微生物资源及开发利用.ppt

微生物资源及开发利用,山东省农业科学院岳寿松2011.6.12威海,第三届特种医学暨三省省际联合（山东-河南-湖北）微生态学学术会议,提要,肠道微生物资源从极端微生物到太空微生物废弃资源的微生物转化,肠道微生物,人类肠道细菌超1000万亿细菌数量是人体细胞的10倍,PLoSbiology.November2008(11)6:280LesDethlefsenDavidA.Relmanetal.,斯坦福大学医学院的戴维雷尔曼(DavidRelman)和同事一起，利用一种新型技术焦磷酸测序法(pyrosequencing)，得到了关于人体肠道内菌落数量更为准确的数据。,PLoSbiology,人类肠道内的细菌群落数量是原有认识的10倍以上。,Science328,228(2010)MatamVijay-Kumaretal.,micegeneticallydeficientinToll-likereceptor5(TLR5),acomponentoftheinnateimmunesystemthatisexpressedinthegutmucosaandthathelpsdefendagainstinfection,exhibithyperphagiaanddevelophallmarkfeaturesofmetabolicsyndrome,includinghyperlipidemia,hypertension,insulinresistance,andincreasedadiposity.,MetabolicSyndromeandAlteredGutMicrobiotainMiceLackingToll-LikeReceptor5,肠道微生物系变化导致代谢综合症。,肠道微生物可能影响人类的健康，如胖瘦、糖尿病、胃病、甚至癌症。,缺乏先天性免疫系统中的某种重要成分（TLR5蛋白）的小鼠会产生代谢综合症的标志性特征，如伴有肠道微生物系变化的脂肪积聚的增加及胰岛素抵抗。将突变小鼠的肠道内微生物转移到无菌小鼠的肠道之中会使接受这些微生物的小鼠出现代谢综合症的几种特征。,人肠道微生物菌落基因目录,JunjieQin,RuiqiangLietalAhumangutmicrobialgenecatalogueestablishedbymetagenomicsequencing.Nature|Vol464.4March2010,theIllumina-basedmetagenomicsequencing,assemblyandcharacterizationof3.3millionnon-redundantmicrobialgenes,derivedfrom576.7gigabasesofsequence,fromfaecalsamplesof124Europeanindividuals.Thegeneset,150timeslargerthanthehumangenecomplement,Over99%ofthegenesarebacterial,indicatingthattheentirecohortharboursbetween1,000and1,150prevalentbacterialspeciesandeachindividualatleast160suchspecies,124位健康的、超重的和肥胖的成年人以及炎症患者提取的人肠道微生物菌落的基因目录。比人类基因组大150倍。超过99%为细菌。这些基因大部分是在不同个体之间共享的。,natureVolume:473,Pages:174180Datepublished:(12May2011)DOI:doi:10.1038/nature09944,Enterotypesofthehumangutmicrobiome,identifythreerobustclusters(referredtoasenterotypeshereafter)thatarenotnationorcontinentspecificThisindicatesfurthertheexistenceofalimitednumberofwell-balancedhostmicrobialsymbioticstatesthatmightresponddifferentlytodietanddrugintake.Theenterotypesaremostlydrivenbyspeciescomposition,butabundantmolecularfunctionsarenotnecessarilyprovidedbyabundantspecieshighlightingtheimportanceofafunctionalanalysistounderstandmicrobialcommunities.individualhostpropertiessuchasbodymassindex,age,orgendercannotexplaintheobservedenterotypes,人类肠道微生物系统可拥有特定的分类肠型,肠型分三类：Enterotype1,Enterotype2,Enterotype3.分别为Bacteroides型、Prevotella型和Ruminococcus型,未来医师或可依据肠型的区划，量身打造病患饮食清单以及开处方，甚至至为抗生素寻求替代品。,肠型与种族、性别、体重、健康或年龄等均无关联。,ThinkTwice:HowtheGutsSecondBrainInfluencesMoodandWell-Being,AdamHadhazy|February12,2010|,Acomplex,independentnervoussystemlinesthegastrointestinaltractthathasbeendubbedthesecondbrain,心理过程与消化系统紧密相联的程度超出人们的想象。