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

作　　者：袁风娇[1] 王春娟[1] 李雪晴[1] 李剑芳[1] 邬敏辰 YUAN Fengjiao;WANG Chunjuan;LI Xueqing;LI Jianfang;WU Minchen(School of Food Science and Technology,Jiangnan University,Wuxi 214122 ,China;Wuxi Medical School,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学食品学院,江苏无锡214122 [2]江南大学无锡医学院,江苏无锡214122

出　　处：《食品与生物技术学报》2019年第4期1-7,共7页Journal of Food Science and Biotechnology

基　　金：国家自然科学基金项目(31271811);江苏省普通高校研究生科研创新计划项目(SJLX16-0472)

摘　　要：为探讨碳水化合物结合结构域(CBM)不同融合位置对Aspergillus usamii 5家族β-甘露聚糖酶AuMan5A酶学性质的影响,将Thermotoga maritima 27家族CBM分别融合至其C和N末端,构建出融合酶基因Auman5A-cbm27和cbm27-Auman5A。将Auman5A和融合酶基因分别在Pichia pastoris GS115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。AuMan5A,a glycoside hydrolase family 5 β-mannanase of Aspergillus 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-cbm27 and cbm27-Auman5A,were constructed as designed theoretically by separately linking family 27 CBM of Thermotoga maritime β-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.

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 

分 类 号：TS201.25[轻工技术与工程—食品科学]