黑曲霉WY-6植酸酶的表达、纯化及性质研究

Expression,Purification and Characterization of Aspergillus niger WY-6 Phytase

目的:表达黑曲霉WY-6植酸酶基因及研究重组酶的性质。方法:通过PCR方法从黑曲霉WY-6基因组中扩增出植酸酶基因,并将该基因表达在毕赤酵母中,再利用蛋白质分离纯化技术对重组酶进行纯化,并测定其性质。结果:黑曲霉WY-6植酸酶基因成功表达在毕赤酵母中,重组植酸酶经饱和硫酸铵分级沉淀、超滤和阴离子交换层析步骤后得以纯化,纯化后的植酸酶比活力为147U/mg,分子量为67kDa,两个最适pH分别为3.0和5.5,最适温度为55℃,与胃蛋白酶以0.01的比率(胃蛋白酶/植酸酶,wt/wt)混合作用2h后仍保留70.9%残余活力。结论:获得了具有商业应用潜能的基因工程植酸酶。

Objective： To express Aspergillus niger WY- 6 phytase gene and study characterizations of the recombinant enzyme. Methods： A phytase gene was isolated from the genomic DNA of A. niger WY - 6 by PCR and expressed in Pichia pastoris. The recombinant enzyme was purified using protein purification methods and characterized. Results： The phytase gene from A. niger WY - 6 was successfully expressed in P. pastoris. The recombinant enzyme was purified using a combination of ammonium sulfate fi-actionafion, ultrafiltation and anion exchange chromatography. Its specific activity was 147 U/mg and apparent molecular mass was 67 kDa. The two optimal pH were 3.0 and 5.5, respectively, and the optimum temperature was 55 ℃. Incubated with pepsin at 37 ℃ for 2 h at the ratio（pepsin/phytase, wt/wt）of 0.01, the enzyme retained 70.9% of its initial phytase activity. Conclusion： A gene - engineering phytase with potential commercial interest was obtained.