Exploring the bioactive potential of peptides derived from the RuBisCO protein in Caulerpa racemosa: an in silico approach

Abstract

Caulerpa racemosa harbors a rich reservoir of bioactive peptides derived from RuBisCO, a photosynthetic enzyme with promising therapeutic potential. This study aimed to systematically identify and characterize bioactive peptides from C. racemosa RuBisCO using a multi-step in silico pipeline. Simulated proteolysis using 33 enzymes predicted peptides with 35 different biological activities using BIOPEP-UWM. In addition to traditional database screening, further computational filtering was conducted using physicochemical profiling (ExPASy ProtParam), bioactivity prediction (PeptideRanker), toxicity and allergenicity evaluation (ToxinPred, AllergenFP), and structure-based molecular docking against relevant therapeutic targets—angiotensin-I converting enzyme (ACE, PDB: 1O8A) and xanthine oxidase (XO, PDB: 3NRZ). Four peptides with high predicted bioactivity scores (>0.75) showed strong binding affinity (−169.00 to −252.29 kcal/mol) and favorable confidence scores, suggesting their possible use as dual-action therapeutic agents—with both antihypertensive and antioxidant effects. This integrative in silico approach demonstrates the therapeutic relevance of C. racemosa peptides and provides a framework for peptide prioritization prior to experimental validation.

Keywords

• Bioinformatics
• Computational
• Proteomics
• Seagrapes

References

1. Adams, C., Sawh, F., Green-Johnson, J., Jones Taggart, H., & Strap, J. (2020). Characterization of casein-derived peptide bioactivity: Differential effects on angiotensin-converting enzyme inhibition and cytokine and nitric oxide production. Journal of Dairy Science, 103(7), 5805-5815. https://doi.org/10.3168/jds.2019-17976
2. Akbarian, M., Khani, A., Eghbalpour, S., & Uversky, V. N. (2022). Bioactive Peptides: Synthesis, Sources, Applications, and Proposed Mechanisms of Action. International Journal of Molecular Sciences, 23(3). https://doi.org/10.3390/ijms23031445
3. Amin, M., Chondra, U., Mostafa, E., & Alam, M. (2021). Green seaweed Ulva lactuca, a potential source of bioactive peptides revealed by in silico analysis. Informatics in Medicine Unlocked, 33, 101099. https://doi.org/10.1016/j.imu.2022.101099
4. Andreatta, R. H., H. Liem, R. K., & Scheraga, H. A. (1971). Mechanism of Action of Thrombin on Fibrinogen, I. Synthesis of Fibrinogen-like Peptides, and Their Proteolysis by Thrombin and Trypsin. Proceedings of the National Academy of Sciences of the United States of America, 68(2), 253-256. https://doi.org/10.1073/pnas.68.2.253
5. Baharuddin, N. A., Halim, N. R. A., & Sarbon, N. M. (2016). Effect of degree of hydrolysis (DH) on the functional properties and angiotensin I-converting enzyme (ACE) inhibitory activity of eel (Monopterus sp.) protein hydrolysate. International Food Research Journal, 23(4), 1424-1431.
6. Bhat, Z. F., Kumar, S., & Bhat, H. F. (2015). Bioactive peptides of animal origin: A review. Journal of Food Science and Technology, 52(9), 5377-5392. https://doi.org/10.1007/s13197-015-1731-5
7. Capurso, G., Traini, M., Piciucchi, M., Signoretti, M., & Arcidiacono, P.G. (2019). Exocrine pancreatic insufficiency: prevalence, diagnosis, and management. Clinical and Experimental Gastroenterology, 12:129-139. https://doi.org/10.2147/CEG.S168266
8. Cermeño, M., Kleekayai, T., Amigo-Benavent, M., Harnedy-Rothwell, P., & FitzGerald, R.J. (2020). Current knowledge on the extraction, purification, identification, and validation of bioactive peptides from seaweed. Electrophoresis, 41(20), 1694–717. https://doi.org/10.1002/elps.202000153.

