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

Abstract:

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.