N-糖基化对重组木聚糖酶XynA酶学特性的影响

为实现木聚糖酶的高效表达,扩大实际生产应用,本实验将木聚糖酶基因xynA在毕赤酵母中进行重组表达,同时采用糖基化抑制剂衣霉素处理毕赤酵母细胞,以此来探讨N-糖基化对木聚糖酶XynA酶学性质的影响。结果表明,SDS-PAGE电泳图谱显示木聚糖酶XynA发生了N-糖基化修饰,分子量约为45 kDa,酶活为406.6 U/mL,最适pH及反应温度分别为5.0和65 ℃;而添加不同浓度衣霉素处理后的木聚糖酶最适pH不变,最适反应温度下降5 ℃,随着衣霉素浓度的增加,去糖基化的木聚糖酶酶活力、分泌量及温度稳定性均有所下降,且当衣霉素添加浓度为15 μg/mL时,剩余酶活为53.6%。去糖基化的木聚糖酶耐受胃蛋白酶的能力较糖基化的有所增强,Na+、K+、Li+、Ca2+、Al3+、EDTA相对于糖基化的木聚糖酶表现出了一定的激活作用。以上结果说明N-糖基化作为一种重要的翻译后修饰对木聚糖酶XynA的分泌及热稳定性有促进作用。

Influence of N-glycosylation on enzymic properties of recombinant Xylanase A

In this study, we expressed the gene of xynA in Pichia pastoris GS115 in order to realize the efficient expression and expand the application of xylanase in practical production. Meanwhile we treated yeast cells with inhibitor tunicamycin to analyze the effects on the expression and secretion of xylanase as well as the enzymatic properties. Results showed that the SDS-PAGE indicated that the xylanase was glycosylated, the molecular mass of glycosylated recombinant XynA was about 45 kDa and its activity was 406.6 U/mL. The recombinant XynA exhibited optimal activity at pH5.0 and 65℃. However, recombinant XynA treated with different concentration tunicamycin showed the same optimal pH but lower optimal temperature of 5℃. The activity, secretion and thermostability of recombinant XynA all decreased through the increased concentration of tunicamycin, when the concentration of tunicamycin was 15 μg/mL, 53.6% of the enzyme activity was left. But the deglycosylated recombinant XynA had a higher tolerance of pepsin compared with glycosylated recombinant XynA, Na+, K+, Li+, Ca2+, Al3+, EDTA revealed the activity treated with tunicamycin. The above results show that N-glycosylation plays a key role in the secretion and thermostability of xylanase expressed by Pichia pastoris GS115.