摘要
【目的】以重组大肠杆菌表达的枯草芽孢杆菌(Bacillus subtilis)L-异亮氨酸双加氧酶(L-isoleucine dioxygenase,IDO)为研究对象,考察其催化L-异亮氨酸(L-Ile)羟基化反应的影响因素,构建IDO催化合成羟基氨基酸的反应体系。【方法】通过Ni-NTA亲和层析法从重组大肠杆菌(Escherichia coli)BL21/p ET28a-ido中纯化获得重组IDO,以L-Ile为底物,考察重组IDO催化羟基化反应的影响因素,并进一步针对耦联反应优化α-酮戊二酸(α-KG)在重组IDO酶促转化体系中的添加浓度。【结果】基于重组IDO催化L-Ile羟基化的活性测定,计算该酶Km为0.247 mmol/L,kcat为1.260 s-1,kcat/Km为5.101 L/(mmol·s),与其他同源酶动力学参数比较分析表明,重组IDO的底物亲和性及催化效率较高。重组IDO催化反应的最适温度为20°C、最适p H为7.0;在35°C以下较为稳定;反应体系中Fe2+最适浓度为1 mmol/L。重组IDO可催化不同L-氨基酸反应,对L-异亮氨酸、L-正亮氨酸、L-甲硫氨酸的活性较高。通过优化α-KG浓度,反应体系中添加30 mmol/Lα-KG时,可将底物浓度提高至70 mmol/L,产物4-羟基异亮氨酸(4-HIL)的摩尔产率达66.20%,表明α-KG作为反应耦联辅因子,其浓度对重组IDO催化L-Ile羟基化具有显著影响。【结论】重组IDO的底物亲和性、催化效率、最适催化条件、稳定性等基本性质有利于催化L-Ile羟基化反应。在其催化反应体系中,α-KG作为反应耦联辅因子,对酶促转化效果影响显著。研究结果为4-HIL及其他羟基氨基酸的酶促转化提供了研究基础。
[Objective] In order to construct a biocatalytic system to synthesize 4-hydroxyl isoleucine（4-HIL）, L-isoleucine（L-Ile） dioxygenase（IDO） from Bacillus subtilis expressed in recombinant Escherichia coli was purified and characterized. [Methods] The recombinant IDO was purified by Ni-NTA affinity chromatography from recombinant Escherichia coli BL21/p ET28a-ido. The enzyme was the characterized by using L-Ile as the substrate. As the necessary cofactor of IDO, the concentration of α-ketoglutaric acid（α-KG） in the enzymatic system was further optimized to improve the catalytic efficiency. [Results] Kinetic parameters of the enzyme were obtained as Km0.247 mmol/L, kcat 1.260 s^-1, and kcat/Km 5.101 L/（mmol·s）. Compared with other homologous enzymes, the recombinant IDO had higher substrate affinity and catalytic efficiency. The recombinant IDO was more active at 20 ℃and p H 7.0, and more stable at the temperatures below 35 ℃. The optimal concentration of Fe^2＋ was 1 mmol/L in the catalytic system. The recombinant IDO was active to a variety of L-amino acids, of which L-isoleucine, L-norleucine, and L-methionine were more suitable for the IDO catalyzing hydroxylation. By optimization of α-KG concentration in enzymatic catalysis, 4-HIL with the yield of 66.20% was achieved from 70 mmol/L L-Ile by adding 30 mmol/L α-KG in the reaction system. Thus, the addition of α-KG as the reaction-coupled cofactor had a significant impact on the reaction efficiency of recombinant IDO-mediated L-Ile hydroxylation. [Conclusion] This study provides the basis for enzymatic conversion of 4-HIL and other hydroxylated amino acids.
出处
《微生物学通报》
CAS
CSCD
北大核心
2017年第3期505-512,共8页
Microbiology China
基金
国家高技术研究发展计划项目(No.2015AA021004
2011AA02A209
2011AA02A210)
国家自然科学基金项目(No.21376107
21336009)
江苏省自然科学基金项目(No.BK20151124)
高等学校学科创新引智计划项目(111计划)(No.111-2-06)
江苏省六大人才高峰计划项目(No.2015-NY-007)
江苏高校优势学科建设工程项目~~
