Investigating the Antioxidant Capacity of Lunasin Expressed in Aspergillus oryzae

Yıl 2024, , 67 - 76, 30.05.2024

Elif Karaman Cem Albayrak Serdar Uysal

https://doi.org/10.26650/EurJBiol.2024.1454771

Öz

Objective: Lunasin is a bioactive protein that possesses anti-carcinogenic, anti-inflammatory, and antioxidant properties. Traditional isolation methods are resource-intensive, and chemical synthesis faces cost and environmental issues. This study aims to achieve cost-effective lunasin expression in Aspergillus oryzae with a focus on exploring its antioxidant properties in vitro.
Materials and Methods: The expression vector carrying four lunasin sequences fused with amylase and an 8xHis-tag was introduced into pyrG auxotrophic A. oryzae. Subsequently, the recombinant protein was purified using metal affinity chromatography. The study uses sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot analyses, and size-exclusion chromatography to evaluate the composition and purity of the protein, a linoleic acid assay to demonstrate the inhibitory effect on lipid peroxidation, and the 2,2’-azinobis-[3-ethylbenzothiazoline-6-sulfonic acid] ABTS) assay to evaluate the radical scavenging activity.
Results: SDS-PAGE and western blot analyses confirmed sustained lunasin expression in A. oryzae, appearing in both fusion and non-fusion forms. Yields were 5.8 mg/L for non-fusion and 4 mg/L for fusion lunasin expression. Moreover, 0.1 μM non-fusion lunasin surpassed α-tocopherol and butylated hydroxyanisole (BHA; p < 0.05) in reducing lipid peroxidation at 4 and 72 h. Unlike the fusion lunasin, the non-fusion lunasin displayed concentration- and time-independent inhibitory effects on linoleic acid peroxidation as well as significant ABTS scavenging activity (p < 0.05).
Conclusion: The study has shown for the first time A. oryzae to efficiently express and secrete both fusion and non-fusion lunasin proteins in a soluble form, with the non-fusion lunasin exhibiting superior antioxidant effectiveness compared to the fusion lunasin. The findings underscore A. oryzae’s potential as a promising host for producing functional lunasin with antioxidant properties, opening avenues for broader applications in biotechnology and bioactive peptides.

