来源于Thermomyces sp.的植酸酶基因在毕赤酵母中的组成型表达

目的]研究植酸酶基因在毕赤酵母中的组成型表达。方法]用PCR方法从嗜热真菌(Thermomyces sp.)中扩增到去除信号肽和内含子后约1.4 kb的phyT基因编码区片段,并对该片段进行克隆与序列测定。结果]序列相似性分析表明,克隆的植酸酶基因无信号肽和内含子,与报道的嗜热真菌Thermomyces lanuginosus植酸酶基因相似性最高,DNA序列相似性为95%。从验证后的转化子筛选得到了表达嗜热真菌植酸酶的重组毕赤酵母菌株p-phy T,SDS-PAGE分析显示其分子量约为45 kD,重组毕赤酵母成功表达植酸酶。与野生型菌株相比,结果显示重组植酸酶酶活性提高了12.6%,最适温度65℃,75℃仍有64%以上酶活活性;最适pH值为5.5。结论]嗜热真菌植酸酶基因用组成型质粒能在毕赤酵母中表达。

Constitutive Expression of the phytase Gent from Thermonyces sp. in Pichia pastoris

Objective]The purpose was to study constitutive expression of the Phytase gene from Thermomyces sp.in Pichia pastoris.Method]The Phytase gene from Thermomyces sp.without intron and signal peptide was amplified and cloned.The gene fragment was about 1.4 kb and had no intron and signal peptide.Result]Sequence similarity analysis indicated Phytase gene cloned was similar to the Phytase gene from Thermomyces lanuginosis.DNA sequence similarity was 95%.The recombined plasmid p-phy T was transformed to Pichia pastoris GS115 by electroporation.The molecular weigh of the recombined enzyme was about 45 kD by SDS-PAGE analysis,and Phytase was successfully expressed by recombined Pichia.Compared with wild-type strains,the enzyme activity of recombined Phytase increased by 12.6%,and optimal temperature and pH value were 65 ℃ and 5.5.The remaining enzyme activity was more than 64% at 75 ℃.Conclusion] The Phytase gene from Thermomyces sp.can be expressed with constitutive plasmid in Pichia pastoris.