Izvestiya of Saratov University.

Physics

ISSN 1817-3020 (Print)
ISSN 2542-193X (Online)

For citation:

Larionova O. S., Drevko Y. B., Tychinin N. D., Krylova L. S., Drevko B. I., Larionov S. V. Optimization of methods for isolation and identification of peptides isolated from Hermetia illucens larvae. Izvestiya of Saratov University. Physics , 2024, vol. 24, iss. 2, pp. 150-160. DOI: 10.18500/1817-3020-2024-24-2-150-160, EDN: YJIWQU

This is an open access article distributed under the terms of Creative Commons Attribution 4.0 International License (CC-BY 4.0).
Published online: 
28.06.2024
Full text:
download
(downloads: 69)
Language: 
Russian
Heading: 
Biophysics and medical physics
Article type: 
Article
UDC: 
60:543.645.6:591.342.5
DOI: 
10.18500/1817-3020-2024-24-2-150-160
EDN: 
YJIWQU

Optimization of methods for isolation and identification of peptides isolated from Hermetia illucens larvae

Autors: 
Larionova Olga Sergeevna, Saratov State University of Genetics, Biotechnology and Engineering named after N. I. Vavilov
Drevko Yaroslav Borisovech, Saratov State University of Genetics, Biotechnology and Engineering named after N. I. Vavilov
Tychinin Nikolay D., Saratov State University of Genetics, Biotechnology and Engineering named after N. I. Vavilov
Krylova Lyubov S., Saratov State Agrarian University named after N.I. Vavilov
Drevko Boris I., Saratov State Agrarian University named after N.I. Vavilov
Larionov Sergey Vasilievich, Saratov State University of Genetics, Biotechnology and Engineering named after N. I. Vavilov
Abstract: 

Background and Objectives: The development of resistance of microorganisms to existing antibacterial agents requires constant updating of existing drugs and research in the search for alternative sources of active substances. In recent years, the problem of the emergence of microorganisms resistant to all existing antimicrobial drugs has become systematic and requires significant attention from researchers to search for alternative sources of active substances. The main problem in the development of drugs based on antimicrobial peptides is the search for optimal solutions in the preparation of these substances. Therefore, optimization and search for methods of isolation, analysis and control of protein fractions of water-soluble peptides used for the subsequent development of antibacterial drugs based on them is an urgent task. Materials and Methods: Optimal conditions and methods have been selected for the preparative production of water-soluble peptides isolated from the biomass of Hermetia illucens larvae. Optimization and search of methods for isolation, analysis and control of protein fractions of these water-soluble peptides will ensure the accuracy of the results and obtain optimal amounts of protein fractions. Results: It has been found that the use of molecular sieves makes it possible to obtain a mixture of three peptides with a difference in chromatographic retention time of less than 1 minute, which has been confirmed by three parallel experiments on the isolation and purification of peptides. During HPLC it has been noted that protein fractions 1 and 2 have similar values and the first and third protein fractions have a smaller difference in retention time, which is why there is no complete separation of these chromatographic peaks. Comparison of the percentage of the area of the peptides obtained allows us to talk about the possibility of obtaining peptides of the same size from H. illucens larvae by HPLC, and in combination with DLS to obtain protein fractions with very similar physicochemical and physical characteristics, since this type of chromatography separates substances according to their size. Conclusion: The use of high-performance liquid chromatography makes it possible to establish the reproducibility of the method of isolation of antimicrobial peptides by cold extraction with water and further stages of protein purification, salting and molecular sieve chromatography, which, in correlation with DLS analysis, makes it possible to reliably identify the peptides obtained, and the developed technology of isolation and purification makes it possible to produce these proteins on an industrial scale at low cost.

