融合β-甘露聚糖酶基因的构建、表达及酶学性质的分析

宇佐美曲霉 （Aspergillus usamii） YL-01-78的糖苷水解酶5家族β-甘露聚糖酶 （AuMan5A） 是一种仅含催化域 （CM） 的单结构蛋白。为改善其酶学性质，基于理性设计将海栖热胞菌 （Thermotoga maritima） MSB8的27家族碳水化合物结合域 （CBM27）融合至AuMan5A的C-端，并采用重叠PCR技术构建融合酶基因Auman5A-cbm27。分别将Auman5A-cbm27和Auman5A在毕赤酵母GS115中进行表达，对重组表达产物reAuMan5A-CBM27和reAuMan5A进行纯化和酶学性质测定。结果表明，reAuMan5A-CBM27和reAuMan5A的最适温度均为68 ℃；两者分别在68和60 ℃及以下稳定。同时，前者较后者具有更广泛的pH稳定范围。reAuMan5A-CBM27对角豆胶的Km值由融合前的1.7 mg/mL降至0.7 mg/mL，表明AuMan5A的底物亲和力得到了提高。
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