The Generation, Purification And Decolorizing Dye Of Laccase Produced By Curvularia Lunata

In this study,an fungi is isolated from corn leaves,the leaves infected yellow macular disease.The fungi with laccase activity was detected using syringaldazine,guaiacol and ABTS.The fungi was identified as Curvularia lunata by Observation of colony morphology and analysis of ITS1-5.8S-ITS2 rDNA region sequence.The strain numbered jqh-100.C,lunata is curvlaria belong to Phragmosporoideae of Fungi Imperferti,it sickens plants of gramineae,and it has been used for industrial production of hydrocortisone.but research on the laccase of C.lunata have no report on the magazine at home and abroad.In Studies in this thesis were focused on the fermentation process of laccase by C.lunata,the enzymatic characteristic of laccase,and laccase from C.lunata was purified and the laccase decolorize dye.Production of the laccase by C.lunata was investigated using the single-factor experiment and the orthogonal design experiment method.The optimal conditions were as follows:initial pH6.0,culture temperature 35℃,seed age 60h,rotating speed 150 rpm/min,150 mL culture medium per 250 mL flask.Media components were as follows:soybean sprout juice 200 g/L, sucrose 20 g/L,glucose 5 g/L,peptone 3 g/L,ammonium nitrate 1.2 g/L,CuSO₄ 0.05 mmol/L,MnSO₄ 0.05 mmol/L,MgSO₄ 0.05 mmol/L,tween80 0.2 mL/L,guaiacol 0.1 mmol/L.The activity of laccase reached 2.8 U/mL,increased about 5 folds than that of original fermentation.Arrived at peak of the laccase activity in the fermentation 72h.Laccase from C.lunata was purified using ammonium sulfate salting-out,DEAE-Sepharose FF anion-exchange chromatography and Sephadex G-75 gel filtration.The purification fold was 2.14 and recovery of total laccase activity was 22.76%.The molecular weight of the purified laccase was 83.8 kDa as estimated by 15%(W/V) SDS-PAGE gel.The pH profile for laccase activity against ABTS showed a peak of maximum activity at pH2.8.The Km value(0.069 mmol/L) was found for ABTS.The enzyme was stable at pH range from 2.6 to 3.8.The optimum temperature of the purifie enzyme was observed at 30℃.The enzyme was stable at 30℃for 1 h in the pH range from 4.0～6.4.There was litte loss of activity during preincubatio of the enzyme for 1h at temperature up to 40℃,but the activity vanished beyond 60℃.The pH profile for laccase activity against" syringaldazine showed a peak of maximum activity at pH5.0.The Km value(0.149 mmol/L) was found for syringaldazine. Through 12 kinds of metal ions on the laccase activity studies have shown:bivalent and trivalence iron ion restrain entirely laccase activity,copper ion promote evidently the enzyme reaction. lunata and dye had high degradation effect as the laccase purified on the congo red,chrome azurol,neutal red,methylene blue and alizarin red.The Co-culture system through cell absorption and degradation of laccase,alizarin red so that the decolorization rate of 90%.Pure laccase has copper at the conditions exist for the decoloration of alizarin red significantly better results.This result showed that the laccase produced by Curvularia lunata had industrial application potential on the dye degradation.