Enzymatic production of L-phenylalanine through transaminase reaction.

Abstract

To develop an enzymatic process for L-phenylalanine production through transaminase reaction, we selected from our bacterial culture collection a Citrobacter freuindii strain as an excellent producer of the enzyme. The cells of this bacterium rapidly and efficiently formed L-phenylalanine from phenylpyruvic acid and an amino donor amino acid. With L-glutamic acid as the amino donor, L-phenylalanine production reached 30mg/ml in 1h and 89mg/ml in 24h incubation. The conversion yield to form L-phenylalanine from phenylpyruvic acid was 50 to 80%. With L-aspartic acid as amino donor, the rate of L-phenylalanine production is lower (i.e. 8mg/ml in 1h and 30mg/ml in 4h) but the conversion yield was much higher (80 to 100%). With L-aspartic acid as the amino donor, oxalacetic acid, a putative reaction product, appeared in small amounts. Instead, a much larger amount of pyruvic acid, a well-known decarboxylation product of oxalacetic acid, appeared. From these facts it was concluded that the reaction, which should be reversible, inclines almost completely toward L-phenylalanine formation when L-aspartic acid is used as the amino donor, because oxalacetic acid (the reaction product and also the substrate for the reverse reaction) is eliminated through decarboxylation to form pyruvic acid and other metabolites. The Citrobacter strain had a high aspartase activity to form L-aspartic acid from fumaric acid and ammonia. Coupling the two activities of the transaminase and the aspartase, an enzymatic process for L-phenylalanine production from phenylpyruvic acid and ammonium fumarate was established.