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首页> 外文期刊>Molecular biology reports >Comparison of enzymatic activity of two linoleic acid isomerases expressed in E-coli
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Comparison of enzymatic activity of two linoleic acid isomerases expressed in E-coli

机译：大肠杆菌中表达的两种亚油酸异构酶的酶活性比较

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Conjugated linoleic acid (CLA) refers to a group of positional and geometric isomers of octadecadienoic fatty acid with conjugated double bonds. CLA possesses many important physiological functions and it can be produced from linoleic acid (LA) by LA isomerases. In this report, we first cloned the genes encoding LA isomerases: C12 isomerases and C9 isomerase, then transformed the recombinant plasmids into Escherichia coli TOP10 and induced E. coli with IPTG (isopropylthio-beta-d-galactoside) to express the recombinant proteins. Next, we purified the isomerases using a HisTrap (TM) HP column, followed with the analysis by SDS-PAGE or Western blot. Finally, we compared their enzymatic activity by biotransformation of LA into CLA. Plasmids containing LA isomerase genes were successfully constructed. LA isomerases were found expressed in E. coli, and the molecular weight was 64 KD for C12 LA isomerase and 55 KD for C9 LA isomerase. The enzyme activity (9.93 +/- A 0.01 U/ml for C12 LA isomerase and 8.12 +/- A 0.02 U/ml for C9 LA isomerase) of both LA isomerases reached the highest when IPTG concentration is 0.2 mM and the induction time is 18 h. After purification, C9 LA isomerase was enriched in peak 4 and C12 LA isomerase was enriched in peak 3. Optimum pH for C9 LA and C12 LA isomerases were 7.5 and 7.0 separately, and optimum temperatures was 37 A degrees C for highest concentration of CLA. The work may provide theoretical significance for an effective production process of CLA for the medical and nutritional purposes.
机译：共轭亚油酸（CLA）是指带有共轭双键的十八碳二烯酸的位置和几何异构体。 CLA具有许多重要的生理功能，可以通过亚油酸异构酶由亚油酸（LA）产生。在本报告中，我们首先克隆了编码LA异构酶的基因：C12异构酶和C9异构酶，然后将重组质粒转化到大肠杆菌TOP10中，并用IPTG（异丙硫基-β-d-半乳糖苷）诱导大肠杆菌表达重组蛋白。接下来，我们使用HisTrap（TM）HP柱纯化异构酶，然后通过SDS-PAGE或Western blot分析。最后，我们通过将LA转化为CLA来比较它们的酶促活性。成功构建了含有LA异构酶基因的质粒。发现LA异构酶在大肠杆菌中表达，并且对于C12LA异构酶，分子量为64KD，对于C9LA异构酶，分子量为55KD。当IPTG浓度为0.2 mM且诱导时间为30min时，两种LA异构酶的酶活性（C12 LA异构酶为9.93 +/- A 0.01 U / ml，C9 LA异构酶为8.12 +/- A 0.02 U / ml）最高。 18小时纯化后，C9 LA异构酶富集在峰4中，C12 LA异构酶富集在峰3中。对于C9 LA和C12 LA异构酶，最适pH分别为7.5和7.0，对于CLA最高浓度，最适温度为37°C。这项工作可能为医学和营养目的有效生产CLA提供理论依据。

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