9. Contreras, M.delM., Gómez-Sala, B., Martín-Alvarez, P. J., Amigo, L., Ramos, M., & Recio, I. (2010). Monitoring the large-scale production of the antihypertensive peptides RYLGY and AYFYPEL by HPLC-MS. Analytical and bioanalytical chemistry, 397(7), 2825–2832. https://doi.org/10.1007/s00216-010-3647-2
10. Coscueta, E. R., Batista, P., Gomes, J. E., da Silva, R., & Pintado, M. M. (2022). Screening of novel bioactive peptides from goat casein: In silico to in vitro validation. International Journal of Molecular Sciences, 23(5), 2439. https://doi.org/10.3390/ijms23052439
11. Daliri, E.B., Oh, D.H., & Lee, B.H. (2017). Bioactive Peptides. Foods, 26, 6(5):32. https://doi.org/10.3390/foods6050032.
12. Dimitrov, I., Naneva, L., Doytchinova, I., & Bangov, I. (2014). AllergenFP: Allergenicity prediction by descriptor fingerprints. Bioinformatics, 30(6), 846-851. https://doi.org/10.1093/bioinformatics/btt619
13. Du, Z., & Li, Y. (2022). Review and perspective on bioactive peptides: A roadmap for research, development, and future opportunities. Journal of Agriculture and Food Research, 9, 100353. https://doi.org/10.1016/j.jafr.2022.100353
14. Ducrocq, M., Boire, A., Anton, M., Micard, V., & Morel, M. (2020). RuBisCO: A promising plant protein to enrich wheat-based food without impairing dough viscoelasticity and protein polymerisation. Food Hydrocolloids, 109, 106101. https://doi.org/10.1016/j.foodhyd.2020.106101
15. Erdmann, K., Cheung, B. W., & Schröder, H. (2008). The possible roles of food-derived bioactive peptides in reducing the risk of cardiovascular disease. The Journal of Nutritional Biochemistry, 19(10), 643-654. https://doi.org/10.1016/j.jnutbio.2007.11.010
16. Garcia-Vaquero, M., Meaney, S., & Tiwari, B. K. (2022). Bioactive Peptides from Algae: Traditional and Novel Generation Strategies, Structure-Function Relationships, and Bioinformatics as Predictive Tools for Bioactivity. Marine Drugs, 20(5). https://doi.org/10.3390/md20050317
17. Grácio, M., Oliveira, S., Lima, A., & Boavida Ferreira, R. (2023). RuBisCO as a protein source for potential food applications: A review. Food Chemistry, 419, 135993. https://doi.org/10.1016/j.foodchem.2023.135993
18. Graszkiewicz, A., Żelazko, M., & Trziszka, T. (2010). Application of pancreatic enzymes in hydrolysis of egg-white proteins. Polish Journal of Food and Nutrition Sciences, 60(1), 57-61.
19. Gupta, S., Kapoor, P., Chaudhary, K., Gautam, A., Kumar, R., Drug Discovery Consortium, O. S., & Raghava, G. P. S. (2013). In Silico Approach for Predicting Toxicity of Peptides and Proteins. PLOS ONE, 8(9), e73957. https://doi.org/10.1371/journal.pone.0073957
20. Hanwell, M.D., Curtis, D.E., Lonie, D.C., Vandermeersch, T., Zurek, E., & Hutchison, G. R. (2012). Avogaro: an advanced semantic chemical editor, visualization, and analysis platform. Journal of Cheminformatics, 4, 17. https://doi.org/10.1186/1758-2946-4-17
21. Hirata, H., Sonoda, S., Agui, S., Yoshida, M., Ohinata, K., & Yoshikawa, M. (2007). Rubiscolin-6, a δ opioid peptide derived from spinach Rubisco, has anxiolytic effect via activating σ1 and dopamine D1 receptors. Peptides, 28, 1998–2003. https://doi.org/10.1016/j.peptides.2007.07.024
22. Hirota, T., Ohki, K., Kawagishi, R., Kajimoto, Y., Mizuno, S., Nakamura, Y., & Kitakaze, M. (2007). Casein hydrolysate containing the antihypertensive tripeptides Val-Pro-Pro and Ile-Pro-Pro improves vascular endothelial function independent of blood pressure-lowering effects: contribution of the inhibitory action of angiotensin-converting enzyme. Hypertension research: official journal of the Japanese Society of Hypertension, 30(6), 489–496. https://doi.org/10.1291/hypres.30.489
