction could be consumed as the carbon source for bacteria metabolism.

Fig. 2
figure 2

GPC chromatograms of molecular weight distribution and FE-SEM images of changes in the control sample and AL treated by strain Q18. The sample lacking bacteria were analyzed as the control

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Table 1 GPC analysis of molecular weight distribution in the control sample and alkali lignin treated by strain Q18
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GC–MS analysis of degraded AL

GC–MS was used to analyze the AL degradation compounds with low molecular weight. After treatment with Pseudomonas sp. Q18, the appearance of new peaks and reduction of peaks indicated the generation of new metabolic products [26]. The numbers of peaks changed (Fig. S2), and the aromatic compounds identified from the peaks of degradation for 0 (control), 3, and 7 days are shown in Table 2. There were 12, 9, and 17 aromatic compounds detected in the control, 3 and 7 day samples, respectively.

Table 2 Aromatic compounds identified as TMS derivatives in the control sample (0 day) and the degraded alkali lignin by strain Q18 for 3 and 7 days
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According to Table 2 and Fig. 3, the high molecular weight compounds, i.e., Compound 25 (1,2-benzenedicarboxylic acid, dinonyl ester) (RT = 29.689 min) and Compound 26 (1,2-benzenedicarboxylic acid, ditridecyl ester) (RT = 29.935 min), detected in all samples, were the main compounds in the control sample. Additionally, in the control sample, a small proportion of aromatic monomer compounds were found, like Compounds 12, 14, 20, and 28. These compounds were considered to be the basic units that built the lignin structure, and may have derived from the industrial process.

Fig. 3
figure 3

Proportion of absorption peaks area of aromatic compounds in the control (0 day) (a), third day (b), and seventh day (c) samples treated by strain Q18

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In the 3 day sample, the complex aromatic compounds, Compound 16 (17.94%), Compound 24 (12.72%), and Compound 25 (8.84%), were decreased compared with the control. However, the relative content of Compound 13 (30.08%) was significantly increased. Most of the low molecular weight aromatic monomers (i.e., Compounds 12 and 14) disappeared. The results were the same as those observed in the previous literature of co-culture of Bacillus subtilis and Klebsiella pneumonia for Kraft lignin degradation [62]. It indicated that lignin was degraded by strain Q18. Meanwhile, the massive consumption and catabolism of aromatic compounds showed that the strain Q18 had the ability to utilize the low molecular weight compounds as a carbon or energy source [48, 62, 70]. Lignin depolymerization, aromatic catabolism, and co-product generation occurred simultaneously.

Lignin is a branched polymer of high complexity that consists of substituted phenylpropane units joined by C–C and ether linkages [5, 33, 47]. After incubation for 7 days (Fig. 3c), the relative content of these high molecular weight compounds (Compounds 16 and 24) was still decreased and some typical lignin degradation compounds were identified (Table 2). Compared with the control, a significant increase in the number of peaks was found after 7 day treatment, which was consistent with other studies [16, 57]. 1-(4-hydroxy-3,5-dimethoxyphenyl)-ethanone (Compound 22, S unit), a typical lignin monomer unit, was found in the 7 day sample and its relative content accounted for 18.51%, as well as Compound 15 (2.56%). The G unit monomer, 1-(4-hydroxy-3-methoxyphenyl)-ethanone (Compound 18, 4.05%) and 2-methoxy-4-vinylphenol (Compound 14, 1.39%) appeared as degraded products in the cultures.

In the degradation process, the amounts of organic acids and esters increased, such as oxalic acid, ethyl acetate, and 3-acetyloxybutanoic acid ethyl ester [9, 31]. This increase possibly reflects chemical reaction among microbial primary metabolites or the intermediate cleavage of lignin after degradation.

