嗜热毛壳菌锰超氧化物歧化酶基因在毕赤酵母中的表达及其酶学性质  

Expression of manganese superoxide dismutase gene from Chaetomium thermophilum in Pichia pastoris and its enzymic properties

作　　者：张丽青[1] 咸洪泉[2] 张庆乐[1] 

机构地区：[1]泰山医学院化学院,山东泰安271016 [2]青岛农业大学生命科学院

出　　处：《环境与健康杂志》2015年第5期422-425,共4页Journal of Environment and Health

基　　金：山东省自然科学基金青年基金(ZR2012CQ035);教育部大学生创新创业训练计划(201410439003);泰安市科技局一般项目(201440777-27)

摘　　要：目的克隆嗜热毛壳菌(Chaetomium thermophilum)锰超氧化物歧化酶(CtMnSOD)基因,进行毕赤酵母真核表达、纯化重组CtMnSOD蛋白,并分析其酶学性质。方法提取嗜热毛壳菌总RNA,根据CtMnSOD基因全长编码序列(登录号为EF569987)的开放阅读框设计合成引物,并进行逆转录-聚合酶链式反应(RT-PCR),扩增产物经双酶切后连接入pPIC9K载体,重组质粒pPIC9K/CtMnSOD经SacⅠ线性化后,采用电穿孔法将其导入毕赤酵母GS115中,并加入甲醇诱导表达,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)结合考马斯亮蓝染色检测表达产物;重组CtMnSOD经硫酸铵沉淀和DEAE-Sepharose Fast Flow阴离子交换纯化;并通过氯化硝基四氮唑蓝(NBT)法测定酶学性质。结果 RT-PCR扩增产物约为684 bp。菌落PCR、双酶切及测序结果显示,重组质粒pPIC9K/CtMnSOD构建成功。SDS-PAGE结果显示,经甲醇诱导获得约22.7 k Da的可溶性重组蛋白。酶活力分析结果表明,工程菌酶活力达906.23 U/ml;该重组酶的最适反应温度为60℃,最适反应pH为6.0;在70℃的条件下保温30 min后仍具有71%的酶活力。结论 CtMnSOD在毕赤酵母中高效分泌表达,该酶具有较高的热稳定性,具有较好的工业应用价值。Objective To clone the protein thermostable manganese superoxide dismutase （CtMnSOD） gene from Chaetomium thermophilum,perform eukaryotic express in Pichia pastoris, purify CtMnSOD, analyze the enzymic properties of recombinant protein. Methods Total RNA was extracted from Chaetomium therrnophilum. Open reading frame of target gene was amplified with a pair of specific primers designed according to the coding sequence of CtMnSOD gene （GenBank accession No： EF569987） by RT-PCR. The RT-PCR product was digested with restrict enzymes and ligated into a pPIC9K vector. The recombinant plasmid pPIC9K/CtMnSOD was then linearized by Sac I and transformed into Pichia pastoris GSll5 by electroporation, and induced to express by methanol treatment. The expression products were analyzed via SDS-PAGE followed by coomassie blue staining. The recombinant protein was purified by ammonium sulfate precipitation, DEAE-Sepharose Fast Flow anion exchange,and its enzyme activity was analyzed by nitrotetrazolium blue chloride （NBT） method. Results The product of RT-PCR was 684 bp. The recombinant plasmid pPIC9K/CtMnSOD was confirmed successfully by colony-PCR, double restrict enzymes digestion and DNA sequencing. The CtMnSOD proteins were successfully expressed in Pichia pastoris and the molecular weight was approximately 22.7 kDa, the enzyme activity reached 906.23 U/ml. The recombinant CtMnSOD exhibited optimum catalytic activity at pH 6.0 and 60 ℃ respectively. The enzyme was thermostable and could still keep 71% activity at 70 ℃ for 30 min. Conclusion CtMnSOD gene can be successfully expressed in Pichia pastoris and the recombinant CtMnSOD was thermostable thus showing a potential for commercial uses.

关 键 词：锰超氧化物歧化酶 嗜热毛壳菌 嗜热酶 毕赤酵母 

分 类 号：R117[医药卫生—公共卫生与预防医学]