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

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.

Expression of a new laccase from Moniliophthora roreri at high levels in Pichia pastoris and its potential application in micropollutant degradation

“…To our knowledge Mrl2 expression achieved the highest level of a fungal laccase in P.   pastoris reported so far with 281,000 U l −1 , which corresponds to 1.05 g Mrl2/l medium at a specific activity of 248 U mg −1 . Similar laccase yield in P. pastoris was only reported for a bacterial laccase from Thermus thermophilus with 1.2 g l −1 (Liu et al 2015a). Specific activity of this enzyme (1.12 U mg −1 ) was, however, much lower compared to Mrl2.…”

Characterization of a Novel Thermostable Enzyme from <b><i>Thermus</i></b> sp. 2.9 with Phospholipase and Acyltransferase Activities

“…The activity of PLP_2.9 even increased for short times, which is a phenomenon already reported for some thermophilic enzymes [Liu et al, 2015]. Incubation at higher temperatures caused an accelerated decrease of activity.…”

Plackett-Burman Design for rGILCC1 Laccase Activity Enhancement in Pichia pastoris: Concentrated Enzyme Kinetic Characterization

“…Nonetheless, a higher affinity to ABTS is reported in comparison to other substrates such as syringaldazine or guaiacol among others, with lower oxidation velocity and higher K m values [4, 5, 35]. …”