硫矿硫化叶菌MTSase和MTHase基因的克隆与表达

Cloning and Expression of MTSase and MTHase from Sulfolobus solfataricus in E.coli

从嗜热硫矿硫化叶菌(Sulfolobussolfataricus)ATCC35092的基因组中用PCR方法扩增得到编码MTSase和MTSase的基因,分别将其插入原核表达载体pTrc99a中,并转入大肠杆菌BL21(DE3),进行诱导表达。MTSase和MTHase酶活产率达到了31·3U/g(wetcell)和403U/g(wetcell)。在75℃,pH5·0条件下,两酶联合作用转化淀粉生产海藻糖,当淀粉浓度为15%,DE值为10时,海藻糖转化率最高为53·6%。

The genes of maltooligosyl trehalose synthase （MTSase） and maltooligosyl trehalose tetrahydmlase （MTHase） from Sulfolobus solfataricus ATCC 35092 were amplified using PCR. The expression plasmids, pTrc99a-MTSase and pTrc99a-MTHase, were constructed by inserting these two DNA fragments into E. coli expression vector pTrc99a. The specific activity of MTSase and MTHase in E. coli BL21 （ DE3 ） at optimal fermentation conditions reached 31.3U/g （wet cell） and 403U/g （wet cell）, respectively. The biotransformation of partially hydmlyzed starch to trehalose catalyzed by MTSase and MTHase was carried out at 75℃ and pH 5.0. The highest yield of trehalose （ca. 53.6% ） was gained when the original starch concentration was 15% （w/v） and the DE value was 10.