生物技术通报 ›› 2018, Vol. 34 ›› Issue (4): 127-132.doi: 10.13560/j.cnki.biotech.bull.1985.2017-0629

• 研究报告 • 上一篇    下一篇

大球盖菇漆酶的分离纯化及酶学性质研究

钱磊1, 张业尼2, 陈雪2, 刘金福2, 张志军1, 刘建华1   

  1. 1. 天津市林业果树研究所,天津 300384;
    2. 天津农学院食品科学与生物工程学院,天津300384
  • 收稿日期:2017-07-28 出版日期:2018-04-20 发布日期:2018-05-04
  • 作者简介:钱磊,男,博士,助理研究员,研究方向:食用菌生物化学与分子生物学;E-mail:qianl1982@sina.cn
  • 基金资助:
    天津市农业科学院院长基金(16009),天津市科技计划项目(16ZXZYNC00170),天津市科技计划项目(17ZXBFNC00160),天津市蔬菜现代农业产业技术体系创新团队(ITTVRS2017008)

Purification and Enzymatic Characterization of Laccase from Stropharia rugosoannulata

QIAN Lei1, ZHANG Ye-ni2, CHEN Xue2, LIU Jin-fu2, ZHANG Zhi-jun1, LIU Jian-hua1   

  1. 1. Tianjin Research Institute of Forestry and Pomology,Tianjin 300384;
    2. College of Food Science and Biotechnology,Tianjin Agricultural University,Tianjin 300384
  • Received:2017-07-28 Published:2018-04-20 Online:2018-05-04

摘要: 从大球盖菇Sr-01菌株液体发酵液中分离漆酶,研究温度、pH和金属离子对酶活的影响。采用硫酸铵分级沉淀、Q-Sepharose阴离子交换层析和Superdex 200凝胶过滤层析对大球盖菇漆酶进行分离纯化。以ABTS[2,2-连氮基-双-(3-乙基苯并二氢噻唑啉-6-磺酸)]为底物,分光光度法测定酶活。结果表明,纯化后的漆酶比活力为152.79 U/mg,回收率为35.8%。SDS-PAGE显示该漆酶为单体蛋白,相对分子质量约40 kD。该漆酶的最适反应温度和pH分别为35℃和4.0,Mg2+、Cu2+对酶活有激活作用,Fe2+、Cd2+、Hg2+则有显著抑制作用。在最优反应条件下,纯化后的漆酶比活力可达222.93 U/mg。

关键词: 大球盖菇, 漆酶, 纯化, 酶学性质

Abstract: The laccase from the fermentation liquid of Stropharia rugosoannulata strain Sr-01 was isolated and the effects of temperature,pH and metal ions on laccase activity and stability were assayed. The laccase was purified by ammonium sulfate fractional precipitation,anionic exchange chromatography on Q-Sepharose,and gel filtration chromatography on Superdex 200. The laccase activity was measured by spectrophotometry with ABTS[2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate)]as the substrate. The results showed that the specific activity of the purified laccase was 152.79 U/mg and the recovery was 35.8%. SDS-PAGE analysis demonstrated that it was a monomeric protein with the molecular weight of 40 kD. The optimal reaction temperature and pH of the laccase were 35℃ and 4.0,respectively. The laccase activity was stimulated by Mg2+ and Cu2+,whereas it was significantly inhibited by Fe2+,Cd2+ and Hg2+. Under the optimal reaction conditions,the specific activity of the purified laccase achieved 222.93 U/mg.

Key words: Stropharia rugosoannulata, laccase, purification, enzymatic characterization