【作者】 闫志勇；

【导师】 王斌；

【作者基本信息】 青岛大学 ， 病原生物学， 2007， 博士

【摘要】 海洋微生物是以海洋生物、水体为正常栖居环境的一类微生物,由于所处的特殊生活环境,使其具有产生生物活性物质的巨大潜力,已成为生物活性物质开发的重要来源。双齿围沙蚕是一种主要生活在海边潮间带的海洋生物,近年来研究人员已从其体内分离出多种生物活性物质。鉴于其可以生活在污染严重的环境,并能帮助改善和治理环境污染,故推测其体内可能具有多种生物活性物质或特殊的微生态菌群,以帮助其拮抗外界污染环境的损害。本研究对双齿围沙蚕消化道菌群进行了分析,筛选出了部分生物活性菌株,并对获得的嗜麦芽寡养单胞菌蛋白酶的酶学特征和纤溶活性进行了研究。本课题从不同来源双齿围沙蚕的消化道中共分离出17株细菌,其中有6株至少对金黄色葡萄球菌、大肠埃希菌、铜绿假单胞菌和白色念珠菌等指示菌中的1种有抑制作用,3株细菌至少对工种肿瘤细胞（HeLa细胞和HepG2细胞）有细胞毒活性。菌株Y1、Y7均同时具有不同程度程度的抑菌活性和细胞毒性,经形态生理、16S rRNA基因序列鉴定,分别为E.aurantiacum和s.marisflavi。奶粉平板法筛选发现5株细菌能产生蛋白酶,其中D2菌株具有较强的产酶能力。纤维蛋白平板法检测显示,菌株Y5、D2株均具有体外纤溶解活性。沙蚕的消化道活性菌株总分离率高于海水等。在对从双齿围沙蚕消化道内分离出的具有体外纤维蛋白溶解活性的菌株Y5进行鉴定时发现,其形态为革兰阴性直杆状,无芽孢和鞭毛,大小为0.5μm～0.8μm×1.5μm～3.0μm;生长温度范围为4℃～40℃;具有嗜盐性,NaCl耐受范围为1%～13%;氧化酶、过氧化氢酶、精氨酸双水解试验阳性,不还原硝酸盐;可利用葡糖糖、麦芽糖为唯一碳源;精氨酸脱羧实验阳性;细胞主要脂肪酸成分为C_18:1ω7c,C_16:1ω7c,C₁6:0),C_10:03-OH。DNA（G+C）mol%为47.5%。其16S rRNA基因序列与9种已确定的海单胞菌同源性为93%～97%,且都位于系统发生树的同一类群中,并与M.aquimarina和M vaga共同构成一个独立的进化枝。根据菌株Y5的生理学、遗传学和系统发生特征确定,菌株Y5属于海单胞菌属,但与已报道的该属其他菌种的某些特征不完全一致,可能为该菌属的一个未定种/新种,暂命名为沙蚕海单胞菌（M.aibuhitensis sp.nov.）,这也是首次从我国海域分离出该属细菌菌株。此外,该菌株具有一定纤溶活性。菌株D2同时具有强产蛋白酶能力和体外纤溶活性,经鉴定为嗜麦芽寡养单胞菌,并对其产蛋白酶条件和蛋白酶学特征进行了研究。Lowry法检测显示该菌株产酶能力为1104U/mL,最佳产酶条件为pH 8.0、25℃培养48h。酪蛋白酶图谱法和凝胶成像分析证实其蛋白酶分子量约为42 kDa,在培养上清液中纯度大于92%。经过饱和硫酸铵沉淀、DEAE-Sepharose阴离子交换层析、Superdex G-75凝胶过滤层析等步骤纯化获得的D2蛋白酶（嗜麦芽寡养单胞菌蛋白酶,SMP）,比活性达到1478.0U/mg。该酶活性的最适pH值为9.0,是一种碱性蛋白酶;最适温度为60℃,在55℃以下及pH6～9的环境中具有较好的稳定性。SMP的等电点为9.17。Cu²⁺、Ca²⁺、Mg²⁺、K⁺等金属离子对SMP活性有明显促进作用,EDTA和PMSF能强烈抑制酶活力,但对尿素、SDS、DTT等变性剂有较好的耐受性,因此可能是一种金属离子依赖性的丝氨酸碱性蛋白酶。在氨基酸序列测定时发现其N末端被修饰,故未能得到该酶的N-末端序列。随后改用ESI-MS/MS质谱分析,获得的其两个内部肽段序列经过检索未发现与其序列一致性高的蛋白,提示SMP可能是一种新的蛋白酶。鉴于SMP良好的稳定性和较宽的温度、pH活性范围,以及菌株D2的高产酶特性,该株有望成为新的蛋白酶生产资源。本研究还对SMP的纤溶活性及机制进行了研究和初步探讨。体外实验（试管凝块法和纤维蛋白平板法）和体内实验（大鼠动静脉旁路血栓模型）均证实SMP同时具有溶解纤维蛋白原和纤维蛋白的活性,且活性呈一定的量效关系,因此,具有抗凝和溶栓双重作用。此外,SMP可不依赖于纤溶酶原的激活直接溶解纤维蛋白,能在实验动物体内可迅速发挥溶栓活性,并直接溶解已经形成的血栓。SMP对实验动物体内的t-PA和PAI-1活性无明显影响,其溶栓活性不通过依赖t-PA,或抑制PAI-1来发挥。综上所述,本课题对双齿围沙蚕消化道菌群进行了研究,对生物活性菌株进行了筛选和鉴定,并对分离出的嗜麦芽寡养单胞菌蛋白酶进行了酶学特征和体内外纤溶活性进行了研究和探索,取得部分成果已经申请国家发明专利,以期将来能够转化成为新的蛋白酶或抗栓药物的生产资源。更多还原

【Abstract】 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.更多还原