Laboratory of Chemical Biology and State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjia St., Nanjing, Jiangsu Province, 210009, China.
Appl Microbiol Biotechnol. 2013 Oct;97(19):8487-94. doi: 10.1007/s00253-013-5117-1. Epub 2013 Jul 28.
Enantiomerically pure L-homophenylalanine (L-HPA) is a key building block for the synthesis of angiotensin-converting enzyme inhibitors and other chiral pharmaceuticals. Among the processes developed for the L-HPA production, biocatalytic synthesis employing phenylalanine dehydrogenase has been proven as the most promising route. However, similar to other dehydrogenase-catalyzed reactions, the viability of this process is markedly affected by insufficient substrate loading and high costs of the indispensable cofactors. In the present work, a highly efficient and economic biocatalytic process for L-HPA was established by coupling genetically modified phenylalanine dehydrogenase and formate dehydrogenase. Combination of fed-batch substrate addition and a continuous product removal greatly increased substrate loading and cofactor utilization. After systemic optimization, 40 g (0.22 mol) of keto acid substrate was transformed to L-HPA within 24 h and a total of 0.2 mM NAD(+) was reused effectively in eight cycles of fed-batch operation, consequently giving an average substrate concentration of 510 mM and a productivity of 84.1 g l(-1) day(-1) for L-HPA. The present study provides an efficient and feasible enzymatic process for the production of L-HPA and a general solution for the increase of substrate loading.
对映体纯 L-高苯丙氨酸(L-HPA)是合成血管紧张素转换酶抑制剂和其他手性药物的关键构建块。在开发的 L-HPA 生产工艺中,已证明使用苯丙氨酸脱氢酶的生物催化合成是最有前途的途径。然而,与其他脱氢酶催化反应类似,该过程的可行性受到底物加载不足和必不可少辅助因子成本高的显著影响。在本工作中,通过组合遗传修饰的苯丙氨酸脱氢酶和甲酸脱氢酶,建立了一种高效、经济的 L-HPA 生物催化工艺。分批进料底物添加和连续产物去除的组合大大增加了底物加载和辅助因子的利用。经过系统优化,在 24 小时内将 40 g(0.22 mol)酮酸底物转化为 L-HPA,在 8 个分批进料操作循环中有效重复使用了 0.2 mM NAD(+),从而使 L-HPA 的平均底物浓度达到 510 mM,产率达到 84.1 g·l(-1)·天(-1)。本研究为 L-HPA 的生产提供了一种高效可行的酶法工艺,为增加底物负荷提供了一种通用的解决方案。