The Hydrolysis Of Laminarin And Agarose By Flavobacteria From Alga

There are many kinds and complex chemical structures of algal polysaccharides,which are decomposed by abundant CAZymes in the marine biochemical cycle,forming one of the largest cycles in the carbon cycle of the Earth.And the oceanic ecosystem itself has the largest algal polysaccharides lyase gene pool,namely the CAZymes gene pool.The main microbial source of the CAZymes gene pool is marine Flavobacteriaceae.Our research group isolated a large number of marine bacteria belonging to the family Flavobacteriaceae that can utilize different algal polysaccharides from the surface of the algae samples.Bioinformatics analysis was used to predict the metabolic pathways of algal polysaccharides in the genus Tamlana.And the molecular mechanism of T.laminarinivorans PT2-4 hydrolyzing laminarin was studied by using the proteomics method.A.agarilytica ZC1 preserved in the laboratory was used as the research object.The agarose catabolism pathway was confirmed through the prokaryotic expression of the target gene and product verification.The principal results were as following.(1).Abundant algae samples were collected from different sites,and nearly 400 strains were isolated,including 24 genera of Flavobacteriaceae,and 80 species.The sequence similarity of 16S r RNA of strain CW2-9 and T.sedimentorum JCM 19808 was 96.3%.The sequence similarity of the 16S r RNA gene of strain PT2-4 and strain 62-3 and T.sedimentorum JCM 19808 was 98.4%and 97.99%,respectively.The main fatty acids were iso-C_15:0,iso-G-C_15:1 and iso-C_17:0 3-OH.The respiratory quinone was MK6.They were named Tamlana fucoidanivorans CW2-9,Tamlana laminarinivorans PT2-4 and Tamlana sargassums 62-3,respectively.(2).Strains in the genus Tamlana were divided into 2 branches at the genomic level according to the analysis of the phylogenetic relationship.Those genomes of Tamlana sp.EP2-2,T.crocina HST1-43,T.carrageenivorans UJ94,T.agarivorans JW-26 and T.fucoidanivorans CW2-9 were relatively low in conservation and had a rich variety of hydrolases,including hydrolases that hydrolyze mannoglycan,rhamnoglycan and xylan,etc.T.laminarinivorans PT2-4,T.sargassums 62-3,Tamlana sp.S2-14,T.nanhaiensis FHC16 and T.sedimentotum JCM 19808 had the conservative laminarin PUL and alginate PUL.(3).Compared with other strains in the genus Tamlana,T.laminarinivorans PT2-4 had a strong hydrolysis ability to utilize laminarin.Proteomics analysis and RT-q PCR verification showed that gene 1092 in strain PT2-4 was significantly up-regulated,and recombinant 1092 could hydrolyze laminaritetraose and laminarihexaose into disaccharides and monosaccharides.The metabolic pathway of laminarin in T.laminarinivorans PT2-4 was elucidated.The catalytic domain of gene 1092 was confirmed to be the GH16 domain by the methods of gene truncation and site-directed mutation.In the GH16 domain,when Asn364,Arg400 and Glu452 were replaced with alanine,gene 1092 lost the activity of hydrolyzing laminaritetraose and laminarihexaose.When Gly361 and His466 were replaced with alanine,the activity of hydrolyzing laminaritetraose and laminarihexaose of gene 1092 was significantly altered.(4).Three metabolic pathways of agarose in strain A.Agarilytica ZC1 were predicted through analysis of extracellular oligosaccharide,prediction of the signal peptide of agarases and cell location prediction.E.coli Rosetta(DE3)-p ET22b(+)-aga575-NH852 was successfully constructed by molecular biological method,and the hydrolytic products were NAOSs with different degrees of polymerization,3,6-anhydro-L-galactose and D-galactose.