Application of a Novel Alkali-Tolerant Thermostable DyP-Type Peroxidase from <i>Saccharomonospora viridis</i> DSM 43017 in Biobleaching of Eucalyptus Kraft Pulp

<div><p><i>Saccharomonospora viridis</i> is a thermophilic actinomycete that may have biotechnological applications because of its dye decolorizing activity, though the enzymatic oxidative system responsible for this activity remains elusive. Bioinformatic analysis revealed a DyP-type peroxidase gene in the genome of <i>S. viridis</i> DSM 43017 with sequence similarity to peroxidase from dye-decolorizing microbes. This gene, <i>svidyp</i>, consists of 1,215 bp encoding a polypeptide of 404 amino acids. The gene encoding <i>Svi</i>DyP was cloned, heterologously expressed in <i>Escherichia coli</i>, and then purified. The recombinant protein could efficiently decolorize several triarylmethane dyes, anthraquinonic and azo dyes under neutral to alkaline conditions. The optimum pH and temperature for <i>Svi</i>DyP was pH 7.0 and 70°C, respectively. Compared with other DyP-type peroxidases, <i>Svi</i>DyP was more active at high temperatures, retaining>63% of its maximum activity at 50–80°C. It also showed broad pH adaptability (>35% activity at pH 4.0–9.0) and alkali-tolerance (>80% activity after incubation at pH 5–10 for 1 h at 37°C), and was highly thermostable (>60% activity after incubation at 70°C for 2 h at pH 7.0). <i>Svi</i>DyP had an accelerated action during the biobleaching of eucalyptus kraft pulp, resulting in a 21.8% reduction in kappa number and an increase of 2.98% (ISO) in brightness. These favorable properties make <i>Svi</i>DyP peroxidase a promising enzyme for use in the pulp and paper industries.</p></div

Multiple sequence alignment of the <i>Svi</i>DyP amino acid sequence with other members of DyP-type peroxidases family.

<p>The sequence alignments were performed using ClustalW (Thompson et al. 1994). Perfect match residues are displayed in white on black shade. The unique GXXDG motif is boxed. The conserved residues of the active site are indicated by asterisks. Residues indicated by triangles are residuals important for coordinating the heme at the active site.</p

Phylogenetic tree analysis.

<p>Phylogenetic tree showing the relationship between <i>Svi</i>DyP and other isolated DyPs belonging to subfamilies A–D: MYspDyP from <i>Mycobacterium</i> sp. (GI: 126434170), <i>Tfu</i>DyP from <i>Thermobifida fusca</i> (GI: 71917209), Sco3963 from <i>Streptomyces coelicolor</i> A3(2) (GI: 541881521), DypA from <i>Rhodococcus jostii</i> (GI: 499917154), <i>Bs</i>DyP from <i>Bacillus subtilis</i> (GI: 732344), EfeB/YcdB from <i>Escherichia coli</i> (GI: 2506638), <i>Po</i>DyP from <i>Pseudomonas putida</i> (GI: 501229266), <i>Pa</i>DyP from <i>Pseudomonas aeruginosa</i> (GI: 94829180), TyrA from <i>Shewanella oneidensis</i> (GI: 119390160), <i>Bt</i>DyP from <i>Bacteroides thetaiotaomicron</i> (GI: 109158102), DypB from <i>R. jostii</i> (GI: 330689635), DyP2 from <i>Amycolatopsis</i> sp. (GI: 409973917), AnaPX from <i>Nostoc</i> sp. (GI: 75704119), <i>Sav</i>DyP from <i>Streptomyces avermitilis</i> (GI: 29604188), <i>Mx</i>DyP from <i>Myxococcus xanthus</i> (GI: 108465542), Msp1 from <i>Marasmius scorodonius</i> (GI: 261266601), <i>Aau</i>DyPI from <i>Auricularia auricula-judae</i> (GI: 433286646), TAP from <i>Termitomyces albuminosus</i> (GI: 20386144), and Cpop21 from <i>Polyporaceae</i> sp. (GI: 2160705). Confidence was evaluated with 1000 bootstrap replicates (values are indicated at branch points).</p

Effect of various agents on <i>Svi</i>DyP activity.

<p>UD, undetectable activity.</p><p>EDTA, ethylenediaminetetraacetic acid.</p><p>SDS, sodium dodecyl sulfate.</p><p>Effect of various agents on <i>Svi</i>DyP activity.</p

SDS–PAGE and western blotting analysis and spectral characteristics of purified <i>Svi</i>DyP.

<p><b>a</b>. SDS–PAGE and western blotting analysis. Lanes: M, molecular mass standard; (1) SDS–PAGE analysis of purified <i>Svi</i>DyP. (2) western blot analysis of the purified <i>Svi</i>DyP recognized by an anti-His6 tag mouse monoclonal antibody. <b>b</b>. Spectral characteristics of purified <i>Svi</i>DyP, as determined by hemoprotein assay. Purified <i>Svi</i>DyP (1 mg ml⁻¹) in 25 mM phosphate buffer (pH 7.0) was scanned at 300–800 nm to identify the Soret band.</p

Physicochemical properties of enzyme-treated eucalyptus kraft pulp.

a<p>The control samples and the test samples were incubated in the selected buffer at the set temperature with and without corresponding enzyme.</p><p>Physicochemical properties of enzyme-treated eucalyptus kraft pulp.</p

Degradation circles produced by <i>Saccharomonospora viridis</i> on azure B screening plates.

<p>Degradation circles produced by <i>Saccharomonospora viridis</i> on azure B screening plates.</p

Enzymatic activity of purified <i>Svi</i>DyP towards various dyes^a.

a<p>The total volume of the enzyme reaction mixture was 3 ml.</p>b<p>Molar extinction coefficient at maximum absorption wavelength (λmax) of each dye.</p>c<p>Activity was calculated as specific activity in U mg⁻¹ (1 U = 1 µmol/min).</p>d<p>Relative activity was defined as activity towards brilliant green.</p>e<p>AQ, anthraquinone.</p>f<p>AZ, azo.</p>g<p>TM, triarylmethane.</p><p>Enzymatic activity of purified <i>Svi</i>DyP towards various dyes^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110319#nt101" target="_blank">a</a>}.</p

Purification of recombinant <i>Svi</i>DyP.

<p>Purification of recombinant <i>Svi</i>DyP.</p

Morphology of bleached pulp fiber surface.

<p>Scanning electron micrograph of unbleached (control) eucalyptus kraft pulp (<b>a</b>). Scanning electron micrograph of MnP treated eucalyptus kraft pulp (<b>b</b>). Scanning electron micrograph of <i>Svi</i>DyP treated eucalyptus kraft pulp (<b>c</b>, <b>d</b>).</p