杂优-2平菇漆酶的分离纯化及酶学性质  

Isolation,Purification and Characterization of Laccase from Pleurotus ostreatus Heterosis-2

作　　者：廖海君 李蕊伽[1] 陶敏[1] 白亚娟[1] 唐菁[1] 唐云明[1] LIAO Haijun LI Ruijia TAO Min BAI Yajuan TANG Jing TANG Yunming＊(Key Laboratory of Eco-environments in Three Gorges Reservoir Region, Ministry of Education, Chongqing Sweetpotato EngineeringResearch Center, School of Life Science, Southwest University, Chongqing 400715, China)

机构地区：[1]西南大学生命科学学院,重庆市甘薯工程研究中心,三峡库区生态环境教育部重点实验室,重庆400715

出　　处：《食品科学》2016年第19期147-153,共7页Food Science

基　　金：中央高校基本科研业务费专项资金项目(XDJK2016C110)

摘　　要：将杂优-2平菇菌丝进行液体培养,发酵液经硫酸铵分级沉淀、DEAE(diethylaminoethyl)-Sepharose fast flow层析和Superdex-200 prep grade层析等方法纯化,获得了电泳纯的杂优-2平菇漆酶,并对纯化的漆酶进行了部分酶学性质研究。结果显示,杂优-2平菇漆酶比活力为115 U/mg,分子质量约为244.0 k D,亚基分子质量约为85.6 k D。最适反应p H值和最适反应温度分别为5.0和55℃,在p H 6.0~8.0及40~55℃范围内稳定性较好;最适条件下,以2,2’-联氮-二(3-乙基苯并噻唑-6-磺酸)为底物的Km值为2.1 mmol/L,最大反应速率(vmax)为0.117μmol/(min·L)。Fe^(2+)、抗坏血酸对该酶活性具有完全抑制作用,乙二胺四乙酸、Ag^+、Mg^(2+)、Li^+对该酶活性影响较小;草酸、甲醇、正丁醇、K^+、Ca^(2+)、Ba^(2+)、Zn^(2+)、Cd^(2+)、Pb^(2+)、Mn^(2+)、Co^(2+)对该酶活性有不同程度的抑制作用;Cu^(2+)激活作用不明显;尿素、乙醇、异丙醇对该酶活性具有激活作用。An electrophoretically pure laccase from the fermentation broth of Pleurotus ostreatus heterosis-2 was obtainedthrough ammonium sulfate fractionation, DEAE-Sepharose fast flow chromatography and Superdex-200 prep gradechromatography. The results indicated that the specific activity of the purified enzyme reached 115 U/mg. The relativemolecular weight of the laccase was approximately 244.0 kD, with a subunit molecular mass of roughly 85.6 kD. Theenzymatic properties showed that the optimum pH and temperature for the laccase were 5.0 and 55 ℃, respectively. Theenzyme was stabled at pH 6.0–8.0 and 40–55 ℃, and its apparent Km and vmax were 2.1 mmol/L and 0.117 μmol/（min·L）,respectively. Fe^2＋ and ascorbic acid could completely inactivate the laccase, whereas the enzyme activity was slightlyaffected by EDTA, Ag^＋, Mg^2＋ and Li^＋. Cu^2＋ had little activating effect on laccase activity. The enzyme activity of laccasecould be activated by urea, ethanol, and isopropanol, and inhibited by oxalic acid, methanol, n-butanol, K^＋, Ca^2＋, Ba^2＋,Zn^2＋, Cd^2＋, Pb^2＋, Mn^2＋, and Co^2＋.

关 键 词：杂优-2平菇 漆酶 分离纯化 酶学性质 

分 类 号：Q946.5[生物学—植物学]