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

Abstract

l-Amino acid deaminase (l-AAD) from Proteus myxofaciens was cloned and over-expressed in Escherichia coli K12. This enzyme has a broad substrate specificity, working on both natural and unnatural l-amino acids. Of the 20 naturally occurring l-amino acids, l-AAD prefers amino acid substrates that have aliphatic, aromatic or sulfur-containing side chains; those with charged side chains (–CO2− or –NH3+) are poor or non-substrates. Enzyme activity was monitored using a microtiter-plate-based assay, which measures the formation of phenylpyruvic acid from l-phenylalanine. The reaction has an absolute requirement for O2, releases NH3 and does not produce H2O2. Substrate comparisons were carried out by using an O2 electrode to measure the O2 utilization rates. Studies on partially purified enzyme show a pH optimum of 7.5 with a subunit molecular weight of approximately 51 kDa. Additional purification and characterization strategies will be presented. The use of whole cells containing l-AAD will be discussed to prepare chiral pharmaceutical intermediates.