Shewanella oneidensis PKA 1008 유래 조효소에 의한 알긴산과 참모자반(Sargassum fulvellum) 분해당의 특성 및 면역 조절능

Abstract

Alginate is a high-molecular-weight polysaccharide whose physicochemical properties limit bioavailability. Therefore, depolymerization into low-molecular-weight oligosaccharide is essential. Enzymatic degradation offers substrate specificity and low by-products. Sargassum fulvellum, a brown seaweed abundant along Korean coasts, provides a source of bioactive polysaccharides for high-value applications. In this study, physicochemical characteristics and immunomodulatory properties of alginate and S. fulvellum extract degradation products generated by an alginate lyase present in the crude enzyme extracted from Shewanella oneidensis PKA 1008 were evaluated. Alginate and S. fulvellum extract was incubated with the crude enzyme extract (1:1 v/v) at 30℃ for 0–72 h. The pH, color values, reducing sugar content, viscosity were monitored, and thin‐layer chromatography (TLC) was performed to characterize the degradation products. Immunomodulatory activity was assessed by measuring nitric oxide (NO) and cytokine secretion in RAW 264.7 macrophages (TNF-α, IL-6), as well as by evaluating proliferation, cytokine release (IL-2, IFN-γ, TNF-α, IL-4), and natural killer (NK) cell activation in murine splenocytes in vitro and in vivo. In alginate, with increasing reaction time, pH and viscosity progressively decreased while reducing sugars and color values all increased. TLC analysis confirmed dimer and trimer formation at 72 h. Macrophage assay showed no cytotoxicity at any time, and high-concentration samples markedly enhanced NO production and elicited dose-dependent TNF-α secretion, whereas IL- 6 remained undetectable. In vitro splenocyte treatment, 48 h product promoted splenocyte cell viability and cytokine(IL-2, IFN-γ, TNF-α) release at low- doses. In vivo oral administration, 12 h product maximized proliferation, IL-2 and IL-4; 48 h product peaked in IFN-γ and NK-cell activation. For S. fulvellum, the 72 h treatment yielded the lowest pH and viscosity along with highest reducing sugar content. TLC analysis confirmed the formation of dimer and trimer at 72 h. Macrophage assay showed no cytotoxicity at any time, and a dose-dependent decrease in NO production and cytokines release. In vitro, splenocytes treated with the products showed a concentration-dependent increase in IL-2 and IFN-γ secretion that peaked with the 48 h product, while TNF-α release was highest with the 0 h product. In vivo oral administration, 48 h product maximized splenocyte proliferation, IL-2 and IFN-γ secretion, as well as NK-cell activation. Based on these results, the alginate lyase in S. oneidensis PKA 1008 crude enzyme extract was confirmed to produce maximally depolymerized alginate and S. fulvellum extract at 72 h, while degradation products at 12 h and 48 h demonstrated significant immunomodulatory effects. These findings support the potential application as immunomodulatory agents.