Abstract
Reactive Black 5 (RB5) is a typical refractory azo dye. Widespread utilization of RB5 has caused a variety of environmental and health problems. The enzymatic degradation of RB5 can be a promising solution due to its superiority as an eco-friendly and cost-competitive process. Bacterial CotA-laccase shows great application prospect to eliminate hazardous dyes from wastewater. However, efficient decolorization of RB5 CotA-laccase generally requires the participation of costly, toxic mediators. In the present study, we modified the amino acids Thr415 and Thr418 near the type 1 copper site and the amino acid Gln442 at the entrance of the substrate-binding pocket of Bacillus pumilus W3 CotA-laccase to boost its RB5 decolorization activity based on molecular docking analysis and site-saturation mutagenesis. Through the strategies, two double site mutants T415D/Q442A and T418K/Q442A obtained demonstrated 43.94 and 52.64% RB5 decolorization rates in the absence of a mediator at pH 10.0, respectively, which were about 3.70- and 4.43-fold higher compared with the wild-type CotA-laccase. Unexpectedly, the catalytic efficiency of the T418K/Q442A to ABTS was enhanced by 5.33-fold compared with the wild-type CotA-laccase. The mechanisms of conferring enhanced activity to the mutants were proposed by structural analysis. In summary, the mutants T415D/Q442A and T418K/Q442A have good application potentials for the biodegradation of RB5.
Key points
• Three amino acids of CotA-laccase were manipulated by site-saturation mutagenesis.
• Decolorization rate of two mutants to RB5 was enhanced 3.70- and 4.43-fold, respectively.
• The mechanisms of awarding enhanced activity to the mutants were supposed.
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Funding
This work was supported by the National Natural Science Foundation of China (31472003), a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, the 111 Project (No. 111-2-06), and the Jiangsu province “Collaborative Innovation Center for Advanced Industrial Fermentation” industry development program.
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M.H. and G.Z.B. conceived and designed the research, interpreted the data, finalized conclusions, and wrote the manuscript. M.H. conducted experimental work. X.K.Z., W.Y.J., and Y.N. provided advice and assistance with molecular docking analyses and data analysis. L.X.R. provided assistance with data analysis. All authors read and approved the manuscript.
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Ma, H., Xu, KZ., Wang, YJ. et al. Enhancing the decolorization activity of Bacillus pumilus W3 CotA-laccase to Reactive Black 5 by site-saturation mutagenesis. Appl Microbiol Biotechnol 104, 9193–9204 (2020). https://doi.org/10.1007/s00253-020-10897-1
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DOI: https://doi.org/10.1007/s00253-020-10897-1