Huang Sen, Shi Feng
State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China.
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China.
Biotechnol Lett. 2018 Aug;40(8):1227-1235. doi: 10.1007/s10529-018-2566-8. Epub 2018 Jun 5.
L-isoleucine dioxygenase (IDO) specifically transforms L-isoleucine (Ile) to 4-hydroxyisoleucine (4-HIL), and 4-HIL is a promising drug for diabetes. To enhance the activity and catalytic efficiency of IDO, we used directed evolution and site-specific mutagenesis.
The IDO gene (ido) derived from Bacillus weihenstephanensis was cloned and expressed in Escherichia coli. Directed evolution using error prone (EP)-PCR and site-specific mutagenesis were conducted. Two improved mutants were obtained after one round of EP-PCR, with Ido exhibiting a 2.8-fold increase in activity. Two improved mutants were obtained through site-specific mutagenesis, with Ido showing a 170% increase in activity. Although the activity of the combined mutant Ido (0.95 ± 0.08 U/mg) was slightly higher than that of the wild-type Ido, its catalytic efficiency was 2.4-fold and 3.0-fold higher than Ido with Ile and α-ketoglutaric acid as substrates. After biotransformation of Ile by E. coli BL21(DE3) expressing Ido and Ido, 66.50 ± 0.99 mM and 26.09 ± 1.85 mM 4-HIL was synthesized, respectively, in 24 h.
Ido had a higher enzyme activity and catalytic efficiency and can therefore be used as a more suitable candidate for 4-HIL production.
L-异亮氨酸双加氧酶（IDO）可将L-异亮氨酸（Ile）特异性转化为4-羟基异亮氨酸（4-HIL），4-HIL是一种很有前景的糖尿病治疗药物。为提高IDO的活性和催化效率，我们采用了定向进化和定点诱变技术。
克隆了源自维氏芽孢杆菌的IDO基因（ido）并在大肠杆菌中表达。利用易错（EP）-PCR进行定向进化并进行定点诱变。一轮EP-PCR后获得两个改良突变体，Ido活性提高了2.8倍。通过定点诱变获得两个改良突变体，Ido活性提高了170%。虽然组合突变体Ido（0.95±0.08 U/mg）的活性略高于野生型Ido，但其以Ile和α-酮戊二酸为底物时的催化效率分别比Ido高2.4倍和3.0倍。用表达Ido和Ido的大肠杆菌BL21（DE3）对Ile进行生物转化后，24小时内分别合成了66.50±0.99 mM和26.09±1.85 mM的4-HIL。
Ido具有较高的酶活性和催化效率，因此可作为更适合生产4-HIL的候选酶。
