多位点突变提高大肠杆菌植酸酶AppA热稳定性

Improving the thermostability of Escherichia coli phytase AppA by multipoint mutation

为提高大肠杆菌植酸酶AppA(一种已得到广泛应用的饲料添加剂,其酶活高,pH作用范围广,但热稳定性较差)热稳定性,在appA基因上组合突变了W46E、Q62W、A73P、K75C、S146E、R159Y、N204C、Y255D、Q258N和Q349N等10个位点,突变后的基因命名为appAM10。将野生型appAppA和突变体appAM10ppAM10基因转入毕赤酵母GS115进行表达,并对重组蛋白AppA和AppAM10进行了酶学性质研究与比较。结果表明:AppAM10分子量约为55kDa,比活性为3022U/mg,AppAM10的K_m=330μmo1/L,V_(max)=3147U/mg。AppAM10的最适pH值为4.5,最适反应温度为65℃。与野生型的AppA的最适pH无明显区别,比AppA的最适温度提高了5℃。经90℃孵育15min后,AppA完全失活,而AppAM10仍残留17.5%的活性,T_m值提高约8℃,说明上述10个位点的组合突变有利于提高大肠杆菌植酸酶AppA热稳定性,其中与N-糖基化有关的N204C、Q258N和Q349N3个位点可能起着重要作用。

In order to enhance the thermostability of Escherichia coli phytase AppA, an important feed supplement with the favorable properties of high specific activity and broad pH optimum, ten residue substitutions of W46E, Q62W, A73P, K75C, S146E, R159Y, N204C ,Y255D, Q258N and Q349N were sequentially introduced into the gene appA, named the appAMlO after mutation. The recombinant genes of appA and appAMlO were expressed in pichia pastoris GS115 and the enzymology properties of them were analyzed. The results showed that the purified AppAM10 had a specific activity of 3 022 U/mg and a molecular weight of approximately 55 kDa. The optimum pH of AppAM10 consistent with AppA was 4.5. However, the optimum temperature of AppAM10 was 65 ℃ ,5 ℃ higher than that of AppA. Meanwhile ,the Km and Vmax of AppAM10 were 330 μmol/L and 3147 U/mg respectively. AppAM10 exhibited 17.5 % residual activity after treatment at 90 ℃ for 15min（none for AppA） and about 8 ℃ increase in the melting temperature （Tm ）. In a word, compared with the wild type AppA, AppAM10 showed an enhancement in thermostability significantly. According to the results above, the ten residue substitutions were found beneficial in promoting the thermostability of the phytase AppA. Especially, N204C, Q258N and Q349N associated with N-glycosylation may play an important role in improving the enzymology properties.