Refolding, characterization, and dye decolorization ability of a highly thermostable laccase from Geobacillus sp. JS12

Autor: Jong-Hun Park, Sung-Jong Jeon
Rok vydání: 2020
Předmět:
Zdroj: Protein Expression and Purification. 173:105646
ISSN: 1046-5928
DOI: 10.1016/j.pep.2020.105646
Popis: A putative laccase gene (lacG) from Geobacillus sp. JS12 was cloned and expressed as a fusion protein with six histidine residues in Escherichia coli BL21 (DE3) cells, and the protein was primarily found in inclusion bodies. The resulting insoluble proteins were solubilized with 6 M guanidine HCl and refolded using an on-column refolding procedure. Ni-chelation affinity chromatography found the laccase to be a 30 kDa monomeric protein. Spectrophotometry and electron paramagnetic resonance (EPR) analysis indicated LacG as a multi-copper oxidase, with the usual laccase copper sites, Type 1, 2, and 3 Cu(II). The optimum pH for enzymatic activity was 3.0, 6.0, and 6.5 with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), guaiacol and 2,6-dimethoxyphenol (2,6-DMP) as the substrate, respectively. The recombinant protein displayed high thermostability, with a heat inactivation half-life of approximately 2 h at 95 °C, and an optimum temperature of 80 °C with 2,6-DMP. Catalytic efficiency (kcat/Km) showed that guaiacol and 2,6-DMP were highly oxidized by the enzyme. The enzymatic reaction was significantly enhanced by Co2+ and Mn2+, while activity was strongly inhibited in the presence of Fe2+, Zn2+, and thiol compounds. LacG decolorized 43% of Congo red and 14% of Malachite green, and the addition of ABTS as a redox mediator dramatically increased the dye decolorization efficiency.
Databáze: OpenAIRE
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