23. Hsiao, N. W., Tseng, T. S., Lee, Y. C., Chen, W. C., Lin, H. H., Chen, Y. R., Wang, Y. T., Hsu, H. J., & Tsai, K. C. (2014). Serendipitous discovery of short peptides from natural products as tyrosinase inhibitors. Journal of chemical information and modeling, 54(11), 3099–3111. https://doi.org/10.1021/ci500370x
24. Ito, K., Hosoya, T., Yamazaki-Ito, T., Terada, Y., & Kawarasaki, Y. (2021). Dipeptidyl peptidase IV inhibitory dipeptides contained in hydrolysates of green tea grounds. Journal of Food Technology Research, 27(2), 329-334. https://doi.org/10.3136/fstr.27.329
25. Kang, A., Kim, J., Jin, K., & Choi, J. (2023). Antioxidant, Collagenase Inhibitory, and Antibacterial Effects of Bioactive Peptides Derived from Enzymatic Hydrolysate of Ulva australis. Marine Drugs, 21(9), 469. https://doi.org/10.3390/md21090469
26. Karami, Z., & Akbari-adergani, B. (2019). Bioactive food derived peptides: A review on correlation between structure of bioactive peptides and their functional properties. Journal of Food Technology Research, 56(2), 535-547. https://doi.org/10.1007/s13197-018-3549-4
27. Karlsson, I., Samuelsson, K., Ponting, D. J., Törnqvist, M., Ilag, L. L., & Nilsson, U. (2016). Peptide Reactivity of Isothiocyanates – Implications for Skin Allergy. Scientific Reports, 6(1), 1-12. https://doi.org/10.1038/srep21203
28. Kaur, A., Pati, P.K., Pati, A.M., & Nagpal, A.K. (2020). Physico-chemical characterization and topological analysis of pathogenesis-related proteins from Arabidopsis thaliana and Oryza sativa using in-silico approaches. PLoS ONE, 15(9): e0239836. https://doi.org/10.1371/journal.pone.0239836
29. Kose, A. (2021). In Silico Bioactive Peptide Prediction from The Enzymatic Hydrolysates of Edible Seaweed RuBisCO Large Chain. Turkish Journal of Fisheries and Aquatic Sciences, 21, 615-625. http://doi.org/10.4194/1303-2712-v21_12_04
30. Langyan, S., Khan, F. N., Yadava, P., Alhazmi, A., Mahmoud, S. F., Saleh, D. I., & Kee Zuan, A. T. (2021). In silico proteolysis and analysis of bioactive peptides from sequences of fatty acid desaturase 3 (FAD3) of flaxseed protein. Saudi Journal of Biological Sciences, 28(10), 5480-5489. https://doi.org/10.1016/j.sjbs.2021.08.027
31. Meng, Y., Zhang, X., Mezei, M., & Cui, M. (2011). Molecular Docking: A powerful approach for structure-based drug discovery. Current Computer-Aided Drug Design, 7(2), 146. https://doi.org/10.2174/157340911795677602
32. Minkiewicz, P., Iwaniak, A., & Darewicz, M. (2019). BIOPEP-UWM Database of Bioactive Peptides: Current Opportunities. International Journal of Molecular Sciences, 20(23). https://doi.org/10.3390/ijms20235978
33. Mostashari, P., Marszałek, K., Aliyeva, A., & Mousavi Khaneghah, A. (2022). The Impact of Processing and Extraction Methods on the Allergenicity of Targeted Protein Quantification as Well as Bioactive Peptides Derived from Egg. Molecules, 28(6), 2658. https://doi.org/10.3390/molecules28062658
34. Nakamura, Y., Yamamoto, N., Sakai, K., Okubo, A., Yamazaki, S., & Takano, T. (1995). Purification and characterization of angiotensin I-converting enzyme inhibitors from sour milk. Journal of dairy science, 78(4), 777–783. https://doi.org/10.3168/jds.S0022-0302(95)76689-9
35. Natesh, R., Schwager, S., Sturrock, E, Acharya, K. R. (2003). Crystal structure of the human angiotensin-converting enzyme–lisinopril complex. Nature 421, 551–554 (2003). https://doi.org/10.1038/nature01370
