Construction and evaluation of a novel bifunctional phenylalanine-formate dehydrogenase fusion protein for bienzyme system with cofactor regeneration.

Phenylalanine dehydrogenase (PheDH) plays an important role in enzymatic synthesis of L-phenylalanine for aspartame (sweetener) and detection of phenylketonuria (PKU), suggesting that it is important to obtain a PheDH with excellent characteristics. Gene fusion of PheDH and formate dehydrogenase (FDH) was constructed to form bifunctional multi-enzymes for bioconversion of L-phenylalanine coupled with coenzyme regeneration. Comparing with the PheDH monomer from Microbacterium sp., the bifunctional PheDH-FDH showed noteworthy stability under weakly acidic and alkaline conditions (pH 6.5-9.0). The bifunctional enzyme can produce 153.9 mM L-phenylalanine with remarkable performance of enantiomers choice by enzymatic conversion with high molecular conversion rate (99.87 %) in catalyzing phenylpyruvic acid to L-phenylalanine being 1.50-fold higher than that of the separate expression system. The results indicated the potential application of the PheDH and PheDH-FDH with coenzyme regeneration for phenylpyruvic acid analysis and L-phenylalanine biosynthesis in medical diagnosis and pharmaceutical field.