CBM融合位置对AuMan5A酶学性质的影响

Effect of Fusion Position of Carbohydrate-Binding Module on the Enzymatic Properties of AuMan5A

DOI:10.3969/j.issn.1673-1689.2019.04.001

中文关键词: β-甘露聚糖酶 碳水化合物结合域 融合位置 酶学性质

英文关键词: β-mannanase,carbohydrate-binding module,fusion position,enzymatic properties

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作者

单位

袁风娇

江南大学 食品学院江苏 无锡 214122

王春娟

江南大学 食品学院江苏 无锡 214122

李雪晴

江南大学 食品学院江苏 无锡 214122

李剑芳

江南大学 食品学院江苏 无锡 214122

邬敏辰

江南大学 无锡医学院江苏 无锡 214122

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中文摘要:

为探讨碳水化合物结合结构域(CBM) 不同融合位置对Aspergillus usamii5家族β-甘露聚糖酶AuMan5A酶学性质的影响,将Thermotoga maritima27家族CBM分别融合至其C和N末端,构建出融合酶基因Auman5A-cbm27cbm27-Auman5A。将Auman5A和融合酶基因分别在Pichia pastorisGS115中表达,分析比较表达产物AuMan5A、AuMan5A-CBM27和CBM27-AuMan5A的酶学性质。结果表明:AuMan5A、AuMan5A-CBM27和CBM27-AuMan5A的最适温度Topt分别为70、70 ℃和60 ℃;其中AuMan5A-CBM27在68 ℃及以下保温1 h残余酶活均大于85%,与原酶相比,其热稳定性提高了约8 ℃,而CBM27-AuMan5A在58 ℃及以下保持稳定,与原酶相比,降低了2 ℃。3种酶的最适pH均为4.0;AuMan5A 在pH值2.5~7.5范围内保持稳定,2种融合酶在pH 2.0~9.0内均能保持稳定。与AuMan5A相比,AuMan5A-CBM27和CBM27-AuMan5A对角豆胶的Km分别降低了45.0%和26.3%。通过比较3种酶的酶学性质,证实CBM不同融合位置对AuMan5A酶学性质具有重要影响。

英文摘要:

AuMan5A,a glycoside hydrolase family 5 β-mannanase ofAspergillus usamii,only consists of a catalytic domain. To explore the effect of fusion position of carbohydrate-binding module on the enzymatic properties of AuMan5A,two fusion genes,Auman5A-cbm27andcbm27-Auman5A,were constructed as designed theoretically by separately linking family 27 CBM ofThermotoga maritimeβ-mannanase into C- and N-termini of AuMan5A. After Auman5A and two fusion genes were expressed in Pichia pastoris GS115,enzymatic properties of the expressed products,AuMan5A,AuMan5A-CBM27 and CBM27-AuMan5A,were analyzed and compared. The results showed that the temperature optima(Topt) of three β-mannanases were 70,70 and 60 ℃,respectively. AuMan5A-CBM27 was thermostable at 68 ℃, while AuMan5A and CBM27-AuMan5A were at 60 and 58 ℃. Besides,three β-mannanases displayed the same pH optimum of 4.0. AuMan5A was stable at a pH range of 2.5~7.5,while AuMan5A-CBM27 or CBM27-AuMan5A was at pH 2.0~9.0. Compared with that of AuMan5A,Km values of AuMan5A-CBM27 and CBM27-AuMan5A towards locust bean gum decreased by 45.0% and 26.3% respectively. It was demonstrated in this work that the fusion of CBM27 and its position into AuMan5A played significant roles in its enzymatic properties.

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