Characterization of an α-amino-ɛ-caprolactam racemase with broad substrate specificity from Citreicella sp. SE45
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α-Amino-ε-caprolactam (ACL) racemizing activity was detected in a putative dialkylglycine decarboxylase (EC 220.127.116.11) from Citreicella sp. SE45. The encoding gene of the enzyme was cloned and transformed in Escherichia coli BL21 (DE3). The molecular mass of the enzyme was shown to be 47.4 kDa on SDS–polyacrylamide gel electrophoresis. The enzymatic properties including pH and thermal optimum and stabilities were determined. This enzyme acted on a broad range of amino acid amides, particularly unbranched amino acid amides including l-alanine amide and l-serine amide with a specific activity of 17.5 and 21.6 U/mg, respectively. The K m and V max values for d- and l-ACL were 5.3 and 2.17 mM, and 769 and 558 μmol/min.mg protein, respectively. Moreover, the turn over number (K cat) and catalytic efficiency (K cat/K m ) of purified ACL racemase from Citreicella sp. SE45 using l-ACL as a substrate were 465 S−1 and 214 S−1mM−1, respectively. The new ACL racemase from Citreicella sp. SE45 has a potential to be used as the biocatalytic application.
KeywordsACL racemase PLP-dependent enzyme d-amino acids Enzyme characterization Citreicella sp. SE45
This work was financially supported by the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program (Grant No. PHD/099/2551) and the Japan Student Services Organization (JASSO). This research was partly supported by Grant-in-Aids for Scientific Research (A) (23248015) and (B) (26292041) to Y. Asano from the Japan Society for the Promotion of Science (JSPS). This work was also partly supported by the Exploratory Research for Advanced Technology (ERATO) Asano Active Enzyme Molecule Project of Japan Science and Technology Agency (JST). The author greatly appreciates Asst. Prof. Ken-ichi Fuhshuku for the preparation of chiral ACLs and enantiomerically pure amino acid amides. This work was performed at Toyama Prefectural University.
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Conflict of interest
The authors declare that they have no completing interests.
This study does not contain any experiment with human participants or animals performed by any of the authors.
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