Identification and Characterization of an Efficient Phenylalanine Ammonia-Lyase from Photorhabdus luminescens

Abstract

A putative aromatic amino acid ammonia-lyase gene (named Pl-pal) was discovered in Photorhabdus luminescens DSM 3368. BLAST and phylogenetic analyses predicted that this enzyme is a histidine ammonia-lyase, whereas sequence alignment suggested that it is more likely a phenylalanine ammonia-lyase (PAL). This gene was amplified from P. luminescens and expressed in Escherichia coli BL21(DE3). The function of Pl-PAL (58 kDa) was characterized by in vitro enzymatic reactions with l-phenylalanine (l-Phe), l-tyrosine (l-Tyr), l-histidine (l-His), and l-tryptophan (l-Trp). Pl-PAL can convert l-Phe and l-Tyr to trans-cinnamic acid and p-coumaric acid, respectively, but had no function on l-His and l-Trp. The optimum temperature and pH were determined to be 40 °C and 11.0, respectively. Under the optimal conditions, Pl-PAL had a kcat/Km value of 0.52 s−1 mM−1 with l-Phe as the substrate, while only 0.013 s−1 mM−1 for l-Tyr. Therefore, the primary function of Pl-PAL was determined to be PAL. The Pl-pal-harboring E. coli strain was used as a whole-cell biocatalyst to produce trans-cinnamic acid from l-Phe. The overall molar conversion rate and productivity were 65.98% and 228.10 mg L−1 h−1, respectively, after the cells were repeatedly utilized 7 times. This work thus provides a promising strain for industrial production of trans-cinnamic acid.