Characterization Of Alginate Lyases From Microbulbifer Sp. ALW1 And Pseudomonas Syringae

In this study,a novel alginate-degrading marine bacterium ALW1 was isolated from rotten brown alga.Strain ALW1 was identified via 16S rRNA gene sequence and biochemical reactions.An extracellular alginate lyase from this strain was purified to homogeneity by steps of ammonium sulfate precipitation,DEAE-Sepharose chromatography,and Sephacryl S-100 HR chromatography.Then characterization of the alginate lyase and antioxidant activity of the enzymatic hydrolysate was conducted.Moreover,the alginate lyase gene from Pseudomonas syringae was cloned,expressed and purified.Identification of the alginate lyase and antioxidant activity of the enzymatic hydrolysate was also performed.The major conclusions are shown as follows.?1?16S rRNA phylogenetic,phenotypic and biochemical analysis showed that the alginate-degrading bacterium ALW1 belonged to the genus Microbulbifer sp..?2?Microbulbifer sp.ALW1 alginate lyase was purified to homogeneity with electrophoretic grade.Substrate specificity analysis showed that the purified strain ALW1alginate lyase showed activities towards both polyguluronate and polymannuronate.This alginate lyase was optimally active at 45°C and pH 7.0.It was stable at temperature below 40°C and showed good stability over a broad pH range of 5.0-9.0.Na⁺was found to significantly enhance the enzyme activity,while Ba²⁺,Ni²⁺,Zn²⁺,Co²⁺,Cu²⁺,Fe³⁺and Al³⁺suppressed the enzymatic activity.Except for EDTA,strain ALW1 alginate lyase had good resistance to other detected inhibitors and detergents.?3?The alginate oligosaccharides obtained by degradation of alginate by Microbulbifer sp.ALW1 alginate lyase displayed reducing power and the scavenging abilities towards ABTS⁺radical and hydroxyl radical with IC₅₀ of 1.23 mg/ml and 3.35 mg/ml,individually.?4?The cloned alginate lyase gene from Pseudomonas syringae was 1137 bp,encoding 378amino acid residues.The results showed that the deduced amino acid sequence of this gene shared high identities with the alginate lyase sequences from other bacterial strains.So the cloned gene was predicted to encode an alginate lyase.The theoretical molecular weight and pI of the alginate lyase were 42.5 kDa and 8.15,respectively.The three-dimensional structure of Pseudomonas syringae alginate lyase was constructed by homology modeling and presented?-helix rich structure.?5?Heterologous expression of the alginate lyase from Pseudomonas syringae in E.coli and purification of the recombinant protein by Ni-NTA chromatography were conducted.The recombinant alginate lyase showed activity towards polymannuronate.And it was optimally active at 30°C and pH 7.0.It was stable at temperature below 50°C and showed good stability over a broad pH range of 3.0-11.0.K⁺,Na⁺,Li⁺and Ca²⁺had no significantly influence on the enzyme activity,while Ba²⁺,Ni²⁺,Zn²⁺,Co²⁺,Cu²⁺,Fe³⁺and Mn²⁺suppressed the enzymatic activity.Except for SDS and DTT,the recombinant alginate lyase had good resistance to other detected inhibitors and detergents.?6?The alginate oligosaccharides obtained by degradation of alginate by recombinant alginate lyase displayed reducing power and the scavenging abilities towards ABTS⁺radical and hydroxyl radical with IC₅₀ of 1.56 mg/ml and 0.56 mg/ml,individually.Microbulbifer sp.ALW1 was a novel type of extracellular biofunctional alginate lyase-producing strain.It had provided a foundation for further study of alginate lyase as well as its application in the production of alginate-oligosaccharide.Cloning,expression,and characterization of the alginate lyase from Pseudomonas syringae lay a good foundation for further researches of structure,function and directed evolution of this enzyme.