含油酸培养基培养对重组解脂耶氏酵母全细胞催化合成反10,顺12-共轭亚油酸的影响  

Influence of medium containing oleic acid on synthesis of the trans10,cis12-conjugated linoleic acid by whole-cell catalysis using recombinant Yarrowia lipolitca

作　　者：宋宇航[1] 倪丽娟[1] 张白曦[1] 陈卫[1] 张灏[1] 

机构地区：[1]江南大学食品科学与工程学院

出　　处：《食品与发酵工业》2016年第10期1-7,共7页Food and Fermentation Industries

基　　金：江苏省自然科学基金青年基金项目(BK20150144)

摘　　要：亚油酸异构酶(PAI)在解脂耶氏酵母细胞内成功表达,以添加外源c9,c12-亚油酸(c9,c12-LA)为底物,全细胞催化合成反10,顺12-共轭亚油酸(trans10,cis12-conjugated linoleic acid,t10,c12-CLA)。解脂耶氏酵母在分别含0.0(对照组)、0.1、0.5、1.0、5.0、10.0 g/L油酸的培养基中生长,收集菌体转移至磷酸盐缓液中进行全细胞催化,比较各组菌体的t10,c12-CLA产率,含5.0 g/L油酸组产物产率最高,达到1.34 g/g,是不含油酸组的1.4倍左右。通过PAI酶活分析及Western blot检测PAI表达量,结果表明,培养基中油酸含量越高,重组解脂耶氏酵母的PAI表达量越低;对比不含油酸组和含5.0 g/L油酸组全细胞催化不同时间的t10,c12-CLA产量,含5.0 g/L油酸组t10,c12-CLA产量最高点的时间比不含油酸组提前约20 h;透射电子显微镜观察2组的菌体状态,含5.0 g/L油酸组细胞壁和细胞膜间隙扩大,该变化可能增加了全细胞催化过程中底物和产物进出细胞的速率,从而提高t10,c12-CLA的转化效率。The linoleic acid isomerase（ PAI） has been successfully expressed in Yarrowia lipolytica to produce trans10,cis12-conjugated linoleic acid（ t10,c12-CLA） by whole-cell catalysis using exogenous free linoleic acid（ LA） as substrate. Whole cells were cultivated in medium containing oleic acid of different concentration（ 0. 0,0. 1,0. 5,1. 0,5. 0,10. 0 g / L）,and catalyzed in phosphate buffer. The highest yield of t10,c12-CLA was 1. 34 g / g from culture containing 5. 0 g / L oleic acid,which was 1. 4 times of yield in experiment group without oleic acid. The PAI relative enzyme activity was analyzed and protein expression was checked by western blot. Results showed that the medium with more oleic acid leaded to lower expression of PAI. As for the production of t10,c12-CLA at different whole-cell catalysis time,the time of highest t10,c12-CLA production of experiment group with 5. 0 g / L oleic acid was 20 h ahead of that of control group with 0. 0 g / L oleic acid. We also investigated cell condition by transmission electron microscopy（ TEM）. Results showed that cells cultivated in the medium containing 5. 0 g / L oleic acid displayed the wider space between cell wall and cell membrane,which might improve transporting / exporting speed of substrates and products between cells and environments,and finally increase conversion rate of t10,c12-CLA.

关 键 词：重组解脂耶氏酵母 油酸 反10 顺12-共轭亚油酸 转化效率 

分 类 号：TS201.3[轻工技术与工程—食品科学]