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 Tenebrio 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. Optimum of the proteolytic activity was observed at pH 9.0. The enzyme was stable at the alkaline pH range, and in the presence of BSA also in the acidic region. Peptidase was inhibited by synthetic inhibitors such as PMSF, TPCK, 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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About the authors

V. F. Tereshchenkova

Lomonosov Moscow State University, Faculty of Chemistry

Author for correspondence.
Email: elp@belozersky.msu.ru
Russian Federation, Moscow

N. I. Zhiganov

Lomonosov Moscow State University, Faculty of Biology

Email: elp@belozersky.msu.ru
Russian Federation, Moscow

A. S. Gubaeva

Lomonosov Moscow State University, Faculty of Chemistry

Email: elp@belozersky.msu.ru
Russian Federation, Moscow

F. I. Akentyev

National Research Center “Kurchatov Institute”; “Kurchatov Genome Center”, National Research Center “Kurchatov Institute”

Email: elp@belozersky.msu.ru
Russian Federation, Moscow; Moscow

Ya. E. Dunaevsky

Lomonosov Moscow State University, A.N. Belozersky Research Institute of Physico-Chemical Biology

Email: elp@belozersky.msu.ru
Russian Federation, Moscow

D. G. Kozlov

National Research Center “Kurchatov Institute”

Email: elp@belozersky.msu.ru
Russian Federation, Moscow

M. A. Belozersky

Lomonosov Moscow State University, A.N. Belozersky Research Institute of Physico-Chemical Biology

Email: elp@belozersky.msu.ru
Russian Federation, Moscow

E. N. Elpidina

Lomonosov Moscow State University, A.N. Belozersky Research Institute of Physico-Chemical Biology

