Diversity Analysis Of Culturable Bacteria In Intertidal Sediment Of Qingdao And Enteromorpha Prolifera Polysaccharide Degrading Bacteria And The Enzymatic Properties Of A PL37 Family Enteromorpha Prolifera Polysaccharide Lyase

The intertidal zone is located at the frontiers of the land and ocean intersection areas and contains rich microbial resources.The intertidal sediment environment is one of the main coastal environments.The exploitation and analysis of microbial resources in the intertidal sediment will help to enhance the cognition of marine microbial species diversity and promote researches on metabolic characteristics,ecological functions and application of marine microorganisms.Enteromorpha prolifera polysaccharide is a non-cellulose water-soluble sulfated heteropolysaccharide which has high content in Enteromorpha prolifera.It has a variety of physiological activities and high application value.Screening of bacteria and exploiting of enzymes that can efficiently degrade Enteromorpha prolifera polysaccharides will provide technical support for efficient and high value utilization of the Enteromorpha prolifera and Enteromorpha prolifera polysaccharide resources.In this thesis,a total of 58 bacterial strains were isolated from Qingdao intertidal sediment samples and the diversity of these culturable strains was analyzed.All these strains belonged to three classes of the Proteobacteria and Bacteroidetes phyla,i.e.Gammaproteobacteria(49 strains,84.5%),Alphaproteobacteria(5 strains,8.6%)and Flavobacteria(4 strains,6.9%).The strains belonging to the genera Vibrio(43.1%)and Marinobacter(25.9%)in the Gammaproteobacteria class were dominant among the isolated bacteria.Among them,totally five strains were found to potentially represent new bacterial species.One of them,strain SM1973T,was then subjected to a polyphasic taxonomic study to determine its taxonomic position.Strain SM1173T was a Gram-stain-negative,rod-shaped,flagellated and aerobic bacterium.The strain grew at 15-37?(optimum at 25?)and with 0-5.5%(w/v)NaCl(optimum with 1.5-2.0%NaCl).It reduced nitrate to nitrite and hydrolyzed Tween 80 and aesculin but was not able to hydrolyze starch,gelatin or DNA.The main polar lipids of strain SM1973T included phosphatidylethanolamine(PE),phosphatidylglycerol(PG)and diphosphatidylglycerol(DPG).The major fatty acids of strain SM1973T was summed feature 3(C16:1?7c and/or C16:1?6c,48.8%),C16:0(25.9%)and summed feature 8(C18:1?6c and/or C18:1?7c,10.5%).The major respiratory quinone of strain SM1973T was Q-9.The genomic DNA G+C content of strain SM1973T was 41.1 mol%.In the phylogenetic trees constructed based on 16S rRNA gene sequence,strain SM1973T formed an independent branch in the cluster formed by members of the order Oceanospirillales.All these polyphasic data indicate that strain SM1973T belongs to a novel genus within the order Oceanospiril lales,for which the name Arenisedimentibacter gen.nov.is proposed and represents a new species in the new genus,for which the name Arenisedimentibacter marinus sp.nov.is proposed.In this thesis,totally 26 Enteromorpha prolifera polysaccharide(EPP)degrading bacterial strains were isolated from Enteromorpha prolifera samples collected from the Qingdao coastal area using media with EPP as the only carbon source.The Shewanella(11 strains,42.3%)and Alkalihalobacillus(7 strains,26.9%)strains were dominant among the isolated bacteria.Through measuring the ratio(D/d)of the degradation circle diameter to colony diameter on plates containing a soild medium with EPP as the sole carbon source,the EPP degradation ability of the isolated strains was compared and three strains {Pseudomonas sp.W2,Mesobacillus sp.E139 and E134)were found to have strong EPP degradation ability(D/d),which laid a foundation for mining high-efficiency EPP-degrading enzymes from them.In this thesis,a gene ht-34 encoding a polysaccharide lyase(HT-34)belonging to the PL37 family was mined from the genomic data of Echinicola Pacifica HT-3 isolated from the surface of Enteromorpha prolifera.The enzyme HT-34 was then heterologously expressed and purified.The results from experiments testing enzyme properties showed that the recombinant enzyme was a EPP-specific lyase,with the optimum reaction temperature to EPP being 40?,the optimum reaction pH to EPP being 8.5 and the enzyme activity was highest without NaCl.