食品科学

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杂优-2平菇漆酶的分离纯化及酶学性质

廖海君,李蕊伽,陶 敏,白亚娟,唐 菁,唐云明*   

  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
  • 出版日期:2016-10-15 发布日期:2016-12-01

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

LIAO Haijun, LI Ruijia, TAO Min, BAI Yajuan, TANG Jing, TANG Yunming*   

  1. Key Laboratory of Eco-environments in Three Gorges Reservoir Region, Ministry of Education, Chongqing Sweetpotato Engineering Research Center, School of Life Science, Southwest University, Chongqing 400715, China
  • Online:2016-10-15 Published:2016-12-01

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 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。

关键词: 杂优-2平菇, 漆酶, 分离纯化, 酶学性质

Abstract:

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+.

Key words: Pleurotus ostreatus heterosis-2, laccase, isolation and purification, enzymatic properties

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