少孢节丛孢菌几丁质酶AO-492对线虫的降解作用研究  

Degradation of Nematodes by Chitinase AO-492 from Arthrospora oligospora

作　　者：张嘉华 张慧梅 马喜喜 孙焱森 李若冰 李柠杏 才学鹏[2] 乔军[1] 孟庆玲[1] ZHANG Jia-hua;ZHANG Hui-mei;MA Xi-xi;SUN Yan-sen;LI Ruo-bing;LI Ning-xing;CAI Xue-peng;QIAO Jun;MENG Qing-ling(Department of Animal Science and Technology,Shihezi University,Shihezi 832003;China Institute of Veterinary Drug Control,Beijing 100081)

机构地区：[1]石河子大学动物科技学院,石河子832003 [2]中国兽医药品监察所,北京100081

出　　处：《生物技术通报》2024年第5期261-268,共8页Biotechnology Bulletin

基　　金：国家自然科学基金项目(32260888,32060801);新疆维吾尔自治区研究生科研创新计划项目(XJGRI2015038)。

摘　　要：【目的】为探究捕食线虫真菌少孢节丛孢菌几丁质诱导过程中分泌蛋白AO-492的生物学功能。【方法】对少孢节丛孢菌几丁质酶AO-492主要结构域编码区进行基因克隆及分子特征分析,并在毕赤酵母中进行表达。利用镍柱亲和层析法纯化重组蛋白ReAO-Z492,采用NAG检测法分析了该重组蛋白在不同温度、pH及金属离子条件下的酶学活性,并将其作用于秀丽隐杆线虫及虫卵分析其生物学功能。【结果】几丁质酶AO-492有信号肽,无跨膜结构域,含有两个几丁质结合结构域和一个糖苷水解酶18家族结构域,并含有糖苷水解酶18家族几丁质酶高度保守的底物结合位点-SVGGWT-和水解酶活性位点-FDGGDLDWE-,含有典型的TIM桶形分子结构。系统进化分析显示,该蛋白与坚粘孢单顶孢几丁质酶(EPS35099.1)的亲缘关系相对最近。SDS-PAGE和Western Blot分析表明,ReAO-Z492分子量约为59 kD,可与小鼠抗少孢节丛孢菌多克隆抗体发生特异性反应。ReAO-Z492最适温度为40℃,最适pH为7.0;Mg^(2+)对其酶活有促进作用,而Ag^(+)、Cu^(2+)、Fe^(3+)和Zn^(2+)有抑制作用。ReAO-Z492对秀丽隐杆线虫体壁及其虫卵卵壳有较强的降解活性。【结论】少孢节丛孢菌几丁质酶 AO-492 对秀丽隐杆线虫及虫卵具有较强的降解作用。【Objective】To explore the biological function of secreted protein AO-492 in the process of chitin induction from nematodetrapping fungus Arthrobotrys oligospora.【Method】The coding region of the main domain of chitinase AO-492 from A.oligospora was cloned and analyzed,and expressed in Pichia pastoris.The recombinant protein ReAO-Z492 was purified by nickel column affinity chromatography.The enzymatic activity of the recombinant protein under different temperature,pH and metal ion conditions was analyzed by NAG assay,and its biological function was analyzed by acting on Caenorhabditis elegans and eggs.【Result】The chitinase AO-492 had a signal peptide,no transmembrane domain,contained two chitin binding domains and a glycoside hydrolase 18 family domain,and the highly conserved substrate binding site of the glycoside hydrolase 18 family chitinase-SVGGWT-and the hydrolase active site-FDGGDLDWE-,as well as a typical TIM barrel molecular structure.Phylogenetic analysis showed that the protein had the closest genetic relationship with chitinase(EPS35099.1).SDS-PAGE and Western Blot analysis showed that the molecular weight of ReAO-Z492 was about 59 kD,which specifically reacted with mouse polyclonal antibody against A.oligospora.The optimal temperature and pH of ReAO-Z492 were 40℃and 7.0,respectively.Mg^(2+)promoted the enzyme activity,while Ag^(+),Cu^(2+),Fe^(3+)and Zn^(2+)inhibited it.ReAO-Z492 had strong degrading activity on the body wall and egg shell of C.elegans.【Conclusion】The chitinase AO-492 of A.oligospora has a strong degradation effect on C.elegans and its eggs.

关 键 词：少孢节丛孢菌 几丁质酶 ReAO-Z492 酶学活性 捕食线虫真菌 

分 类 号：S85[农业科学—兽医学]