Stently threatens the environment and public overall health [13,14]. The fungal laccases from
Stently threatens the environment and public health [13,14]. The fungal laccases from Trametes sp. 48424, Cerrena sp., and T. asperellum, too as bacterial laccases from Bacillus pumilus, Bacillus sp. FNT, and Sulfitobacter indolifex, were demonstrated to decolorize MG below mesothermal circumstances [159]. Nevertheless, the temperature of wastewater released in the dyeing course of action is always above 50 C [20], plus a greater temperature normally suggests larger decolorization velocity [21]. As a way to stay away from the extra cooling method to lessen the price and take complete advantage from the high temperature from the dyeing wastewater to fulfill the maximum decolorization rate within a quick period, laccases with high optimal temperatures and great thermostability are expected. The DUF152 laccases, a brand new subfamily from the bacterial laccases, were characterized in 2006. The molecular weights (about 30 kDa) and amino acid sequences in the DUF152 laccases are fairly unique from these of standard laccases (5030 kDa) [22]. The isolated DUF152 laccases RL5 from a metagenome expression library on the bovine rumen, Tfu1114 from Thermobifida fusca, and LaclK from Kurthia huakuii had high optimal temperatures (above 60 C) and showed superb thermostability [224]. Their possible to decolorize various dyes like poly-R 478, ethyl violet, and methylene blue was demonstrated [22,24]. In addition to, due to its good thermostability, Tfu1114 was Metalaxyl-M medchemexpress incorporated into the cellulosome, drastically enhancing the capability to hydrolyze the unpretreated wheat straw [25]. As a result, the DUF152 laccases showed wonderful prospective to treat high-temperature dyeing wastewater. Herein, a novel member of DUF152 laccases, Ghlac, was characterized from Geothermobacter hydrogeniphilus, and its putative copper binding web site was identified. Moreover, Ghlac variant Mut2 with improved thermostability was engineered, and its capability of decolorizing MG at higher temperatures was assessed. After Mut2 therapy, the toxicity of MG to bacteria and plants was evaluated to promote its practical application. 2. Final results and Discussion 2.1. Sequence Evaluation, Expression, Purification, and Mutation of Ghlac The open reading frame of Ghlac encoding an uncharacterized protein containing the consensus sequences of DUF152 laccases was discovered within the thermophile G. hydrogeniphilus. Ghlac includes 263 residues having a theoretical molecular weight of 29.0 kDa. Various sequence Bopindolol In Vivo alignment showed that Ghlac shares 22.6 , 30.2 , and 34.0 identities to LaclK, RL5, and Tfu1114, respectively (Figure 1A). The putative copper binding internet sites (N41, H78, C119, and H136) had been conserved in Ghlac [22]. The structure model indicated that Ghlac includes a comparable structural fold to the DUF152 member GsYlmD (Figure 1B). As aforementioned, we recommended that Ghlac is often a putative functional laccase. To verify this, Ghlac was cloned, expressed, and purified employing Ni-NTA chromatography (Figure 1C). The molecular weight of your purified homogeneous Ghlac corresponded to the predicted size (Figure 1C). The activity assay showed that Ghlac could oxidize two,2 -azino-bis(3ethylbenzthiazoline)-6-sulfonate (ABTS), the common substrate of laccases. Km and kcat of Ghlac had been 1.3 mM and 125.7 min-1 , respectively (Figure 2A), which are comparable to those from the DUF152 laccases Tfu1114 and LaclK as well as the standard laccase pLacSi from S. indolifex [18,23,24].Int. J. Mol. Sci. 2021, 22,Int. J. Mol. Sci. 2021, 22,3 of3 ofFigure 1. Structure analysis and puri.
