亲和层析法纯化重组大肠杆菌表达的亚油酸异构酶  

Purification of Linoleic Acid Isomerase from Recombinant E.coli by Affinity Chromatography

作　　者：洛雪 李越 张爽[2] 岳喜庆[1] 张兰威[3] Luo Xue;Li Yue;Zhang Shuang;Yue Xiqing;Zhang Lanwei(College of Food Science, Shenyang Agricultural University, Shenyang 110866;College of Food Science and Engineering, Ocean University of China, Qingdao 266100, Shandong;College of Food Science, Northeast Agricultural University, Harbin 150030)

机构地区：[1]沈阳农业大学食品学院,沈阳110866 [2]东北农业大学食品学院,哈尔滨150030 [3]中国海洋大学食品科学与工程学院,山东青岛266100

出　　处：《中国食品学报》2018年第1期88-94,共7页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家自然科学基金-青年基金项目(31701623,31601493)

摘　　要：亚油酸异构酶能够催化亚油酸(LA)生成不同类型共轭亚油酸(CLA),共轭亚油酸是一类由不同位置和几何异构体组成的有两个共轭双键的十八碳二烯酸,具有多种生理活性,且广泛存在于多种食物中。目前报道的丙酸杆菌虽能够生产t10,c12-CLA,但含量较低。为提高CLA的产量,以痤疮丙酸杆菌的亚油酸异构酶基因为研究对象,利用PCR扩增亚油酸异构酶基因到大肠杆菌中表达,采用亲和层析的方法分离纯化重组大肠杆菌发酵液中的亚油酸异构酶。优化的亲和层析条件:上样量10 m L,流速0.25 m L/min。酶被纯化了13.22倍,比活力达12.30 U/(mg蛋白),回收率为94.84%。经SDS-PAGE检测,该蛋白分子质量为55 ku。采用气相色谱检测该蛋白催化LA生成t10,c12-CLA,酶活为(34.775±0.07)U/m L,该酶为亚油酸异构酶。Linoleic acid isomerase（LAI） can catalyze linoleic acid（LA） to generate different types of conjugated linoleic acid（CLA）. CLA is a kind of octadecadienoic acid with different position and geometric isomers contained two conjugated double bonds. It widely exists in a variety of food sand has many biological activities. However, different types of CLA present different physiological activity. Propioni bacterium can catalyze LA to t10, c12-CLA, but the content of CLA was very low. In this study, lai of Propioni bacterium acnes will be investigated and introduced into E. coli for expression. To purify the linoleic acid isomerase from recombinant, affinity chromatography method was employed.Affinity chromatographic conditions（flow rate and the sample volume） were structured and optimized. The optimized sample volume and flow rate were 10 m L and 0.25 m L/min, respectively. Enzyme was purifiedby 13.22-fold the specific activity and there cover rate were 12.30 U/mg protein and 94.84%. The protein had a relative mass of about 55 ku analyzed by SDS-PAGE. The result of GC showed that the protein catalytic LA to t10, c12-CLA with enzyme activity of（34.775±0.07） U/m L, so this protein was linoleic acid isomerase.

关 键 词：亲和层析 亚油酸异构酶 气相色谱 生物转化 

分 类 号：TS201.3[轻工技术与工程—食品科学]