Study Of Alginate Lyase Fro Marine Bacteria Vibrio Sp. QY101

Alginates are linear polysaccharides in which p-D-mannuronic acid (M) and a-L-guluronic acid (G) are (l,4)-linked to form blocks of consecutive G residues (G blocks), consecutive M residues (M blocks), and alternating M and G residues (MG blocks). Alginate and alginate-derived oligosaccharides have been applied as raw materials in pharmaceutical, food, textile, agricultural and oil industries. Studies have shown that alginate oligosaccharides exhibit high activity on antitumor, anti-AIDS and so on. Alginate lyases catalyze the depolymerization of alginates by p-elimination of the 4-O-glycosidic bond. Alginate lyase can not only be used as useful biological tool to prepare alginate oligosaccharides, but also can be used in combination with other chemotherapeutics in the treatment of cystic fibrosis (CF) patients who are infected with alginate-producing Pseudomonas aeruginosa. Otherwise, alginate lyases have been applied successfully to the extraction of protoplasts for basic study of a variety of algal species. However, there is no alginate lyase purified to high levels of purity and yield for the tightly combination between alginate lyase and alginate substrate resulting the difficulties of purification. In conclusion, it is important to search novel alginate lyase having high activity.In our study, more than 50 alginate lyase-producing marine bacterium strains have been isolated by selective medium, in which strain QY101 has more activity and degrade alginate faster compared with other alginate lyases. Using morphologic observation, physiological and biochemical methods and 16S rRNA methods,marine bacteria QY101 has been identified as Vibrio sp., and named as Vibrio sp. QY101.Extracellular alginate lyase secreted by Vibrio sp. QY101 has been purified to homogeneity by a combination of ammonium sulfate precipitation, DEAE-Sepharose Fast Flow anion-exchange chromatography and Superdex 75 gel filtration chromatography. Its molecular mass is 39 kD as determined by SDS-PAGE analysis. The enzyme has an optimal temperature of 30 C for its activity, and is most active at pH 7.5. The thermal and pH stability are 0-30"C, and pH 6.5-8.5, respectively. The enzyme activity is stimulated by 0.5 mol/L NaCl, 1.0 mmol/L Ca2+ or 5.0 mmol/L Mn2+, and inhibited by 5.0 mmol/L Ni2+, 1.0 mmol/L Fe2+ or 1.0 mmol/L EDTA. Preliminary analysis on substrate specificity shows that this alginate lyase have both poly- a 1,4-Z-guluronate and poly- 3 1,4-D-mannuronate substrate activity. More relative analysis shows that this alginate lyase can degrade O-acetylated alginate of Pseudomonas aeruginosa ATCC9027, which is different from the alginate lyase reported. It is likely that this alginate lyase would be used in degrading O-acetylated alginate in bacteria biofilms to make the bacteria more sensitive to the antibiotics that it was resistant before.There is another fraction having alginate lyase activity during above experiments. Since it is little to get by chromatography methods, a strategy combined degenerate PCR and long range-inverse PCR (LR-IPCR) has been used to clone the gene alyVI encoding another alginate lyase of Vibrio sp. QY101. Gene alyVI is composed of a 1014 bp open reading frame encoding 338 amino acid residues. The calculated molecular mass of alyVI product is 38.4 kDa. With a signal peptide cleaved off, the mature protein is of 34 kDa. The alyVI gene has been cloned into pET-24a(+) plasmid and AlyVI has been purified from culture supernatants to electrophoretic homogeneity using Ni2+ affinity chromatography.AlyVI is most active at pH7.5 and 40 C in the presence of 1 mM ZnCh. A 9-amino-acid consensus region (YXRESLREM), which is only found in polyguluronate lyases, is also observed in the amino-terminal region of AlyVI. Though AlyVI can degrade both M block and G block, the difference of Km of AlyVI to different substrate supports the hypothesis that this region is related to substrate recognization.In conclusion, though alginate lyase is difficult to purify, an effective method system to purify alginate lyase ha...