Isolation And Identification Of Alginate Lyase Producing Strain I1~T And Characterization Of Its Enzymes

Brown algae is an important marine resource in China,which is rich in various nutrients and bioactive substances.Alginate is an important marine organic carbon source,which mainly exists in the cell walls of hundreds of brown algae.The degradation product of alginate-alginate oligosaccharides is a kind of multifunctional oligosaccharides with unique biological activity,which has been widely used in food and pharmaceutical industry and has attracted more and more attention.Efficient alginate lyase is a key tool for the production of functional oligosaccharides from alginate.Therefore,it is of great significance and value to discover and characterize different types of new alginate lyase browning for the high-value development and utilization of alginate.In this study,32 strains of alginate-degrading bacteria were preliminarily screened from the intestinal contents of sea cucumber,which were distributed in 4 phyla and 15 genera.The enzyme activity of strain I1T was the highest,reaching 32.6 U/mL.Through morphological observation,physiological and biochemical characteristics determination and phylogenetic analysis,strain I1T was classified and identified as a new species of Tamlana,named Tamlana flavus sp.nov.The optimal fermentation conditions of alginate-degrading strain I1T were obtained as follows:pH 7.0,temperature 28℃,sodium alginate mass fraction 2.5%,NaCl mass fraction 3%.When the main nitrogen source was yeast powder,the maximum enzyme activity was 42.99 U/mL,which was 1.3 times higher than that before optimization.The enzyme production conditions of strain I1T were optimized to obtain the optimal enzyme production conditions.At pH 7.0,temperature 28℃,sodium alginate concentration 2.5%,NaCl concentration 3%and yeast powder as the main nitrogen source,the maximum enzyme activity was 42.9 U/mL after 3 days of continuous culture,which was 1.3 times higher than that before optimization.The whole genole of strain I1T was sequenced,and five alginate lyase genes were annotated,and the sequences of these genes were analyzed.Phylogenetic analysis showed that Aly-I1,Aly-I2 and Aly-I5 belonged to PL7 family,Aly-I3 belonged to PL6 family,and Aly-I4 belonged to PL 17 family.Two alginate lyase genes Aly-I4 and Aly-I5 were heterologously expressed,and the enzymatic properties of the recombinant enzymes were characterized.Among them,the recombinant enzyme rAly-I5 showed high activity.The optimum temperature and pH of rAly-I5 were 30℃ and 8.5,respectively,and it had more than 70%activity in the wide pH range of 5.0-11.0.At NaCl concentration of 1M-3M,the highest enzyme activity remained above 70%.The recombinant enzyme rAly-I5 has obvious metal ion tolerance,and has degradation effects on alginate,polyM and polyG.The degradation product is mainly oligosaccharides,which is an endoenzyme.Therefore,rAly-I5 has high catalytic activity,good pH stability,metal ion tolerance and salt tolerance,which means that rAly-I5 is a promising alginate lyase in industry.