Journal
PEERJ
Volume 8, Issue -, Pages -Publisher
PEERJ INC
DOI: 10.7717/peerj.10267
Keywords
Dye degradation; Bacteria laccase; Error-prone PCR; Site-directed
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Background. Dye wastewater increases cancer risk in humans. For the treatment of dyestuffs, biodegradation has the advantages of economy, high efficiency, and environmental protection compared with traditional physical and chemical methods. Laccase is the best candidate for dye degradation because of its multiple substrates and pollution-free products. Methods. Here, we modified the laccase gene of Bacillus licheniformis by error-prone PCR and site-directed mutagenesis and expressed in E. coli. The protein was purified by His-tagged protein purification kit. We tested the enzymatic properties of wild type and mutant laccase by single factor test, and further evaluated the decolorization ability of laccase to acid violet, alphazurine A, and methyl orange by spectrophotometry. Results. Mutant laccase Lac(eP69) and D500G were superior to wild type laccase in enzyme activity, stability, and decolorization ability. Moreover, the laccase D500G obtained by site-directed mutagenesis had higher enzyme activity in both, and the specific activity of the purified enzyme was as high as 426.13 U/mg. Also, D500G has a higher optimum temperature of 70 degrees C and temperature stability, while it has a more neutral pH 4.5 and pH stability. D500G had the maximum enzyme activity at a copper ion concentration of 12 mM. The results of decolorization experiments showed that D500G had a strong overall decolorization ability, with a lower decolorization rate of 18% for methyl orange and a higher decolorization rate of 78% for acid violet. Conclusion. Compared with the wild type laccase, the enzyme activity of D500G was significantly increased. At the same time, it has obvious advantages in the decolorization effect of different dyes. Also, the advantages of temperature and pH stability increase its tolerance to the environment of dye wastewater.
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