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

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。

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

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.