α-酮戊二酸依赖型双加氧酶催化特性及反应耦联辅因子对其催化羟基化反应的影响  

Characterization of α-ketoglutaric acid-dependent dioxygenase and the effect of reaction-coupled cofactor on dioxygenase-catalyzed hydroxylation

作　　者：文方[1] 聂尧[1] 穆晓清[1] 徐岩 

机构地区：[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

出　　处：《微生物学通报》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~~

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.

关 键 词：双加氧酶 异亮氨酸 4-羟基异亮氨酸 酶学特性 羟基化反应 

分 类 号：Q55[生物学—生物化学]