The Research On Alginate Lyase Genes In Vibrio Alginolyticus ATCC17749

Alginate lyases belong to Polysaccharide lyases family and catalyze the depolymerization of alginate into oligoalginates by eliminative cleavage of the glycosidic bonds between the uronate residues.In recent years,attention has been paid to the oligoalginates because of their useful and medically important functions.Besides,alginate lyase is feasible for the therapy of cystic fibrosis and other infectious diseases caused by Pseudomonas aeruginosa.Thus,it is more important to research the alginate lyases.According to the published genome sequence of V.alginolyticus40B in the database,we cloned five putative alginate lyase genes from V.alginolyticus ATCC17749.and then constructed the expression plasmids of every single gene. These plasmids were transformed into E.coli BL21(DE3),and then induced to express the recombinant proteins.The qualitative and quantitative determination of alginate lyase activity of the recombinant proteins demonstrates that the genes algVl,algV2,algV3,algV5were all alginate lyase genes:ALGV1,ALGV2and ALGV3were extracellular enzyme;ALGV5was intracellular enzyme. The enzyme units of every recombination strains can reach to644.1U/L,4347U/L,1011U/L and245.3U/L, respectively. Among these strains, pET28a-algV2/BL21(DE3) has potential value for industrial application.The optimal pH was around8.6in glycine-NaOH buffer,and the optimal temperature was30℃.In the presence of0.3M NaCl,the activity was increased to2.2times. KCl,Fe3+, Ca2+could strongly activated the enzyme;Ba2+,Mn2+could slightly activated the enzyme.;Mg2+, Zn2+, Cu2+ions inhibited the enzyme,and Cu2+could strongly inhibited the ALGV2activity. Depolymerization of alginate by ALGV2in100mL reaction system can be lasted for8.5h.After purification,the products were identified by ESI-MS to be trisaccharides (MW:528Da).