Isolation Of Marine Protease-producing Bacteria And Characterization Of The Extracellular Protease Of Vibrio Pomeroyi 12613-9

Oceans cover approximately 70%of the earth's surface and contain microorganisms with both high abundance and diversity,which are considered as an important source of enzymology research materials.Microbial proteases play a key role in the global biogeochemical cycles.Thus,studying the marine microorganism-sourced proteases is crucial for understanding the mechanisms of global biogeochemical cycles.Antarctica's extreme climatic conditions make it one of the harshest habitats,which however,is the home of some of the most unique microbial groups on earth.Thus,microorganisms in Antarctica may possess the ability to produce unique enzymes that adapt to extreme environments.In this thesis,the diversity of protease-producing bacteria from Atlantic Ocean sediments and seawater samples and the surface of Nacella polaris collected from Antarctica were investigated.Furthermore,P9,a metalloprotease secreted by an Atlantic surface strain V.pomeroyi 12613-9,were isolated,purified,and heterologously expressed.Its enzymatic characteristics and collagenolytic mechanism were also studied.1.Isolation and diversity analysis of cultivable protease-producing bacteria from the surface of Nacella polaris collected from AntarcticaWe isolated protease-producing bacteria strains firom the surface of Nacella polaris collected near the Great Wall Station in Antarctica.Totally 26/5 protease-producing bacteria strains were isolated with skim milk/gelatin medium.All the strains generated relatively small hydrolytic circles.16S rDNA sequence analysis showed that all the strains belong to the genus Pseudoalteromonas.These results indicated that Pseudoalteromonas is the predominant group of protease-producing bacteria at the surface of Nacella polaris from Antarctica.2.Isolation and diversity analysis of cultivable protease-producing bacteria from the Atlantic Ocean sediments and seawater samplesTotally 151 protease-producing bacteria strains were isolated from the sediments and seawater samples from the Atlantic Ocean with screening media.16S rDNA sequence analysis showed that the strains belong to 4 individual genus:Pseudoalteromonas,Vibrio,Alteromonas and Sulfitobacter.While Pseudoalteromonas and Vibrio were the predominant groups in the surface seawater samples,the predominant protease-producing bacteria gradually changed from Pseudoalteromonas to Alteromonas as the sampling depth goes deeper.The bacterial protease diversity was investigated by detecting the hydrolysis ability of the proteases against different proteins(casein,gelatin and elastin),which was represented by the hydrolytic circle/colony diameter ratio.Most extracellular proteases from these strains could hydrolyze casein and gelatin significantly.Alteromonas and Pseudoalteromonas were the predominant groups in the sediments,and Vibrio and Pseudoalteromonas were the predominant groups in seawater.Comparatively,proteases produced by Vibrio strains have higher caseinase and gelatinase activities,indicating that the proteases produced by Vibrio may play an important role in ocean surface nitrogen cycling.3.Purification,heterologous expression,and characterization of P9 from V.pomeroyi 12613-9V.pomeroyi 12613-9 is a protease-producing strain isolated from an Atlantic surface seawater sample.A M4 family metalloprotease was isolated and purified from 12613-9's fermentation broth,and designated as P9.P9 was heterologously expressed in Escherichia coli BL21(DE3)and purified.Both wild-type and recombinant P9 were characterized.Substrate specificity analysis,showed that P9 could hydrolyze casein,gelatin and collagen,with highest activity toward gelatin.With gelatin as the substrate,the optimum pH of both wild-type and recombinant P9 was 6.0,and the optimum temperature was both 40?.P9 showed the highest activity in a buffer without NaCl,but it remained more than 50%activity in 4 M Nael,showing its high salt tolerance.The inhibitor assay indicated that Zn2+ and metalloproteinase inhibitor o-P can inhibit the acticity of both wild-type and recombinant P9.These results indicated that P9 has evolved over time to adapt to high salt concentration of marine environment.4.Study of the collagenolytic mechanism of P9Substrate specificity analysis showed that P9 can degrade soluble collagen(gelatin)signaficantly,and had a small activity against insoluble bovine collagen Type ? fibers.Atomic force microscopy observation showed that P9 treated collagen fibers were loosened,and microfibers were exposed,but were not degraded.Biochemical assay showed that after insoluble collagen type ? fibers was treated with P9,the content of glycosaminoglycans released in the supernanant was increased,with no significant change on the content of free amino acids released in the supernanant.SDS-PAGE analysis showed that Decorin and Fibronectin,which play a role in the cross link of collagen fibers,were degraded by P9 at different levels.Furthermore,through HPLC,we found that protease P9 was active against the synthetic peptides from bovine type ? collagen.This provides an explanation of the higher activity of P9 against gelatin because when insoluble collagen is heated and converted to gelatin,its stable triple helix structure is destroyed,and becomes single strands,which is vulnerable to P9.These results suggest that when collagen is treated by P9,only the glycoproteins can be degraded by P9,but its collagen monomers can not.This lays a foundation for further research on the structure and function of this protease.In conclusion,studying the diversity of marine protease-producing bacteria and P9's mechanism of action against collagen is crucial for understanding the mechanisms of global biogeochemical cycles.Meanwhile,it can also prove beneficial developing new protease and microbial resources.