【摘 要】
：
大肠杆菌植酸酶appA,属于组氨酸酸性磷酸酶家族,是一种酶活高,pH作用范围广的饲料添加剂.植酸酶appA上存在三个糖基化位点NVTl39-14l、NVS204-206和NWT317-319,分别位于α-螺旋、β-片层和无规则卷曲上.为探讨N-糖基化对植酸酶appA酶学性质的影响,本研究将植酸酶基因appA在毕赤酵母中进行了表达,利用定点突变技术构建了植酸酶去糖基化突变体appA-1NQ,appA
【机 构】
：
山西大学生物技术研究所,化学生物学与分子工程教育部重点实验室,山西太原,030006
【出 处】
：
中国生物化学与分子生物学会2016年全国学术会议
论文部分内容阅读
大肠杆菌植酸酶appA,属于组氨酸酸性磷酸酶家族,是一种酶活高,pH作用范围广的饲料添加剂.植酸酶appA上存在三个糖基化位点NVTl39-14l、NVS204-206和NWT317-319,分别位于α-螺旋、β-片层和无规则卷曲上.为探讨N-糖基化对植酸酶appA酶学性质的影响,本研究将植酸酶基因appA在毕赤酵母中进行了表达,利用定点突变技术构建了植酸酶去糖基化突变体appA-1NQ,appA-2NQ,appA-3NQ和糖基化引入突变体appA-2QN,对突变前后的植酸酶进行了纯化和酶学性质研究与比较.结果表明：野生型植酸酶appA-WT分子量约为53kDa,酶活为3197 U/mg,Km=480 μmol/L,Vmax=3 848 U/mg.野生型植酸酶appA-WT的最适pH值为4.5,最适反应温度为60 ℃,与植酸酶糖基化突变体的最适pH无明显区别,appA-2QN的最适温度提高了5 ℃,Tm值提高约5 ℃.经75 ℃孵育15 min后,appA-3NQ完全失活,appA-WT保留11％的酶活,而appA-2QN仍残留23％的酶活.用不同比例的蛋白酶处理2h后,appA-2QN具有较强的抗蛋白酶的能力,并且随着糖基化个数的减少,植酸酶的抗蛋白酶的能力大幅下降.胃蛋白酶/植酸酶质量比为1.2处理2h后,appA-2QN和appA-WT仍保留60％以上的酶活,而appA-3NQ已失活.胰蛋白酶/植酸酶质量比为1.2处理2h后,appA-3NQ已完全失活,appA-WT保留31％的酶活,而appA-2QN保留40％以上的酶活.综上所述,N-糖基化不仅对植酸酶appA的稳定性有促进作用,而且引入糖基化可以增强植酸酶抗蛋白酶降解能力.
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