烟曲霉植酸酶基因的克隆及在毕赤酵母的高效表达

Cloning of Phytase Gene from Aspergillus fumigatus and Its Expression in Pichia pastoris

根据文献报道的烟曲霉植酸酶序列,设计引物以烟曲霉CCTCCAF93044的总DNA为模板进行梯度PCR,扩增出了1条约1.4 kb的带,将该片段进行DNA序列测定,其序列与已报道的烟曲霉植酸酶序列完全一致,但只编码完整的成熟植酸酶序列。将该片段克隆到毕赤酵母分泌型表达载体pPIC9K上,获得表达质粒pHBM108。该质粒转化毕赤酵母KM71所得重组子经PCR验证后进行诱导表达,其中一重组子经144 h诱导,植酸酶表达量至少为1.25 mg/ml,比同类报道的表达量高出60％以上,以植酸钠为底物测得酶活为11.17U/ml。

Based on the published sequence of the Aspergillus fumigatus phytase gene, a 1. 4 kb DNA fragment containing the coding region of the phytase gene was cloned by touchdown PCR from A. fumigatus CCTCC AF93044 that was isolated in China. Sequencing shows that this fragment is the same as the published,but it has only encoded mature phytase. This 1. 4 kb DNA fragment was inserted into the Pichia pastorix expression vector, and pHBM108 was obtained. Then the pHBM108 was introduced into P. pastoris KM71 ,and one clone was selected for induced expression from hundreds of trans-formants after PCR confirmation. SDS-PAGE shows that extracellar phytase amounts to 1. 25 mg/ml, 60% higher than the published. Extracellar phytase activity is 11. 15 U/ml.