It was reported that more than 160 kinds of single phenyl ring compounds could be obtained from lignin, such as p-coumaryl alcohol, coniferyl alcohol, sinapyl alcohol, and their derivatives [68]. However, in this study and previous literatures, less than 20 single phenyl ring aromatic products were identified, because more compounds might be in very small amounts outside the GC–MS scale [70]. Benzenepropanoic acid, syringic acid, and ferulic acid were found in kraft lignin degraded by Acetoanaerobium sp. WJDL-Y2 detected by GC–MS [16]. Some aromatic compounds like vanilic acid, coniferyl, and sinapyl alcohols were important intermediates or products, and their presence in the alkali lignin degradation production could be related to the oxidation of lignin [38, 44, 46, 57]. In a recent study, vanillic acid was determined to be a highly abundant aromatic metabolite (44.2%) of alkali lignin after 5 days of treatment by Bacillus ligniniphilus L1 [70].

Bioinformatic analysis of PmDyP

Combined with the analysis of aromatic compounds and the reported metabolic pathways [70], a DyP-type peroxidase was thought to be present in Pseudomonas sp. Q18. The primers of PmDyP were designed according to the sequence of the hypothetical protein of DyP-type peroxidase reported in NCBI, especially the sequences of related Pseudomonas sp. strains. The results of PCR amplification showed that PmDyP could be amplified using the primers designed according to the sequences of the DyP genes of Pseudomonas sp. JY-Q. Then, sequence of PmDyP was cloned into pET-28a(+) and transformed into E. coli DH5α. Analysis of the nucleotide sequence of PmDyP indicated that the cloned DNA contained a single open reading frame of 864 bp which encoded a protein monomer of 287 amino acids with a deduced theoretical pI of 4.71 [18, 29]. The cloned enzyme contained a signature DyP-type peroxidase motif after analysis of protein domain databases (Fig. S3).

According to the previous literature, DyPs were divided into four (A–D) subfamilies with bacterial and fungal enzymes phylogenetically [35, 49]. With the construction of a phylogenetic tree and the maximum likelihood analysis of reported DyPs (Fig. 4), PmDyP was found to be closest to PpDyP (GI: 501229266), which was reported to belong to the B-type subfamily [49]. However, 20 amino acids were different in these proteins, which may lead to the different activity. Most homology analysis has focused on primary, secondary and tertiary structure homology. Some homologous proteins have been analyzed, though most of the proteins remain theoretical or their function is unknown. This study chose two well-studied and shared proteins (DyPPa and TyrA) that are homologous to PmDyP in primary structure. Compared with the reported primary sequences of DyPs, PmDyP shared highly conserved amino acid residues (Fig. 5), such as Asp132 (D132), His197 (H197), Arg214 (R214), and the GXXDG motif which played a significant role in the formation of the catalytic centers and the functions of the DyP-type peroxidase family. PmDyP was identified with 48.67 and 60.88% amino acid homology to DyPPa and TyrA, respectively. Like the tertiary structure of DyPPa and TyrA, PmDyP showed a typical spatial structure. The predicted structure of PmDyP includes two domains, four-stranded antiparallel β-sheets and peripheral α-helices [29, 55]. The structural characteristics supported the hypothesis that PmDyP was a member of the DyP-type peroxidase family. As reported previously, D132, H197, R214, and GXXDG motifs were thought to be related to the heme peroxidase catalytic cycle process and form the activity center. The bent degree of R242 and R245 in TyrA and BtDyP, respectively, lead to different activities, suggesting that the state of R214 might influence enzyme activity [17, 49, 54].

