嗜热厌氧菌Caldicellulosiruptor sp.F32中降解β-1,3-1,4葡聚糖水解酶的协同性分析及糖基化对F32EG5热稳定性影响

β-glucan degrading hydrolases from Caldicellulosiruptor sp. F32 and influence of glycosylation on F32EG5 thermostability

摘要:

[目的] 通过研究来自极端嗜热厌氧菌Caldicellulosiruptor sp.F32中3个可降解β-1，3-1，4葡聚糖（β-葡聚糖）的糖苷水解酶，解析其在降解β-葡聚糖过程中协同作用，以及异源表达的糖基化修饰对β-葡聚糖酶F32EG5热稳定性的影响，为该系列水解酶的应用提供考据。[方法] 通过大肠杆菌异源表达β-葡聚糖酶F32EG5和Lam16A-GH，以及β-葡萄糖苷酶BlgA，利用DNS、TLC等方法检测其在β-葡聚糖降解过程中的协同性及底物耐受能力。随后，利用毕赤酵母对F32EG5进行异源表达，以及对糖基化修饰的p-F32EG5进行酶学对比。[结果] β-葡聚糖酶F32EG5和Lam16A-GH单独作用于底物时，水解产物不同。但混合使用时，低聚合度寡糖的比例增加。β-葡萄糖苷酶BlgA分别与F32EG5和Lam16A-GH复配时，均展示出良好的协同效应和底物耐受能力。此外，利用毕赤酵母异源表达的p-F32EG5，没有明显改变其最适pH和最适温度，但在超高温下（80-90℃）的热稳定性和催化效率相对于未被糖基化的F32EG5提高2倍以上。[结论] 葡萄糖糖苷水解酶BlgA分别与β-葡聚糖酶F32EG5、Lam16A-GH复配，在水解β-葡聚糖过程中表现出良好的协同性和底物耐受能力，同时毕赤酵母异源表达的糖基化修饰能提高在超高温环境下的热稳定性能，有利于酶制剂生产造粒过程中的酶活保留，从而使F32EG5具备应用化潜力。

Abstract:

[Objective] Three β-glucan degrading glycoside hydrolases from an extreme thermophilic anaerobic bacterium Caldicellulosiruptor sp. F32 were investigated, including the synergistic effect. The effect of glycosylation on β-glucanase F32EG5 thermostability was also studied.[Methods] Two β-glucanases (F32EG5, Lam16A-GH) and β-glucosidase (BlgA) were heterologously expressed in E. coli. The synergistic effect of all these enzymes on β-glucan degradation was evaluated by 3,5-Dinitrosalicylic acid (DNS) and Thin-layer chromatography (TLC) assays including substrate tolerant abilities. Furthermore, the glycosylated p-F32EG5 was expressed in Pichia pastoris, and compared with F32EG5 from E. coli.[Results] F32EG5 and Lam16A-GH released oligosaccharides with different degrees of polymerization (DP) after hydrolyzing β-glucan. The proportion of low-DP oligosaccharides was increased, when two enzymes used together. BlgA showed excellent synergistic effect with F32EG5 and Lam16A-GH, respectively. Although the glycosylated p-F32EG5 from Pichia pastoris did not obviously change its optimum pH and temperature when compared with E. coli-expressed F32EG5, both thermal stability and catalytic efficiency were found two-folds higher than those of E. coli-expressed F32EG5 at the extreme-high temperature (80-90℃).[Conclusion] F32EG5 and Lam16A-GH showed excellent synergistic effect and substrate tolerant abilities with BlgA. The heterologous glycosylation by Pichia pastoris could improve the thermal stability of F32EG5 under extreme thermophilic environment, which was a benefit during the granulation process of enzyme.