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In Silico Identification for α-Amino-ε-Caprolactam Racemases by Using Information on the Structure and Function Relationship

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Abstract

In silico identification for enzymes having desired functions is attractive because there is a possibility that numerous desirable enzymes have been deposited in databases. In this study, α-amino-ε-caprolactam (ACL) racemases were searched from the NCBI protein database. Four hundred thirteen fold-type I pyridoxal 5′-phosphate-dependent enzymes which are considered to contain sequences of ACL racemase were firstly obtained by submitting the sequence of ACL racemase from Achromobacter obae to the database. By identifying Lys241 as a key amino acid residue, 13 candidates for ACL racemase were selected. Then, putative ACL racemase genes were synthesized as codon-optimized sequences for expression in Escherichia coli. They were subcloned and expressed in E. coli BL21 and underwent His-tag purification. ACL and amino acid amide racemizing activities were detected among ten of the candidates. The locus tags Oant_4493, Smed_5339, and CSE45_2055 derived from Ochrobactrum anthropi ATCC49188, Sinorhizobium medicae WSM 419, and Citreicella sp. SE45, respectively, showed higher racemization activity against d- and l-ACLs rather than that of ACL racemase from A. obae. Our results demonstrate that the newly discovered ACL racemases were unique from ACL racemase from A. obae and might be useful for applications in dynamic kinetic resolution for d- or l-amino acid production.

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Acknowledgments

This work was financed by the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program (Grant No. PHD/099/2551) and Japan Student Services Organization (JASSO). The authors greatly appreciated Asst. Prof. Ken-ichi Fuhshuku for the preparation of the chiral ACLs and enantiomerically pure amino acid amides.

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Authors and Affiliations

  1. Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Hat-Yai, 90112, Thailand

    Wisarut Payoungkiattikun & Aran H-Kittikun

  2. Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama, 939-0398, Japan

    Wisarut Payoungkiattikun, Seiji Okazaki, Shogo Nakano, Atsutoshi Ina & Yasuhisa Asano

  3. Asano Active Enzyme Molecule Project, ERATO, JST, 5180 Kurokawa, Imizu, Toyama, 939-0398, Japan

    Seiji Okazaki, Shogo Nakano, Atsutoshi Ina & Yasuhisa Asano

Authors
  1. Wisarut Payoungkiattikun
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  2. Seiji Okazaki
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  3. Shogo Nakano
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  4. Atsutoshi Ina
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  5. Aran H-Kittikun
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  6. Yasuhisa Asano
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Correspondence to Aran H-Kittikun or Yasuhisa Asano.

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Payoungkiattikun, W., Okazaki, S., Nakano, S. et al. In Silico Identification for α-Amino-ε-Caprolactam Racemases by Using Information on the Structure and Function Relationship. Appl Biochem Biotechnol 176, 1303–1314 (2015). https://doi.org/10.1007/s12010-015-1647-6

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  • DOI: https://doi.org/10.1007/s12010-015-1647-6

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