大肠杆菌植酸酶的原核表达及酶学性质

Characteristics of Phytase from Escherichia coli

将大肠杆菌植酸酶appA基因重组于表达载体pET32a中,导入大肠杆菌Origami(DE3)构建工程菌Origami(DE3)-pET32a-appA.对表达的appA重组植酸酶经Ni柱亲和纯化、SDS-PAGE分析表明,纯化后条带单一,酶的纯度较高.对其酶学性质的研究表明,该酶反应的比活力为3.36×106U/mg;最适pH为4.5;最适温度为60℃;在80℃和85℃的耐干热能力超过耐湿热能力;在37℃下,Mg2+﹑Ca2+﹑Mn2+对酶活性有促进作用,Co2+﹑Cu2+﹑K+对酶活性有不同程度的抑制作用,而Fe3+和Zn2+对酶活性有强烈的抑制作用;此外,该酶还具有非常好的抗胃蛋白酶水解的能力和部分抗胰蛋白酶水解的能力.

Gene appA was cloned into prokaryotic expression vector pET32a to construct pET32a-appA and gene appA was successfully expressed in Escherichia coli Origami （DE3）. Recombinant phytase was purified and its enzymatic characterization was studied. The results of SDS-PAGE showed higher purity of the enzyme after purified by nickel-chelating sepharose chromatography. Its specific activity was 3.36×10^6 U/mg, and optimal pH and temperature for its activity were 4.5 and 60 ℃, respectively. The dry thermal stability of phytase was stronger than wet thermal stability at 80℃ and 85 ℃. The enzyme activity was activated by Mg^2＋, Ca^2＋ and Mn^2＋, and inhibited by Co^2＋, Cu^2＋ and K^＋. However, it almost lost activity when influenced by Fe^3＋ and Zn^2＋. In addition, it had strong ability to resist hydrolyzation by pepsin and partial ability to resist hydrolyzation by trypsin.