Heterologous Expression Of Laccase Gene From Bacillus Pumilus LC01 In Pichia Pastoris And Characterization Of The Recombinant Enzyme

Laccase(EC 1.10.3.2 p-diphenol:dioxygen oxidoreductase)is a copper-containing oxidoreductase,which is widely distributed in nature.It can catalyze a variety of substrates.The free radicals produced by the oxidation substrate can undergo non-enzymatic reactions.Laccases show great potential in practical applications in many industrial fields.However,bacterial laccase has defects such as low production level in practical applications.To obtain high level production of bacterial laccase with good stability and efficient decolorization ability,the strain LCO1 with laccase activity screened at the early stage was identified by physiological and biochemical experiments and 16S rDNA,and its growth characteristics were further studied.The amino acid composition,molecular weight,isoelectric point were analyzed by sequence analysis and bioinformatics software.The laccase gene from strain LCO1 was amplified by PCR and cloned into the expression vector pPICZaA.The constructed vector pPICZ?A-lac was transfonned into P.pastoris.The positive P.pastoris strain was induced by methanol to produce the recombinant laccase that simplified the following purification process.The length of 16S rDNA sequence of strain LCO1 is 1512 bp.The strain is identified as Bacillus pumilus LCO1.The laccase has 510 amino acids and a molecular weight of 58.71.The laccase has 25.88%extended strand,60.78%random coil,5.88%beta brige and 7.45%alpha helix.The optimum growth temperature of strain LCO1 is 30??the optimum growth pH is 7,and the optimum NaCl concentration is 5%,which has a positive effect on the growth of LCO1 strain.The maximum activity of strain LCO1 spore laccase reached 450 U/g on the third day of incubation.The expression of recombinant laccase reached a maximum of 1390 U/L after 7 days of incubation.Then the recombinant enzyme was purified by DEAE-Sepharose FF ion exchange chromatography and Sephadex G-75 gel chromatography.The purified recombinant laccase can oxidize different substrates.The optimum pH for 2,2'-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid ammonium salt),syringaldazine and 2,6-dimethoxyphenol as substrates is 3,6.8 and 7.4 respectively.The recombinant laccase was highly stable at pH 9 after 10 days with totally initial enzyme activity retained.The optimum temperature of the recombinant laccase is 70?,which maintains high catalytic activity at 50-80?.the enzyme retained 36%of its initial activity after 7 h incubation at 70?.recombinant laccases have better tolerance to organic solvents.In the decolorization of remazol brilliant blue R,reactive black 5 and indigo carmine,The purified enzyme could efficiently decolorize the tested dyes in the presence of acetosyringone.More than 90%of the tested dyes were decolorized within 6 h at pH 9.0,indicating the recombinant laccase is an ideal candidate for treatment of dye effluents.