天然产物研究与开发 ›› 2020, Vol. 32 ›› Issue (2): 296-304.doi: 10.16333/j.1001-6880.2020.2.014

• 开发研究 • 上一篇    下一篇

一株木蹄层孔菌SO3高产漆酶发酵工艺及部分酶学特性研究

谢玉清1,2,陈竞1,2,代金平1,2,杨新平1,2,古丽努尔·艾合买提1,2,王志方1,2,王小武1,2,冯蕾1,2*   

  1. 1新疆农业科学院微生物应用研究所;2新疆特殊环境微生物重点实验室,乌鲁木齐830091
  • 出版日期:2020-02-29 发布日期:2020-04-23
  • 基金资助:

    国家自然科学基金(2166038);新疆维吾尔自治区重点研发项目(2017B01003);新疆维吾尔自治区重点研发项目(2017B01001);新疆维吾尔自治区公益性科研院所基本科研业务经费资助项目(KY2017065)

Fermentation techonology of Fomes fomentarius SO3 producing laccase and partial enzymatic properties

XIE Yu-qing1,2,CHEN Jin1,2 ,DAI Jin-ping1,2,YANG Xin-ping1,2,GuliAhmat1,2,WANG Zhi-fang1,2,WANG Xiao-wu1,2,FENG Lei1,2*   

  1.  1Institute of Microbiology,Xinjiang Academy of Agricultural Sciences;2Xinjiang Key Laboratory of Special Enviromental Microbiology,Urumqi 830091,China
  • Online:2020-02-29 Published:2020-04-23

摘要: 从采自新疆阿勒泰山的木蹄层孔菌(Fomes fomentarius)中筛选获得一株高产漆酶的SO3菌株,对其产酶发酵工艺进行优化,并对部分酶学特性进行了研究。结果表明:SO3菌株产漆酶的最佳碳、氮源分别为麦麸和酵母粉; Mn2+ 、Ca2+、Zn2+和Mg2+的添加对SO3产漆酶有一定的促进作用,而Cu2+具有明显的抑制作用;没食子酸和单宁酸可使产酶高峰期提前2天;添加ABTS可使酶活由6 736 U/L提高至8 470 U/L;正交实验L9(34)优化培养基最佳组分为:酵母粉0.5%、麦麸2.5%、葡萄糖0.5%,磷酸二氢铵0.5%,漆酶活性达到10 863 U/L。酶学特性结果表明:SO3菌漆酶最适反应温度为50 ℃,且在80 ℃仍具有漆酶活性,最适反应pH为3.0,在40 ℃,pH 4.0~5.0之间,酶活性最稳定;同时研究表明Mn2+和Zn2+对酶稳定性有一定的促进作用,Co2+、Cd2+、Cr2+和Pb2+对酶的稳定性具有明显的破坏作用;对菌体及发酵液进行了双向电泳检测,测定其等电点为4.1,获得了1个纯化酶蛋白质谱序列。结果表明,该蛋白为LaccaseE(Trametes sp. 420)。SO3菌株具有潜在的应用价值。

关键词: 木蹄层孔菌, 漆酶, 发酵工艺优化, 酶学特性, 质谱测序

Abstract: Strain Fomes fomentarius SO3 with high-yield laccase was screened from the Alter Mountains in Xinjiang.We optimized the fermentation technology and studied the partial enzymatic properties.The experimental result showed that the optimal carbon and nitrogen sources were wheat bran and yeast extract powder.Adding Mn2+,Ca22+,Zn2+ and Mg2+ could promote the yield of laccase ,the effect of Cu2+ was adverse.Gallic acid and tannic acid made enzyme production peak two days ahead and ABTS may increase enzyme activity from 6 736 U/L to 8 470 U/L,orthogonal experiment L9(34) proved that optimum medium consists of 0.5% yeast extract powder,2.5 % wheat bran,0.5 % glucose and 0.5 %(NH4)H2PO4,in this case,enzyme activity could reach to 10 863 U/L.The study of enzymatic properties showed optimal reaction temperature of laccase is 50 ℃ and there was still activity at 80 ℃,optimal reaction pH was 3.0 and enzyme activity was stable when pH was 4.0-5.0 and temperature was under 40 ℃.At the same time,Co2+,Cd2+,Cr2+nd Pb2+ could damage enzyme stability.Bidirectional electrophoresis was performed on the bacteria and fementation liquid in which isoelectric point of laccase was determined to be 4.1.A pure enzyme protein sequcence was obtained by mass spectrometry that showed the protein to be LaccaseE (Trames sp. 420).

Key words: Fomes fomentarius SO3, laccase, optimizing fermentation technology, enzymatic properties, mass spectrometry sequencing

中图分类号: 

Q939.97