Enhancing the decolorization activity of Bacillus pumilus W3 CotA-laccase to Reactive Black 5 by site-saturation mutagenesis

Appl Microbiol Biotechnol. 2020 Nov;104(21):9193-9204. doi: 10.1007/s00253-020-10897-1. Epub 2020 Sep 12.


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.

Keywords: Bacillus pumilus; CotA-laccase; Dye decolorization; Reactive Black 5; Site-saturation mutagenesis.

MeSH terms

  • Bacillus pumilus* / genetics
  • Bacterial Proteins / genetics
  • Coloring Agents
  • Laccase* / genetics
  • Molecular Docking Simulation
  • Mutagenesis
  • Naphthalenesulfonates


  • Bacterial Proteins
  • Coloring Agents
  • Naphthalenesulfonates
  • Laccase
  • Remazol black B