Shewanella oneidensis PKA 1008 유래 조효소에 의한 알긴산 분해패턴과 미야베모자반(Sargassum miyabei) 추출 분해물의 항염증 특성

Abstract

Alginate lyases have gained significant attention as biocatalysts for the preparation of functional oligosaccharides. Alginate lyases have become the focus of numerous studies aimed at the efficient utilization of alginate, with one approach being its degradation into smaller molecules. Crude enzyme was prepared from strain for further research. Sargassum miyabei belongs to the brown algae and contains various polysaccharides, including alginate, fucoidan, and mannitol. This study aimed to elucidate the alginate degradation patterns of a crude enzyme extracted from Shewanella oneidensis PKA 1008 and examine the characteristics of extracts prepared by incubating this enzyme with S. miyabei. To determine substrate specificity, alginate, polyM, and polyG were incubated at 30°C for 0-48 h in a 1:1 (v/v) ratio with the crude enzyme, and changes in reducing sugars and thin-layer chromatography (TLC) of the reactants were measured. The reducing sugar of polyM showed the maximum increment, reaching 171.72 μg/mL at 12 h, while polyG increased slightly. In comparison to alginate, the activity of polyM was observed to be 76 times that of alginate, while the activity of polyG demonstrated a value of 2.9 times that of alginate. After 12 h of incubation, polyM was mainly degraded to trisaccharides, while polyG degraded to a mixture of di-, tri-, and tetrasaccharides. To confirm the precise degradation patterns, purified alginate oligosaccharides (DP2-6) were reacted with the crude enzyme. In the case of mannuronic acid oligomers, hexamer (DP6) was degraded to a trisaccharide. In the case of guluronic acid oligomers, tetramer (DP4) was degraded to disaccharides, pentamer (DP5) to di- and trisaccharides, and hexamer (DP6) to di-, tri-, and tetrasaccharides. To characterize the brown algae extract, S. miyabei and the crude enzyme were mixed and reacted at 30°C for 0-72 h. The reducing sugar of S. miyabei increased significantly to 2421.60 μg/mL at 72 h. Significant decreases in pH and viscosity were observed at 72 h compared to initial measurements, and TLC revealed that di-, tri-, and tetrasaccharides were the primary products. These results indicated that the crude enzyme from Shewanella oneidensis PKA 1008 exhibits specificity for polyM and partial activity against polyG. The minimum substrates that could act were M6 and G4 also G5 and G6 could be degraded, suggesting that guluronate may have a broader substrate selectivity. It can act on both types of polysaccharides and be effectively degraded polysaccharides contained in S. miyabei.