糖基化影响真菌植酸酶的胰蛋白酶耐受性

Glycosylation of fungal phytase affects its resistance to trypsin

通过重叠聚合酶链式反应,将黑曲霉植酸酶（Aspergillus niger NRRL 3135phytase）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 13073phytase）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pGAPZαA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.

Using overlapping PCR technique,the DNA sequences encoding protein regions 238-337and 382-444fromAspergillus niger NRRL 3135 phytase gene were replaced by corresponding Aspergillus fumigatus ATCC 13073 phytase sequences.The constructed Fragments-replaced phytase gene was inserted in pGAPZαA plasmid,and transformed into Pichia pastoris.Secreted Fragments-replaced phytase with phytase activity was purified through ion-exchange combined with gel filtration chromatography.While compared with Aspergillus niger NRRL 3135 phytase,Fragments-replaced phytase showed significantly improved resistance to tryptic digestion.However,after the N-glycosylation removed by PNGase thoroughly,the sensitivity of Fragments-replaced phytase to trypsin was recovered.These results indicated that the N-glycosylation modification of the replacing regions in Aspergillus fumigatus ATCC 13073 phytase enhanced the trypsin resistance of Aspergillus niger NRRL 3135 phytase expressed in Pichia pastoris.