Key words: 
water-soluble peptides
high-performance liquid chromatography
ultraviolet detector
dynamic light scattering
Acknowledgments: 
The research was supported by the Russian Science Foundation (project No. 22-26-00167, https://rscf.ru/project/22-26-00167/).
Reference: 
  1. Wright G. D. QA Antibiotic resistance where does it come from and what can we do about it. BMC Biology, 2010, vol. 8, pp. 123. https://doi.org/10.1186/1741-7007-8-123
  2. Arias C. A., Murray B. E. Emergence and management of drug-resistant enterococcal infections. Expert Rev. Anti. Infect. Ther., 2008, vol. 6, no. 5, pp. 637–655. https://doi.org/10.1586/14787210.6.5.637
  3. Martens E., Demain A. L. The antibiotic resistance crisis, with a focus on the United States. J. Antibiot., 2017, vol. 70, no. 5, pp. 520–526. https://doi.org/10.1038/ja.2017.30
  4. Payne D. J., Gwynn M. N., Holmes D. J., Pompliano D. L. Drugs for bad bugs: Confronting the challenges of antibacterial discovery. Nat. Rev. Drug Discov., 2007, vol. 6, pp. 29–40. https://doi.org/10.1038/nrd2201
  5. Tommasi R., Brown D. G., Walkup G. K., Manchester J. I., Miller A. A. ESKAPEing the labyrinth of antibacterial discovery. Nat. Rev. Drug Discov., 2015, vol. 14, pp. 529–542. https://doi.org/10.1038/nrd4572
  6. Manniello D., Moretta A., Salvia R., Scieuzo C., Lucchetti D., Vogel H., Sgambato A., Falabella P. Insect antimicrobial peptides: Potential weapons to counteract the antibiotic resistance. Cel. Mol. Life Sci., 2021, vol. 78, no. 9, pp. 4259–4282. https://doi.org/10.1007/s00018-021-03784-z
  7. Lu H. L., Leger R. S. Insect immunity to Entomopathogenic fungi. Adv. Genet., 2016, vol. 94, pp. 251–285. https://doi.org/10.1016/bs.adgen.2015.11.002
  8. Hultmark D., Steiner H., Rasmuson T., Boman H. G. Insect immunity. Purification and properties of three inducible bactericidal proteins from hemolymph of immunized pupae of Hyalophora cecropia. Eur. J. Biochem., 1980, vol. 106, pp. 7–16. https://doi.org/10.1111/j.1432-1033.1980.tb05991
  9. Ursic-Bedoya R., Buchhop J., Joy J. B., Durvasula R., Lowenberger C. Prolixicin: A novel antimicrobial peptide isolated from Rhodnius prolixus with differential activity against bacteria and Trypanosoma cruzi. Insect Mol. Biol., 2011, vol. 20, pp. 775–786. https://doi.org/10.1111/j.1365-2583.2011.01107
  10. Vilcinskas A. Anti-infective therapeutics from the Lepidopteran model host Galleria mellonella. Curr. Pharm. Des., 2011, vol. 17, pp. 1240–1245. https://doi.org/10.2174/138161211795703799
  11. Vonkavaara M., Pavel S. T. I., Hölzl K., Nordfelth R., Sjöstedt A., Stöven S. Francisella is sensitive to insect antimicrobial peptides. J. Innate Immun., 2013, vol. 5, pp. 50–59. https://doi.org/10.1159/000342468
  12. Kruse T., Kristensen H. H. Using antimicrobial host defense peptides as anti-infective and immunomodulatory agents. Expert Rev. Anti. Infect. Ther., 2008, vol. 6, no. 6, pp. 887–895. https://doi.org/10.1586/14787210.6.6.887
  13. Chernysh S., Kim S. I., Bekker G., Pleskach V. A., Filatova N. A., Anikin V. B., Platonov V. G., Bulet P. Antiviral and antitumor peptides from insects. Proc. Natl. Acad. Sci. USA, 2002, vol. 99, pp. 12628–12632. https://doi.org/10.1073/pnas.192301899
  14. Thomas S., Andrews A. M., Hay N. P., Bourgoise S. The anti-microbial activity of maggot secretions: Results of a preliminary study. J. Tissue Viability, 1999, vol. 9, no. 4, pp. 127–132. https://doi.org/10.1016/s0965-206x(99)80032-1
  15. Bexfield A., Nigam Y., Thomas S., Ratcliffe N. A. Detection and partial characterization of two antibacterial factors from the excretions/secretions of the medicinal maggot Lucilia sericata and their activity against methicillin-resistant Staphylococcus aureus (MRSA). Microbes Infect., 2004, vol. 6, no. 14, pp. 1297–1304. https://doi.org/10.1016/j.micinf.2004.08.011
