中文摘要：

为比较在不同宿主中表达的大肠杆菌植酸酶突变体app AM8的性质,将app AM8基因分别在大肠杆菌BL21（DE3）和毕赤酵母GS115中进行表达,对纯化后的植酸酶进行酶学性质分析与比较.结果显示：（1）在大肠杆菌中表达的app AM8（E-app AM8）和酵母中表达的app AM8（P-app AM8）的最适p H值均为4.5,最适反应温度均为65℃,与野生型的app A的最适p H无明显区别,比app A的最适温度高了5℃;（2）经SDS-PAGE检测,E-app AM8分子量（Mr）大小为47×10^3,而P-app AM8为53×10^3左右,由于在酵母中的糖基化修饰电泳显示为两条带;（3）在60-80℃之间,E-app AM8的热稳定性性明显下降,而且在70℃处理15 min后,E-app AM8残余活性仅为4%,而P-app AM8仍保留50%的残余活性,表明酵母表达的植酸酶经过糖基化修饰后提高了酶的热稳定性.（4）E-app AM8的Km=0.245 mmol/L,Vmax=3196 U/mg,P-app AM8的Km=0.36 mmol/L,Vmax=3333 U/mg.本研究表明,糖基化修饰对植酸酶app AM8的热稳定性起着重要作用,8个位点的突变对该植酸酶的稳定性也起到了一定作用.

英文摘要：

To compare the property of phytase produced in different host cells,the recombinant phytase appAM8 gene was expressed in Escherichia coli（E-appAM8） and Pichia pastoris（P-appAM8）.The results showed that both phytases have the same optimum pH of 4.5 and reaction temperature of 65℃.The molecular weight of E-appAM8 and P-appAM8 was estimated to be about 47×10^3 and 53×10^3 respectively by SDS-polyaerylamide gel electrophoresis.The protein expressed in P.pastoris was modified by glycosylation,showing two diffuse bands on SDS-PAGE.When heated to 60- 80℃,the thermo stability of E-appAM8 was obviously lower than that of P-appAM8.After treatment at 70℃ for 15 min,the relative activity of phytase E-appAM8 remained only 4%,while that of the P-appAM8 reached about 50% under the same condition,suggesting that glycosylation of phytase appAM8 improved its thermo stability.The Km of E-appAM8 and P-appAM8 was 0.245 mmol/L and0.36 mmol/L,and their Vmax was 3 196 U/mg and 3 333 U/mg,respectively.The results suggested that glycosylation plays an important role in the thermal stability of phytase appAM8.Point mutations of appAM8 also affect its stability.