毕赤酵母表达的嗜热蓝状菌β-葡萄糖苷酶N-糖基化修饰的作用与功能

Effect of N-glycosylation on enzymatic properties of β-glucosidase from Talaromyce leycettanus

【目的】研究N-糖基化对来源于嗜热蓝状菌β-葡萄糖苷酶(β-glucosidase,Bgl3A)的酶学性质影响。【方法】采用定点突变技术构建了3个去N-糖基化的突变体T44A、S228A、S299A,并分别在毕赤酵母GS115中表达纯化。【结果】与野生型Bgl3A相比,突变体S228A分泌蛋白产量极低,仅能微量检测到p NPG活性;突变体T44A和S299A的最适pH和最适温度没有改变,分别为4.0和75°C,但二者的T_m值和70°C下的热稳定性都明显优于野生型。以p NPG为底物时,突变体S299A和T44A的催化效率分别降低了14.5%和70.0%;以纤维二糖为底物时,T44A的催化效率基本不变,而S299A的催化效率提高了1.1倍。【结论】Bgl3A不同位点的N-糖基化修饰对酶的分泌和酶学性质的影响具有明显差异。其中,N226位的N-糖基化在维持酶的表达和功能方面至关重要,而去除N297位点的N-糖基化可以提高酶的热稳定性及对纤维二糖的催化效率。

[Objective]To investigate the effect of N-glycosylation on the enzymatic properties ofβ-glucosidase Bgl3A from Talaromyce leycettanus.[Methods]Site-directed mutagenesis was conducted to construct three N-glycosylation-removing mutants T44A,S228A and S299A,and the gene products were expressed in Pichia pastoris GS115.[Results]In comparison with wild type Bgl3A,the mutant S228 A was low in protein secretion,with trace activity against p NPG,while mutants T44 A and S299 A showed similar optimal pH and temperature,i.e.pH 4.0 and 75℃,but had higher Tm values and greater thermostability at 70℃.When using p NPG as the substrate,mutants S299 A and T44 A had decreased catalytic efficiencies(kcat/Km)of 14.5%and 70%,respectively;for cellobiose,T44A retained almost the same catalytic efficiency,while S299 A showed an improvement of 1.1-fold.[Conclusion]N-glycosylation modification at different sites of Bgl3A had different effect on the secretion and enzymatic properties.Among them,S228 is essential for maintaining the expression and function of the enzyme,whereas S299 can increase the enzyme thermostability and catalytic efficiency on cellobiose.