Email: elp@belozersky.msu.ru
Russian Federation, Moscow

References

  1. de Souza A.N., Martins M.L. // Braz. J. Microbiol. 2001. V. 32. № 4. P. 271–275. https://doi.org/10.1590/S1517-83822001000400003
  2. Zambare V.P., Nilegaonkar S.S., Kanekar P.P. // World J. Microbiol. Biotechnol. 2007. V. 23. P. 1569–1574. https://doi.org/10.1007/s11274-007-9402-y
  3. Abidi F., Limam F., Nejib M.M. // Process Biochem. 2008. V. 43. № 11. P. 1202–1208. https://doi.org/10.1016/ j.procbio.2008.06.018
  4. Kumar C.G., Takagi H. // Biotechnol. Adv. 1999. V. 17. № 7. P. 561–594. https://doi.org/10.1016/s0734-9750(99)00027-0
  5. Singh J., Vohra R., Sahoo D. // Biotechnol. Lett. 1999. V. 21. P. 921–924. https://doi.org/10.1023/A:1005502824637
  6. Anwar A., Saleemuddin M. // Arch. Insect. Biochem. Physiol. 2002. V. 51. № 1. P. 1–12. https://doi.org/10.1002/arch.10046
  7. Vinokurov K.S., Elpidina E.N., Oppert B., Prabhakar S., Zhuzhikov D.P., Dunaevsky Y.E., Belozersky M.A. // Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 2006 b. V. 145. № 2. P. 138–146. https://doi.org/10.1016/j.cbpb.2006.05.004
  8. Sanatan P.T., Lomate P.R., Giri A.P., Hivrale V.K. // BMC Biochem. 2013. V. 14. https://doi.org/10.1186/1471-2091-14-32
  9. Sharifi M., Chitgar M.G., Ghadamyari M., Ajamhasani M. // Romanian Journal of Biochemistry. 2012. V. 49. № 1. P. 33–47.
  10. Mahdavi A., Ghadamyari M., Sajedi R.H., Sharifi M., Kouchaki B. // J. Insect Sci. 2013. V. 13. https://doi.org/10.1673/031.013.8101
  11. Zou Z., Lopez D.L., Kanost M.R., Evans J.D., Jiang H. // Insect Mol. Biol. 2006. V. 15. № 5. P. 603–614. https://doi.org/10.1111/j.1365–2583.2006.00684.x
  12. Choo Y.M., Lee K.S., Yoon H.J., Lee S.B., Kim J.H., Sohn H.D., Jin B.R. // Eur. J. Entomol. 2007. V. 104. № 1. P. 1–7. https://doi.org/10.14411/eje.2007.001
  13. Jiang H., Vilcinskas A., Kanost M.R. // Adv. Exp. Med. Biol. 2010.V. 708. P. 181–204. https://doi.org/10.1007/978-1-4419-8059-5_10
  14. Kanost M.R., Jiang H. // Curr. Opin. Insect Sci. 2015. V. 11. P. 47–55. https://doi.org/10.1016/j.cois.2015.09.003
  15. Cao X., Jiang H. // Insect Biochem. Mol. Biol. 2018. V. 103. P. 53–69. https://doi.org/10.1016/j.ibmb.2018.10.006
  16. Cao X., Gulati M., Jiang H. // Insect Biochem. Mol. Biol. 2017. V. 88. P. 48–62. https://doi.org/10.1016/j.ibmb.2017.07.008
  17. Lin H., Xia X., Yu L., Vasseur L., Gurr G. M., Yao F., Yang G., You M. // BMC Genomics. 2015. V. 16. Article 1054. https://doi.org/10.1186/s12864-015-2243-4
  18. Elpidina E.N., Tsybina T.A., Dunaevsky Y.E., Belozersky M.A., Zhuzhikov D.P., Oppert B. // Biochimie. 2005. V. 87. № 8. P. 771–779. https://doi.org/10.1016/j.biochi.2005.02.013
  19. Sato P.M., Lopes A.R., Juliano L., Juliano M.A., Terra W.R. // Insect Biochem. Mol. Biol. 2008. V. 38. № 6. P. 628–633. https://doi.org/10.1016/j.ibmb.2008.03.006
  20. Perona J.J., Tsu C.A., Craik C.S., Fletterick R.J. // Biochemistry. 1997. V. 36. № 18. P. 5381–5392. https://doi.org/10.1021/bi9617522
  21. Bown D.P., Wilkinson H.S., Gatehouse J.A. // Insect Biochem. Mol. Biol. 1997. V. 27. № 7. P. 625–638. https://doi.org/10.1016/s0965-1748(97)00043-x
  22. Tsu C.A., Perona J.J.., Schellenberger V., Turck C.W., Craik C.S. //J. Biol. Chem. 1994. V. 269. № 30. P. 19565–19572.
  23. Tsu C.A., Craik C.S. // J. Biol. Chem. 1996. V. 271. № 19. P. 11563–11570. https://doi.org/10.1074/jbc.271.19.11563
  24. Whitworth S.T., Blum M.S., Travis J. // J. Biol. Chem. 1998. V. 273. № 23. P. 14430–14434. https://doi.org/10.1074/jbc.273.23.14430
  25. Houben-Weyl Methods of Organic Chemistry: Synthesis of Peptides and Peptidomimetics, 4th Ed. V. E22a. In: / Eds. Goodman M., Toniolo C., Moroder L., Felix A. Stuttgart, NY: Thieme, 2004. 785 p.
  26. Горбунов А.А., Акентьев Ф.И., Губайдуллин И.И., Жиганов Н.И., Терещенкова В.Ф., Элпидина Е.Н., Козлов Д.Г. // Биотехнология. 2020. Т. 36. № 6. С. 136–145.
  27. Frugoni J.A.C. // Gazz. Chem. Ital. 1957. V. 87. P. 403–407.
  28. Elpidina E.N., Semashko T.A., Smirnova Y.A., Dvoryakova E.A., Dunaevsky Y.E., Belozersky M.A., et al. // Anal. Biochem. 2019. V. 567. P. 45–50. https://doi.org/10.1016/j.ab.2018.12.001
  29. Krahn J., Stevens F.C. // Biochemistry. 1970. V. 9. № 13. P. 2646–2652. https://doi.org/ 10.1021/bi00815a013
  30. De Vonis Bidlingmeyer U., Leary T.R., Laskowski M.Jr. // Biochemistry. 1972. V. 11. № 17. P. 3303–3310. https://doi.org/10.1021/bi00767a028
  31. Vinokurov K.S., Elpidina E.N., Oppert B., Prabhakar S., Zhuzhikov D.P., Dunaevsky Y.E., Belozersky M.A. // Comp. Biochem. Physiol. B Biochem. Mol. Biol. 2006 a. V. 145. № 2. P. 126–137. https://doi.org/10.1016/j.cbpb.2006.05.005
  32. Zhu Y.C., Baker J.E. // Arch. Insect. Biochem. Physiol. 2000. V. 43. № 4. P. 173–184. https://doi.org/10.1002/(SICI)1520-6327(200004) 43:4<173:: AID-ARCH3>3.0.CO;2-8
  33. Mazumdar-Leighton S., Broadway R.M. // Insect Biochem. Mol. Biol. 2001. V. 31. № 6–7. P. 633–644. https://doi.org/10.1016/s0965-1748(00)00168-5
  34. Herrero S., Combes E., Van Oers M.M., Vlak J.M., de Maagd R.A., Beekwilder J. // Insect Biochem. Mol. Biol. 2005. V. 35. № 10. P. 1073–1082. https://doi.org/10.1016/j.ibmb.2005.05.006
  35. Tamaki F.K., Padilha M.H., Pimentel A.C., Ribeiro A.F., Terra W. R. // Insect Biochem. Mol. Biol. 2012. V. 42. № 7. P. 482–490. https://doi.org/10.1016/j.ibmb.2012.03.005
  36. Matsuoka T., Kawashima T., Nakamura T., Yabe T. // Biosci. Biotechnol. Biochem. 2017. V. 81. № 7. P. 1401–1404. https://doi.org/10.1080/09168451.2017.1318698
  37. Gráf L., Szilágyi L., Venekei I. Chapter 582 – Chymotrypsin. Handbook of Proteolytic Enzymes. 3 Ed. In: / Eds. Rawlings N. D., Salvesen G. Academic Press, 2013. P. 2626–2633. https://doi.org/10.1016/B978-0-12-382219-2.00582-2
  38. Botos I., Meyer E., Nguyen M., Swanson S.M., Koomen J.M., Russell D.H, Meyer E.F. // J. Mol. Biol. 2000. V. 298. № 5. P. 895–901. https://doi.org/10.1006/jmbi.2000.3699
  39. Perona J.J., Craik C.S. // Protein Sci. 1995. V. 4. № 3. P. 337–360. https://doi.org/10.1002/pro.5560040301

