淀粉液化芽孢杆菌β-1,3-1,4葡聚糖酶基因的克隆和表达

Cloning and Expression of β-1,3-1,4-glucanase Gene from Bacillus amyloliquefaciens

提取淀粉液化芽孢杆菌(Bacillusamyloliquefaciens)基因组,通过PCR克隆了该菌的β-1,3-1,4葡聚糖酶基因全长。结果显示,该基因全长843bp,ORF为717bp,编码239个氨基酸,计算相对分子质量为26800,等电点为6.07。经Blast分析,该序列与基因库中的淀粉液化芽孢杆菌(M15674)同源性最高(97%)。该基因已被GenBank接受(AY205562.1)。用BamH和Xho双酶切目的片段和表达载体pET-30a(+)后相连接,构建了重组表达载体pET-amy,并导入BL21细菌中表达,酶学特性表明SDS-PAGE电泳在26000左右有表达蛋白带,该工程菌总酶活达90.74U/mL,是出发菌的3倍,最适温度在60℃左右,pH值在4～10之间。该工程菌可作为酶活高的理想材料,用以构建耐热性好和酶活高的杂合基因工程菌。

A gene encoding β-glucanase from Bacillus amyloliquefaciens has been cloned and expressed in Escherichia coli.The whole length of the gene is 843 bp,including an ORF of 717 bp,which encodes 239 amino acids.The enzyme shows a molecular size of ~26 800 and pI of 6.07.The nucleotide sequence of the gene share high degree of sequence similarity to that of B.amyloliquefaciens and B.subtilis accessed in GenBank,and their similarities are 97% and 94% respectively.The gene has been registered in GenBank(Accession number is AY205562.1).The gene from recombinant cloning plasmid was subcloned into BamHⅠ and XhoⅠ site of expression plasmid pET-amy in E.coli.The recombinant expression plasmid was transformed into E.coli strain BL21.The result of SDS-PAGE showed that about ~26 000 protein of the β-1,3-1,4-glucanase was expressed in E.coli strain BL21.The enzyme activity of E.coli expressed strain was 90.74 U/mL,which was about 3-fold higher than that of original strain.The pH and temperature at which the highest activity was found were 5 and 60℃ respectively.It′s good material for gene engineeting to construct highly active and thermophiamy enzymatic gene.