Determination Of Taxonomic Position Of Strain C3212 And Study Of Agarases Produced By Agarivorans Sp.D1326

BackgroundThere are considerable diversities in terms of microbial resources and enzyme resources with special functions in marine environment.Agarase is a kind of glycoside hydrolase mainly produced by marine microorganisms.Agarases have potential applications in food industry,cosmetics,and medical fields because they produce oligosaccharides with remarkable activities.The existing agarase showed low hydrolysis efficiency and poor stability,which greatly limits its application.ObjectivesIn order to enrich agarase resources,the culturable bacteria habited in marine environment were tried to be isolated and purified and the agarase-producing strains wrere screened from the purified strains.Then,agarases produced by agarase-positive strains were mined by using combination of genomic and secretomic analysis.Methods1.Four kinds of medium were used to isolate and purify culturable bacteria from seawater and seaweeds samples collected from the Yellow Sea.16 S rRNA gene sequences analysis was used to identify novel species candidates.The exact taxonomic position of the novel species candidates was determined by using a polyphasic taxonomy approach.Lugol's iodine staining and observation of agar plate degradation were used to screen agarase-producing strains.2.Using a combination of genomics and secretomics to excavate agarase produced by agarase-producing strain D1326,and DNAMAN,DNAstar,SignalP-5.0 Server and other bioinformatics software and servers were used to analyze agarase gene sequences.The novel agarases were chosen for further research.3.Heterologous expression of novel agarases Aga1459,Aga2820 and Aga3584 in E.coli using molecular biological techniques.The optimal agarse-producing conditions were obtained by single-factor experiments and orthogonal design.4.The fusion proteins rAga1459,rAga2820 and rAga3584 with histidine tag were purified by nickel column affinity chromatography,and The DNS method was used to measure enzyme activity to characterize the recombinant agarase.Results1.A total of 143 strains were isolated and purified.After deduplication,71 different species were obtained,of which 6 strains were novel species candidates.The results of the phylogenetic analysis(16S rRNA gene sequences and genomic DNA sequences),phenotypic and chemotaxonomic studies supported the taxonomic position of the novel species,Leucothrix sargassi C3212.The strain Agarivorans sp.D1326 with strong agarase activity was screened for agarase study.2.The genomic sequence analysis of strain D1326 showed that the strain contains 9 agarase encoding genes,combined with the results of mass spectrometry analysis,three agarase encoding genes aga1459,aga2820,and aga3584 were selected for further research.The aga1459,aga2820 and aga3584 genes encode three novel agarases of the family GH50,GH118 and GH50,respectively.The theoretical molecular weights are 110.0 kDa,47.7 kDa and 31.7 kDa,and the amino acids number is 996,447,and 309.All of them contain a signal peptide,except for Aga2820.3.The aga1459,aga2820,and aga3584 were heterologously expressed by strains 1459-28a-BL(DE3)plyss,2820-28a-BL(DE3)plyss and 3584-28a-BL(DE3)plyss,respectively.The optimal fermentation conditions are: induction temperature 20 ?,induction time 36 h,induction time 3 h,and IPTG concentration 0.01 mM;induction temperature 12 ?,induction time 30 h,induction time 2 h,and IPTG concentration 0.015 mM.mL;induction temperature 16 ?,induction time 24 h,induction time 1 h,and IPTG concentration 0.01 mM.4.The recombinant proteins rAga1459,rAga2820 and rAga3584 were purified by nickel column affinity chromatography.The optimal reaction temperature of rAga1459,rAga2820 and rAga3584 were 35 ?,30 ? and 45 ?,respectively,the optimal reaction pH were 6,9 and 6,the Km and Vmax values were 2.946 mg/mL and 0.035 mg/min,10.585 mg/mL and 0.084 mg/min and 30.273 mg/mL and 0.423 mg/min,respectively.Conclusions1.Six novel species bacterial candidate were obtained from the coastal area of the Yellow Sea,and the taxonomic position of the novel species Leucothrix sargassi C3212 was determined,which enriched the marine bacterial resources.2.Three novel agarases belong to the GH50 and GH118 families were obtained,which greatly enriched the agarase resource library.