Enhancing the decolorization activity of Bacillus pumilus W3 CotA-laccase to Reactive Black 5 by site-saturation mutagenesis.
Appl Microbiol Biotechnol
; 104(21): 9193-9204, 2020 Nov.
Article
em En
| MEDLINE
| ID: mdl-32918582
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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MEDLINE
Assunto principal:
Lacase
/
Bacillus pumilus
Idioma:
En
Revista:
Appl Microbiol Biotechnol
Ano de publicação:
2020
Tipo de documento:
Article