Analysis Of Microbial Diversity In The Intestine Of Sea Cucumber And Studies On Alginate Lyase Of Strain S12~T

Alginate is abundant in the cell wall of brown algae.It is one of renewable carbohydrates on the earth,which hold significant reserves.Alginate and its degradation product,oligosaccharide,have good biological activity and economic value,so they have broad development prospects.The ocean environment contains abundant microbiological resources.The extreme environmental conditions of the ocean have enabled marine microbes to evolve unique and various metabolic pathways so that they show the ability of producing a variety of enzymes with high potential value for utilization.The gut microbes of marine animals not only live in the intestines of animals,but also could assist the host to digest food.Therefore,the intestinal microbes of marine animals are natural resources bases of degrading enzymes,but there are few related studies.In this thesis,the intestinal microbes of sea cucumber are the main research objects."Isolation and culture of intestinal microbes and diversity analysis-screening of functional strains-expression and characterization of alginate lyase"is the main line.The results are as follows:1.Isolation and culture of the microorganisms from sea cucumber's intestinal contents,and analyse the intestinal microbial diversity based on the culture and 16S rRNA gene high-throughput sequencing.A total of 226 strains of bacteria were isolated from the gut contents of the sea cucumber,distributed in 4 phyla,49 genera and 102 species.The phyla Proteobacteria and Firmicutes dominated the culturable bacteria of all samples.At the genus level,the most abundant cultural group is the genus Bacillus.The results of 16S rRNA high-throughput sequencing showed that the microbial abundance and diversity were the highest in the sediment around the sea cucumber,followed by the sea cucumber intestine and finally the water sample around the sea cucumber.The dominant groups in the sea cucumber gut are phyla Proteobacteria,Chloroflexi,Actinobacteria,Firmicutes and Bacteroides,which account for about 90%of the intestinal flora.The microbial composition of sea cucumber in the gut is more similar to the microbial composition of sediments,but the proportion of phyla Firmicutes,Chloroflexi and Proteobacteria in the intestine is higher than that in sediments,while the proportion of phyla Bacteroides and Actinobacteria is slightly lower.The proportion of phyla Verrucomicrobia and Bacteroidetes in water samples is higher.2.Screening moree than 100 strains that show the potential of producing alginate lyase from the intestine of the sea cucumber,and verifying and evaluating some potential high-yield strains.A total of 102 strains which showed potential of aproducing alginate lyase were screened from the cultured bacteria isolated from two samples of sea cucumbers' gut.Among them,there are 31 strains whose hydrolyzed circles were greater than or equal to 10 mm,and strains with high-yielding potential were mainly distributed in Vibrio and Bacillus.After the second screening for partial strains,16 strains are determined to possess the ability of degrading alginate,but all of them show a weaker activity compared with strain s12T which was isolated by our laboratory before.The enzyme activity of the selected strain 6-3-04 could reach more than 90%of the strain s12T in the second screening,but is generally lower than the strain s12T throughout the fermentation experiment.3.Heterologous expression,purification and characterization of four alginate lyase genes of strain s12T.The four potential alginate lyase genes alg3,alg7,alg9 and alg10 of strain s12T,which showed high-efficiency degrading ability of alginate,were heterologously expressed.Under the given expression conditions,the expression products of genes alg3,alg7 were mainly precipitated and weak alginate lyase activity was detected.The genes alg9 and alg10 were expressed in a soluble manner.The activity of expression product of gene alg9 was weak,however a significant alginate-degradation activity was detected for the expression product of gene alg10.The product was named recombinant alginate lyase rAlg10.The recombinant enzyme rAlg10 was purified using an affinity chromatography with nickel column and its enzymatic properties were examined.The recombinant enzyme rAlg10 is a bifunctional enzyme with Poly-G preference.The optimum temperature of the recombinant enzyme rAlg10 is 40 ?,the optimum pH is 7.4,and the optimum salinity is 300 mM NaCl.Other common metal ions could reduce the degradation activity of the enzyme.The specific activity of recombinant alginate lyase rAlg10 under the optimum condition is 106.1U/mg.4.Polyphasic taxonomic studies of three potential new strains isolated from marine environment.The taxonomic positions of strains E4404T,YLY04T and SEH01T were determined by a polyphasic taxonomic studies including phylogenetics,genomics,phenotypic and chemical taxonomic characteristics.The strain E4404T is determined to represent a new species of the genus Vibrio,the family Vibrionaceae,the order Vibrionales,the class Gammaproteobacteria in the phylum Proteobacteria,for which the name Vibrio albus sp.nov.is proposed.The type strain of Vibrio albus sp.nov.is E4404T(=MCCC 1H00197T= KCTC 52890T).The strain YLY04-T is determined to represent a new species of the genus Pelagivirga,the family Rhodobacteraceae,the order Rhodobacterales,the class a-Proteobacteria in the phylum Proteobacteria,for which the name Pelagivirga dicentrarchi sp.nov.is proposed.The type strain of Pelagivirga dicentrarchi sp.nov.is YLY04T(=MCCC 1H00334T=KCTC 62452T).The strain SEH01T belongs to the family Bradymonadaceae,the order Bradymonadales,the class Deltaproteobacteria in the phylum Proteobacteria,and a new genus named Lujinxingia gen.nov.is found to cover strains B210T and SEH01T.The name Lujinxingia sediminis sp.nov.is proposed for the strain SEH01T.The type strain of Lujinxingia sediminis sp.nov.is SEH01T(=KCTC 42950T =DSM 101859T).