A Novel <i>Lentinula edodes</i> Laccase and Its Comparative Enzymology Suggest Guaiacol-Based Laccase Engineering for Bioremediation

<div><p>Laccases are versatile biocatalysts for the bioremediation of various xenobiotics, including dyes and polyaromatic hydrocarbons. However, current sources of new enzymes, simple heterologous expression hosts and enzymatic information (such as the appropriateness of common screening substrates on laccase engineering) remain scarce to support efficient engineering of laccase for better “green” applications. To address the issue, this study began with cloning the laccase family of <i>Lentinula edodes</i>. Three laccases <i>perfectio sensu stricto</i> (Lcc4A, Lcc5, and Lcc7) were then expressed from <i>Pichia pastoris</i>, characterized and compared with the previously reported Lcc1A and Lcc1B in terms of kinetics, stability, and degradation of dyes and polyaromatic hydrocarbons. Lcc7 represented a novel laccase, and it exhibited both the highest catalytic efficiency (assayed with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) [ABTS]) and thermostability. However, its performance on “green” applications surprisingly did not match the activity on the common screening substrates, namely, ABTS and 2,6-dimethoxyphenol. On the other hand, correlation analyses revealed that guaiacol is much better associated with the decolorization of multiple structurally different dyes than are the two common screening substrates. Comparison of the oxidation chemistry of guaiacol and phenolic dyes, such as azo dyes, further showed that they both involve generation of phenoxyl radicals in laccase-catalyzed oxidation. In summary, this study concluded a robust expression platform of <i>L. edodes</i> laccases, novel laccases, and an indicative screening substrate, guaiacol, which are all essential fundamentals for appropriately driving the engineering of laccases towards more efficient “green” applications.</p></div

Biodegradation of (A) ANT and (B) BaA by the recombinant laccases and their LMSs.

<p>The reaction was performed in 1×McIlvanie buffer (pH 4) at 30°C by using 5 µg of enzyme with or without 1 mM HBT or TEMPO. Residual amount of PAHs was resolved by high-performance liquid chromatography (Waters) equipped with a C-18 reverse-phase column (4.6 mm×250 mm, Grace) at ambient temperature and quantified by a photodiode array detector at 252 nm. Results shown are the average of three independent experiments ± S.D.</p

Specific dye decolorization rate of (A) the recombinant laccases, (B) their laccase-HBT system, and (C) laccase-TEMPO system.

<p>The reaction was performed in 1×McIlvanie buffer (pH 4) at 30°C by using 5 µg of enzyme with or without 1 mM HBT or TEMPO. The specific decolorization rate was defined as the amount of dye decolorized in one hour by one mg of protein under the assay condition. Detailed decolorization profiles are displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066426#pone.0066426.s007" target="_blank">Fig. S7</a>. Results shown are the average of three independent experiments ± S.D.</p

A rooted maximum likelihood phylogenetic tree of basdiomycete laccases <i>perfectio sensu stricto</i>.

<p>Laccases of <i>L. edodes</i> are in red, whereas others are individually colored. Numbers at nodes are bootstrap percentages (≥50%) from 100 replicates. Branch lengths here did not represent evolutionary changes. The tree was rooted by using two ascomycetous sequences [<i>Chaetomium globosum</i> (Cg) and <i>Neurospora crassa</i> (Nc)]. Cc: <i>Coprinopsis cinerea</i>; Fm: <i>Fomitiporia mediterranea</i>; Sc: <i>Schizophyllum commune</i>; Ha: <i>Heterobasidion annosum</i>; Sh: <i>Stereum hirsutum</i>; Po: <i>Pleurotus ostreatus</i>; Gs: <i>Ganoderma spp.</i>; Ds: <i>Dichomitus squalens</i>; Tv: <i>Trametes versicolor</i>; Cs: <i>Ceriporiopsis subvermispora</i>; Ps: <i>Punctularia strigosozonata</i>. Numbers following the abbreviations refer to JGI protein IDs in the respective fungal genome.</p

A heat map displaying the association between activity on benchmark substrates, dyes, and PAHs.

<p>Clustering was performed by MeV v4.8 based on the Pearson’s correlation, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066426#pone-0066426-t004" target="_blank">Table 4</a>. +H: with 1 mM HBT; +T: with 1 mM TEMPO.</p

Kinetic parameters^a of recombinant laccases on benchmark substrates (mean ± SD).

a<p>Apparent values. Parameters of Lcc1A and Lcc1B were obtained from our previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066426#pone.0066426-Wong1" target="_blank">[2]</a>.</p>b<p>One U was defined as the number of µmol of respective oxidized product formed in one minute under standard assay condition.</p>c<p>No measurable activity.</p>d<p>Not determined.</p

Correlation^a of activity on benchmark substrates, dye decolorization^b and PAH degradation^c.

a<p>Good (|C| ≥0.9) and fair association (0.5<|C|<0.9) were bold and underlined, respectively.</p>b<p>Initial decolorization rate.</p>c<p>Degradation % at 24 hours.</p>d<p>Applications substrates without decolorization/degradation were excluded.</p>e<p>1 mM mediator.</p

Electrophoretic analyses of Lcc4A, Lcc5, and Lcc7.

<p>(A) SDS-PAGE of recombinant laccases. Proteins were denatured before being resolved in 12% (w/v) SDS-PAGE followed by CBBR staining. (B) Zymogram of recombinant laccases. Native proteins were resolved in 12% (w/v) SDS-PAGE followed by an activity staining with 1 mM ABTS in accordance with Srinivasan <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066426#pone.0066426-Srinivasan1" target="_blank">[23]</a>.</p

Amino acid sequence alignments of recombinant laccases of <i>L. edodes</i> with other fungal laccases.

<p>Lt: <i>Lentinus tigrinus</i> (PDB: 2QT6); Pc: <i>Pycnoporus cinnabarinus</i> (PDB: 2XYB); Rl: <i>Rigidopours lignosus</i> (PDB: 1V10); Tt: <i>Trametes trogii</i> (PDB: 2HRG); Tv: <i>Trametes versicolor</i> (PDB: 1GYC). Predicted signal peptides are red-underlined. Conserved residues are blue-shaded. Unique residues between Lcc1A, Lcc4A, Lcc5, and Lcc7 are red-shaded. Predicted N-glycosylation sites of recombinant laccases are black-circled. Signature sequences (L1–L4) of fungal laccases are annotated. Substrate-binding loops are green-boxed. Residues with known functions (reviewed by Giardina <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066426#pone.0066426-Giardina1" target="_blank">[4]</a>) are arrowed.</p

Firmicutes phylogeny and principal component analysis (PCA) plot based on Unifrac distances between the Firmicutes sequences in control and NASH subjects.

<p>(A) The Firmicutes phylogeny was reconstructed from the OTU representative sequences in the control and NASH samples, and their relative abundance was indicated by gradient color from red to blue. (B) PCA plot of controls and NASH patients. The percentage of variation explained by each principal component was indicated in the parenthesis.</p