地芽孢杆菌Y565-5分离鉴定及其木糖异构酶基因xylA的克隆表达和酶学性质

Cloning, expression and characterization of xylose isomerase, XylA from Geobacillus sp. Y565-5

摘要:

从甘肃玉门油田地表土中分离到一株嗜热木糖利用菌, 地芽孢杆菌Y565-5。利用PCR方法从该菌株中克隆得到一个木糖异构酶基因, xylA。该基因开放阅读框长1 182 bp, 编码394个氨基酸, XylA氨基酸序列与Geobacillus sp. Y412MC52相似性达到99%。将xylA基因克隆到原核表达载体pET-28a(+)上, 得到重组质粒pET-28a(+)-xylA, 然后将此重组质粒转化至BL21(DE3)中, 经IPTG诱导后, 通过SDS-PAGE电泳检测出明显的45 kD (相对分子质量)特异性蛋白质条带, 并且通过半胱氨酸咔唑法检测出表达产物具有木糖异构酶的活性。对其酶学性质的研究发现, XylA最适温度为90 °C, 最适pH值为8.0。

Abstract:

Xylose-utilizing and thermophilic Geobacillus sp. Y565-5 was isolated from surface soil of an oilfield in Yumen Town, Gansu Province, China. A xylose isomerase (XylA) gene was cloned from the strain by PCR. The open reading frame of xylA (1 182 bp) encoded a protein of 394 amino acids, which showed high sequence homology (99% identity) with that of Geobacillus sp. Y412MC52. The intact coding region was subcloned into pET28a(+) vector and expressed in Escherichia coli BL21(DE3). The molecular weight of the recombinant protein was 45 kD based on SDS-PAGE and its xylose isomerase activity was detected through cysteine welts thiazole method after the induction of isopropyl β-D-1-thiogalactopyranoside? (IPTG). The optimum temperature and pH for the partially purified recombinant XylA activity were 90 °C and pH 8.0, respectively.