a College of Biotechnology, Tianjin Engineering Lab of Efficient and Green Amino Acid Manufacture, National and Local United Engineering Lab of Metabolic Control Fermentation Technology, Tianjin University of Science and Technology , Tianjin , China.
b Linghua Group Limited , Shandong , China.
Bioengineered. 2018 Jan 1;9(1):72-79. doi: 10.1080/21655979.2017.1304872. Epub 2017 Apr 28.
L-Isoleucine dioxygenase (IDO) specifically converts L-isoleucine(L-Ile) to 4-hydroxyisoleucine(4-HIL). To obtain IDO with improved activity, a strategy was developed that is dependent on the restoration of succinate-minus E. coli cell growth by the coupling of L-Ile hydroxylation and the oxidation of α-ketoglutarate(α-KGA) to succinate. Five mutants were obtained with this strategy, and the characteristics of IDO, which exhibited the highest activity, were studied. The catalytic efficiency, thermal stability and catalytic rate of IDO were significantly improved compared with those of wild-type IDO. Moreover, an efficient method for the biotransformation of 4-HIL by resting cells expressing IDO was developed, with which 151.9 mmol of 4-HIL/L (22.4 g/L) was synthesized in 12 h while the substrates seldom exhibited additional consumption.
L-异亮氨酸双加氧酶（IDO）特异性地将 L-异亮氨酸（L-Ile）转化为 4-羟基异亮氨酸（4-HIL）。为了获得活性提高的 IDO，开发了一种策略，该策略依赖于通过 L-Ile 羟化和 α-酮戊二酸（α-KGA）氧化为琥珀酸的偶联来恢复琥珀酸盐缺失的大肠杆菌细胞生长。使用该策略获得了五个突变体，并研究了表现出最高活性的 IDO 的特性。与野生型 IDO 相比，IDO 的催化效率、热稳定性和催化速率均得到显著提高。此外，还开发了一种通过表达 IDO 的静息细胞进行 4-HIL 生物转化的有效方法，在 12 小时内合成了 151.9 mmol/L（22.4 g/L）的 4-HIL，而底物很少表现出额外的消耗。