  16. Huberman L., Gollop N., Mumcuoglu K. Y., Block C., Galun R. Antibacterial properties of whole-body extracts and haemoloymph of Lucilia sericata maggots. J. Wound Care., 2007, vol. 16, no. 3, pp. 123–127. https://doi.org/10.12968/jowc.2007.16.3.27011
  17. Jaklic D., Lapanje A., Zupancic K., Smrke D., Gunde-Cimerman N. Selective antimicrobial activity of maggots against pathogenic bacteria. J. Med. Microbiol., 2008, vol. 57 (pt. 5), pp. 617–625. https://doi.org/10.1099/jmm.0.47515-0
  18. Arora S., Lim C. S., Baptista C. Antibacterial activity of Lucilia cuprina maggot extracts and its extraction techniques. Int. J. Integr. Biol., 2010, vol. 9, no. 1, pp. 43–48.
  19. Arora S., Baptista C., Lim C. S. Maggot metabolites and their combinatory effects with antibiotic on Staphylococcus aureus. Ann. Clin. Microbiol. Antimicrob., 2011, vol. 10, no. 6, pp. 1–8. https://doi.org/10.1186/1476-0711-10-6
  20. Barnes K. M., Gennard D. E., Dixon R. A. An assessment of the antibacterial activity in larval excretion/ secretion of four species of insects recorded in association with corpses, using Lucilia sericata Meigen as the marker species. Bull. Entomol. Res., 2010, vol. 100, no. 6, pp. 635–640. https://doi.org/10.1017/S000748530999071X
  21. Masiero F. S., Aquino M. F. K., Nassu M. P., Pereira D. I. B., Leite D. S., Thyssen P. J. First record of larval secretions of Cochliomyia macellaria (Fabricius, 1775) (Diptera: Calliphoridae) inhibiting the growth of Staphylococcus aureus and Pseudomonas aeruginosa. Neotrop. Entomol., 2017, vol. 46, no. 1, pp. 125–129. https://doi.org/10.1007/s13744-016-0444-4
  22. El-Bassiony G. M., Stoffolano J. G. In vitro antimicrobial activity of maggot excretions/secretions of Sarcophaga (Liopygia) argyrostoma (Robineau[1]Desvoidy). Afr. J. Microbiol. Res., 2016, vol. 10, no. 27, pp. 1036–1043. https://doi.org/10.5897/AJMR2016.8102
  23. Schweizer M., Green B. E., Segal K. I., Lodges D. Burcon Nutrascience MB Corp. Soluble Canola Protein Isolate Production. Patent no. 2475036 RF, 2013 (in Russian).
  24. Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J. Protein measurement with Folin phenol reagent. J. Biol. Chemistry, 1951, vol. 193, no. 1, pp. 265–275.
  25. Larionova O. S., Drevko Ya. B., Khanadeev V. A., Gorbunova S. V., Kozlov E. S., Larionov S. V. Analysis of protein fractions of water-soluble peptides by dynamic light scattering. Izvestiya of Saratov University. Physics, 2023, vol. 23, iss. 1, pp. 37–45 (in Russian). https://doi.org/10.18500/1817-3020-2023-23-1-37-45
  26. Hong P., Koza S., Bouvier E. S. P. A Review size-exclusion chromatography for the analysis of protein biotherapeutics and their aggregates. J. Liq. Chromatogr. RT, 2012, vol. 35, pp. 2923–2950. https://doi.org/10.1080/10826076.2012.743724
  27. Patten P. A., Schellekens H. The immunogenicity of biopharmaceuticals: Lessons learned and consequences for protein drug development. Dev. Biol. (Basel), 2003, vol. 112, pp. 81–97.
  28. Rosenberg A. S. Effects of protein aggregates: An immunologic perspective. AAPS J., 2006, vol. 8, pp. 501–507. https://doi.org/10.1208/aapsj080359
  29. Philo J. S. A critical review of methods for size characterization of non-particulate protein aggregates. Curr. Pharm. Biotechnol., 2009, vol. 10, pp. 359–372. https://doi.org/10.2174/138920109788488815
  30. Striegel A., Yau W. W., Kirkland J. J., Bly D. D. Modern size-exclusion liquid chromatography: Practice of gel permeation and gel filtration chromatography. 2nd ed. New York, Wiley, 2009. 512 p.
  31. Wu C. S. Handbook of size-exclusion chromatography and related techniques. New York, Marcel Dekker, 2003. 720 p.
  32. Khlebtsov B. N., Pylaev T. E., Khanadeev V. A., Khlebtsov N. G. Application of absorption and dynamic light scattering spectroscopy in studies of gold nanoparticle + DNA systems. Izvestiya of Saratov University. Physics, 2017, vol. 17, iss. 3, pp. 136–149 (in Russian). https://doi.org/10.18500/1817-3020-2017-17-3-136-149
Received: 
30.10.2023
Accepted: 
21.02.2024
Published: 
28.06.2024
  • 223 reads