Heterologous expression of bacterial CotA-laccase, characterization and its application for biodegradation of malachite green

Chiu-Min Cheng; Anil Kumar Patel; Reeta Rani Singhania; Cheng-Hsian Tsai; Shen-Yi Chen; Chiu-Wen Chen; Cheng Di Dong

doi:10.1016/j.biortech.2021.125708

Heterologous expression of bacterial CotA-laccase, characterization and its application for biodegradation of malachite green

Bioresour Technol. 2021 Nov:340:125708. doi: 10.1016/j.biortech.2021.125708. Epub 2021 Aug 3.

Authors

Chiu-Min Cheng¹, Anil Kumar Patel², Reeta Rani Singhania², Cheng-Hsian Tsai³, Shen-Yi Chen⁴, Chiu-Wen Chen², Cheng Di Dong⁵

Affiliations

¹ Department and Graduate Institute of Aquaculture, National Kaohsiung University of Science and Technology, Taiwan.
² Department of Marine Environmental Engineering, National Kaohsiung University of Science and Technology, Taiwan.
³ Department of Chemical and Materials Engineering, National Kaohsiung University of Science and Technology, Taiwan.
⁴ Department of Safety, Health, and Environmental Engineering, National Kaohsiung University of Science and Technology, Taiwan.
⁵ Department of Marine Environmental Engineering, National Kaohsiung University of Science and Technology, Taiwan. Electronic address: cddong@nkust.edu.tw.

PMID: 34391187
DOI: 10.1016/j.biortech.2021.125708

Abstract

Malachite green (MG) is used as fungicide/parasiticide in aquaculture, its persistence is detrimental as it exhibits carcinogenic effects to aquatic organisms. Bacterial laccase evaluated as the best enzyme at extreme condition for aquatic MG removal. Study aims to increase laccase concentration, CotA-laccase from Bacillus subtilis was cloned and overexpressed in Escherichia coli. Optimal catalysis for purified CotA-laccase were at pH 5.0, 60 °C, and 1 mM of (2,2-azino-di-[3-ethylbenzothiazoline-sulphonate-(6)]) with Km and Kcat 0.087 mM and 37.64 S^-1 respectively. MG biodegradation by CotA-laccase in clam and tilapia pond wastewaters and cytotoxic effect of biodegraded products in grouper fin-1 cells were determined. MG degradation by CotA-laccase was equally efficient, exhibiting upto 90-94% decolorization at freshwater and saline conditions and treated solution was non-toxic to GF-1 cells. Thus, recombinant-CotA-laccase could be an environmentally-friendly enzyme for aquaculture to remove MG, thereby effective to reduce its accumulation in aquatic organisms and ensuring safe aquaculture products.

Keywords: ABTS; CotA-laccase; Malachite green; Overexpression; Recombinant-CotA.

MeSH terms

Bacillus subtilis
Bacterial Proteins
Coloring Agents
Escherichia coli / genetics
Hydrogen-Ion Concentration
Laccase* / genetics
Rosaniline Dyes* / toxicity

Substances

Bacterial Proteins
Coloring Agents
Rosaniline Dyes
malachite green
Laccase