Penicillin amidohydrolase [EC 3.5.1.11] was used to resolve stereoisomers of a β-amino acid ester (ethyl 3-amino-5-(trimethylsilyl)-4-pentynoate) by phenylacetylation. After screening commercially available sources of the immobilized enzyme, one was found to be significantly more efficient, and this was developed at 1-L scale reaction. The effects of phenylacetic acid concentration, β-amino acid ester concentration, and pH on bioconversion rates and side reactions were examined. The enzymatic reaction was monitored off-line by naphthoylation of samples and chiral analytical chromatography. The best conditions for the bioconversion were pH 5.7, 28 °C, and 14 000 units of enzyme activity per liter. The phenylacetic acid concentration was set at 50 g/L (0.37 M), and the amine at 100 g/L (0.47 M). Under these conditions, yields of the desired (S)amino acid ester were on the order of 90% with ee's of 95% or greater in less than 12 h. This process, along with a slight modification, was tested through 15 cycles at 0.4-L scale, and was scaled to 70 L. Recycle results extrapolated to approximately 25 reaction cycles before the enzyme lost 50% of its initial activity. Through three runs at 70 L, overall yield of (S)-amine was 42.7 ( 0.6%, overall yield of (R)-phenylacetyl amide was 47.2 ( 1.8%. The average ee of the amine (two runs) was 98.1 ( 0.4%, for the amide the ee was 99.5 ( 0.2%. Experimental Section I. Analytical. ReVersed Phase (RP-HPLC) Chromatography. Aqueous samples for RP-chromatography were diluted into an equal volume of acetonitrile, except for amine