The Isolation, Optimization And Decolorization Of Dye Of Laccase Highly Produced By Myrothecium Verrucaria

Soil samples were collected in Liangshui Nature Reserve, China and enriched in medium with lignin-sodium salfacid as sole carbon source. Guaiacol-PDA plates were used to separate laccase-producing fugi. A high-yield strain NF-05was screened after testing with2,2’-azino-bis(3-ethylbenz-thiazoline-6-sufonic acid)(ABTS) and4-hy-droxy-3,5-dimethoxybenzaldthyde azine (SGZ). NF-05was identified as Myrotheium verrucaria based on morphological characteristics and sequence analysis of rDNA-ITS. Enzyme activity reached the peak of8375.9U/L. The studies in this thesis were focused on the Bilirubin oxidase utilizing on medicine and electrical chemistry, while the resources of laccase high-yield Myrothecium verrucaria which could be applied to environment protecting field are very rare.The medium composition and ferment condition of the laccase produced by Myrotheium verrucaria NF-05was initially optimized by using the single-factor experiment. According to the results of single-factor experiment, a Plackett-Burman design and Response surface methodology (RSM) were applied to further optimizion of the fermentation conditions of NF-05for laccase production. The medium composition and ferment condition of the laccase produced by Myrotheium verrucaria NF-05were as follows:CuSO40.09mmol/L, Glucose62.89g/L, CoCl20.25mmol/L, Fe2(SO4)30.15mmol/L, Peptone14.0g/L, KH2PO43.0g/L, NH4C11.0g/L, MgSO4-7H2O1.5g/L, Potato juice200g/L, Vitamin solution0.5mL/L Inoculation way using the fungus cookie, inoculating volume6%. plating age15d, liquid volume60mL/250mL, agatation120r/min, initial pH6.0and culture temperature28℃. The yield of laccase produced by NF-05in the optimal ferment condition increased4.38times as compared with that in the initial medium8375.9U/L and reached45082.6U/L.The decolorization ability of four main kinds dyes were studied by Myrotheium verrucaria NF-05self-immobilizing its mycelium during the growth process. The decolorization ability of the NF-05was tested in using different dyes. The decolorization effect of the triphenylmethane dyes were most obvious among all the consequence(the decolorization of malachite green and crystallization purple is66.9%、63.2%), so ascertain the malachite green to be the main target for the study of self-immobilize mycelium. The results of the effect of the additional carbon、nitrogen resource and metal element on the decolorization of self-immobilize mycelium show that the ability of decolorization of NF-05self-immobilize mycelium was sharply strengthened after adding5g/L additional carbon and3g/L additional nitrogen. Moreover, Cu2+, Fe3+and Co2+have obvious promotion effect on dye decolorization, whereas Mg2+、Al3+and Na+showed no obvious effect. In addition, Ca2+presented an obviously inhibitory effect. The maximum load of dyes experiment showed that with malachite green concentration75mg/L, self-immobilize mycelium using5cycles demonstrated a good stability, the total decolorization load of unit self-immobilize mycelium is highest for18.05mg. Furthermore, it is suitable for choosing this to deal with dyes liquid waste. This study is a beneficial attemption in utilizing fungus to deal with dyes liquid waste, it constucted a theory foundation in model estabilishing for the Myrotheium verrucaria NF-05using for biologic membrane method in the treatment of dye-containing wastewater.