Properties of .ALPHA.-amino-.EPSILON.-caprolactam racemase from Achromobacter obae.

Abstract

a-Amino-e-caprolactam racemase, which occurs in the cytoplasmic fraction of Achromobacter obae, has been purified to homogeneity. It has a monomericstructure with a molecular weight of approximately 50,000. The absorption spectrum of the enzymeexhibits maximaat 280 and 412 nm at pH 7.3, and is independent of pH from 6.0 to 8.0. One mole ofpyridoxal 5'-phosphate is bound per mol of the enzyme.Incubation of the enzymewith hydroxylamineresulted in the formation of the apoenzyme. d-and L-a-Amino-a-caprolactamsare the only substrates. The maximum activity is found at pH 8.8 for both the isomers. Michaelis constants are as follows: 8him for D-a-amino-ecaprolactam, 6 mMfor L-a-amino-e-caprolactam and 2. 1 x 10~7 m for pyridoxal 5'-phosphate. The enzyme is inhibited significantly by CuSO4, HgCl2, thiol reagents such as N-ethylmaleimide and /?-chloromercuribenzoate, and carbonyl reagents (e.g., phenylhydrazine and hydroxylamine). aAmino-e-caprolactam racemase catalyzes the a-proton exchange of the substrate with deuteron during racemization in deuterium oxide. Various racemases have been demonstrated in bacteria and actinomycetes, but only a few enzymes have been purified to homogeneity for enzymological studies: pyridoxal 5'-phosphate dependent amino acid racemases,6~8) imino acid racemases with no cafactor requirement,9'10) an ATP dependent phenylalanine racemase11] and a Mg++ dependent non-amino acid racemase, mandelate racemase.12) aAmino-e-caprolactam racemase is unique amongracemases in that it acts exclusively on a synthetic non-carboxylic compound i.e., an intramolecular cyclic amide of L-lysine.5) We have shown that this enzyme requires pyridoxal S'-phosphate as a coenzyme.5) Thus, the enzyme is the only pyridoxal 5'-phosphate enzyme acting on an a-carboxyamide derivative of an amino acid. Wehere describe the localization of the enzyme in bacterial cells and the physicochemical and enzymological properties of the enzyme purified from Achromobacter obae.

Hydrolysis of Amides

“…In this process, racemic a-amino-e-caprolactam (ACL, 39) is L-selectively hydrolyzed to Llysine 40 by the action of the L-ACL lactamase from Cryptococcus laurantii, whereas the remaining D-ACL is in situ racemized by the ACL racemase from Achromobacter obae (Scheme 15.6). More recently, Asano and Yamaguchi showed that this ACL racemase, in contrast to earlier reports [125], is also able to racemize linear a-H-a-amino acid amides [176], although with at least 35-fold lower specific activity than for ACL [177]. The A. obae ACL racemase has been combined with the D-aminopeptidase from Ochrobactrum anthropi C1-38 (Section 15.4.1.3) for the synthesis of D-alanine from Land DL-alanine amide in near stoichiometric amounts [177,178].…”

“…The ACL specific racemase has been studied in much greater detail [123][124][125][126][127][128][129]. Recently, the crystal structure of this racemase in its native form and in complex with e-caprolactam has been solved [130].…”

New Enzymatic Method of Chiral Amino Acid Synthesis by Dynamic Kinetic Resolution of Amino Acid Amides: Use of Stereoselective Amino Acid Amidases in the Presence of α-Amino-ε-Caprolactam Racemase

“…The use of L-␣-amino-ε-caprolactam (ACL)-hydrolase in the presence of ACL racemase is a typical example of L-lysine production (1,2,12). L-cysteine is produced by hydrolases acting on amino-⌬ 2 -thiazoline-4-carboxylic acid in the presence of a racemase (23).…”

Use of Enzymes in the Synthesis of Amino Acids

“…This ACL racemase has been developed by scientists at Toray Industries for the industrial production of L-lysine from D,L-ACL [26,55]. In contrast to earlier reports [56], Asano and Yamaguchi recently reinvestigated this ACL racemase and found that it is also able to racemize linear a-H-a-amino acid amides, although with at least 35-fold lower specific activity than for ACL [57]. Furthermore, they showed that by combining this enzyme with the D-aminopeptidase from O. anthropi C1-38 [45], synthesis of near stoichiometric amounts of D-amino acids from L-amino acid amides is possible [58].…”