Preparation Of Alginate Oligosacchrides With Alginate Lyase

Alginic acid oligosaccharide is a functional oligomeric uronic acid that can be used in promoting plant and animal growth. It also has many other physiological activities. The preparation of alginate oligosaccharides was discussed in this study.Alginate hydrolysates were separated by gradient elution with aqueous acetic acid (Dowex 1×8). L-guluronic acid and D-mannuronic acid can be successfully purified by HPLC from the hydrolysis of sodium alginate, purity 99%. Then they were used as the standard to detect the L-guluronic acid and D-mannuronic acid in sodium alginate and seaweed. The HPLC detection result showed that L-guluronic acid and D-mannuronic acid in sodium alginate were 11.48%, 63.06% and 4.67%, 21.65% in seaweed. Alginate oligosaccharides which obtained by Dalian Institute of Chemical Physics, Chinese Academy of Sciences was used as samples. TLC, HPLC and FACE detection methods were compared. The results showed that three methods can be used in separation and detection on the alginate oligosaccharides. In this study TLC and HPLC were mainly selected as the experimental method. TLC solvent system was n-butanol: formic acid: water (4: 6: 1, v: v: v). HPLC chromatography methods were as follows: Chromatography System: Waters 2695; Detector: Waters PAD UV detector; Column: TSK-GEL DEAE-2SW 4.6 mm×250 mm (Tosoh Corp., Tokyo, Japan); Mobile phase: H2O-0.25 M NaCl within the gradient in 60min elution; Flow rate: 1.0 ml/min; Detection temperature: Room temperature; Detection time: 60min.Crude enzyme solution was obtained through fermentation the Streptomyces.violaceoruber IFO15732. The enzyme activity reached the highest in the eighth day at 88.00 U/mL using DNS method. The major enzyme protein molecular weights were 23,000 Da and 27,000 Da. Sodium alginate were degradated by the enzyme. It can be proved by TLC and ESI-TOF-MS that mono-uronic acid and DP of Alginic acid oligosaccharides between two to eight were contained. The results showed that the crude extract was with the alginate lyase activity. It can be used in degradation sodium alginate and the main products were mono-uronic acid and alginate oligosaccharides with the degree of polymerization 2-8.