Cho Byung-Kwan, Seo Joo-Hyun, Kang Tae-Won, Kim Byung-Gee
Interdisciplinary Program for Biochemical Engineering and Biotechnology, School of Chemical Engineering, and Institute of Molecular Biology and Genetics, Seoul National University, Seoul, Korea.
Biotechnol Bioeng. 2003 Jul 20;83(2):226-34. doi: 10.1002/bit.10661.
L-Homophenylalanine (L-HPA) was asymmetrically synthesized from 2-oxo-4-phenylbutyric acid (2-OPBA) and L-aspartate using a recombinant aromatic amino acid transaminase (AroAT). To screen microorganisms having such an L-specific AroAT with a relaxed substrate inhibition in the asymmetric synthesis of unnatural amino acids, enrichment cultures were performed in a minimal media containing 50 mM L-HPA as a sole nitrogen source. To reduce the intracellular background synthetic activity by amino acid pools in the cells, a two-step screening method was used. The putative AroAT (i.e., AroATEs) from the screened Enterobacter sp. BK2K-1 was cloned, sequenced, and overexpressed in E. coli cells. The activity of the overexpressed AroATEs was 314-fold higher than that of the wild-type cell. The substrate specificities of the enzyme and homology search revealed that the cloned transaminase is true AroAT. The AroATEs showed a substrate inhibition by 2-OPBA from 40 mM in the asymmetric synthesis, which made it difficult to perform batch asymmetric synthesis of L-HPA at high concentrations of 2-OPBA. To avoid the substrate inhibition by 2-OPBA, intermittent addition of the solid-state substrate was attempted to obtain a high concentration of L-HPA. By using the cell extract (75 U) obtained from the recombinant E. coli harboring the AroATEs gene, the asymmetric synthesis of L-HPA at 840 mM of 2-OPBA resulted in >94% of conversion yield and >99% ee of L-HPA of optical purity. Due to the low solubility (<2 mM) of L-HPA in the reaction buffer, synthesized L-HPA was continuously precipitated in the reaction media, which drives the reaction equilibrium towards the product formation. After full completion of the reaction, L-HPA of high purity (>99% ee) was easily recovered by simple pH shift of the reaction media. This method can permit very efficient asymmetric synthesis of other unnatural amino acids using a single transaminase reaction.
使用重组芳香族氨基酸转氨酶（AroAT），以2-氧代-4-苯基丁酸（2-OPBA）和L-天冬氨酸为原料不对称合成了L-高苯丙氨酸（L-HPA）。为了筛选在非天然氨基酸不对称合成中具有这种对L-特异性且底物抑制作用较弱的AroAT的微生物，在含有50 mM L-HPA作为唯一氮源的基本培养基中进行了富集培养。为了降低细胞内氨基酸池的背景合成活性，采用了两步筛选法。从筛选出的肠杆菌属BK2K-1中克隆、测序了假定的AroAT（即AroATEs），并在大肠杆菌细胞中进行了过量表达。过量表达的AroATEs的活性比野生型细胞高314倍。该酶的底物特异性和同源性搜索表明，克隆的转氨酶是真正的AroAT。在不对称合成中，AroATEs对2-OPBA的底物抑制作用在40 mM时就出现了，这使得在高浓度2-OPBA下进行L-HPA的分批不对称合成变得困难。为了避免2-OPBA的底物抑制作用，尝试间歇添加固态底物以获得高浓度的L-HPA。使用从携带AroATEs基因的重组大肠杆菌中获得的细胞提取物（75 U），在840 mM 2-OPBA条件下进行L-HPA的不对称合成，转化率>94%，L-HPA光学纯度>99% ee。由于L-HPA在反应缓冲液中的溶解度较低（<2 mM），合成的L-HPA在反应介质中持续沉淀，这推动反应平衡向产物生成方向移动。反应完全完成后，通过简单改变反应介质的pH值，即可轻松回收高纯度（>99% ee）的L-HPA。该方法可以通过单一转氨酶反应非常高效地不对称合成其他非天然氨基酸。
