Candida Cloacae长链脂肪酸醇氧化酶的结构域谱分析(英文)

Mapping domains of long-chain fatty acid alcohol oxidase of candida cloacae

目的 分析酵母CandidaCloacae中长链脂肪酸醇氧化酶的酶活性结构域。方法 利用RT PCR技术 ,从酵母Candidacloacae细胞中克隆长链脂肪酸醇氧化酶的 5个不同长度的cDNA片段。扩增这些片段的上游引物5’端引入了NheI限制性内切酶位点和起始密码ATG ,下游引物 5’端引入了NheI限制性内切酶位点。扩增产物经NheI消化后 ,被克隆进T7启动子控制下的表达质粒pET 17b中 ,转化大肠杆菌BL2 1(DE3)菌株。结果 转化大肠杆菌BL2 1(DE3)菌株表达出 5个不同长度的长链脂肪酸醇氧化酶的羧基端肽段 ,其氨基酸残基数分别为5 35AA(5 0KD)、4 73AA(43KD)、4 11AA(38KD)、30 6AA(2 6KD)和 2 16AA(18KD)。与完整的长链脂肪酸醇氧化酶(6 97AA)相比较 ,相当于它们分别从氨基端被切除 16 2、2 2 4、2 88、391和 4 81个氨基酸 ,其酶活性分别下降了 18.8%、2 0 .6 %、88.6 %、91.4 %和 99.7%。结论 结果表明随着长链脂肪酸醇氧化酶的氨基端被切除的氨基酸数增加 ,其酶活性下降 ,尤其是切除 2 2 4 2 88氨基酸序时 ,酶活性下降更明显 。

Objective to investigate domains function of long chain fatty acid alcohol oxidase at the DNA level. Methods Five fragments of Lc FAO gene of Candida cloacae were prepared with reverse transcription polymerase chain reaction, whose 5' ends of sense primers contained Nhe I restriction site and initiator ATG; the 5' ends of antisense primers contained Nhe I restriction site. These fragments were cloned into pET17b plasmid under T7 promoter and then transformed into E.coli BL21(DE3) cells. Results The BL21(DE3) cells yielded carboxyl terminal domains of Lc FAO, which were respectively 535AA (50KDa),473AA(43KDa), 411AA (38KDa), 306AA(26KDa), 216AA(18KDa), corresponding to the 162, 224, 288, 391, 481AA deletions from amino terminus. As compared with intact Lc FAO (697AA, 64Kda), the specific activities of Lc FAO(50Kda),Lc FAO(43Kda), Lc FAO(38Kda), Lc FAO(26Kda) and Lc FAO(18Kda) were as much as 99.7%, 91.4%, 88.6%, 20.6% and 18.8% specific activity of intact Lc FAO, respectively. Above carboxyl terminal half of Lc FAO(38KDa) possesses the catalytic activity. Further deletion into the amino terminal domain renders the polypeptide deactive. Especially, when the 224～288AA sequence of amino terminus was deleted, the descent of specific activity was more remarkable. Conclusions The results indicated that the 224～288AA sequence of amino terminus of Lc FAO may play an important role in the specific activities of enzyme. With this strategy, dissection and expression of a proteins function domain is possible at the DNA level.