来源于不同表达菌株的β-葡聚糖酶酶学性质比较  

Comparison of enzymatic properties of β-glucanase from different expression strains

作　　者：汪凌旭 易卓林 罗惠波 黄丹 赵海 WANG Lingxu;YI Zhuoling;LUO Huibo;HUANG Dan;ZHAO Hai(Sichuan University of Science&Engneering,Yibin 644000,China;Key Laboratory of Liquor Making Bio-Technology&Application of Sichuan Province,Yibin 644000,China;Key Laboratory of Environmental and Applied Microbiology of Chinese Academy of Sciences,Chengdu 610041,China;Environmental Microbiology Key Laboratory of Sichuan Province,Chengdu 610041,China)

机构地区：[1]四川轻化工大学,宜宾644000 [2]酿酒生物技术及应用四川省重点实验室,宜宾644000 [3]中国科学院环境与应用微生物重点实验室,成都610041 [4]环境微生物四川省重点实验室,成都610041

出　　处：《应用与环境生物学报》2022年第5期1312-1317,共6页Chinese Journal of Applied and Environmental Biology

基　　金：四川省教育厅科技重点研究项目(2019YJ0475);中国轻工业浓香型白酒固态发酵重点实验室开放基金项目(2019zd035)资助。

摘　　要：β-葡聚糖酶是大曲中重要的水解酶系,在大曲发酵过程中起着水解原料细胞壁和释放胞内淀粉的作用.NFEg16A是首个从浓香型大曲中挖掘到的β-葡聚糖纯酶,其表达系统为大肠杆菌表达系统Escherichia coli BL21.NFEg16A耐热性极好,但酶活却很低.为了提升该酶的酶学性质,成功实现了该酶在Pichia pastoris X33中的异源表达,并将来源于不同表达系统的NFEg16A酶学性质进行对比,从糖基化的角度分析产生这种差异的原因.结果表明,来源于毕赤酵母表达系统的β-葡聚糖酶NFAmy16A-Pichia相较于来源于大肠杆菌表达系统的β-葡聚糖酶NFAmy16A-E. coli,比活提升约40%. NFAmy16A-E. coli最适反应条件为pH 7和70℃;在弱酸性的条件下pH稳定性较好;在50℃时热稳定性最好. NFAmy16A-Pichia最适反应条件为pH 5.5和65℃;在中性条件下pH稳定性最好;且温度耐受范围很广,在30-90℃下热稳定性都很好.对NFAmy16A的糖基化位点进行预测,发现该酶有5个糖基化位点,分别为4个O-糖基化位点(164、168、187、240)和1个N-糖基化(295)位点.本研究表明,可能是因为NFAmy16A在毕赤酵母中发生糖基化反应,从而造成了蛋白质结构的变化从而导致其酶学性质发生改变.Beta-glucanase is an important hydrolytic enzyme in Daqu that plays a key role in hydrolyzing the cell wall of raw materials and releasing intracellular starch during the fermentation process of Daqu. NFEg16A is the first purified β-glucanase from Nong-flavor Daqu, and its expression system is Escherichia coli BL21.NFEg16A shows good heat resistance and low enzymatic activity. To improve its enzymatic properties, this study successfully expressed the NFEg16A gene in Pichia pastoris X33 and compared the enzymatic properties of NFEg16A derived from these two expression systems. The reasons for these different properties were further investigated based on glycosylation. The specific activity of NFEg16A-P. pastoris was improved by approximately 40% compared to that of NFEg16A-E. coli. NFEg16A-E. coli showed optimal activity at pH 7and 70 ℃, with good stability under weak acid conditions and the best thermal stability at 50 ℃. Meanwhile,NFEg16A-P. pastoris showed optimal activity at pH 5.5 and 65 ℃, with the best pH stability under neutral conditions and good thermal stability over a wide range, from 40 to 90 ℃. Five glycosylation sites of NFEg16A were predicted: four possible O-glycosylation sites(164, 168, 187, and 240) and one N-glycosylation site(295sites). Therefore, it is reasonable to speculate that NFEg16A may be glycosylated in P. pastoris, which would change its protein structure, resulting in changes in its enzymatic properties.

关 键 词：Β-葡聚糖酶 NFEg16A Escherichia coli BL21 Pichia pastoris X33 酶学性质 糖基化 

分 类 号：TS261.1[轻工技术与工程—发酵工程]