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Recombinant Chymotrypsin-like Peptidase from Tenebrio molitor with a Non-Canonical Substrate-Binding Site

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Abstract

We characterized an alkaline chymotrypsin-like serine peptidase from the yellow mealworm Te-nebrio molitor with a non-canonical substrate-binding subsite for its possible application as a component (an additive) in various biological products. The enzyme was obtained as a recombinant preparation. Purification was carried out using affinity chromatography on Ni2+-NTA agarose. The specificity constants (kcat/KM) for the chymotrypsin substrates, Glp-AAF-pNA, Suc-AAPF-pNA, and Ac-Y-pNA, were 7, 4.2, and 0.9 (µM min)–1, respectively. The optimum of the proteolytic activity was observed at pH 9.0. The enzyme was stable in the alkaline pH range, and in the presence of BSA, also in the acidic region. Peptidase was inhi-bited by synthetic inhibitors such as PMSF, TPCK, and chymostatin, while EDTA, E-64, and pepstatin had no effect on the enzyme activity. The purified enzyme showed high stability over time in the presence of BSA. The short life cycle of the insect and the production of a large number of peptidases in the midgut with high catalytic activity and stability can make T. molitor an excellent alternative source of industrially important enzymes for application as components (additives) in various biological products (e.g., stain removers, detergents, etc.).

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ACKNOWLEDGMENTS

The authors honor the memory of their colleague, Professor Irina Yuryevna Filippova, who directed and supervised this research until her untimely passing away.

Funding

This study was supported by the Russian Science Foundation, grant no. 22-24-00553, https://rscf.ru/project/22-24-00553/.

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Authors and Affiliations

  1. Moscow State University, Faculty of Chemistry, 119991, Moscow, Russia

    V. F. Tereshchenkova & A. S. Gubaeva

  2. Moscow State University, Faculty of Biology, 119991, Moscow, Russia

    N. I. Zhiganov

  3. National Research Center Kurchatov Institute, 123182, Moscow, Russia

    F. I. Akentyev & D. G. Kozlov

  4. Kurchatov Genomic Center, National Research Center Kurchatov Institute, 123182, Moscow, Russia

    F. I. Akentyev

  5. Moscow State University, A.N. Belozersky Research Institute of Physico-Chemical Biology, 119991, Moscow, Russia

    Ya. E. Dunaevsky, M. A. Belozersky & E. N. Elpidina

Authors
  1. V. F. Tereshchenkova
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  2. N. I. Zhiganov
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  8. E. N. Elpidina
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Contributions

Conceptualization, E.N. Elpidina; Data curation, D.G. Kozlov; Formal analysis, Ya.E. Dunaevsky; Investigation, V.F. Tereshchenkova, N.I. Zhiganov, A.S. Gubaeva and F.I. Akentyev; Methodology, V.F. Tereshchenkova; Validation, M.A. Belozersky; Writing (original draft), V.F. Tereshchenkova; Writing (review and editing), Ya.E. Dunaevsky and E.N. Elpidina. All authors have read and agreed to the published version of the manuscript.

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Correspondence to E. N. Elpidina.

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Tereshchenkova, V.F., Zhiganov, N.I., Gubaeva, A.S. et al. Recombinant Chymotrypsin-like Peptidase from Tenebrio molitor with a Non-Canonical Substrate-Binding Site. Appl Biochem Microbiol 60, 420–430 (2024). https://doi.org/10.1134/S0003683824603652

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