摘要
为研究近暗散白蚁(Reticulitermes perilucifugus)β-葡萄糖苷酶RpBgl7,利用生物信息学分析β-葡萄糖苷酶RpBgl7的家族及结构特点,构建RpBgl7的重组表达载体pCold-TF-RpBgl7,在大肠杆菌中过量表达并纯化,分析其最适反应条件和酶动力学参数。结果表明,RpBgl7属于糖苷水解酶1家族,具有2个保守的谷氨酸催化残基Glu187和Glu394。RpBgl7在BL21(DE3)中过量表达,经过镍离子亲和层析1步纯化可得到高纯度RpBgl7重组蛋白。以4-硝基苯基-β-D-吡喃葡萄糖苷为底物,测得RpBgl7的最适pH值为6.0,最适温度为70℃;动力学参数Km=0.13 mmol/L,kcat=3.15 s^-1。RpBgl7可耐受Na^+、K^+、Mg^2+、Cu^2+和Ba^2+,而Ca^2+、Hg^2+和Fe^3+明显抑制RpBgl7的酶活力。
To characterize theβ-glucosidase RpBgl7 from Reticulitermes perilucifugus,it was analyzed by bioinformatics methods to reveal its belonged family and structure.The recombinant vector pCold-TF-RpBgl7 was transferred into E.coli and overexpressed.RpBgl7 was purified to study its optimal conditions and kinetics.The results showed that,RpBgl7 belonged to glycoside hydrolase family 1,and contained 2 conserved catalytic residues Glu187 and Glu394.RpBgl7 overexpressed in BL21(DE3)was purified in one step using nickel chelating affinity chromatography with high purity.The optimal pH value of RpBgl7 was 6.0 and the optimum temperature was 70℃.The Km and kcat of RpBgl7 with p-nitrophenyl-β-D-glucopyranoside as substrate were 0.13 mmol/L and 3.15 s^-1,respectively.RpBgl7 could be tolerant to Na^+,K^+,Mg^2+,Cu^2+and Ba^2+,but it was strongly inhibited by Ca^2+,Hg^2+and Fe^3+.
作者
毛国涛
刘茜
王方园
苏丽娟
张宏森
宋安东
MAO Guotao;LIU Qian;WANG Fangyuan;SU Lijuan;ZHANG Hongsen;SONG Andong(College of Life Sciences,Henan Agricultural University,Zhengzhou 450002,China)
出处
《河南农业科学》
北大核心
2020年第7期68-74,共7页
Journal of Henan Agricultural Sciences
基金
国家自然科学基金项目(31800050)。
关键词
近暗散白蚁
Β-葡萄糖苷酶
大肠杆菌宿主
融合表达
纯化
酶学性质
金属离子耐受性
Reticulitermes perilucifugus
β-glucosidase
E.coli host
Fusion expression
Purification
Enzymatic characteristics
Metal ion tolerance
