Shewanella oneidensis PKA 1008 이 생성한 알긴산 분해 효소의 정제 및 특성

Abstract

Recently polysaccharides of marine algae was extensively studied due to their various biological functions including antitumor, antiviral, anticoagulant and anti-inflammatory activities. Although polysaccharides of marine algae are known to possess numerous beneficial properties, their industrial applications have been limited due to low inclusion efficiency and high cost of manufacturing involved in chemical hydrolysis. In addition, the smell has been a limiting factor in its application in the food, cosmetic and pharmaceutical industries. Alginic acid is one of polysaccharide and a co-polymer of D-mannuronic acid and L-guluronic acid. Alginic acid have found application in pharmaceutical and food industries owing to their ability to form viscous solutions at relatively low concentration and to form gels with Ca2+. These physical properties are strongly influenced by the ratio and arrangement of the mannuronic acid and guluronic acid residues in the polymer. And many studies investigated that alginate oligosaccharide had many biological functions including antitumor, antibacterial and antiviral. So to improve alginic acid using, numerous studies were conducted to screening of alginate degrading bacteria and purification of alginate degrading enzyme. Alginate-degrading enzyme was purified in the past decade from Streptomyces sp., Sphingomoans sp., Pseudomonas atlantica, Alginovibrio aquatilis, Azotobacter vinelandii, Vibrio sp., but it is not found that high activity of alginate degrading enzyme applied to industry. This study was conducted to screening the alginate-degrading microorganisms and purification and characterization of alginate degrading enzyme. We isolated marine bacteria produced extracellular alginate-degrading enzyme. And the results of identification through 16S rRNA sequence analysis, the marine bacteria belonged to the genus Shewanella oneidensis strain and it was named Shewanella oneidensis PKA 1008. Its optimal culture condition was measured by spectrophotometry and alginate-degrading ability was measured by reducing sugar assay and viscomerty. The optimal culure conditions for growth of Shewanella oneidensis strain were pH 9, 2% NaCl, 30℃ and 24 hr incubation time. And optimal conditions of alginate-degrading ability of crude enzyme were pH 9, 30℃, 7% alginic acid and 48 hr incubation time. Alginate-degrading enzyme produced by Shewanella oneidensis PKA 1008 was purified by ammonium sulfate salting, dialysis, sephadex G-25 column, sephadex G-100, DEAE-sephadex column and sephacrly S-200 HR column.