Supplementary files

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2. Fig. 1. Sequence of rProSerP38. R| – place of processing by trypsin (cleavage of propeptide). Amino acid residues of the active center (HDS) are marked in red, and substrate-binding subsite S1 (GGD) – in blue.

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3. Fig. 2. Dependence of the activity of the processed preparation of the recombinant propeptidase rProSerP38 T. molitor on the processing time. Error bars reflect the confidence interval.

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4. Fig. 3. Stability of the mature form of the recombinant T. molitor peptidase rSerP38. Error bars represent the confidence interval.

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5. Fig. 4. Stability of processed recombinant peptidase rProSerP38 of T. molitor. Error bars represent confidence intervals.

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6. Fig. 5. Purification of the processed preparation of the recombinant propeptidase rProSerP38 T. molitor by metal-chelate affinity chromatography on Ni2+-NTA agarose: 1 – enzyme activity on the substrate Suc-AAPF-pNA, 2 – imidazole concentration gradient (mM).

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7. Fig. 6. Result of post-electrophoretic testing of activity for the fluorogenic substrate Suc-AAPF-AMC: 1 – rProSerP38 preparation before processing; 2 – rProSerP38 peptidase preparation processed by trypsin; 3 – pooled eluate fractions 16–18 before desalting; 4 – pooled fractions 16–18 after desalting and concentration.

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8. Fig. 7. Stability of processed and purified T. molitor rProSerP38 peptidase preparation. Error bars represent confidence intervals.

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9. Fig. 8. Stability of the mature form of the recombinant T. molitor peptidase rSerP38 in the presence of BSA.

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10. Fig. 9. Effect of pH on the activity of recombinant peptidase rSerP38 T. molitor.

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11. Fig. 10. Effect of pH on the stability of recombinant peptidase rSerP38 T. molitor (7 nM): a) without BSA, incubation for 30 min; b) in the presence of BSA (0.62 μM).

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12. Fig. 11. Activity of recombinant peptidase rSerP38 T. molitor towards various substrates.

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13. Fig. 12. Dependence of the rate of the enzymatic reaction of hydrolysis of substrates by rSerP38 peptidase: a) Glp-AAF-pNA (0.1–1.0 μM); b) Suc-AAPF-pNA (0.1–1.0 mM); c) Ac-Y-pNA (0.1–3.0 mM).

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