Study On Alginate Utilization Ability Of Algae-epiphytic Alteromonas Spp.

Algal polysaccharides,as an important carbon resource in the ocean,play an important role in the carbon cycle of earth.And the study on usage of algal polysaccharides is a major focus of research.As one of the three major marine bacteria,Alteromonas spp.possesses the ability to utilize a variety of algal polysaccharides.In this study,the protein sequences of various species of Alteromonas spp.obtained from NCBI database were analyzed by sequence alignment.Then,the species and abundances of polysaccharide lyases(PLs)in Alteromonas spp.were compared.Moreover,serval stains of Alteromonas spp.were screened from the algae.The abilities of different strains to utilize various algal polysaccharides were compared.The mechanism of alginate hydrolysis was explored by analyzing the function and properties of related enzymes.The main results obtained are as follows:(1)Through the analysis of carbohydrate active enzymes(CAZymes)of different species of Alteromonas spp.,it was found that several strains of Alteromonas stellipolaris,Alteromonas addita,Alteromonas australica and Alteromonas portus contained a large number of PL6 and PL7 family enzymes,and the strains of Alteromonas macleodii contained a large number of PL6 and PL7 family enzymes,and the other strains of Alteromonas spp.had no or less enzymes compared to that in these two families.It is speculated that the difference in the abundance of PL6 and PL7 family enzymes is similar to the ability of Alternomonas spp.to utilize alginate.Furthermore,7 species(26 strains)of Alteromonas spp.were screened from Sargassum horneri,Sargassum hemiphyllum and Ulva lactuca.Enzyme activity assay experiments showed that compared with Alteromonas mediterranea,Alteromonas stellipolaris had a stronger ability to utilize alginate.The draft gene analysis showed that the number of PL6,PL7 and PL18 family enzymes of A.stellipolaris was significantly higher than that of A.mediterranea.(2)Under the condition of sole carbon source culture,RT-qPCR was used to verify the expression of nine alginate utilization related genes from A.stellipolaris T18.The results showed that the expressions of Aly0191,Aly3318,Aly3321 and Aly3764 were significantly up-regulated.Furthermore,the recombinants of these four enzymes were constructed,and the degradation modes and products were explored when the substrates were alginate,poly MG,poly M and poly G.The recombinant Aly0191 can degrade alginate into oligosaccharides with different degrees of polymerization(DP).It can almost completely degrade poly MG into disaccharides to hexasaccharides.,and a small amount of monosaccharides are produced.But the degrading ability of poly M and poly G is weak.The recombinant Aly3318 can degrade alginate,poly MG and poly M into oligosaccharides,the main product is disaccharides to hexasaccharides,and a small amount of monosaccharides are produced.But the degrading ability of poly G is relatively weak.The recombinant Aly3321 can degrade alginate into oligosaccharides with different DP,but the ability is weak.Only a small amount of poly MG can be degraded by the recombinant Aly3764.Therefore,it is speculated that the difference in the ability to utilize alginate between A.stellipolaris and A.mediterranea is mainly due to the difference in the number and expression of PL7 and PL18 family enzymes they possess,respectively.(3)Subsequently,the sequence analysis and enzymatic properties of the alginate lyase Aly3318 obtained from the A.stellipolaris T18 were carried out.The optimal reaction temperature of recombinant Aly3318 is 20?,indicating it is a cold-adapted enzyme,it has poor thermal stability and will be rapidly inactivated at higher temperatures.Its optimal reaction p H is 7.0.When the concentration of Na Cl is 700m M,the enzyme activity is the highest.Na⁺,K⁺,Li⁺,NH₄⁺,Ca²⁺,Mg²⁺can promote the recombinant Aly3318.Mn²⁺,Co²⁺,Ba²⁺,Ni²⁺,Cu²⁺,Zn²⁺,Hg²⁺,Fe²⁺,Fe³⁺,Al³⁺,SDS and EDTA can inhibit the recombinant Aly3318.The recombinant Aly3318 had the ability to utilize poly MG,alginate,poly M and poly G,and the ability decreased in turn.The recombinant Aly3318 can degrade alginate into oligosaccharides at a lower temperature.The main product is disaccharides to hexasaccharides,and a small amount of monosaccharide is produced.The specific activity of recombinant Aly3318 is 399.58U/mg.Through the above research,the ability of Alteromonas spp.to utilize alginate and the difference in the ability of the strains to degrade alginate were verified.At the same time,the enzymatic properties of a cold adapted alginate lyase were studied.These results contribute to the research of Alteromonas spp.and alginate degradation.