摘要
【目的】旨在用毕赤酵母高效表达灰盖鬼伞过氧化物酶。【方法】借助DNAworks 3.1软件设计、优化引物,用自己构建的基因合成、定点突变平台合成了毕赤酵母密码子偏好性的灰盖鬼伞过氧化物酶基因,测序后构建在表达载体pPICZαA上,整合于巴斯德毕赤酵母GS115染色体,来自酿酒酵母的α因子作为信号肽序列指导重组蛋白的分泌表达。从82个PCR检测为阳性的酵母转化子中筛选出6株高Zeocin抗性的菌进行表达,选表达酶活性最高的作为实验菌株命名为CIP/GS115。【结果】以ABTS为底物时,CIP/GS115在甲醇诱导第4天酶活最高达到487.5U/mL,是目前摇瓶培养诱导表达灰盖鬼伞过氧化物酶活性最高报道。纯化后的酶最适反应温度为25℃,45℃酶反应速度是最适温度时的61.5%,在低于40℃时比较稳定,超过45℃稳定性迅速下降。最适反应pH为5.0,在p H 4.5-6.5之间比较稳定。以不同的底物研究纯酶底物特异性发现最适底物的顺序是:ABTS>愈创木酚>2,6-二甲氧苯酚>2,4-二氯苯酚>苯酚。【结论】灰盖鬼伞过氧化物酶在毕赤酵母中的高效分泌表达和高的特殊活性为该酶在废水处理、染料脱色等方面的工业化应用奠定了一定基础。
[Objective] The aim of our study is to express Coprinus cinereus peroxidase（ CIP） in Pichia Pastori efficiently.[Methods] We synthesized CIP gene with P.pastori codon bias by our Gene Synthesis and site-specific mutagenesis platform,using DNAWorks 3.1 program to design and optimize primers.Then,we sequenced the PCR products,inserted the correct gene into expression vector p PICZαA and transformed the linearized p PICZαA-Cip DNA into P.pastori GS115.We integrated CIP gene into the genome of P.pastori,using the α-mating factor from Sacchoramyces cerevisiae as signal peptide to direct the secretion of the recombinant protein.To obtain transformants with high CIP activity,we checked transformants by nested PCR and stained 82 positive ones on YPD agar plate with 1000 mg / L Zeocin.Then,we got 6 transforments with high resistance to Zeocin and expressed them in small scale; the one exhibiting the highest activity was chosen as engineered strain and named CIP / GS115.[Results]We purified CIP from culture medium after induction with ethanol,the maximum activity reached 487.5 U / mL on the 4thday.The purified CIP exhibited maximal activity at p H 5.0 and 25℃ with ABTS as substrate.The enzyme had 61.5% of the maximal activity at 45℃ and was stable below 40℃.However,the stability was drastically reduced above 45℃.The recombinant CIP remained stable between p H 4.5 and 6.5.We studied the substrate specificity on different substrates with the purified enzyme,and the optimal substrates were in the order of ABTS 〉2,6- Dimethoxyphenol 〉guaiacol 〉2,4- Dichlorophenol phenol.[Conclusion]The highly secretory expression of CIP and high special activity lay the good foundation for it＇s industrial applications in waste water treatment,decolouration of dyestuffs.
出处
《微生物学报》
CAS
CSCD
北大核心
2015年第3期321-329,共9页
Acta Microbiologica Sinica
基金
国家自然科学基金项目(31300669)
河南省科技厅科技攻关项目(112102210385
142102210479)
南阳师范学院高层次人才科研启动项目(zx20110007)
河南省教育厅科学技术重点研究项目(13A18081
14B180003)~~
关键词
灰盖鬼伞过氧化物酶
基因合成
毕赤酵母表达
Coprinus cinereus peroxidase （CIP）, gene synthesis, expression in Pichia pastoris
