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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 ,推测此肽段对长链脂肪酸醇氧化酶酶活性起重要作用
Objective To analyze the enzyme activity domain of long chain fatty acid alcohol oxidase in yeast CandidaCloacae. Methods Five different length cDNA fragments of long chain fatty acid alcohol oxidase were cloned from yeast Candidacloacae cells by RT-PCR. The NheI restriction enzyme site and the start codon ATG were introduced at the 5 ’end of the upstream primer for amplification of these fragments, and the NheI restriction enzyme site was introduced at the 5’ end of the downstream primer. The amplified product was digested with NheI, cloned into the expression plasmid pET 17b under the control of the T7 promoter, and transformed into E. coli BL21 (DE3) strain. Results The E. coli BL21 (DE3) strain was transformed into E. coli BL21 (DE3), and its five amino acid residues were 5 35 AA (5 0KD), 4 73AA (43KD) and 4 11AA (38KD), 30 6AA (26KD) and 2 16AA (18KD). Compared with the intact long-chain fatty alcohol alcohol oxidase (6 97AA), they were equivalent to 16,224,288,391 and 4,81 amino acids excised from the amino terminus, respectively, and their enzymatic activities decreased by 18.8% , 2.06%, 88.6%, 91.4% and 99.7% respectively. Conclusion The results showed that the enzymatic activity of long-chain fatty acid alcohol oxidase decreased with the increase of the number of amino acids excised, especially when the amino acid sequence of 2 2 4 2 88 was excised. Chain fatty acid alcohol oxidase activity plays an important role