Screening Of Auricularia Auricular Strains Of Hich Laccase,Optimization Of Fermentation Conditions And Enzymatic Characteristics

Laccase (Laccase, EC1.10.3.2) is a type of polyphenol oxidases contained copper ion. It was not only playing an important role in lignin degradation process,but also a key enzyme on the carbon and energy metabolism of edible fungus. It also had a direct impact on mushroom production and quality.This article explored the principle on efficient production of laccase,screened the high-yield strains of laccase,researched and analysised the optimization and enzymatic characteristics. Main results are as follows:1.25kinds of Auricularia Auricular all Heilongjiang were vaccinated on the guaiacol mediums. LY,Fangzheng and JGDQ were screened with the name of high yield strains by the color circle diameter.The strains were compared with each other on enzymatic properties. Temperature stability and pH stability of the strain LY are better than the other two strains.The laccase activity of LY was115U/L in the ninth day.The optimal temperature was45℃and the half-life was over50min at55℃. The optimal reaction pH was4.0.The activity could remain50%for4h at pH5.5. The activity could be all right at pH6.5for4h and the half-life could be2h at pH7.5.So LY was high yield strain.2. The medium of wild black Auricularia Auricular was added by different sources of carbon,nitrogen,metal ion,controlled the pH, volume of medium,temperature and proportion of medium volume,used single factor experiment and uniform design experiment,measured laccase activity(ABTS) and dry weight of hyphae. The results were as follows:com flour23.5g/L, glucose23.5g/L, NH4C11.5g/L, bean powder3g/L, KH2PO42.9g/L,MgSO41.8g/L, CuSO4·5H2O0.009g/L, distilled water1L.The proper environment condition:pH6.5,volume of medium50mL/250mL,28℃proportion of medium volume4%.The laccase activity could reach67U/L by DPS. After a two-step enzyme production conditions, the results were as follows:corn flour28.1g/L, glucose28.1g/L, NH4Cl4.7g/L, bean powder9.3g/L, KH2PO43.7g/L,MgSO40.9g/L, CuSO4·5H2O0.006g/L, distilled water1L.The proper environment condition:pH6.5,volume of medium50mL/250mL,28℃proportion of medium volume4%.The laccase activity could reach98U/L with DPS.3. The result of Native-PAGE for laccase crude extract showed that three kinds of Isozymes existed in LY. The laccase was precipitated by Ammonium sulfate and purified by DEAE-Sepharose fast flow anion column chromatography to have electrophoretic homogeneity LacB.The purification factor was4.53, enzyme recovery was47.93%. LacB was detected by SDS-PAGE with an apparent molecular weight of64.7KD. The optimal pH and temperature for LacB activity were4.0and45℃. The LacB catalyzed the oxidation of ABTS with a Michaelis constant of0.02mmol/L. LacB was stable in the25-55℃(pH4.0) and pH2.5-6.5(30℃).Various metal ions showed different effects on the LacB activity.The activity was enhanced by Cu2+、K+and Mg2+, specially the Mg2+and inhibited by Fe2+、Zn2+and Mn2+, specially the Fe2+. LacB could make the obvious decolorization of synthetic dyes crystal violet without adding ABTS.The decolorization rate was reaching89.8%for24h.