灵芝EIM-40漆酶的分离纯化及酶学性质  

Purification and properties of laccase from Ganoderma EIM-40

作　　者：何国斌[1] 蔡少丽[1] 柯崇榕[1] 黄平[1] 张明亮[1] 黄建忠[1] 

机构地区：[1]福建师范大学生命科学学院福建省现代发酵技术工程研究中心工业微生物教育部工程研究中心,福州350108

出　　处：《生物加工过程》2012年第2期34-39,共6页Chinese Journal of Bioprocess Engineering

基　　金：福建省发改委产业化资助项目(闽发改投资[2009]958号)

摘　　要：采用冻干浓缩、(NH4)2SO4盐析、Hi Trap phenyl(FF)疏水层析和Q Sepharose Fast Flow离子交换层析对灵芝EIM-40发酵液进行分离纯化,获得纯化漆酶,纯化倍数为14.6,回收率为5.3%。SDS-PAGE银染的结果为单一条带,相对分子质量约为6.53×104。以愈创木酚和2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(ABTS)为催化底物进行酶学性质研究,最适pH分别为4.8和4.5,最适温度分别为55和50℃,2种底物在pH 4.0～5.0范围内,温度低于50℃时,酶的稳定性都很好。以愈创木酚为底物,Km=645.0μmol/L;以ABTS为底物,Km=22.2μmol/L。Cu2+对该酶起激活作用,Fe2+、Ca2+、Ba2+则完全抑制酶的活性。After the crude laccase from Ganoderma sp. EIM40 was purified using lyophilization, fraction- al ammonium sulfate precipitation, HiTrap phenyl （FF） hydrophobic chromatography and Q Sepharose Fast Flow Ion-exchange chromatography, a final yield of 5.3% and a specific activity of 14.6-fold were achieved. SDS-PAGE analysis by silver staining revealed a single band with molecular weight of 6.53 × 104. The optimal reaction conditions of the laccase toward Guaiacol and ABTS as substrate in- clude 55 ℃ and 50 ℃ of reaction temperature and pH d. 8 and pH 4.5. When the temperature was below 50 ℃ and the pH was in the range of 4. 0 to 5.0, the laccase exhibited excellent stability in the two sub- strates. The Km for oxidizing guaiacol and ABTS were 645.0 and 22.2 umol/L, respectively. Laccase could be enhanced by Cu2＋ and inhibited by Fe2＋ , Ca2＋ , and Ba2＋.

关 键 词：漆酶 灵芝 分离纯化 酶学性质 

分 类 号：Q554[生物学—生物化学]