Preparative Coupled Enzymatic Synthesis of L-Homophenylalanine and 2-Hydroxy-5-oxoproline with Direct In Situ Product Crystallization and Cyclization

A continuous in situ crystallization concept is presented for the coupled preparative synthesis of L-homophenylalanine and 2-hydroxy-5-oxoproline (a cyclized form of alpha-ketoglutarate) using the alpha-transaminase from Megasphaera elsdenii. The process consists of a spontaneous reactive crystallization step of the enantiopure amino acid itself and a parallel spontaneous cyclization of the deaminated cosubstrate in solution. In parallel, these effects significantly improve the overall productivity of the biocatalytic reaction. Batch, repetitive, and fed-batch processes were investigated, and the fed-batch option proved to be the most viable option. The fed-batch process was subsequently used for a coupled synthesis approach at the gram scale. In total, >18 g of chemically pure L-homophenylalanine and >9 g of 2-hydroxy-5-oxoproline were isolated. This optimized process allows for the design of effective transaminase-catalyzed reactions at a preparative scale utilizing standard (fed-)batch-mode crystallizers.