Discovery of amino acid amides as new substrates for α-amino-ɛ-caprolactam racemase from Achromobacter obae

Amino acid amide racemizing activity was discovered in α-amino-ɛ-caprolactam (ACL) racemase (EC 5. 1. 1. 15) from Achromobacter obae. The nucleotide sequence of 1305 bp ACL racemase gene was designed for expression in Escherichia coli and synthesized in a reaction with 46 oligonucleotides by assembly PCR technique. The gene was expressed under the control of the lac promoter, and ACL racemase was purified from E. coli JM109/pACL60 harboring the gene for the enzyme. The enzyme catalyzed the racemization of newly discovered substrates such as 2-aminobutyric acid amide, alanine amide, threonine amide, norvaline amide, norleucine amide, leucine amide, methionine amide, serine amide, and phenylalanine amide. The relative activity towards l-2-aminobutyric acid amide was 2.7% that for l-ACL (350 U/mg), followed by l-alanine amide by 2.1%. Kinetic parameters for the two d- and l-alanine-isomers of amino acid amides (2-aminobutyric acid amide and alanine amide) and l-ACL were obtained from Lineweaver-Burk plots. The Kcat values for l-ACL, d- and l-2-aminobutyric acid amide, and d- and l-alanine amide were calculated to be 360, 40, 13, 9.8 and 7.2 S−1, respectively. The Km values for l-ACL, d- and l-2-aminobutyric acid amide, and d- and l-alanine amide were shown to be 10.1, 3.5, 1.1, 3.4 and 2.5 mM, respectively. The l-enantiomer of 2-aminobutyric acid amide (22.5 μmol) was completely racemized by ACL racemase (136 μg) in 100 min, and the same amount of l-alanine amide in 120 min. The catalytic efficiency of the newly discovered racemization reaction of 2-aminobutyric acid amide was calculated for be fairly high at about 33% that for l-ACL, while that for alanine amide was about 8.1%. Here, we report that amino acid amides act as new substrates for the ACL racemase.