摘要
阿拉伯糖苷酶 /木糖苷酶是木聚糖类半纤维素生物降解和转化所必需的酶类。本文在国内首次报道对该酶的研究 :通过PCR从产乙醇热厌氧杆菌ThermoanaerobacterethanolicusJW2 0 0克隆出编码高度热稳定性阿拉伯糖苷酶 /木糖苷酶的基因 ,与组氨酸标签融合 ,以高拷贝质粒pAlter Ex1在大肠杆菌中得到高效表达 ;基因表达产物通过热处理和亲和层析柱纯化后 ,酶纯度达电泳均一。纯化重组酶稳定性检测表明 ,得到的阿拉伯呋喃糖苷酶在pH4 .2～ 8.2之间酶活力稳定 ,75℃的半衰期为 1h ;β 木糖苷酶在pH 5 .0～ 8.2之间有较高的稳定性 ,酶 1h半衰期温度为 84℃。
Thermoanaerobacter ethanolicus JW200 grows between 37～78℃, and isolated from analkaline hot spring in Yellowstone Park. The gene of arabinofuranosidase/xylosidase was obtained from a genome DNA of T. ethanolicus, cloned and expressed in E. coli JM109(DE3). Two primer were designed on the basis of nucleotide sequence of xar gene. The xar gene fragments were successfully amplified by PCR reaction under the suitable condictions. E. coli JM109(DE3) competent cells were transformed by electroporation with the ligated xar-pAlter-Ex1 withPstⅠ-XhoⅠsite. The translated protein was soluble α-arabinofuranosidase and β-xylosidase activities, and the C-terminal 6-His tag was introduced in the recombinase. The recombinant protein was purified by the heat treatment and immobilized metal affinity chromatography. The purified enzyme presented as a single protein band on SDS-PAGE with a molecular weight of 85 kD. The optimum activity of arabinofuranosidase was found be at pH 6.0 and 80℃,at the pH range of 4.2～8.2, the arabinofuranosidase was stable and had 1h half-life at 75℃. At the pH range of 5.0～8.2, the β-xylosidase was stable with an half-life of 1h at 84℃.
出处
《中国农业大学学报》
CAS
CSCD
北大核心
2003年第5期9-13,共5页
Journal of China Agricultural University
