Decolorization of different dyes by LacTT.

<p>(a) Decolorization of synthetic dyes by LacTT at 70°C for 24 h. (b) Decolorization of RBBR by LacTT with different concentrations of NaCl at 70°C for 24 h. The reactions were performed in 50 mM Na₂HPO₄–NaH₂PO₄ buffer (pH 7.5, but pH 8.0 for CR decolorization), 50 mg/L dye, 10 μM CuSO_4, and purified LacTT (40 U/L). The error bars represent the standard deviation.</p

Primers used for plasmid construction and qPCR analysis.

<p>Primers used for plasmid construction and qPCR analysis.</p

Effect of temperature on the activity and stability of the purified laccase.

<p>(a) Optimal temperature (open circles) was determined at pH 7.5 by using guaiacol as the substrate. Residual activity (closed circles) was determined after incubation at 40°C–90°C for 1 h. (b) Residual activity was determined after incubation at 70°C, 80°C, and 90°C, respectively, for 0–4 h. The graphs display the average values from triplicate measurements. The error bars represent the standard deviation.</p

Time course of laccase production during shake flask cultivation and 10-L fed-batch fermentation.

<p>(a) Progress curves constructed in the induction phase of shake flask cultivation for the determination of the cell density of <i>P</i>. <i>pastoris/LacTT</i> (closed square) and <i>P</i>. <i>pastoris/</i>(<i>LacTT</i>)₄ (open square), volumetric activity of <i>P</i>. <i>pastoris/LacTT</i> (closed circle) and <i>P</i>. <i>pastoris/</i>(<i>LacTT</i>)₄ (open circle), specific activity of <i>P</i>. <i>pastoris/LacTT</i> (closed triangle) and <i>P</i>. <i>pastoris/</i>(<i>LacTT</i>)₄ (open triangle). (b) Cell density (closed square), volumetric activity (closed circle), and specific activity (closed triangle) of <i>P</i>. <i>pastoris/</i>(<i>LacTT</i>)₄ in fed-batch fermentation. (c) SDS-PAGE image of LacTT supernatant expressed in <i>P</i>. <i>pastoris/</i>(<i>LacTT</i>)₄ in fed-batch fermentation. Lane M: protein marker; Lane 1: 0.5 mg/mL BSA; Lane 2: 0.3 mg/mL BSA; Lane 3: 0.1 mg/mL BSA; Lane 4: Enzyme supernatant was 1:10 diluted. The error bars represent the standard deviation.</p

Overexpression of a Novel Thermostable and Chloride-Tolerant Laccase from <i>Thermus thermophilus</i> SG0.5JP17-16 in <i>Pichia pastoris</i> and Its Application in Synthetic Dye Decolorization

<div><p>Laccases have been used for the decolorization and detoxification of synthetic dyes due to their ability to oxidize a wide variety of dyes with water as the sole byproduct. A putative laccase gene (<i>LacTT</i>) from <i>Thermus thermophilus</i> SG0.5JP17-16 was screened using the genome mining approach, and it was highly expressed in <i>Pichia pastoris</i>, yielding a high laccase activity of 6130 U/L in a 10-L fermentor. The <i>LacTT</i> open reading frame encoded a protein of 466 amino acid residues with four putative Cu-binding regions. The optimal pH of the recombinant LacTT was 4.5, 6.0, 7.5 and 8.0 with 2,2'-azino-bis(3-ethylbenzothazoline-6-sulfonic acid) (ABTS), syringaldazine (SGZ), guaiacol, and 2,6-dimethoxyphenol (2,6-DMP) as the substrate, respectively. The optimal temperature of LacTT was 90°C with guaiacol as the substrate. LacTT was highly stable at pH 4.0–11.0 and thermostable at 40°C–90°C, confirming that it is a pH-stable and thermostable laccase. Furthermore, LacTT also exhibited high tolerance to halides such as NaCl, NaBr and NaF, and decolorized 100%, 94%, 94% and 73% of Congo Red, Reactive Black B and Reactive Black WNN, and Remazol Brilliant Blue R, respectively. Interestingly, addition of high concentration of NaCl increased the RBBR decolorization efficiency of LacTT. These results suggest that LacTT is a good candidate for industrial applications such as dyestuff processing and degradation of dyes in textile wastewaters.</p></div

Effect of pH on the activity (a) and stability (b) of purified LacTT at 90°C.

<p>(a) ABTS, pH 3.0–6.0; SGZ, pH 5.5–8.5; guaiacol, pH 6.5–10.0; 2, 6-DMP, pH 5.0–10.0. (b) Investigation of the pH stability of LacTT by measuring the enzyme activity at 90°C with guaiacol as the substrate. The data represent the average values from triplicate measurements. The error bars represent the standard deviation.</p

SDS-PAGE, zymogram analyses and glycosylation analyses of the purified LacTT.

<p>(a) The gel stained with Coomassie Brilliant Blue R-250. Lane M: protein marker; Lane 1: purified LacTT after heat denaturation; Lane 2: purified LacTT without heat denaturation. (b) The zymogram stained with 0.1 mM SGZ and 2 mM guaiacol. Lane M: protein marker; Lane 1: unheated purified LacTT stained with 0.1 mM SGZ; Lane 2: unheated purified LacTT stained with 2 mM guaiacol; Lane 3: heat-denatured LacTT stained with 0.1 mM SGZ; Lane 4: heat-denatured LacTT stained with 2 mM guaiacol. (c) Deglycosylation analyses of the purified laccase. Lane 1: purified LacTT; Lane 2: purified LacTT was deglycosylated by PNGase F; Lane M: protein marker.</p

Effects of halides on LacTT activity.

<p>The enzyme was incubated in 50 mM Na₂HPO₄-NaH₂PO₄ (pH 7.5, supplemented with 10 μM CuSO₄), using guaiacol as the substrate. The error bars represent the standard deviation.</p