化工进展

• 生物与医药化工 • 上一篇    下一篇

新型异亮氨酸双加氧酶及其重组大肠杆菌合成羟基异亮氨酸

付敏杰1, 聂尧1, 穆晓清1, 徐岩1,2, 肖荣3   

  1. 1江南大学生物工程学院工业生物技术教育部重点实验室,江苏 无锡 214122;2江南大学食品生物技术国家重点实验室,江苏 无锡 214122;3罗格斯大学高级生物技术与医学中心,新泽西州 08854,美国
  • 出版日期:2014-11-05 发布日期:2014-11-01

A novel isoleucine dioxygenase and its expression in recombinant Escherichia coli for synthesis of 4-hydroxyisoleucine

FU Minjie1,NIE Yao1,MU Xiaoqing1,XU Yan 1,2,XIAO Rong3   

  1. 1School of Biotechnology,Key Laboratory of Industrial Biotechnology of Ministry of Education,Jiangnan University,Wuxi 214122,Jiangsu,China; 2State Key Laboratory of Food Science and Technology,Jiangnan University,Wuxi 214122,Jiangsu,China; 3Center for Advanced Biotechnology and Medicine,Rutgers University,New Jersey 08854,USA
  • Online:2014-11-05 Published:2014-11-01

摘要: 异亮氨酸双加氧酶(IDO)可特异性的转化底物L-异亮氨酸(L-Ile)生成4-羟基-L-异亮氨酸(4-HIL),该产物具有促进胰岛素分泌的功能,可用于抗糖尿病、降胆固醇等。本研究结合了酶标显色和薄层层析(TLC)的方法从自然界中筛到了具有IDO活性的菌株,并将该菌株中的目的基因ido克隆到大肠杆菌中,获得重组表达菌株,并且验证该菌具有IDO的转化功能。本研究优化了转化反应体系和条件,同时通过30℃过夜温育菌体细胞的方法,使该菌株全细胞转化合成4-HIL的产率达到85%以上。

关键词: 异亮氨酸双加氧酶, 异亮氨酸, 羟基异亮氨酸, 生物转化, 重组表达

Abstract: Isoleucine dioxygenase (IDO) could hydroxylyze L-isoleucine (L-Ile) to produce 4-hydroxy-L-isoleucine (4-HIL),which increases glucose-induced insulin release and is usually used in pharmaceuticals with antidiabetic and cholesterol-reducing properties. The strain with dioxygenase activity was screened from nature via combination of reaction coloration assayed by spectrophotometer and thin-layer chromatography (TLC). The gene encoding IDO (ido) was cloned and expressed in Escherichia coli and the constructed recombinant strain exhibited the function of isoleucine hydroxylation. The reaction conditions were optimized and the yield of transformation with the recombinant whole cells was increased to more than 85% by incubating the cells overnight at 30℃. The newly identified IDO and involved recombinant whole cells would be promising to prepare 4-HIL with high efficiency.

Key words: isoleucine dioxygenase, isoleucine, hydroxyl isoleucine, biotransformation, recombinant expression