Phenylpyruvic acid (PPA) is an important organic acid that has a wide range of applications. In this study, the membrane-bound l-amino acid deaminase (l-AAD) gene from Proteus mirabilis KCTC 2566 was expressed in Escherichia coli BL21(DE3) and then the l-AAD was purified. After that, we used the purified enzyme and the recombinant E. coli whole-cell biocatalyst to produce PPA via a one-step biotransformation from l-phenylalanine. l-AAD was solubilized from the membrane and purified 52-fold with an overall yield of 13 %, which corresponded to a specific activity of 0.94 +/- 0.01 mu mol PPA min(-1)center dot mg(-1). Then, the biotransformation conditions for the pure enzyme and the whole-cell biocatalyst were optimized. The maximal production was 2.6 A +/- 0.1 g center dot L-1 (specific activity of 1.02 A +/- 0.02 mu mol PPA min(-1)center dot mg(-1) protein, 86.7 A +/- 5 % mass conversion rate, and 1.04 g center dot L-1 center dot h(-1) productivity) and 3.3 A +/- 0.2 g L-1 (specific activity of 0.013 A +/- 0.003 mu mol PPA min(-1)center dot mg(-1) protein, 82.5 A +/- 4 % mass conversion rate, and 0.55 g center dot L-1 center dot h(-1) productivity) for the pure enzyme and whole-cell biocatalyst, respectively. Comparative studies of the enzymatic and whole-cell biotransformation were performed in terms of specific activity, production, conversion, productivity, stability, need of external cofactors, and recycling. We have developed two eco-friendly and efficient approaches for PPA production. The strategy described herein may aid the biotransformational synthesis of other alpha-keto acids from their corresponding amino acids.