内生解淀粉芽孢杆菌TB2内切β-1，4-葡聚糖酶基因的克隆和原核表达分析

Cloning and Expression of β-1,4-endoglucanase Gene from Endophytic Bacillus amyloliquefaciens TB2

以内生解淀粉芽孢杆菌TB2菌株基因组为模板,克隆了该菌的β-1,4-内切葡聚糖酶基因的ORF。测序结果表明该ORF全长1 500 bp,编码499个氨基酸,分子量为55.07 ku,等电点为7.83。Blast同源性分析结果表明,该序列与GenBank登录的1株枯草芽孢杆菌(Bacillus subtilis M28332)纤维素酶核苷酸序列的同源性最高(98%),其氨基酸同源性也达98%,已被GenBank收录(Accession number)。用BamHⅠ和XhoⅠ双酶切目的片段和表达载体pET-29a(+)后相连接,构建重组表达载体pET-glu,并导入BL21细菌中表达。酶学特性表明:SDS-PAGE电泳在59 ku左右有融合蛋白带,该酶的最适反应温度为50℃,最适反应pH为6.4,为中性纤维素酶。实验结果为进一步研究内生解淀粉芽孢杆菌TB2纤维素酶的功能和应用打下了基础。

A β-1,4-endoglucanase gene was cloned from endophytic bacteria strain TB2 and expressed in Escherichia coll. The whole length of the gene is 1 500 bp, which encodes 499 amino acids. The molecular weight of the enzyme is 55.07 ku and the pI is 7.83. The nucleotide and amino acid sequences of the gene share high homology with those of Bacillus subtilis cellulase （M28332） accessed in GenBank, and both similarities are 98 %. The gene has been registered in GenBank （Accession number is EU022559）. The β-1,4-endoglucanase gene was ligated to carrier pET-29a （＋） using BamHIand XhoI to construct expression plasmid pET-glu which was expressed in E.coli BL 21. The SDS-PAGE showed that a fusion protein of about 59 ku was expressed in E.coli BL 21. The optimum temperature and pH for reaction of this fusion enzyme were found at 50 ℃ and 6.4 respectively, indicating that the enzyme belonged to neutral enzyme. This result might contribute to further study on endophytic bacteria cellulase from B, amyloliquefaciens TB2.