Tani Yasushi, Miyake Ryoma, Yukami Ryoichi, Dekishima Yasumasa, China Hideyasu, Saito Shigeki, Kawabata Hiroshi, Mihara Hisaaki
Department of Biotechnology, College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga, 525-8577, Japan.
Appl Microbiol Biotechnol. 2015 Jun;99(12):5045-54. doi: 10.1007/s00253-014-6308-0. Epub 2014 Dec 31.
L-Pipecolic acid is a key component of biologically active molecules and a pharmaceutically important chiral building block. It can be stereoselectively produced from L-lysine by a two-step bioconversion involving L-lysine α-oxidase and ∆(1)-piperideine-2-carboxylae (Pip2C) reductase. In this study, we focused on an L-lysine α-oxidase from Scomber japonicus that was originally identified as an apoptosis-inducing protein (AIP) and applied the enzyme to one-pot fermentation of L-pipecolic acid in Escherichia coli. A synthetic gene coding for an AIP was expressed in E. coli, and the recombinant enzyme was purified and characterized. The purified enzyme was determined to be a homodimer with a molecular mass of 133.9 kDa. The enzyme essentially exhibited the same substrate specificity as the native enzyme. Optimal temperature and pH for the enzymatic reaction were 70 °C and 7.4, respectively. The enzyme was stable below 60 °C and at a pH range of 5.5-7.5 but was markedly inhibited by Co(2+). To establish a one-pot fermentation system for the synthesis of optically pure L-pipecolic acid from DL-lysine, an E. coli strain carrying a plasmid encoding AIP, Pip2C reductase from Pseudomonas putida, lysine racemase from P. putida, and glucose dehydrogenase from Bacillus subtilis was constructed. The one-pot process produced 45.1 g/L of L-pipecolic acid (87.4 % yield from DL-lysine) after a 46-h reaction with high optical purity (>99.9 % enantiomeric excess).
L-哌啶酸是生物活性分子的关键成分和重要的药用手性结构单元。它可以通过两步生物转化从L-赖氨酸立体选择性地产生，这两步生物转化涉及L-赖氨酸α-氧化酶和∆(1)-哌啶-2-羧酸（Pip2C）还原酶。在本研究中，我们聚焦于来自日本鲭的一种L-赖氨酸α-氧化酶，该酶最初被鉴定为凋亡诱导蛋白（AIP），并将该酶应用于大肠杆菌中L-哌啶酸的一锅法发酵。编码AIP的合成基因在大肠杆菌中表达，重组酶被纯化并进行了表征。纯化后的酶被确定为分子量为133.9 kDa的同型二聚体。该酶基本上表现出与天然酶相同的底物特异性。酶促反应的最佳温度和pH分别为70℃和7.4。该酶在60℃以下和pH 5.5 - 7.5范围内稳定，但受到Co(2+)的显著抑制。为了建立从DL-赖氨酸合成光学纯L-哌啶酸的一锅法发酵系统，构建了一种携带编码AIP的质粒、恶臭假单胞菌的Pip2C还原酶、恶臭假单胞菌的赖氨酸消旋酶和枯草芽孢杆菌的葡萄糖脱氢酶的大肠杆菌菌株。经过46小时的反应，该一锅法工艺产生了45.1 g/L的L-哌啶酸（从DL-赖氨酸的产率为87.4%），具有高光学纯度（对映体过量>99.9%）。
