Characterization of Laccase TaLac from Thermus aquaticus and Its Application in Removing Malachite Green Dye

doi:10.13560/j.cnki.biotech.bull.1985.2021-0852

Abstract

Abstract:

To mine the laccases of DUF152 family showing great potential in removing the hazardous dye MG,a candidate from the thermophile Thermus aquaticus（TaLac）was screened by bioinformatics methods,and the recombinant vector pET28a-TaLac was constructed and overexpressed in Escherichia coli. TaLac was purified to study its enzymatic characterization and the ability to decolorize and detoxify MG. The results showed that TaLac contained the conserved copper binding sites of DUF152 family. The purified TaLac with high purity using nickel chelating affinity chromatography catalyzed the oxidation of 2,6-dimethylphenol（DMP）,the substrate of typical laccases. The optimal pH,temperature,and K_m of TaLac were 5.0,60℃,and 0.74 mmol/L,respectively using DMP as the substrate. Besides,TaLac retained 80% of its original activity after incubation for 4 h at 50℃. TaLac tolerated high concentrations of Mg²⁺,Ca²⁺,Mn²⁺,Zn²⁺ and Ba²⁺. Furthermore,TaLac completely decolorized 50 mg/L of MG in 3 h,and completely eliminated the toxicity of MG to Zea mays. Taken together,TaLac is a functional and thermostable laccase of DUF152 family,showing great ability to decolorize and detoxify MG.

Key words: laccase, DUF152 family, Thermus aquaticus, enzymatic characterization, decolorization and detoxification of malachite green

MAO Guo-tao, WANG Jie, WANG Kai, WANG Fang-yuan, CAO Le-yan, ZHANG Hong-sen, SONG An-dong. Characterization of Laccase TaLac from Thermus aquaticus and Its Application in Removing Malachite Green Dye[J]. Biotechnology Bulletin, 2022, 38(4): 261-268.

Figures/Tables 8

Fig.1 Bioinformatic analyses of TaLac A：Sequence alignment of TaLac,GsYlmD（WP_053413740.1）,RL5（CAK32504.1）,Tfu1114（AAZ55152.1）and LaclK（WP_029500662）. The identical residues are highlighted with red,and the possible copper ion binding sites are labelled with black triangles. B：Tertiary structure model of TaLac

Fig.2 Cloning and purification of TaLac A：The expression vector structure of pET28a-TaLac. B：SDS-PAGE of the purified TaLac. 1：Purified TaLac. M：Protein marker

Fig.3 Effects of pH and temperature on the activity of TaLac A：Optimal pH. B：Optimal temperature. C：Thermostability

Fig.4 Substrate specificity of TaLac（A）and kinetic parameters against DMP（B）

Fig. 5 Dependence of TaLac activity on Cu2+

Fig. 6 Effects of metal ions on the activity of TaLac

Fig. 7 Decolorization of MG catalyzed by TaLac

Table 1 Phytotoxicity test of MG and MG decolorized products

参数Parameter	对照组Control group	MG组MG group	MG脱色产物组MG decolorization group
发芽率 Germination/%	100 ^a	33 ^b	100 ^a
胚芽长度 Plumule length/cm	1.50 ± 0.82 ^a	0.40 ± 0.17 ^b	1.55 ± 0.90 ^a
胚根长 Radicle length/cm	5.75 ± 1.60 ^a	1.26 ± 0.64 ^b	5.25 ± 1.98 ^a

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