Gene Cloning,expression And Characterization Of Alginate Lyase From Marine Bacterium Vibrio Sp.QD-5

Alginate lyases,catalyze the degradation of alginate by a?-elimination mechanism,have been isolated from a variety of organisms.Alginate lyase had different characteristics in substrate specificity,mode of action,primary structure and3-dimensional structures.Due to their characteristics,alginate lyases have been classfied into different groups.Alginate is the most abundant?accounting for 40%of the dry weight?polysaccharide in brown algae,which composed of?-D-mannuronic acid?M?and?-L-guluronic acid?G?.The units were linked via?-1,4 glycosidic linkages forming poly?-D-mannuronate?polyM?,poly?-L-guluronic acid?polyG?and polyMG.Alginate oligosaccharides were the depolymerization products of alginate by alginate lyase or physicochemical method,which have widely applicated in food and pharmaceutical industry.Due to the high-efficiency and high-specificity,alginate lyases have been the focus for various fields.In this study,a strain capable of degrading alginate preserved in our laboratory was identified using 16S rDNA sequence.The analysis of 16S rDNA sequence and phylogenetic tree indicated that the strain belongs to Vibrio sp.and was named Vibrio sp.QD-5.The whole genome sequence of this strain was sequenced,and its genome sketch was constructed.The gene cluster analysis showed that there was a cluster of alginate lyase gene in the genome of strain Vibrio sp.QD-5.Using the designed primers,aly-II and aly-IV were cloned.By sequencing,we acquired the sequence information of the gene aly-II and aly-IV.The analysis of the sequences showed that the alginate lyase gene aly-II had a length of 2169 bp encoding 723 amino acids.The aligment of the amino acid sequence showed that Aly-II was a member of polysaccharide lyase family 17?PL17?.The alginate lyase gene aly-IV had a length of 1560 bp encoding 520 amino acids.The aligment of the amino acid sequence showed that Aly-II was a member of polysaccharide lyase family 7?PL7?.In this study a heterologous expression system was constructed for aly-IV and optimized its fermentation and induction conditions.The optimized fermentation medium contained sucrose,yeast extract,?NH₄?₂SO₄,sodium citrate,NaCl,KH₂PO₄,and MgSO₄·7H₂O;its initial pH was 6.0;4.5 mL seed liquid was added to 150 mL liquid fermentation medium that loaded into a 250 mL flask;when the OD₆₀₀ reached1.0,IPTG was added to a final concentration of 0.4 mM;the induction temperature and induction time were 30^oC and 8 hours,respectively.After optimization,the content of the target protein increased nearly 1.58 times.When lactose was used as an inducer,the OD₆₀₀ should also reache 1.0.Lactose was added to a final concentration of 0.5 g/L.When induced at 25 ^oC for 8 hours,the target protein increased nearly 1.83times.In this paper,the expression and characterization of the Aly-IV were studied.Aly-IV was expressed in the form of inclusion bodies in BL21?DE3?.In this study,the inclusion bodies were dissolved in Sarkosyl.The Ni affinity chromatography and gel chromatography were used for the purification of Aly-IV.The specific activity of the purified Aly-IV reached 1256.78 U/mg.The optimal temperature and pH for the enzyme Aly-IV was 35^oC and 8.9,respectively.Its activity almost unchanged when the enzyme incubated in the temperature below 25^oC for 30 min.When the temperature was higher than 25 ^oC,with the increasing of temperature,enzyme activity decreased rapidly.The effects of pH on enzyme activity were not only related to pH,but also to the type of buffer,indicating that the activity of the enzyme was affected by the ionic strength.Studies of the effects on the enzyme activity of metal ions showed that K⁺,Mg²⁺and Ca²⁺could promote the activity of Aly-IV,however,Cu²⁺,Ba²⁺,Al³⁺,Ni²⁺,Zn²⁺,Pb²⁺,EDTA^2-,Co²⁺and Cr₂O₄^2-inhibited the activity of Aly-IV.NaCl also has an effect on the activity of Aly-IV.When the concentration of NaCl was 1 mM-20 mM,the activity of Aly-IV was promoted,and the activity of the enzyme was inhibited when the concentration was higher than 25 mM.Substrate specificity studies showed that Aly-IV could degrade sodium alginate and polyG.According to the degrading products and the changes of viscosity,sodium alginate was degraded by Aly-IV via endo-cleavage.