36. Ngo, D., Wijesekara, I., Vo, T., Van Ta, Q., & Kim, S. (2011). Marine food-derived functional ingredients as potential antioxidants in the food industry: An overview. Food Research International, 44(2), 523-529. https://doi.org/10.1016/j.foodres.2010.12.030
37. Nawaz, M. A., Kasote, D. M., Ullah, N., Usman, K., & Alsafran, M. (2024). RuBisCO: A sustainable protein ingredient for plant-based foods. Frontiers in Sustainable Food Systems, 8, 1389309. https://doi.org/10.3389/fsufs.2024.1389309
38. Okamoto, K., Matsumoto, K., Hille, R., Eger, B. T., Pai, E. F., & Nishino, T. (2004). The crystal structure of xanthine oxidoreductase during catalysis: implications for reaction mechanism and enzyme inhibition. Proceedings of the National Academy of Sciences of the United States of America, 101(21), 7931–7936. https://doi.org/10.1073/pnas.0400973101
39. Ornano, L., Donno, Y., Sanna, C., Ballero, M., Serafini, M., & Bianco, A. (2014). Phytochemical study of Caulerpa racemosa (Forsk.) J. Agarth, an invading alga in the habitat of La Maddalena Archipelago. Natural Product Research, 28(20), 1795-9. https://doi.org/10.1080/14786419.2014.945928
40. Purohit, K., Reddy, N., & Sunna, A. (2023). Exploring the Potential of Bioactive Peptides: From Natural Sources to Therapeutics. International Journal of Molecular Sciences, 25(3), 1391. https://doi.org/10.3390/ijms25031391
41. Roney, M., & Aluwi, M. F. F. M. (2024). The importance of in-silico studies in drug discovery. Intelligent Pharmacy. https://doi.org/10.1016/j.ipha.2024.01.010
42. Roshanak, S., Yarabbi, H., Shahidi, F., Tabatabaei Yazdi, F., Movaffagh, J., & Javadmanesh, A. (2023). Effects of adding poly-histidine tag on stability, antimicrobial activity and safety of recombinant buforin I expressed in periplasmic space of Escherichia coli. Scientific Report, 13(1), 1-16. https://doi.org/10.1038/s41598-023-32782-3
43. Sbroggio, M.F., Montilha, M.S, Figueiredo, Vrg De., Georgetti, S.R, & Kurozawa, L. (2016). Influence of the degree of hydrolysis and type of enzyme on antioxidant activity of okara protein hydrolysates. Food Science and Technology, 36, 375–81. https://doi.org/10.1590/1678-457X.000216.
44. Seppo, L., Jauhiainen, T., Poussa, T., & Korpela, R. (2003). A fermented milk high in bioactive peptides has a blood pressure–lowering effect in hypertensive subjects. The American Journal of Clinical Nutrition, 77(2), 326-330. https://doi.org/10.1093/ajcn/77.2.326
45. Shao, Y., Zhang, Y., Liu, J., & Tu, Z. (2021). Investigation into predominant peptide and potential allergenicity of ultrasonicated β-lactoglobulin digestion products. Food Chemistry, 361, 130099. https://doi.org/10.1016/j.foodchem.2021.130099
46. Stefano, E. D., Agyei, D., Njoku, E. N., & Udenigwe, C. C. (2018). Plant RuBisCO: An Underutilized Protein for Food Applications. Journal of the American Oil Chemists' Society, 95(8), 1063-1074. https://doi.org/10.1002/aocs.12104
47. Stennicke, H. R., & Breddam, K. (2012). Glutamyl Endopeptidase II. Handbook of Proteolytic Enzymes, 2556-2558. https://doi.org/10.1016/B978-0-12-382219-2.00566-4
48. Thiviya, P., Gamage, A., Suranjith, N., Merah, O., & Madhujith, T. (2022). Seaweeds as a Source of Functional Proteins. Phycology, 2(2), 216-243. https://doi.org/10.3390/phycology2020012
49. Udenigwe, C. C., & Aluko, R. E. (2012). Food protein-derived bioactive peptides: production, processing, and potential health benefits. Journal of food science, 77(1), R11–R24. https://doi.org/10.1111/j.1750-3841.2011.02455.x
