融合β-甘露聚糖酶基因的构建、表达及酶学性质的分析  被引量:2

Expression and Enzymatic Properties Analysis of a Fusion β-Mannanase

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作  者:王春娟[1] 李剑芳[1] 唐诗涵[1] 董运海[1] 邬敏辰[2] 

机构地区:[1]江南大学食品学院,江苏无锡214122 [2]江南大学无锡医学院,江苏无锡214122

出  处:《食品与生物技术学报》2016年第11期1135-1141,共7页Journal of Food Science and Biotechnology

基  金:国家自然科学基金项目(31271811)

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。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 (A uman5A -cbm27) constructed by the overlapping PCR was expressed in Pichia pastoris GSll5. 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.

关 键 词:Β-甘露聚糖酶 催化域 碳水化合物结合域 热稳定性 底物亲和力 

分 类 号:Q55[生物学—生物化学] Q78

 

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