| One bacterial strain designated as Microbulbifer sp.ALW1 was isolated from the rotten brown alga sample and was revealed to be able to degrade Laminaria japonica thallus fragments into the finest particles rapidly.As the cell density of Microbulbifer sp.ALW1 increased,the shape of the cells changed from being rod-to coccoid-shaped.Strain ALW1 was capable of degrading alginate,laminarin,and fucoidan.To the best of our knowledge,this is the first study to directly demonstrate the existence of laminarase and fucosidase in Microbulbifer species.Two new alginate lyase genes of algl17 and algl6 were cloned from strain ALW1.The algl17 gene contained 2220 bp and encoded a 739-amino acid protein classified into the PL-17family.The algl6 gene contained 2277 bp and encoded a 758-amino acid protein classified into the PL-6 family.The three-dimensional structure of the alginate lyase AlgL17 was constructed by homology modeling and presentedα-barrel and carboxyl-terminalβ-sheet domains structure.The two recombinant enzymes of AlgL17 and AlgL6 were overexpressed and purified from Escherichia coli BL21(DE3)with molecular mass of 85 kDa and 87 kDa,respectively.The alginate lyase AlgL17 enzyme showed activities towards sodium alginate,polyM and alginate oligosaccharide,but very low activity towards polyG.The alginate lyase AlgL6 enzyme showed activities towards sodium alginate,polyM and polyG,low activity towards alginate oligosaccharide.When sodium alginate was used as a substrate,the optimum reaction temperature and pH for the AlgL17 and AlgL6 were both 35oC and pH 8.0,respectively.The AlgL17 was stable at 25oC,but not stable at 30oC and 35oC and AlgL6 was stable at 30oC,but not stable at above 35oC.The AlgL17 showed good stability over a pH range of 5.0-8.0 and AlgL6 showed good stability over a pH range of 5.0-9.0.The AlgL17 activity was increased to1.7 times by adding NaCl to a final concentration of 0.7 mmol/L and the AlgL6 activity was increased to 1.8 times by adding NaCl or KCl to a final concentration of 0.5 mmol/L.The AlgL17 and AlgL6 enzymes could produce 4-deoxy-L-erythro-5-hexoseulose uronic acid(DEH)from sodium alginate,and AlgL17 could also produce DEH from polyM.These indicated that the AlgL17 and AlgL6 enzymes were both exo-type alginate lyases.AlgL17 is a Na~+-activated enzyme and AlgL6 is a Na~+,K~+-activated enzyme. |
