Abstract
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 (LacTT) from Thermus thermophilus SG0.5JP17-16 was screened using the genome mining approach, and it was highly expressed in Pichia pastoris, yielding a high laccase activity of 6130 U/L in a 10-L fermentor. The LacTT 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.
Highlights
Laccases belong to the family of multi-copper oxidases (MCOs) that usually contain four copper atoms and are classified as blue copperPLOS ONE | DOI:10.1371/journal.pone.0119833 March 19, 2015Overexpression of a Laccase and Its Application
The P. pastoris expression vectors pHKFA were constructed in our laboratory
P. pastoris was grown in BMGY/BMMY medium (1% yeast extract, 2% peptone, 100 mM potassium phosphate, 1.34% yeast nitrogen base (YNB), and 1% glycerol or 1% methanol; pH 6.0) at 30°C and 250 rpm
Summary
Laccases (benzenediol: oxygen oxidoreductases, EC 1.10.3.2) belong to the family of multi-copper oxidases (MCOs) that usually contain four copper atoms and are classified as blue copper. The majority of the laccases from fungi such as Paraconiothyrium variabile [12], Pleurotus florida [13], and Polyporus brumalis [14] have low thermostability, lose their activities under alkaline conditions, and are susceptible to high concentrations of chloride [10,15] These disadvantages of fungal laccases limit their practical applications. Bacterial laccases may be attractive alternatives, because many of them present high pH stability and are resistant to high temperature and chlorides [10,16,17] These properties could benefit the applications these enzymes in the decolorization of synthetic dyes from textile industries. The laccase from T. thermophilus SG0.5JP17–16 exhibited outstanding ability to decolorize synthetic dyes
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