Anahtar Kelimeler

Lunasin, Aspergillus oryzae, Antioxidant, Biotechnology

Destekleyen Kurum

Bezmialem Vakif University

Proje Numarası

20220404

Kaynakça

• Galvez AF, de Lumen BO. A soybean cDNA encoding a chromatin-binding peptide inhibits mitosis of mammalian cells. Nat Biotechnol. 1999;17(5):495-500. google scholar
• Hernandez-Ledesma B, Hsieh CC, Dia V, de Mejia EG, de Lumen BO. In: Soybean and Health. Lunasin, a cancer preventive seed peptide. Soybean and health: 2011:145-166. google scholar
• Gonzalez de Mejia EG, Dia VP. Lunasin and lunasin-like peptides inhibit inflammation through suppression of NF-kB pathway in the macrophage. Peptides. 2009; 30(12): 2388-2398. google scholar
• Hsieh CC, Hernandez-Ledesma B, Jeong HJ, Park JH, de Lumen BO. Complementary roles in cancer prevention: Protease inhibitor makes the cancer preventive peptide lunasin bioavailable. PloS One. 2010;5(1):e8890. doi:10.1371/journal.pone.0008890. google scholar
• Hernandez-Ledesma B, de Lumen BO, Hsieh, CC. 1997-2012: fifteen years of research on peptide Lunasin. In: Bioactive Food Peptides in Health and Disease. London, England: InTech Open: 2013: 3-22. doi:10.5772/52368. google scholar
• Chang HC, Lewis D, Tung CY, et al. Soypeptide lunasin in cytokine immunotherapy for lymphoma. Cancer Immunol Immunother. 2014;63:283-295. google scholar
• Fernandez-Tome S, Ramos S, Cordero-Herrera I, Recio I, Goya L, Hernandez-Ledesma B. In vitro chemo-protective effect of bioactive peptide lunasin against oxidative stress in human HepG2 cells. Food Res Inter. 2014;62:793-800. google scholar
• Garaa-Nebot MJ, Recio I, Hernandez-Ledesma B. Antioxidant activity and protective effects of peptide lunasin against oxidative stress in intestinal Caco-2 cells. Food Chem Toxicol. 2014;65:155-161. google scholar
• Fernandez-Tome S, Hernandez-Ledesma B. Current state of art after twenty years of the discovery of bioactive peptide lunasin. Food Res Inter. 2019;116:71-78. google scholar
• Kaufman-Szymczyk A, Kaczmarek W, Fabianowska-Majewska K, Lubecka-Gajewska K. Lunasin and its epigenetic impact in cancer chemoprevention. Inter J Mol Sci. 2023;24(11):9187. doi: 10.3390/ijms24119187 google scholar
• World Health Organization. WHO report on cancer: Setting pri-orities, investing wisely and providing care for all. World Health Organization. 2020 https://iris.who.int/handle/10665/330745. Li-cense: CC BY-NC-SA 3.0 IGO. google scholar
• Vuyyuri SB, Shidal C, Davis KR. Development of the plant-derived peptide lunasin as an anticancer agent. Curr Opin Pharmacol. 2018;41:27-33. google scholar
• Galvez AF, Chen N, Macasieb J, Ben O. Chemopreventive prop-erty of a soybean peptide (lunasin) that binds to deacetylated histones and inhibits acetylation. Cancer Res. 2001;61(20):7473-7478. google scholar
• Jia S, Zhang S, Yuan H, Chen N. Lunasin inhibits cell prolifera-tion via apoptosis and reduces the production of proinflammatory cytokines in cultured rheumatoid arthritis synovial fibroblasts. BioMed Res Inter. 2015;346839. doi:10.1155/2015/346839 google scholar
• Galvez AF. U.S. Patent No. 7,731,995. Washington, DC: U.S. Patent and Trademark Office, 2010. google scholar
• Gu L, Ye P, Li H, et al. Lunasin attenuates oxidant-induced endothelial injury and inhibits atherosclerotic plaque progres-sion in ApoE-/- mice by up-regulating heme oxygenase-1 via PI3K/Akt/Nrf2/ARE pathway. FASEB J. 2019;33(4):4836-4850. google scholar
• Odani S, Koide T, Ono T. Amino acid sequence of a soybean (Glycine max) seed polypeptide having a poly (L-aspartic acid) structure. J Biol Chem. 1987;262(220):10502-10505. google scholar
• He B, Tu Y, Jiang C, Zhang Z, Li Y, Zeng B. Functional genomics of Aspergillus oryzae: Strategies and progress. Microorganisms. 2019;7(103):1-13. google scholar
• Ntana F, Mortensen UH, Sarazin C, Figge R. Aspergillus: A pow-erful protein production platform. Catalysts. 2020;10(9):1064. doi: 10.3390/catal10091064 google scholar
• Karaman E, Eyupoglu AE, Mahmoudi Azar L, Uysal S. Large-scale production of anti-RNase A VHH expressed in pyrG auxotrophic Aspergillus oryzae. Curr Issues Mol Biol. 2023;45(6):4778-4795. google scholar
• Sakai K, Kinoshita H, Nihira, T. Heterologous expression system in Aspergillus oryzae for fungal biosynthetic gene clusters of sec-ondary metabolites. Appl Microbiol Biotechnol. 2012;93(5):2011-2022. google scholar
• Hernandez-Ledesma B, Hsieh CC, de Lumen BO. An-tioxidant and anti-inflammatory properties of cancer pre-ventive peptide Lunasin in RAW 264.7 macrophages. Biochem Biophys Res Commun. 2009;390(3):803-808. google scholar
• Gonzalez de Mejia E, Vasconez M, de Lumen BO, Nelson R. Lunasin concentration in different soybean genotypes, commer-cial soy protein, and isoflavone products. J Agric Food Chem. 2004;52(19):5882-5887. google scholar
• Liu CF, Pan TM. Recombinant expression of bioactive pep-tide lunasin in Escherichia coli. Appl Microbiol Biotechnol. 2010;88(1):177-186. google scholar
• Kyle S, James KA, McPherson MJ. Recombinant production of the therapeutic peptide lunasin. Microb Cell Fact. 2012;29:11-28. google scholar
• Setrerrahmane S, Zhang Y, Dai G, Lv J, Tan S. Efficient pro-duction of native lunasin with correct N-terminal processing by using the pH-induced self-cleavable Ssp DnaB mini-intein system in Escherichia coli. Appl Biochem Biotechnol. 2014;174(2):612-622. google scholar
• Tian Q, Zhang P, Gao Z, Li H, Bai Z, Tan S. Hirudin as a novel fusion tag for efficient production of lunasin in Escherichia coli. Prep Biochem Biotechnol. 2017;47(6):619-626. google scholar
• Zhu Y, Nadia E, Yao Y, Shi Z, Ren G. Tandem repeated expres-sion of lunasin gene in Pichia pastoris and its anti-inflammatory activity in vitro. J Biosci Bioeng. 2018;126(1):1-8. google scholar
• Jeong JB, De Lumen BO, Jeong HJ. Lunasin peptide purified from Solanum nigrum L. protects DNA from oxidative damage by suppressing the generation of hydroxyl radical via blocking fenton reaction. Cancer Lett. 2010;293(1):58-64. google scholar
• Aleksis R, Jaudzems K, Muceniece R, Liepinsh E. Lunasin is a re-dox sensitive intrinsically disordered peptide with two transiently populated a-helical regions. Peptides. 2016;85:56-62. google scholar
• Alves de Souza SM, Fernandes TVA, Kalume DE, T R Lima LM, Pascutti PG, de Souza TLF. Physicochemical and structural properties of lunasin revealed by spectro-scopic, chromatographic and molecular dynamics approaches. Biochim Biophys Acta Proteins Proteom. 2020;1868(8):140440. doi:10.1016/j.bbapap.2020.140440 google scholar