摘要
目的:克隆植酸酶基因phyA,构建毕赤酵母表达载体,转化毕赤酵母,并对重组工程菌的表达产物进行初步酶学性质研究。方法:以植酸酶高产菌株─黑曲霉Z6染色体DNA为模板,PCR扩增得到植酸酶基因phyA,序列鉴定后连接到毕赤酵母穿梭载体pPIC9K上,构建重组质粒pPIC9K-phyA,电击转化毕赤酵母KM71,筛选得到重组转化子。对重组工程菌表达产物进行SDS-PAGE分析和酶活性研究。结果:phyA序列分析表明该基因具有典型的植酸酶活性位点保守序列ArgHisGlyAlaArgTyrPro,与NC-BI已发表的植酸酶基因同源性较高,达到94%以上。该序列已提交GenBank,序列号为DQ318022。重组工程菌KM71-phyA7的PCR扩增证实了植酸酶基因已整合到酵母基因组中,植酸酶能有效分泌和表达,粗酶液酶活可达875U/mL。结论:植酸酶基因phyA在毕赤酵母中成功表达,为今后的定向改组奠定了基础。
Objective： The phyA gene was cloned and the Pichia pastoris expression vector was constructed and transformed into P. pastoris, then the activity of the expressed pnxtuction of the recombinant strain was studied. Method： The phyA gene was amplified by PCR from Aspergillus niger Z6 chromosome DNA. Then phyA was linked with P. pastoris shuttle vector pPIC9K after sequencing. The recombinant plasmid pPIC9K- phyA was constructed and transformed into P. pastoris KM71 by electroporation： The recombinant strain was obtained through plate screening. The expressed production of the recombinant strain was analyzed by SDS- PAGE and enzymatic kinetic analysis. Result： The sequencing result of phyA revealed that it had a typical conservative sequence of phytase active site which contained ArgHisGlyAlaArgTyrPro. By nucleotide blasting in NCBI phyA showed high homology to reported DNA sequence of phytase which reached 94% highest. The nucleotide sequence was submitted to GenBank and the Accession Number is DQ318022. It was proved that phyA had been inserted in the chromosome of P. pastorh KM71 - phyA7 by PCR and phytase could be secreted effectively and expressed highly which crude enzyme reached 875U/mL. Conclusion： The phyA gene was successfully expressed in P. pastoris which laid a foundation of next DNA shuffling.
出处
《生物技术》
CAS
CSCD
2008年第6期18-20,共3页
Biotechnology
基金
济南市留学人员创业计划项目资助("重组表达植酸酶的芽孢杆菌微生物制剂的研制"
2006第5-02号)
