黑木耳漆酶纯化及部分漆酶特性的研究

黑木耳漆酶研究可为漆酶的进一步分离纯化、基因克隆表达和大规模生产应用奠定基础。对黑木耳"黑29"菌株漆酶粗酶液进行硫酸铵分级沉淀后,通过Native SDS-PAGE电泳检测,存在3种漆酶LacA、LacB、LacC,分子量分别为60,34,19 kD。经硫酸铵分级沉淀和DEAE-Sephacel柱层析技术分离得一纯化成分LacC,纯化倍数7.60,酶活性回收4.28%。对LacC的pH、温度、金属离子和Km值等部分酶学性质进行研究发现,该酶氧化ABTS的Km值为1.18×10-6mol/L,催化氧化底物ABTS的最适pH为3.8,在pH 3.0~4.6表现出较强的稳定性;最适反应温度为55℃,低于50℃时有较好的热稳定性;金属离子Ag+对漆酶有激活作用,而Fe3+、Mn2+、Co2+则有抑制作用。

Purification and Characterization of Laccase from Auricularia auricula

Laccase of Auricularia auricula were studied for further separation and purification, gene ex- pression and mass production applications. Native SDS-PAGE for laccase extract by （NH4）2SO4 step clas- sified showed that three kinds of isoenzymes existed in ＂Hei 29＂. The molecular weight of the three isoenzymes was determined, LacA （60 kD）, LacB （34 kD）, LacC （19 kD）. The LacC purifications were purified by ammonium sulphate fractionation, DEAE-Sephacel chromatography. Purification of about 7.60 fold was achieved with an overall yield of 4.28 %. The effects of pH, temperature and metal ion on ＂Hei-29＂ strain of Auricularia auricular laccase activity and stability were studied. Meanwhile, the sub- strate concentration effect of laccase was also studied and Km measured. The results showed that： The LacC catalyzed the value of Km of oxidation of ABTS was 1.18 ×10-6 mol/L. The optimum pH for LacC activity were 3.8, respectively in catalytic reaction of oxidizing ABTS. LacC showed a good stability when the pH of the buffer varied from 3.0 to 4.6. The optimum temperature for LacC activity was 55℃, and the laccase could work continuously under 50℃. Various metal ions showed different effects on the LacC ac- tivity. The activity was enhanced by Ag＋ , and was strongly inhibited by Fe3＋ , Mn2＋ and Co2＋ .