Expression and Enzymatic Properties Analysis of a Fusion β-Mannanase

Abstract:

AuMan5A，which belongs to the glycoside hydrolase family 5 β-mannanase from Aspergillus usamii YL-01-78，only contains a catalytic module （CM）. To improve its enzymatic properties，a fusion β-mannanase （AuMan5A-CBM27） was well designed by fusing a family 27carbohydrate-binding module （CBM27） from Thermotoga maritima MSB8 into the C-terminus of AuMan5A. A fusion gene （Auman5A-cbm27） constructed by the overlapping PCR was expressed in Pichia pastoris GS115. The enzymatic properties of the purified reAuMan5A-CBM27 and reAuMan5A were investigated. The optimal temperatures of both reAuMan5A-CBM27 and reAuMan5A were determined at 68 ℃. They were respectively thermo-stabled at 68 ℃ or 60 ℃ and below. A wider pH tolerant range was observed for reAuMan5A-CBM27，whose Km value to locust bean dropped from 1.7 mg/mL to 0.7 mg/mL. The increase of substrate affinity of AuMan5A was confirmed.