Functional expression of L-lysine alpha-oxidase from Scomber japonicus in Escherichia coli for one-pot synthesis of L-pipecolic acid from DL-lysine
Functional expression of L-lysine alpha-oxidase from Scomber japonicus in Escherichia coli for one-pot synthesis of L-pipecolic acid from DL-lysine
复制标题
鲭鱼L-赖氨酸α-氧化酶在大肠杆菌中的功能表达，用于DL-赖氨酸一锅法合成L-哌啶酸
DOI：
10.1007/s00253-014-6308-0
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
H.
中科院分区：
文献类型：
--
作者：
Tani;Y.;Miyake;R.;Yukami;R.;Dekishima;Y.;China;H.;Saito;S.;Kawabata;H.;and Mihara;H.
L-吡喃甲酸是生物活性分子的关键成分，也是药学上重要的手性构件。它可以通过L-赖氨酸α-氧化酶和∆-1-哌啶-2-羧基(PIP2C)还原酶的两步生物转化来立体选择性地生产。在本研究中，我们研究了一种最初被认为是一种细胞凋亡诱导蛋白的α酶，并将其应用于大肠杆菌一锅发酵生产1-吡喃甲酸。表达了编码AIP的人工合成基因。并对重组酶进行了纯化和鉴定。纯化后的酶为均二聚体，相对分子质量为133.9 kDa。该酶基本上表现出与天然酶相同的底物专一性。酶促反应的最适温度为70℃，最适pH为7.4。该酶在60℃以下、pH值5.5~7.5范围内稳定，但受Co2+的抑制作用明显。建立以赖氨酸为原料，一锅发酵合成光学葛雷尔-吡哌酸的发酵体系。大肠杆菌携带恶臭假单胞菌的AIP、PIP2C还原酶、P。构建了枯草芽孢杆菌葡萄糖脱氢酶。一锅法反应46小时，得到45.1克/L的l-吡喃甲酸(dl-赖氨酸的产率为87.4%)，光学纯度高(对映体过量99.9%)。
l-Pipecolic acid is a key component of biologically active molecules and a pharmaceutically important chiral building block. It can be stereoselectively produced froml-lysine by a two-step bioconversion involvingl-lysine α-oxidase and ∆1-piperideine-2-carboxylae (Pip2C) reductase. In this study, we focused on anl-lysine α-oxidase fromScomber japonicusthat was originally identified as an apoptosis-inducing protein (AIP) and applied the enzyme to one-pot fermentation ofl-pipecolic acid inEscherichia coli. A synthetic gene coding for an AIP was expressed inE. 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 Co2+. To establish a one-pot fermentation system for the synthesis of optically purel-pipecolic acid fromdl-lysine, anE. colistrain carrying a plasmid encoding AIP, Pip2C reductase fromPseudomonas putida, lysine racemase fromP. putida, and glucose dehydrogenase fromBacillus subtiliswas constructed. The one-pot process produced 45.1 g/L ofl-pipecolic acid (87.4 % yield fromdl-lysine) after a 46-h reaction with high optical purity (>99.9 % enantiomeric excess).