Analysis Of Microbial Diversity In The Abdominal Intestine And Studies On The Alginate Lyase From Strain Agarivorans Sp.B2Z047

Alginate is a macromolecular substance widely existing in the cell wall of alginate,which is polymerized by a-L-guluronic acid and ?-D-mannuronic acid.The brown algae production in the world is huge,and it is one of the sustainable energy sources Nowadays,with the depletion of energy sources,the use of alginate lyase to degrade brown algae has become an important issue for solving the energy crisis.According to the current research,many institutions in the world have carried out research on the conversion of alginate to bioethanol,but often encountered many problems with low enzyme activity or incomplete degradation.Therefore,the screening of alginate lyase-producing strains and multifunctional enzymes has become an important work for alginate biotransformation.Based on these questions,this study takes the screening of alginate-degrading bacteria as the main line.Starting from abalones that feed on brown algae,the first investigation is the diversity of intestinal microbial in the abalone Second,the isolated strains are screened for alginate degradation.The optimal conditions of enzyme-producing of the strain Agarivorans sp.B2Z047,and finally the new bacterial species isolated in the previous work were identified by classification.The results of the research are as follows1.Analyze the community structure of abalone intestinal microorganisms based on pure culture method.The abalone rectal contents and visceral mass mixtures were used as samples to analyze the diversity of cultured microorganisms in the digestive tract.The sample was diluted and coated on 2216E solid medium,and cultured in a 20? constant temperature incubator for 7 days before isolating bacteria.A total of 364 strains of bacteria were isolated from the two samples.After 16S rRNA gene sequence online alignment,these bacteria belonged to 4 phyla,16 families,44 genera,and 87 species.Among them,Proteobacteria have the most types of bacteria,with 65 species,Vibrio and Shewanella are dominant species;18 species belong to Bacteroides,and they all belong to Flavobacteriaceae.During the isolation of bacteria,27 strains are potential new species.2.Based on the culture-free method to analyze the community structure of abalone intestinal microorganisms.A total of 6 samples of abalone's rectal contents and visceral mass mixture and 7-day and 15-day enrichment solutions were subjected to 16S rDNA gene high-throughput sequencing and online analysis.The number of effective sequencing of all samples is more than 35,000.All samples have a total of 328 OTUs,which are distributed in 21 phyla,29 classes,74 orders,117 families and 181 genera.During the enrichment process,the microbial flora of the two samples changed differently:with the extension of the enrichment time,the microbial diversity of the visceral samples increased first and then decreased,while the microbial diversity of the rectal samples gradually decreased trend.The predominant bacterial groups of the two original samples are different:in the visceral mass,the Tamlana in the Bacteroidetes accounting for 63.3%is the dominant group,followed by Vibrio(15.0%),Formosa(6.4%);the microbial diversity in the rectum is relatively rich,with the largest number of Mycoplasma(30.6%)in the Tenericutes,followed by Psychromonas(25.6%)and some among the unclassified others(12.1%),Vibrio accounted for only 1.1%.The microbial diversity of abalone rectum is higher than that of visceral mass.3.The isolated bacteria were screened and re-screened to select a strain B2Z047 with strong ability to degrade alginate,and analyzed the gene of alginate lyase.Through preliminary screening,100 strains of 364 strains of bacteria were initially identified as having the ability to degrade alginate,accounting for 27%of the total number of isolates,of which 44%of the strains belonged to Vibrio.Select 30 strains of bacteria with a large hydrolysis circle on the plate culture for rescreening in shake flasks,and obtain multiple strains with strong liquid culture degradation ability of alginate,among which the strain B2Z047 with the strongest degradation ability has an initial enzyme activity of 0.23 U/mL,higher than the enzyme-producing activity of the deposited strains in our laboratory.According to the multiple sequence alignment and identification of 16S rRNA gene,the highest similarity to the 16S rRNA gene of model species is 98.77%.It is a member of Agarivorans and a potential new bacterium.4.Genome sequencing was performed on strain B2Z047,and the alginate lyase gene of the strain was analyzed based on the genome.According to the genome of strain B2Z047,it is predicted that the genome of this strain contains 8 alginate lyase genes.After preliminary identification,alginate lyases belong to the PL6,PL7 and PL5 polysaccharide lyase families,respectively.These enzyme genes all contain signal peptides,and their half-lives when expressed in E.coli and Yeast are all greater than 10 h.In addition to the genes AlgA2,AlgA6,AlgA8 containing a single domain,other enzyme genes contain multiple domains.The prediction results show that all 8 alginate lyases are suitable for heterologous expression 5.The production conditions of strain B2Z047 alginate lyase were optimized,and the optimal culture conditions for shake flask fermentation were determined.The culture and fermentation conditions of strain B2Z047 were optimized.The results of single factor analysis showed that the most suitable carbon source for the enzyme production was sodium alginate;the preference of the bacteria for the use of organic nitrogen source was significantly better than that of inorganic nitrogen source Through the orthogonal test,the optimal medium for enzyme production was determined:sodium alginate 1.1%,yeast powder 0.3%,sodium chloride 1.0%,magnesium sulfate 0.1%,natural sea water 1 L.On this basis,the cultivation temperature,initial pH,shaker speed,shaker bottle liquid volume,and inoculation volume were optimized.The research results laid the foundation for the later experiments6.Polyphasic studies of the three new bacterial species isolated in the work.Strain T58T was isolated from the coast of Weihai and it was Gram-stain-negative After polyphasic taxonomy,it is a new species in Hyunsoonleella with a clear taxonomic status,named Hyunsoonleella(lava,and the standard strain is T58T(=KCTC 72081T=MCCC 1H00359T).Strain WD6-1T was isolated from the Wendeng salt field in Weihai.it was Gram-stain-negative,aerobic,rod-shaped,mobile,with a polar flagellum or a polar prostheca.After identification,the strain was a new species of Marinecauda,named Marinecauda salina,and the standard strain was WD6-1T(=KCTC 62348T=MCCC 1H00282T).Strain XYN52T was isolated from Xingyun Lake in the city of Yuxi,Yunnan Province.Gram staining was negative,short rod-shaped with terminal flagella,mobile,aerobic.It was identified as a new species of Pelagibacterium,named Pelagibacterium lacus,and the standard strain was XYN52T(=KCTC 62845T=MCCC 1H00348T).