Shewanella oneidensis PKA 1008 유래 알긴산 분해효소의 분리 및 산업적 이용가능성

Abstract

Alginate is a linear polysaccharide composed of β-D-mannuronate and α-L-guluronate, arranged in three different ways: poly-β-D-mannuronate, poly-α-L-guluronate, and hetero-polymeric region in which there is random arrangement of the monomer. Alginate oligosaccharide has many biological functions including antioxidant, anti-inflammatory, and anticholesterol activities. So, numerous studies are going on to improve the use of alginate, and one of them is lowering the molecular weight through alginate degradation. Thus, alginate lyases have attracted much attention as biocatalysts for preparation of functional oligosaccharide from alginate. This study was conducted to separate alginate degrading enzyme from Shewanella oneidensis PKA 1008 and investigate its biological activity. The optimal conditions of crude enzyme for alginate degrading activity were pH 9, 30℃, concentration of alginate 7% concentration and 48 h reaction time. Alginate degrading enzyme was purified by ammonium sulfate salting out, DEAE-sephadex, sephadex G-100, and 2nd DEAE-sephadex column chromatography. The total activity and specific activity of purified alginate degrading enzyme were 1.06 U and 25.238 U/mg, respectively. The optimal conditions for the purification of enzyme were pH 9.0, 30℃, and 0.05 M NaCl concentration. The activity of enzyme was inhibited by Ca2+, Mg2+, Ba2+, and Fe2+. To measure biological activity of alginate hydrolysate by crude enzyme from S. oneidensis PKA 1008, alginate and crude enzyme was incubated for 0-60 h and antimicrobial, antioxidant, and antiinflammatory activities were investigated. The degraded alginate was identified by thin layer chromatography. DPPH radical scavenging activity of alginate hydrolysate at 36-60 h was higher than that of alginate. In particular, the alginate hydrolysate at 60 h showed the highest DPPH radical scavenging activity of 82.10% at 5 mg/mL. The alginate hydrolysate showed significant anti-inflammatory effects. The production of NO and pro-inflammatory cytokines (IL-6, TNF-α, and IL-1β) was significantly decreased without cytotoxicity. In particular, NO level of alginate hydrolysate at 36 h were reduced by 55% at 100 μg/mL and the IL-6 and IL 1β production was decreased to 40% and 55% by alginate hydroysate at 60 h of 100 μg/mL. The edema formation in the ICR mouse ear was also reduced compared to that in control. These results indicated that alginate lyase from Shewanella oneidensis PKA 1008 can be useful for the production of alginate oligosaccharide and the alginate hydrolysate by alginate lyase has significant antioxidant and anti-inflammatory effects.