嗜热真菌植酸酶基因的克隆及其在巴斯德毕赤酵母中高效表达  

Cloning and Overexpression of a Gene Encoding Phytase from Thermophilic Fungi in Pichia pasteris

作　　者：张文荟[1] 诸葛斌[1] 沈微[1] 饶志明[1] 方慧英[1] 诸葛健[1] 

机构地区：[1]江南大学教育部工业生物技术重点实验室江南大学生物工程学院工业微生物中心,江苏无锡214122

出　　处：《食品与发酵工业》2009年第12期10-14,共5页Food and Fermentation Industries

基　　金：国家高技术863项目(2006AA020103)资助

摘　　要：根据已报道的植酸酶基因序列设计一对引物,用PCR方法从嗜热真菌(Thermomyces sp.)中扩增获得不含信号肽和内含子的植酸酶基因(phyT),对该1.4kb的片段进行克隆与序列测定。序列分析表明该基因与已报道的嗜热疏绵霉(Thermomyces lanuginosus)的植酸酶基因相似性最高,DNA和氨基酸水平相似性分别达到95%和99%。将phyT基因插入毕赤酵母表达载体pPIC9K中,构建重组质粒pPIC9K-phyT并电转化毕赤酵母(Pichia pastoris GS115)。植酸酶基因成功在毕赤酵母中表达并对重组酶酶学性质进行了研究。诱导表达酶活结果显示与野生型菌株相比,活性提高了67.8%,最适温度65℃,75℃处理20min后仍有70%以上酶活活性。最适pH为6.0。SDS-PAGE分析显示其分子量约55ku以上。Based on the phytase coding sequences from GeneBank, 1.4kb phytase fragment was cloned from Thermomyces sp.. PCR product was sequenced and the result showed that the gene had no intron and signal peptide. Sequence similarity analysis suggested it was similar to Thermomyces lanuginosus phytase gene, and DNA and amino acid sequence similarity was 95% and 99% , respectively. The phytase gene was inserted into pPIC9K to construct pPIC9K-phyT. The secreted recombined plasmid was linearized by BglII and transformed into Pichia pastorisGS115 by electroporation. After methanol induction, phytase was expressed in transformants and showed improving activity and better character than native strain. The enzyme activity was 68.7% higher than parent strain. The optimal temperature and pH value of the recombined phytase activity were 65℃and 6.0, respectively. The enzyme remained high stability after heat treatment at 75℃ for 20 min. SDS-PAGE showed the molecular weigh of the recombined enzyme was above 55 ku.

关 键 词：嗜热真菌 植酸酶 重组毕赤酵母 热稳定性 

分 类 号：Q78[生物学—分子生物学]