Production And Purification Of Laccase From Curvularia Trifol And Its Characterization

Laccase(EC 1.10.3.2) is a copper-containing polyphenol oxidase,widely distributed in plants,fungi and bacteria.Laccase can catalyze degradation of many organic and inorganic substrates,and has a broader range of uses in industry and bioremediation.From the plant of Artemisia,a fungal with laccase activity was isolated,named Z-1.and ITS1-5.8S-ITS2 rDNA region sequence identification showed that it has a closer relationship with Curvularia trifolii,the homology can be reach 100 percent.In this paper,the fermentation conditions optimization of Curvularia trifolii Z-1 extracellular laccase was studied,the laccase was purified,and the its properties was researched.Through single-factor experiment,the best culture conditions was found:volume of inoculation was 1%,medium volume 60%(V/V),the rotation speed was 150 r/rain,broth initial pH was 7.0,the culture temperature was 29℃.Screening test of the medium showed that organic nitrogen sources promote the secretion of laccase greatly than inorganic nitrogen. and the optimal carbon and nitrogen source were glucose and peptone,the best induction is guaiacol.Plackett-Burman design test show that the peak period(72 h fermentation) calcium chloride,manganese sulfate and zinc sulfate is a major factor.By PEG concentration,ion-exchange chromatography and gel filtration,the Curvularia trifoli Z-1 extracellular laccase was purified,the final purification fold is 2.90,the recovery of total activity was 16.01%.Using ABTS as substrate,the purified Curvularia trifoli Z-1 laccase optimum pH is 2.6,and during pH2.4～3.2,laccase maintain its high activity;the pure enzyme in pH6.0～pH8.0 buffer in the more stable,and within the range of pH2.2～pH4.0,activity of laccase declined rapidly.Pure enzyme optimum reaction temperature is 40℃;under the optimum pH,it was found that the lassase stability against temperature below 45℃.through the batch ion exchange chromatography,the Curvularia trifoli Z-1 laccase isoelectric point was 6.5.The purified enzyme can catalyze different substrates including ABTS and variety of dyes. Curvularia trifoli Z-1 laccase degradation test of five kinds of dye such as alizarin red,Congo red showed that,it didn't need the participation of medium.Under 1000 U/L activity of Curvularia trifoli Z-1 laccase,the degradation of Alizarin Red after 70 hours,the degradation efficiency reached 80%,indicated that the Curvularia trifoli strain Z-1 laccase has a strong potential in applications of dye degradation.