凝结芽孢杆菌中耐热木酮糖激酶基因的克隆表达及生信分析  

Cloning, expression and bioinformatics analysis of thermo-stable xylulose kinase gene from Bacillus coagulans

作　　者：聂恬 孟未群 李梦然 张玉明[1] 倪志华[1] NIE Tian;MENG Weiqun;LI Mengran;ZHANG Yuming;NI Zhihua(College of Life Science,Hebei University,Baoding 071002,China)

机构地区：[1]河北大学生命科学学院

出　　处：《中国酿造》2019年第8期142-147,共6页China Brewing

基　　金：河北省教育厅资助科研项目（QN2017013）;河北省自然科学基金项目（C2019201188）;河北大学雄安新区生态服务类本科实践教学改革课题（201801）

摘　　要：该研究以凝结芽孢杆菌(Bacillus coagulans)IPE22为研究对象,通过聚合酶链式反应(PCR)扩增获得木酮糖激酶基因Bc-XK,将其与载体pET-30a连接后,在大肠杆菌(Escherichia coli)BL21(DE3)中进行诱导表达。然后采用镍柱亲和层析纯化重组酶Bc-XK,对基因Bc-XK及其编码的蛋白质进行生信分析。结果表明,纯化后重组酶Bc-XK的酶比活力为(20.56±3.31)U/mg,热稳定性好,在60℃保持活力180 min以上,具有很好的工业应用潜力。Bc-XK基因含有一个1 536 bp的开放阅读框,共编码511个氨基酸,其编码的蛋白质为亲水蛋白,等电点(PI)为5.46,分子质量为56.15 kDa,二级结构中α-螺旋、无规卷曲和延伸链含量丰富,3个催化位点分别为天冬氨酸-8、苏氨酸-11、天冬氨酸-239,高度保守。Using Bacillus coagulans IPE22 as research object,the xylulose kinase gene Bc-XK was obtained from B.coagulans IPE22 by polymerase chain reaction(PCR),which was ligated to the vector pET-30a and induced to express in Escherichia coli BL21(DE3).Then,the recombinase Bc-XK was purified by Ni-chelating affinity chromatography,and the Bc-XK gene and its coding protein were analyzed by bioinformatics method.The results showed that the specific activity of recombinase Bc-XK was(20.56±3.31)U/mg.The recombinase Bc-XK had good thermal stability and could maintain activity at 60℃for more than 180 min,which had good industrial application potential.The Bc-XK gene contained a 1 536 bp open reading frame(ORF),encoding a protein of 511 amino acids.The protein was hydrophilic protein,with isoionic point(pI)5.46,molecular mass 56.15 kDa,abundant alpha-helix,random coil and extended strand in the protein secondary structure,and 3 highly conserved catalytic sites,which were aspartic acid-8,threonine-11 and aspartic acid-239,respectively.

关 键 词：木酮糖激酶 基因 凝结芽孢杆菌 克隆 表达 纯化 生物信息学分析 

分 类 号：TS245.8[轻工技术与工程—制糖工程]