Investigating the Antioxidant Capacity of Lunasin Expressed in Aspergillus oryzae

Year 2024, , 67 - 76, 30.05.2024

Elif Karaman , Cem Albayrak , Serdar Uysal

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

Abstract

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.

Keywords

Lunasin, Aspergillus oryzae, Antioxidant, Biotechnology

Supporting Institution

Bezmialem Vakif University

Project Number

20220404

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