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

作　　者：张洁[1,2] 黄志勇[2] 王钦宏[2] 王永莉[3] 王硕[1] 

机构地区：[1]天津科技大学食品营养与安全重点实验室,天津300457 [2]中国科学院天津工业生物技术研究所,天津300108 [3]中国科学院兰州地质所,甘肃兰州730000

出　　处：《微生物学通报》2011年第8期1147-1154,共8页Microbiology China

基　　金：纤维素乙醇高温发酵与生物炼制子课题(No.KSCX1-YW-11E);中国科学院知识创新工程重要方向项目(KZCX2-YW-Q05-05)

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1182 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。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 ofxylA （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 BL2 I（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-l-thiogalactopyranosid.e （1PTG）. The optimum temperature and pH for the partially purified recombinant XylA activity were 90 ℃ and pH 8.0, respectively.

关 键 词：GEOBACILLUS sp.Y565.5 木糖异构酶 克隆 表达 

分 类 号：Q78[生物学—分子生物学]