N-糖基化对一种新型重组耐高温β-甘露聚糖酶(ReTMan26)稳定性的影响

Effect of N-glycosylation on the stability of a novel recombinant thermostable β-mannanase (ReTMan26)

摘要:

【背景】对来源于嗜热枯草芽孢杆菌(TBS2)的一种新型重组耐高温β-甘露聚糖酶(ReTMan26)基因序列进行分析，该基因中含有3个N-糖基化位点(N8、N26与N255)，经毕赤酵母表达时可进行N-糖基化修饰。【目的】确定N-糖基化对ReTMan26稳定性的影响。【方法】通过构建ReTMan26蛋白质三维结构模型，初步分析N-糖基化对该酶稳定性的影响。在此基础上，利用天然蛋白去糖基化试剂盒除去ReTMan26的N-多糖链，获得去除N-糖基化的耐高温β-甘露聚糖酶(ReTMan26-DG)，并对纯化后的ReTMan26及ReTMan26-DG进行相应的稳定性对比检测。【结果】 ReTMan26与ReTMan26-DG的最适反应pH均为6.0，但在pH 1.5?9.0范围内，ReTMan26的稳定性比ReTMan26-DG有小幅提高。ReTMan26的最适反应温度为60 °C，比ReTMan26-DG高5 °C；ReTMan26经100 °C处理10 min，剩余酶活为58.6%，而ReTMan26-DG经93 °C处理10 min，剩余酶活为58.2%，100 °C处理10 min则完全失活。经胃蛋白酶及胰蛋白酶在37 °C处理2 h后，ReTMan26的剩余酶活分别为70.5%及91.2%，比ReTMan26-DG分别提高了23.7%及25.6%。【结论】N-糖基化可提高ReTMan26的pH稳定性、耐热稳定性及抗蛋白酶消化性能。

Abstract:

[Background] Based on the sequence analysis of a novel recombinant highly thermostable b-mannanase (ReTMan26) from a thermophilic Bacillus subtilis (TBS2), there are 3 N-glycosylation sites (N8, N26 and N255) in the encoding gene of ReTMan26, and ReTMan26 could be N-glycosylated when expressed by Pichia pastoris. [Objective] To determine the effects of N-glycosylation on the stability of ReTMan26. [Methods] Through constructing the three-dimensional structure models, the effects of N-glycosylation on the stability of ReTMan26 were analyzed. Then, the N-deglycosylated ReTMan26 (ReTMan26-DG) was obtained using Native Protein Deglycosylation Kit. After purification, the differences of enzymatic stability between ReTMan26 and ReTMan26-DG were determined. [Results] The optimum reaction pH of ReTMan26 was 6.0, identical with that of ReTMan26-DG, and pH stability of ReTMan26 was slightly higher than that of ReTMan26-DG in pH range between 1.5 and 9.0. The optimum temperature of ReTMan26 was 60 °C, 5 °C higher than that of ReTMan26-DG. ReTMan26 retained 58.6% of its maximum activity after treatment at 100 °C for 10 min. However, ReTMan26-DG retained 58.2% residual activity after treatment at 93 °C and was completely inactivated after treatment at 100 °C for 10 min. After treatment with trypsin or pepsin at 37 °C for 2 h, ReTMan26 retained 91.2% and 70.5% of its baseline activity, 23.7% and 25.6% higher than ReTMan26-DG, respectively. [Conclusion] N-glycosylation could improve the stability of ReTMan26 at different pH, high-temperature and the resistance to digestive proteases.