杂优-2平菇漆酶的分离纯化及酶学性质

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 fastflow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 kD，亚基分子质量约为85.6 kD。最适反应pH值和最适反应温度分别为5.0和55 ℃，在pH 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0.117 μmol/（min·L）。Fe2＋、抗坏血酸对该酶活性具有完全抑制作用，乙二胺四乙酸、Ag＋、Mg2＋、Li＋对该酶活性影响较小；草酸、甲醇、正丁醇、K＋、Ca2＋、Ba2＋、Zn2＋、Cd2＋、Pb2＋、Mn2＋、Co2＋对该酶活性有不同程度的抑制作用；Cu2＋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。

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

An electrophoretically pure laccase from the fermentation broth of Pleurotus ostreatus heterosis-2 was obtained
through ammonium sulfate fractionation, DEAE-Sepharose fast flow chromatography and Superdex-200 prep grade
chromatography. The results indicated that the specific activity of the purified enzyme reached 115 U/mg. The relative
molecular weight of the laccase was approximately 244.0 kD, with a subunit molecular mass of roughly 85.6 kD. The
enzymatic properties showed that the optimum pH and temperature for the laccase were 5.0 and 55 ℃, respectively. The
enzyme 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. Fe2+ and ascorbic acid could completely inactivate the laccase, whereas the enzyme activity was slightly
affected by EDTA, Ag+, Mg2+ and Li+. Cu2+ had little activating effect on laccase activity. The enzyme activity of laccase
could be activated by urea, ethanol, and isopropanol, and inhibited by oxalic acid, methanol, n-butanol, K+, Ca2+, Ba2+,
Zn2+, Cd2+, Pb2+, Mn2+, and Co2+.