长期压力过大，过度紧张、焦虑、抑郁、易怒等不良情绪，均可使胃肠道生理功能发生紊乱。患老年性痴呆症及帕金森氏病的病人中，常在头部和腹部发现同样的组织坏死现象。疯牛病病人通常是大脑受损而出现精神错乱，与此同时肠器官也经常遭到极度损害。,natureVol467|2September2010|doi:10.1038/nature09354,Bacterialcharityworkleadstopopulation-wideresistanceHenryH.Lee,MichaelN.Molla,CharlesR.Cantorandhairwasdegradedobviouslyat84h.,B1,B2,C2,0hHair84h,0hFeather36h,0hWool72h,野生菌接种于羽毛、羊毛、毛发三种培养基，观察对三种底物的降解情况。36小时能将羽毛完全降解，72小时将羊毛完全降解，84小时后对头发有明显降解。,StudiesonfeatherdegradatingandkeratinasepropertiesofS.maltophiliaYHYJ-1,嗜麦芽窄食单胞菌菌YHYJ-1角蛋白酶KerF基因克隆、表达、纯化及酶学性质分析,ProteincontentinfermentedliquidProteinconcentrationwasdeterminedaccordingtoBradford(1976)，usingbovineserumalbumin(BSA)asstandardsubstrate,发酵液蛋白质含量,S.maltophiliaYHYJ-1发酵产酶曲线,Timecourseproductionofkeratinaseinfeathersubstrate,OptimumtemperatureandthermalstabilityofcrudekeratinaseTheoptimumtemperatureofcrudeenzymewas50.Andthekeratinasewasstableat4045.,角蛋白酶的最适反应温度及热稳定性,Effectoftemperatureonactivityofcrudekeratinase,Temperaturestabilityofcrudekeratinase,OptimumpHandpHstabilityofcrudekeratinaseTheoptimumpHofcrudeenzymewas9.0.TheactivityofkeratinasewasstableinthepH6.6and8.4indicatedthatitisacombinationenzyme.,角蛋白酶最适pH值及pH值稳定性嗜麦芽窄食单胞菌角蛋白酶粗酶最适pH值为9.0；在pH8.4时比较稳定，同时在pH6.6时也相对稳定。由这两个峰可以初步推断此角蛋白酶可能为一种复合酶。,EffectofpHonactivityofcrudekeratinase,EffectofpHonstabilityofcrudekeratinase,Cloning,Expression,PurificationandFunctionalAnalysisofS.maltophiliaYHYJ-1keratinasegeneKerF,ExpressionandPurification,CloningofgeneKerF,Functionalanalysisofkeratinase,KerF基因克隆,表达纯化,酶活性质分析,嗜麦芽窄食单胞菌菌YHYJ-1角蛋白酶KerF基因克隆、表达、纯化及酶学性质分析,PrimerdesignP1：5-GAACGAACATTTCATCTTGCT-3；P2：5-GGTCTGAGGAACAGCGTG-3.DesigntheP3,P4withoutsignalpeptideaccordingtoexpressionvectorpET28a（+）:P3：5-AAACCATGGCTCAGGTAACGCAACC-3；NcoP4：5-AAAAAGCTTGTACTGGGCGTTGAGGGTC-3HindIII,嗜麦芽窄食单胞菌R551-3全基因组已测序，基因组序列和注释已登陆GenBank，根据推定的嗜麦芽窄食单胞菌R551-3的角蛋白酶基因序列设计引物P1、P2。测序后针对pET28a（+）表达载体设计去信号肽引物P3、P4。,ExtractionofgenomicDNAExtracttheDNAofStenotrophomonasmaltophiliaasthetemplateforfurtherPCR.,AgarosegeleletrophoresisofgenomicDNAofS.maltophiliaYHYJ-1,嗜麦芽窄食单胞菌总DNA提取提取嗜麦芽窄食单胞菌的染色体DNA，作为下一步PCR扩增的模板。,M:Marker(1KbDNALadder);1:GenomicDNAofS.maltophiliaYHYJ-1,PCRamplificationofkeratinasegeneandconnectionofthetargetgeneandT-vector.角蛋白酶基因KerF的扩增及与T载体的连接,以S.maltophiliaYHYJ-1的基因组DNA为模板，进行PCR扩增，得到一条大小为1980bp左右的特异性条带。连接后的重组质粒pMD18-KerF经EcoRI和HindIII双酶切后出现2条带。