摘要
研究Pseudothermotoga thermarum木聚糖酶Pthxyn的高效表达及在啤酒酿造中的应用。通过构建、扩增重组质粒pET-20b(+)-pthxyn并在大肠杆菌中进行表达和纯化,重组木聚糖酶分子质量为130 kDa,最适pH值与最适反应温度分别为5.5和95℃。重组木聚糖酶在pH 5.0~8.0的情况下保温2 h仍能保持80%的酶活力,在75℃保温1 h仍保持83%活力。在啤酒酿造的糖化阶段,于65℃时将重组木聚糖酶分别加入以大麦、小麦为原料的反应体系中,采用高效液相色谱检测木糖与葡萄糖的产物。以不加重组木聚糖酶为对照,结果表明,以大麦作为反应体系原料时,木糖质量浓度由0.017 g/L升至1.25 g/L,葡萄糖质量浓度由20 g/L增至36.57 g/L;以小麦作为反应体系原料时,木糖质量浓度由0.003 g/L上升到0.708 g/L,葡萄糖质量浓度由21.9 g/L增加到36.63 g/L。结论:本研究获得的重组木聚糖酶具有pH值耐受性广和良好的热稳定性等特点,同时在啤酒糖化阶段与内源性酶产生协同效应使其具备较高的催化效率,因此在啤酒酿造中具有很好的应用前景。
This study aimed to investigate the efficient expression of the xylanase gene Pthxyn of Pseudothermotoga thermarum and its application in beer brewing.The recombinant plasmid pET-20b-pthxyn was constructed and expressed in Escherichia coli.Pthxyn was purified.The molecular mass of the recombinant xylanase was 130 kDa,and the optimum reaction pH and temperature were 5.5 and 95℃,respectively.It retained 80% and 83% of its activity after incubation at pH 5.0-8.0 for 2 h,and at 75℃ for 1 h,respectively.In the saccharification stage of beer brewing,the recombinant xylanase was added to the reaction system consisting of barley or wheat as raw material at 65℃,and the products xylose and glucose were detected by high performance liquid chromatography(HPLC).The results showed that when barley was used as raw material,the xylose content increased from 0.017 to 1.25 g/L,and the glucose content increased from 20 to 36.57 g/L.When wheat was used as the raw material,xylose increased from 0.003 g/L to 0.708 g/L,and the glucose content increased from 21.9 g/L to 36.63 g/L.In summary,the recombinant xylanase is tolerant to a wide range of pH and stable to heat treatment,and can synergize with endogenous enzymes to improve its catalytic efficiency in the saccharification stage of beer brewing,making it have good prospects in beer brewing.
作者
李青飞
刘越
言行
聂新玲
李相前
谭中标
时号
LI Qingfei;LIU Yue;YAN Xing;NIE Xinling;LI Xiangqian;TAN Zhongbiao;SHI Hao(School of Life Science and Food Engineering,Huaiyin Institute of Technology,Huai’an 223001,China)
出处
《食品科学》
EI
CAS
CSCD
北大核心
2023年第10期181-187,共7页
Food Science
基金
江苏省自然科学基金项目(BK20171266)
江苏省生物质转化与过程集成工程实验室开放基金项目(JPELBCPI2018005)。
关键词
木聚糖酶
高效表达
热稳定性
啤酒酿造
xylanase
efficient expression
thermal stability
beer brewing
