Purification and characterization of an L-amino acid deaminase used to prepare unnatural amino acids

L-Amino acid deaminase (L-AAD) from Proteus myxofaciens was cloned and over -expressed in Escherichia coli K12. This enzyme has a broad substrate speci ficity, working on both natural and unnatural L-amino acids. Of the 20 natu rally occurring L-amino acids, L-AAD prefers amino acid substrates that hav e aliphatic, aromatic or sulfur-containing side chains; those with charged side chains (-CO2- or -NH3+) are poor or non-substrates. Enzyme activity wa s monitored using a microtiter-plate-based assay, which measures the format ion of phenylpyruvic acid from L-phenylalanine. The reaction has an absolut e requirement for O-2, releases NH3 and does not produce H2O2. Substrate co mparisons were carried out by using an O-2 electrode to measure the O-2 uti lization rates. Studies on partially purified enzyme show a pH optimum of 7 .5 with a subunit molecular weight of approximately 51 kDa. Additional puri fication and characterization strategies will be presented. The use of whol e cells containing L-AAD will be discussed to prepare chiral pharmaceutical intermediates. (C) 2001 Elsevier Science B.V. All rights reserved.