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A novel type IIb l-asparaginase from Latilactobacillus sakei LK-145: characterization and application

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Abstract

We succeeded in homogeneously expressing and purifying l-asparaginase from Latilactobacillus sakei LK-145 (Ls-Asn1) and its mutated enzymes C196S, C264S, C290S, C196S/C264S, C196S/C290S, C264S/C290S, and C196S/C264S/C290S-Ls-Asn1. Enzymological studies using purified enzymes revealed that all cysteine residues of Ls-Asn1 were found to affect the catalytic activity of Ls-Asn1 to varying degrees. The mutation of Cys196 did not affect the specific activity, but the mutation of Cys264, even a single mutation, significantly decreased the specific activity. Furthermore, C264S/C290S- and C196S/C264S/C290S-Ls-Asn1 almost completely lost their activity, suggesting that C290 cooperates with C264 to influence the catalytic activity of Ls-Asn1. The detailed enzymatic properties of three single-mutated enzymes (C196S, C264S, and C290S-Ls-Asn1) were investigated for comparison with Ls-Asn1. We found that only C196S-Ls-Asn1 has almost the same enzymatic properties as that of Ls-Asn1 except for its increased stability for thermal, pH, and the metals NaCl, KCl, CaCl2, and FeCl2. We measured the growth inhibitory effect of Ls-Asn1 and C196S-Ls-Asn1 on Jurkat cells, a human T-cell acute lymphoblastic leukemia cell line, using l-asparaginase from Escherichia coli K-12 as a reference. Only C196S-Ls-Asn1 effectively and selectively inhibited the growth of Jurkat T-cell leukemia, which suggested that it exhibited antileukemic activity. Furthermore, based on alignment, phylogenetic tree analysis, and structural modeling, we also proposed that Ls-Asn1 is a so-called “Type IIb” novel type of asparaginase that is distinct from previously reported type I or type II asparaginases. Based on the above results, Ls-Asn1 is expected to be useful as a new leukemia therapeutic agent.

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Acknowledgements

We thank Ms. Saho Kambe for the construction of pET21-Ls-asn1/Escherichia coli Rosetta (DE3). We thank Ms. Mai Sakata for her fundamental experiments on Ls-Asn1. We thank Ms. Miki Hatanaka for the construction of pET21-C196S, C264S, C290S, C196S/C264S, C196S/C290S, C264S/C290S, and C196S/C264S/C290S-Ls-asn1.

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Authors and Affiliations

  1. Faculty of Agriculture, Kagawa University, 2393 Ikenobe, Miki, Kagawa, 761-0795, Japan

    Shiro Kato

  2. Department of Microbiological Chemistry, Kobe Pharmaceutical University, 4-9-1 Motoyamakita-machi, Higashinada-ku, Kobe, Hyougo, 658-8558, Japan

    Yuki Masuda & Morichika Konishi

  3. Department of Life Science and Biotechnology, Faculty of Chemistry, Materials and Bioengineering, Kansai University, 3-3-35 Yamate-cho, Suita, Osaka-fu, 564-8680, Japan

    Kazuya Tamura, Kazuya Yamanaka & Tadao Oikawa

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  1. Shiro Kato
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  2. Kazuya Tamura
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  3. Yuki Masuda
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  4. Morichika Konishi
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  5. Kazuya Yamanaka
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  6. Tadao Oikawa
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Contributions

T. O. designed and supervised the studies. S. K., Y. M., and T. O. wrote the manuscript. K. T. helped the preparation of manuscript partly. K. T., Y. M., and M. K. carried out the experiments. S. K. performed the primary structure, phylogenetic, and modeling analyses. S. K. carried out the molecular docking simulation. K. Y. advised the experiments part.

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Correspondence to Tadao Oikawa.

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Communicated by PANKAJ BHATT.

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Kato, S., Tamura, K., Masuda, Y. et al. A novel type IIb l-asparaginase from Latilactobacillus sakei LK-145: characterization and application. Arch Microbiol 206, 266 (2024). https://doi.org/10.1007/s00203-024-03979-5

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  • DOI: https://doi.org/10.1007/s00203-024-03979-5

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