,PCRamplificationofKeratinasegeneKerFM:Marker(DL2000Plus);1:KeratinasegeneKerF,IdentificationofrecombinantplasmidpMD18-KerFbydigestion(EcoRI,HindIII),SequencingandsignalpeptideanalysisofthetargetgeneThekeratinasegene(KerF),includecompletesequenceof1981bpanda1734bpopenreadingframeandencode580aminoacid,wasclonedfromS.maltophilia(GenBankAccessionNo.HM590650).UsingSignalP3.0toassaythesignalpeptideofKerFgene.,KerF基因序列测定与信号肽分析利用DNAman软件和NCBI的BLAST进行序列同源性分析，结果显示，该基因全长1981bp，含有一个1743bp的开放阅读框，编码580个氨基酸。将该基因提交GenBank，获得登录号HM590650。利用SignalP3.0软件分析推导氨基酸的信号肽,ConstructionofpET28-kerFrecombinantplasmid,pET28-kerF重组质粒构建,PCRamplificationofKerFgenewithoutsignalpeptideandidentificationofrecombinantplasmidpET28-kerFbydigestion(NcoI,HindIII).,去信号肽的KerF片段PCR扩增及与pET28表达载体的连接设计引物P3、P4在PCR时将KerF片段的信号肽切除，电泳检测后得到一段与预计结果大小一致的条带（1756bp）。将KerF片段与pET28表达载体连接，提取转化子质粒，进行NcoI和HindIII双酶切鉴定。,PCRamplificationofgeneKerFwithoutsignalpeptide,IdentificationofrecombinantplasmidpET28-kerFbydigestion(NcoI,HindIII),ExpressionofS.maltophiliaKeratinaseGene（KerF）inEscherichiacoli,重组角蛋白酶的表达以重组质粒pET28-kerF转化大肠杆菌BL21（DE3），37振荡培养1h至OD600约为0.1，25，0.1mmol/LIPTG诱导表达，重组质粒pET28-kerF在相对分子量50kD处出现一条特异蛋白带。,SDS-PAGEanalysisoftheexpressionproductatdifferentinducedtimes.1:Marker;1-7:recombinantstraininducedbyIPTGat17h,respectively;8-14:recombinantstrainlysateinducedbyIPTGat17h,respectively.,PurificationofkeratinaseKerFRecombinantKerFwaspurifiedtoelectrophoreticalhomogeneityafternickelaffinitychromatographyandexhibitedahighspecificactivityof726U/mg.,重组角蛋白酶的纯化重组角角蛋白酶末端含有His标签，可采用镍亲和层析法快速纯化。对纯化的角蛋白酶进行蛋白含量测定和酶活测定，得出重组角蛋白酶比酶活力达到26U/mg。,AnalysisofrecombinantexpressedkeratinasebySDS-PAGE.M,Marker;1,purifiedkeratinase;2,recombinantstrainpET28-kerFlysateinducedbyIPTG,OptimumtemperatureandthermalstabilityofkeratinaseKer,Effectoftemperatureonenzymeactivity,Effectoftemperatureonenzymestability,KerF角蛋白酶的最适作用温度和温度稳定性在pH7.4，反应30min的条件下，于2575范围内，每隔5，分别测定酶活，以酶活最高为100%计算相对酶活，由此确定酶的最适作用温度。将酶液于30、35、40、45、50、55下保温处理不同时间，测定残余酶活性。以未保温（4放置）的酶样活性为100%，由此确定酶的热稳定。,OptimumpHandpHstabilityofkeratinasKerFTheoptimumpHwasdeterminedat30usingthefollowingbuffers：Aceticacid/sodiumacetatebuffer(pH4.25.4),Tris-HC1buffer(pH6.08.4),andGlycine/NaOHbuffer(pH9.09.6)Theenzymesolutionwaspreincubatedfor30minindifferentpHbuffer.Thentheresidualactivitywasmeasured,EffectofpHonenzymeactivity,EffectofpHonenzymeactivity,EffectofpHonenzymeactivity,KerF角蛋白酶的最适pH值及pH稳定性,EffectofpHonenzymeactivity,EffectofpHonenzymeactivity,EffectsofpHonenzymeactivity,EffectofpHonenzymestability,EffectofpHonenzymestability,EffectofmetalionsonkeratinaseKerFactivity,金属离子对KerF角蛋白酶活性的影响,Effectofproteinaseinhibitorsandorganicsolventonkeratinaseactivity,有机溶剂、蛋白酶抑制剂等对KerF角蛋白酶活性的影响,Effectofsulfurcompoundsonkeratinaseactivity,含硫化合物对角蛋白酶的影响,SubstratespecificityofkeratinaseRecombin