Enhancement of biocatalytic efficiency by increasing substrate loading: enzymatic preparation of l-homophenylalanine
Enhancement of biocatalytic efficiency by increasing substrate loading: enzymatic preparation of l-homophenylalanine
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DOI：
10.1007/s00253-013-5117-1
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发表时间：
2013-07
影响因子：
5
通讯作者：
Jielin Zhang;Tian Zhu;Xuri Wu;Yijun Chen
中科院分区：
文献类型：
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作者：
Jielin Zhang;Tian Zhu;Xuri Wu;Yijun Chen
对映体纯的高苯丙氨酸（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结构单元。在已开发的用于thel-HPA生产的方法中，使用苯丙氨酸脱氢酶的生物催化合成已被证明是最有前途的路线。然而，类似于其他的酶催化的反应，该过程的可行性显着影响不足的基板加载和不可或缺的辅因子的高成本。本研究通过偶联转基因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。补料分批底物添加和连续产物去除的组合大大增加了底物负载和辅因子利用率。系统优化后，40 g（0.22 mol）酮酸底物在24 h内转化为tol-HPA，在8个循环的补料分批操作中，总共0.2 mM NAD+被有效地重复使用，因此平均底物浓度为510 mM，l-HPA的生产率为84.1 g l-1 day-1。本研究提供了一种高效、可行的酶法生产fl-HPA的工艺，并为提高底物负荷提供了一种通用的解决方案。
Enantiomerically purel-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 thel-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 forl-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 tol-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−1day−1forl-HPA. The present study provides an efficient and feasible enzymatic process for the production ofl-HPA and a general solution for the increase of substrate loading.