Fig. 4
figure 4

Radial phylogram of DyPs. The names of bacterial or fungal strains are indicated in subscript with the protein or abbreviated organism names. Phylogenetic tree comparing PmDyP (bold) with isolated DyPs belonging to subfamilies A–D: DyP A: TfuDyp from Thermobifida fusca (GI: 71917209), Mva from Mycobacterium vanbaalenii PYR-1 (ABM12972.1), Bsu168 from Bacillus subtilis 168 (CAB15852.1), Pde from Paracoccus denitrificans PD1222 (ABL69832.1), Rpa from Rhodopseudomonas palustris BisB18 (ABD87513.1). DyP B: DyPPa from Pseudomonas aeruginosa (GI: 94829180), CteKF-1 from Comamonas testosteroni KF-1 (EED66859.1), PpDyP from Pseudomonas putida (GI: 501229266), YfeX from E. coli K-12 (BAE76711.1), Ddi from Dictyostelium discoideum AX4 (EAL70759.1), Acsp from Acinetobacter sp. ADP1 (CAG67144.1), RjoDypB from Rhodococcus jostii (GI: 330689635), BtDyP from Bacteroides thetaiotaomicron VPI-5482 (2GVK), Cco from Campylobacter concisus 13826 (EAT98288.1). DyP C: Chu from Cytophaga hutchinsonii ATCC 49185 (ABG59511.1), Cysp from Cyanothece sp. PCC 7424 (ACK71272.1), Cvi from Chromobacterium violaceum ATCC 12472 (AAQ59612.1), Oan from Ochrobactrum anthropi ATCC 49185 (ABS17389.1), Pssp from Psychrobacter sp. PRwf-1 (ABQ94167.1), MxDyP from Myxococcus xanthus (GI: 108465542), SavDyP from Streptomyces avermitilis (GI: 29604188), Ana from Anabaena sp. PCC 7120 (BAB77951.1), AmspDyp2 from Amycolatopsis sp. Atcc 39116 (4G2C_B). DyP D: Lbi2 from Laccaria bicolor S238 N-H82 (EDR12662.1), Ppl from Postia placenta Mad-698-R (EED79944.1), Pch from Penicillium chrysogenum Wisconson 54-1255 (CAP99029.1), BjaDyP from Bjerkandera adusta Dec1 (GI: 4760440), AauDyP1 from Auricularia auricula-judae (GI: 433286646), Pos from Pleurotus ostreatus (CAK55151.1), Msp1 from Marasmius scorodonius (GI: 261266601)

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Fig. 5
figure 5

Deduced tertiary structure of PmDyP using I-TASSER with the C-score of 1.16. In addition, the multiple alignment of amino acid sequences for PmDyP, DyPPa, and TyrA. DyPPa from Pseudomonas aeruginosa PKE117 (GU967380), TyrA from Shewanella oneidensis (NP716371)

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Expression and steady-state kinetic characterization

The PmDyP was expressed and purified. As shown in Fig. S4, the PmDyP was expressed as insoluble inclusion bodies. The inclusion bodies were dissolved in denaturation buffer and purified by the Ni–NTA agarose column. After the refolding process, the purified PmDyP was analyzed by SDS-PAGE (Fig. 6), which showed that its molecular weight was about 31.7 kDa. Steady-state kinetic characterization was performed using a range of peroxidase substrates, providing the Km and kcat values, as listed in Table 3 (original data shown in Supporting information, Fig. S5). PmDyP showed catalytic activity with all tested peroxidase substrates. However, according to the previous report, there was no activity to be observed with Mn2+ and guaiacol for TfuDyP. The kcat/Km value of PmDyP was 7000 using ABTS as the substrate, which was higher than the value of TfuDyP [41]. In addition, for guaiacol and Mn2+, PmDyP showed higher values of kcat/Km than Dyp1B, Dyp2B, and DyPA from P. fluorescens Pf-5 [40]. The steady-state kinetic characterization demonstrated the potential peroxide ability of PmDyP for biotechnological applications.

Fig. 6
figure 6

Result of SDS-PAGE analysis of protein. Lane 1, insoluble fraction of sonicated whole cell lysate after 6 h induction at 30 °C with 0.2 mM IPTG; lane 2, soluble fraction of sonicated whole cell lysate after 6 h induction at 30 °C with 0.2 mM IPTG; lane 3, inclusion bodies dissolved in extraction buffer; lane 4, eluate with 250 mM imidazole; M, protein molecular weight marker

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Table 3 Steady-state kinetic parameters for PmDyP
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Conclusion

In summary, Pseudomonas sp. Q18 was proved to have the excellent ability to break down alkaline lignin and native lignocellulosic material. Many kinds of relative aromatic compounds were identified as changing in the process of lignin degradation. Combined with the reported metabolic pathways and the results in this paper, a novel DyP-type peroxidase, PmDyP, was cloned and expressed from Pseudomonas sp. Q18. Steady-state kinetic characterization suggested that PmDyP was a potential peroxidase. Future work should explore more relative metabolites and enzyme characterization combined with new technologies such as genetic engineering and bioinformatics.