【摘要】 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(Au Man5A)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Au Man5A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re Au Man5A-CBM27和re Au Man5A进行纯化和酶学性质测定。结果表明,re Au Man5A-CBM27和re Au Man5A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p H稳定范围。re Au Man5ACBM27对角豆胶的Km值由融合前的1.7 mg/m L降至0.7 mg/m L,表明Au Man5A的底物亲和力得到了提高。更多还原

【Abstract】 Au Man5 A,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(Au Man5A-CBM27) was well designed by fusing a family27carbohydrate-binding module(CBM27) from Thermotoga maritima MSB8 into the C-terminus of Au Man5 A. A fusion gene(Auman5A-cbm27) constructed by the overlapping PCR was expressed in Pichia pastoris GS115. The enzymatic properties of the purified re Au Man5A-CBM27 and re Au Man5 A were investigated. The optimal temperatures of both re Au Man5A-CBM27 and re Au Man5 A were determined at 68 ℃. They were respectively thermo-stabled at 68 ℃ or 60 ℃ and below. A wider p H tolerant range was observed for re Au Man5A-CBM27,whose Kmvalue to locust bean dropped from 1.7 mg/m L to 0.7 mg/m L. The increase of substrate affinity of Au Man5 A was confirmed.更多还原