一种嗜酸普鲁兰芽孢杆菌普鲁兰酶基因在大肠杆菌中的诱导表达与活性表征

Inducible expression and enzymic characterization of the pullulanase gene from Bacillus acidopullulyticus in Escherichia coli

从嗜酸普鲁兰芽孢杆菌基因组中扩增出普鲁兰酶基因Pul A,并将该基因连接到大肠杆菌表达载体p ET-28a中,构建了普鲁兰酶基因的诱导表达载体p ET-28a-Pul A。测序结果表明,普鲁兰酶基因Pul A长度为2766 bp,编码922个氨基酸。将重组载体转化至大肠杆菌BL21(DE3)后,普鲁兰酶基因Pul A在IPTG诱导下获得表达,产生胞内蛋白。SDS-PAGE测定的分子量约为110 k D。细胞超声破碎液酶活为0.45 U/m L。该酶的最适温度为55℃,最适p H为5.0,金属离子对酶活性影响不显著,具有I型普鲁兰酶特性。此重组普鲁兰酶的酶学性质表明此酶具有独特的耐酸性质,具备一定的工业化应用价值。

The pullulanase gene from the Bacillus acidopullulyticus genome has been obtained by PCR amplification. By connecting the gene to the inducible expression plasmid of p ET-28 a,a pullulanase gene inducible expression plasmid of p ET-28a-Pul A was constructed. The pullulanase gene was 2766 bp in length and encoded 922 amino acids. The competent cells of E. coli BL21( DE3) were transformed with the recombinant plasmid,and after inducement by IPTG,the pullulanase was able to be expressed and examined from the inside of the cells. The enzyme activity of the recombinant pullulanase obtained from ultrasonic broken cells was 0. 45 U / m L. SDS-PAGE analysis showed that the molecular weight of the protein was approximately 110 k D. The maximum activity of the enzyme occurred at 55 ℃ and p H 5. 0. Metal ions had no significant influence on the enzyme activity. The enzyme belongs to the type I pullulanase class. The recombinant pullulanase showed excellent acid resistance so that it may be useful for applications of pullulanase in industry.