Improving decolorization of dyes by laccase from Bacillus licheniformis by random and site-directed mutagenesis

Abstract

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 Lacep69and 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 °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.

Shewanella baltica strain JD0705 isolated from the mangrove wetland soils in Thailand and characterization of its ligninolytic performance

“…It is somewhat different from other bacterial laccases. To date, it has been reported that bacterial laccases predominantly come from Bacillus, Pseudomonas and Streptomyces (Christopher et al 2014;Bu et al 2020). Bacterial laccases are generally active and stable at nearnormal human body temperature and a neutral pH.…”

Enhanced Enzyme Activity of Thermoalkaliphilic Laccase (CtLac) of Caldalkalibacillus thermarum Strain TA2.A1 by Random and Site-directed Mutagenesis and Its Application to Lignin Degradation

“…Thus, our results could provide a path to improve catalytic activity of CtLac with further investigation of molecular structure. Indeed, most investigations on mutagenesis to enhance enzyme activity have been performed between well-known laccases with high amino acid identities, such as Bacillus licheniformis and Bacillus subtilis [33][34][35].…”

Improvement of thermoalkaliphilic laccase (CtLac) by a directed evolution and application to lignin degradation

“…Thus, our results could provide a pathway for improving the catalytic activity of CtLac by investigating the molecular structure further. Indeed, most investigations on mutagenesis to enhance enzyme activity have been performed between well-known laccases with high amino acid identities, such as Bacillus licheniformis and Bacillus subtilis (Bu et al 2020 ; Mollania et al 2011 ; Wang et al 2017 ).…”