N-糖基化对植酸酶phy(QF)酶学性质的影响

摘要

为探讨N-糖基化对植酸酶phy（ QF ）酶学性质的影响，将植酸酶基因phy（ QF ）在毕赤酵母中进行了表达，利用脱糖基化酶Endo Hf去除重组植酸酶phy（QF）的糖链，对去除糖链前后的植酸酶phy（QF）进行了纯化和酶学性质研究与比较。结果表明，糖基化的植酸酶phy（QF）分子量约为53 kDa，酶活为3201 U／mg，Km ＝392μmol／L，Vmax ＝3267 U／mg；糖基化的植酸酶phy（ QF ）最适pH值为4．5，最适反应温度为55℃，与去糖基化的植酸酶phy（ QF ）的最适pH值无明显区别，比去糖基化的植酸酶phy（QF）最适温度提高了5℃，Tm值提高约2℃。经70℃孵育10 min后，去糖基化的植酸酶phy（QF）完全失活，而糖基化的植酸酶phy（QF）仍残留13％的活性，说明N-糖基化作为一种重要的翻译后修饰，对植酸酶phy（ QF ）的稳定性有促进作用。%In order to study the effects of N-glycosylation on enzymological properties of phytase phy (QF),the recombinant phytase gene phy(QF) was expressed in pichia pastoris GS115.Phytase phy(QF) was deglycosylated by endoglycosylase Endo H f .Enzymology properties of glycosylated phy (QF)and deglycosylated phy (QF)were an-alyzed.The results showed that the glycosylated phy (QF) had a specific activity of 3 201 U/mg with an MW of ap-proximately 53 kDa and the Km and Vmax of glycosylated phy(QF) were 392 μmol/L and 3 267 U/mg,respectively. The optimal pH value of glycosylated phy(QF) was 4.5 and was consistented with that of deglycosylated phy (QF). However,the optimal temperature of glycosylated phy (QF) was 55 ℃,5 ℃ higher than that of deglycosylated phy (QF) and 2 ℃increased in the melting temperature (Tm).The glycosylated phy(QF) exhibited 13%residual ac-tivity after treatment at 70 ℃for 10 min and deglycosylated phy (QF)was completely deactivated under the condi-tion.As one of posttranslational modifications ,N-glycosylation may play an important role in improving the stability of phytase phy (QF).