Research On Cloning,Expression,Protein Purification And Enzymology Characteristics Of Laccase Gene From Kurthiahuakuii

Laccases have been widely used for the decolorization and detoxification of synthetic dyes due to their ability to catalyze the oxidation of various phenolic and non-phenolic aromatic compounds using molecular oxygen. A putative laccase-like gene(laclK) from Kurthia huakui LAM0618 T was cloned using the genome search approach, and overexpressed in Escherichia coli. A series of studies in laclK gene prokaryotic expression, recombinant LaclK enzymology characteristics and dye decolorization were performed in this study. The main conclusions are as followed:(1) Construction of BL21(DE3)/pET28a-laclK expression strain successfully.(2) The optimal induced expression conditions for LaclK were 0.2mM IPTG and 0.5 mM Cu2+ at16 ℃, as determined by gradient induced of IPTG, copper ion and temperature. The optimum imidazole concentration to elute LaclK was 300 mM.(3) Structural properties: The UV-visible spectrum of the purified LaclK showed that the traditional absorption shoulder around 610 nm and 330 nm were absent, and the ratio of copper atoms/molecule of LaclK was calculated to be 0.86±0.04 by atomic absorption spectroscopy.(4) Enzymology characteristics: The optimal temperature of LaclK was 65 ℃ with ABTS as the substrate. The half-life of LaclK exceeded 83 hours at 80 ℃, ranking it as the most thermophilic laccase-like phenol oxidase reported thus far. LaclK is able to oxidize substrates such as ABTS,2,6-DMP, and L-dopamine. The optimal p H of the recombinant LaclK was 2.5, 6.0 and 7.0 with ABTS,L-dopamine and 2,6-DMP as the substrate, respectively. LaclK was quiet stable at p H 2.0-3.0 and thermostable at 60 ℃-100 ℃. More than 70 % of the original activity retained after 7 days incubation at p H 2.5. Extra Cu2+(0.2 mM) was essential for 2,6-DMP oxidation. The activity was increased in the presence of 1 mM Mg2+, Pb2+, K+, Co2+ or Ca2+, while the addition of Mn2+, Zn2+, Fe2+, or Ag+ reduced the activity. LaclK also exhibited higher stability in the presence of organic solvents. Organic solvent(v/v) had a positive effect on the enzyme activity in the presence of 10 % Methanol, Ethanol, Isopropyl alcohol, Butyl alcohol, Triton x-100 or Dimethyl sulphoxide. The purified LaclK was strongly inhibited by L-Cys, EDTA, DTT and SDS. The turnover numbers(kcat) are approximately two orders of magnitude lower than those reported laccases originating from different organisms. Whereas, its structural and catalytic properties were distinct from other laccases. LaclK may be classified as laccase-like phenol oxidases.(5) Dye decolorization: LaclK was able to decolorize all the tested dyes under the condition of neutral or alkaline with outstanding decolorization capability, with 92% and 94% of Victoriablue B(25μM) and Ethyl violet(25 μM) being decolorized by 60 U/L enzyme after 1 h incubation at 60 ℃ in the presence of ABTS, respectively. Moreover, LaclK exhibited halide ions and organic solvents tolerance abilities. These results indicated the promising application of LaclK in the treatment of dye effluents.