Study Of The Flora In The Alimentary Tract Of Perinereis Aibuhitensis Grube And Its Bioactivity

Marine microbes are kinds of microorgnism that inhabit in marine life or water body.The special living environments promote them producing bioactive compound with enormous latent capacity and thus they are important source in bioactive compound. Perinereis aibuhitensis Grube is one kind of marine life that mainly lives in intertidal zone.To date,researchers have isolated various bioactive compounds in vivo.Respecting of the ability in living at serious pollution surrounding and assisting to improve and manage environmental pollution,we presume that possibly various bioactive compounds or special microbial flora may exist and help to oppose the damage of pollution.This study,we analyzed the microbial intestinal flora of the Grube,screened part of the strain with bioactivity,and investigated the enzymology characteristic and fibrolysis activity.Perinereis aibuhitensis Grube were collected from different resources.17 bacterial strains were isolated from their alimentary tract,in which 5 strains have inhibitory actions to at least one microbial indicator;3 strains were toxic to one sort of tumor cell line.Stain Y1 and Y7 were identified as E.aurantiacum and S.marisflavi respectively,both with degrees of bacteriosatsises and cytotoxicities.5 strains can produce protease on milk plate, among which D2 strain was identified as S.maltophilia with stronger ability in secreting protease.Both Strains Y5 and D2 showed fibrinolytic activities on fibrin plates.At last,we found the active strains have a higher isolating rate than that of in aqua marina.The characterization of bacterial strain Y5 has been performed.Bacterial cells are Gram-negative,rod-shaped,non-spore-forming,which are 0.5μm-0.8μm wide and 1.0μm-4.0μm long.No flagellum was found.Its growth temperature range is between 4℃and 40℃.The organism requires Na⁺ ions,and grows in the presence of 1%to13%(w/v) NaCl.It is positive for oxidase,catalase,and arginine dihydrolase,but negative for nitrate reduction,indole and H₂S production,and negative for gelatin,Tween 80,starch,and urease hydrolysis.D-glucose and maltose are utilized as sole carbon sources.The major fatty acids are C_18:1ω7c,C_16:1ω7c,C_16:0,and C_10:0 3-OH.The DNA G+C content is 47.5 mol%.On the basis of the almost-complete 16S rRNA gene sequence(DQ279852),strain Y5 exhibites 93%-97%similarity with the nine recognized species of Marinomonas,and the phylogenetic analyses using N-J algotithm show that strain Y5 is within the species cluster comprising the genus Marinomonas,and forms a clade with M.aquimarina (AJ843078) and M.vaga(X67025).Therefore,phylogenetic,genetic and physiological properties of the organism confirms that strain Y5 may be a novel species of the genus Marinomonas,for which the name Marinomonas aibuhitensis sp.nov.is proposed.And its fibrinolytic activity may privide a new resource of thrombolytic drug. The high-proteinase-producing bacteria,strain D2(deposited in CGMCC,Store Number:1868#),was identified as S.maltophilia.Lowry method showed its enzyme capability was 1104U/mL,with optimization condition of pH 8.0 in cultivation for 48 h in 25℃.Casease mapping and gel imaging analysis confirmed its molecular weight was 42 kDa with more than 92%purity in supernate fluid in culture.The S.maltophilia protease (SMP) were purified by a stepwise procedure including centrifugation,ammonium sulfate precipitation,gel filtration and ultrafiltration.Enzyme activity is 9 with optimization pH, showing it is kind of alkaline protease,and optimal temperature is 60℃with better stability under 55℃and pH 6-9.The pI is 9.17.Cu²⁺,Ca²⁺,Mg²⁺ and K⁺ has promotion in enzyme activity while EDTA and PMSF strongly inhibit its activity.The enzyme owns better toleration with carbamide,SDS and DTT.Amino acids N-terminus sequencing suggests that its termination is possibly modified.ESI-MS/MS mass spectroscopy acquired two internal peptide sequences and no high homology protein was found which suggested that SMP is possibly a novel protease.Strain D2 maybe a new resource of proteas production.Moreover,we studied and initially explored the fibrolysis activity and its mechanism of SMP in this research.Tests in vitro(test tube clump method and fibrin plate process) and in vivo(thrombus model of rat arteriovenous shut ) all proved that SMP has the activity of dissolve both fibrin and fibrious with certain dose-effect relationship. Therefore,SMP have dual effects in anticoagulation and thrombolysis.In addition,SMP could dissolve fibrin without activation of plasminogen,and show its thrombolysis activity quickly in experimental animal to dissolve thrombus in form directly.SMP has no clear effect on t-PA and PAI-1 in experimental animals.The activity of thrombolysis thus does not play role depending on t-PA or PAI-1.To sum up,this topic studies the microbial population in the alimentary tract of Perinereis aibuhitensis Grube,screens and identifies strain with bioactivity.Also,we studied and explored the isolated S.maltophilia protease in enzymology characteristic and fibrolysis activity.We have applied for national invent patent by part of our results to transform these into production resource in novel protease or thrombolytic drugs.