CBM融合位置对AuMan5A酶学性质的影响

doi:10.3969/j.issn.1673-1689.2019.04.001

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CBM融合位置对AuMan5A酶学性质的影响
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                        10.3969/j.issn.1673-1689.2019.04.001
                    
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Effect of Fusion Position of Carbohydrate-Binding Module on the Enzymatic Properties of AuMan5A

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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。

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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.

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袁风娇,王春娟,李雪晴,李剑芳,邬敏辰. CBM融合位置对AuMan5A酶学性质的影响[J].食品与生物技术学报,2019,38(4):1-7.

YUAN Fengjiao, WANG Chunjuan, LI Xueqing, LI Jianfang, WU Minchen. Effect of Fusion Position of Carbohydrate-Binding Module on the Enzymatic Properties of AuMan5A[J]. Journal of Food Science and Biotechnology,2019,38(4):1-7.

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在线发布日期: 2019-07-03
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