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

## TL;DR

Scientists developed a continuous method to efficiently produce two chemicals, L-homophenylalanine and 2-hydroxy-5-oxoproline, using an enzyme from Megasphaera elsdenii.

## Contribution

A novel coupled enzymatic synthesis with in situ crystallization and cyclization for preparative-scale production of two compounds.

## Key findings

- Fed-batch process proved most viable for the coupled synthesis.
- Over 18 g of L-homophenylalanine and 9 g of 2-hydroxy-5-oxoproline were produced at gram scale.
- The process improves productivity through spontaneous crystallization and cyclization.

## Abstract

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 α-ketoglutarate) using the α-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.