50. Udenigwe, C. C., Okolie, C. L., Qian, H., Ohanenye, I. C., Agyei, D., & Aluko, R. E. (2017). Ribulose-1,5-bisphosphate carboxylase as a sustainable and promising plant source of bioactive peptides for food applications. Trends in Food Science & Technology, 69, 74-82. https://doi.org/10.1016/j.tifs.2017.09.001
51. Valegård, K., Andralojc, P.J., Haslam, R.P., Pearce, F.G., Eriksen, G.K., Madgwick, P.J., Kristoffersen, A.K., van Lun, M., Klein, U., Eilertsen, H.C., Parry, M.A.J., & Andersson, I. (2018). Structural and functional analyses of Rubisco from arctic diatom species reveal unusual posttranslational modifications. Journal of Biological Chemistry, 293(34), 13033-13043. https://doi.org//10.1074/jbc.RA118.003518
52. Wei, L., Ye, X., Sakurai, T., Mu, Z., & Wei, L. (2022). ToxIBTL: Prediction of peptide toxicity based on information bottleneck and transfer learning. Bioinformatics, 38(6), 1514-1524. https://doi.org/10.1093/bioinformatics/btac006
53. Windarto, S., Lee, M.C., Nursyam, H., & Hsu, J.L. (2022). First Report of Screening of Novel Angiotensin-I Converting Enzyme Inhibitory Peptides Derived from the Red Alga Acrochaetium sp. Marine Biotechnology, 24(5), 882-894. https://doi.org/10.1007/s10126-022-10152-w.
54. Windarto, S., M. Kamaludin, D., Bella, W., Sarjito, Pinandoyo, Susilowati, T., Haditomo, A. H. C., & Harwanto, D. (2023). Effect of coconut (Cocos nucifera) water, and aqueous extract of Mung bean (Vigna radiata) sprouts and Moringa (Moringa oleifera) leaf on the growth and nutrition of Caulerpa racemosa. International Journal of Aquatic Biology, 11(6), 513–522. https://doi.org/10.22034/ijab.v11i6.2044
55. Windarto, S., Hsu, J., & Lee, M. (2024a). First report of antioxidant potential of peptide fraction derived from Colaconema formosanum (Rhodophyta) protein hydrolysates. Biocatalysis and Agricultural Biotechnology, 58, 103232. https://doi.org/10.1016/j.bcab.2024.103232
56. Windarto, S., Lee, M., Nursyam, H., & Hsu, J. (2024b). A novel phycoerythrin-derived peptide from Colaconema formosanum: Synthesis, in vitro, and in silico study on angiotensin-converting enzyme (ACE) inhibitory activity. Biocatalysis and Agricultural Biotechnology, 62, 103452. https://doi.org/10.1016/j.bcab.2024.103452
57. Windarto, S., Susilowati, T., Haditomo, A.H.C., & Harwanto, D. (2024c). Effect of exogenous natural plant growth regulators (PGRs) on the morphology, growth, and nutrient of sea grapes (Caulerpa racemosa). Aquaculture International, 32, 3545–3562. https://doi.org/10.1007/s10499-023-01337-8
58. Yan, Y., Zhang, D., Zhou, P., Li, B., & Huang, S. Y. (2017). HDOCK: a web server for protein-protein and protein-DNA/RNA docking based on a hybrid strategy. Nucleic acids research, 45(W1), W365–W373. https://doi.org/10.1093/nar/gkx407
59. Yan, Y., Tao, H., He, J., Huang, S. Y. (2020). The HDOCK server for integrated protein–protein docking. Nature Protocols, 15, 1829–1852. https://doi.org/10.1038/s41596-020-0312-x
60. Zhao, Z., Gui, J., Yao, A., Khanh Le, N. Q., & Heng Chua, M. C. (2022). Improved Prediction Model of Protein and Peptide Toxicity by Integrating Channel Attention into a Convolutional Neural Network and Gated Recurrent Units. ACS Omega, 7(44), 40569-40577. https://doi.org/10.1021/acsomega.2c05881
61. Zloh, M., & Kirton, S. B. (2018). The Benefits of In Silico Modeling to Identify Possible Small-Molecule Drugs and Their Off-Target Interactions. Future Medicinal Chemistry, 10(4), 423–432. https://doi.org/10.4155/fmc-2017-0151