The Screening And Properties Study Of Seaweed Polysaccharides Degrading Enzymes From Marine Bacteria

Seaweed polysaccharides are the main biological active substances in seaweed,and are high molecular carbohydrates formed by a single identical or different glycosyl group by the linkage of glycosidic bonds.Alginate polysaccharides and agarose polysaccharides are two kinds of seaweed polysaccharides which are further studied.The alginate polysaccharides are mainly derived from brown algae such as kelp,and the agarose polysaccharides are mainly derived from red algae such as red cabbage.The oligosaccharides produced by degradating of seaweed polysaccharides possess more unique biological activities,such as improving immunity,anti-inflammatory,anti-tumor,anti-oxidation,anti-virus and so on.Seaweed polysaccharides degrading enzyme is a key class of enzymes in the green production process of algae oligosaccharides,However,most of the seaweed polysaccharides degrading enzymes are rarely applied in industrial production due to poor activity and stability,Therefore,the searching for algae polysaccharides degrading enzymes with high activity and stable nature is of great significance in the future utilization of seaweed polysaccharides.In this study,a strain H1 abundanted in seaweed polysaccharide degrading enzymes obtained in a preliminary screening in our laboratory was used for strain identification and whole genome sequencing.Through functional gene annotation and analysis of carbohydrate active enzymes(CAZy),new alginate lyase and agarase were obtained and their enzymatic properties were characterized.Among them,the new alginate lyase AlgH belongs to the PL7 family.Recombinant enzymes AlgH-? and AlgH-? were obtained through knockout and fusion engineering of the non-catalytic domain,and successfully expressed in Escherichia coli.The enzymatic properties revealed that the enzyme showed the highest enzyme activity at pH 10.0 and temperature 45?,and the domain optimization and modification made its activity and stability significantly improved.The enzyme is a bifunctional alginate lyase which exhibits high activity against poly-guluronic acid(polyG)and polymannuronic acid(polyM),so it can completely degrade alginate to obtain a polymerization degree of 2-6 oligomeric brown algae oligosaccharides.At the same time,the kinetic constants(Km,kcat,etc.)of wild and engineered enzymes were measured,and it was confirmed that the enzyme has higher affinity for sodium alginate and polyG,and the removal of non-catalytic domains will not affect the enzyme substrate specificity.In addition,this paper excavated two new agarases,AgaA and AgaB,and heterologously expressed in E.coli.The expression of AgaA and the purification efficiency were improved by deleting the special repeat sequences of the N-terminus of the protein.It was found that the optimum temperature of AgaA was 60?,the optimum pH was 6.0-7.0,and the enzyme activity remained unchanged after incubation at 50? for 5 hours.AgaA is an endo-type agarase that degrades agarose to form new agaroses,tetrasaccharide and hexaose.AgaB has an optimum temperature and pH of 40? and 7.0,respectively.It is an exo-type agarase that degrades agarose to form a neoagarobiose with uniform polymerization.In addition,through the combination use of these two enzymes,a two-stage double enzyme module was used to degrade agar to produce new agarose.The purity of the new agarose prepared by this method is over 90%,and the substrate conversion rate is close to 94%.In this study,we discovered new alginate lyase and agarase through identification of marine bacterial strains,as well as genome-wide sequencing and functional annotation.Through domain optimization,the heterologous expression level,enzyme activity and thermal stability were improved.The enzymatic properties of alginate lyase AlgH,agarase AgaA and AgaB were studied in detail by enzymatic characterization.On this basis,an efficient method for the production of fucoidan and new agarose by using seaweed polysaccharide degrading enzymes was established,which has the potential to be applied in the industrial production of seaweed oligosaccharides.