糖基化影响真菌植酸酶的胰蛋白酶耐受性

Qingfeng ZHU,Zhilin WANG,Baiyuan CUI,Qi ZHANG,Zhuang CHEN,Shijuan YAN,Jinlong BEI

Journal of HuaZhong Normal University(Natural Sciences)（2016）

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Abstract

通过重叠聚合酶链式反应,将黑曲霉植酸酶(Aspergillus niger NRRL 3135 phytase)第238～337位与第382～444位多肽片段的基因编码序列替换为烟曲霉植酸酶(Aspergillus fumigatus ATCC 13073 phytase)中的对应序列,构建片段置换植酸酶(Fragments-replaced phytase)基因.将插入该基因的pGAPZα A重组质粒转化入毕赤酵母.通过离子交换与凝胶过滤纯化,获得重组酵母分泌的活性片段置换植酸酶.与黑曲霉植酸酶相比,片段置换显著提高了片段置换植酸酶的胰蛋白酶耐受性.而通过脱糖基化酶PNGase彻底去除N-糖基化,可恢复片段置换植酸酶对胰蛋白酶的敏感性.上述结果表明烟曲霉植酸酶对应片段上的N-糖基化修饰可显著提高毕赤酵母表达的黑曲霉植酸酶的胰蛋白酶耐受性.

Key words

phytase,Aspergillus niger,Aspergillus fumigatus,glycosylation,proteolysis

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