Polyphasic Taxonomy Analysis Of Three Novel Marine Bacterial Strains And Studies On Agar-degrading Enzymes Produced By Strain A100~T

Cell walls of red alga are rich in agar.Bacteria attached to these algae contain agarase which degrades agar from polysaccharides into oligosaccharides and monosaccharides support their own growth.Along with the deepening of research on carbohydrate biology,application of agar oligosaccharides has become more and more widely.It has been found that agar oligosaccharide has positive activity on anti-cancer,antioxidant,anti-inflammatory and anti caries.It also has significant physiological activity in inducing cell apoptosis,promoting probiotic growth and preventing diabetes.Therefore,it has great potential in food,feedstuff,cosmetics and medicine.Therefore,several research were carried out with the main line of agarolytic bacteria,which contains the identification of three agarolytic bacterial strains,optimization of fermentation conditions of A100T,whole genome sequencing and bioinformatic study,the extraneous expression of 12 agar enzyme genes and the purification of the recombinant agar enzyme proteins agaA4 and agaA6.In the identification of marine new bacteria,strains B011T and A100T were isolated from the red alga Gracilaria(Gracilaria blodgettir)collected from coastal sea of Lingshui County,Hainan province,both of which can degrade agar.Strain B011T represents a new species belonging to Bacteroidetes,vobacterium,Flavobacterium,Flavobacteraceae and Halobacterium,named Seonamhaeicola marinus sp.nov.,and strain A100T represents a new genus of Bacteroidetes,Flavobacterium,Flavobacterium and Flavobacteraceae,named Agarcorrumpuntur marinu gen.nov.sp.nov.,Strain HQSZ'T was isolated from mud samples Xiaoshi island in Weihai,Shandong Province,and has strong agar degradation ability.It is a new species in the phylum Proteobacteria,Gammaproteobacteria,Cellvibrionales,Cellvibrionaceae,Gilvimarinus.Gilvimarinus marinus sp.nov.was proposed.Strain A100T has strong agar degradation ability.Therefore,the fermentation conditions of agarase were optimized.Through single factor experiment,orthogonal experiment and response surface optimization experiment,the optimum conditions for agar degrading enzyme were determined.Agar powder(0.175 g/L)and fish meal protein(0.6 g/L)were added with the following condition,initial pH at 7.245,temperature at 27 ?,and salinity of 30 g/L,fermentation time 72 h,inoculation amount 2%.Fermentation bottling(250 mL conical bottle)amount was 75 mL.Agarase activity was examined under the condition described,which was 2.169 U/mL.In order to have a more in-depth understanding of strain A100T agarase production,we performed whole genome sequencing.Through the analysis of the whole genome sequence,12 agarase genes were predicted.Bio informatics study suggested that amino acid sequence encoded by the gene of agaAl gene,agaA2,agaA3,agaA4,agaA6,agaA7 belonged to the glycoside hydrolase GH16 family,while amino acid sequence encoded by the gene of digaAXl,agaAX2,agaAX3,agaAX4,and agaAX5 coded enzymes that belonged to ?-neoagarobiose hydrolase family,while amino acid sequence encoded by the gene of agaA5 which no hydrolase family matched.Heterogenous expression of these agar degrading enzyme genes was performed and we found that the recombinant agar degrading enzyme protein of agaA4 and agaA6 were successfully expressed tested by SDS-PAGE,and tests on water agar plate proved high enzyme activity..The recombinant protein coded by agaA4 was purified by Ni+ affinity chromatography,and recombinant protein with high purity was obtained for further research.