초록
<P>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.</P>