[生物医药论文精品a]一株海洋琼胶酶产生菌的分离、筛选和初步鉴定

一株海洋琼胶酶产生菌的分离、筛选和初步鉴定A1A0A2A4A5A3A6A4A8A7A9A4A11A10A12A13A14A15A16A17A18A19A18A20A21A13A14A22A23310014A24A25A26A27A28A29A30A31A32A33A34A35A36A37A38A39A40A41A42A43A44A45A46A47A48A49A50A51A52A53A54A55A51A52A56A57A58A59A60A47A51A52A56A57A61A62A63A64A59A65A66A67A47A68A69A70A71A72A73A71A74A75A76A77A78A62A79A80A58A81A54A82A83A84A85A86A85A86A48A51A52A87A88A89A90A91A47A54A92A93A94A95A96A97A98A99A77A100A101A47A96A97A102A103A104A98A99A77A105A106A47A107A108A109A110A98A99A77A111A106A47A112A99A58DNSA113A114A98A99A77A53A66A115A58A116A117A84A118A119A118A119A120A121A122A123A100A101A106A124A12518A126A51A52A127A128A129A126A47A130A111A106A131A125A89A126A60A53A66A115A132A133A58A121A134A135A129HS0326601A47A136A137A138A129A126A99A77A139A105A140A141A117A47A142A143A144A103A145A87A146A147A148A149A67A135A129A47A129A150A151A152A153A154A84A118A155A118A155A141A55A138A129A126A59A156A157A158A159A47A160A161A137A145A99A89A140A58A162A163A164A165A166A167A168A169A72A47A62A170A171A87A133A60A51A52A53A60A59A129A47A120A172A87A51A52A53A71A51A52A56A57A58A173A174A175A176A164A177A165A81A54A178A117A179A180A84A181A182A183A182A183A184A51A52A127A128A129A185A51A52A53A185A100A101A185A106A124A123A186A100A187A188A189A190A191A192A193A194A189AA190A195A196A188A18900000000200800000000ISOLATION,SCREENINGANDIDENTIFICATIONOFAGARASEPRODUCINGBACTERIAFROMTHESEAWATERLIUMEIYING,MEIJIANFENG,YINGGUOQING,WANGHONGCOLLEGEOFPHARMACEUTICALSCIENCE,ZHEJIANGUNIVERSITYOFTECHNOLOGY,HANGZHOU310014,CHINAABSTRACTOBJECTIVETODEGRADEAGARINTOAGAROOLIGOSACCHARIDEBYTHEAGARASEWITHAHIGHERACTIVITY,THESTRAINSWERESEPARATEDFROMSEAWATERTHEAGAROOLIGOSACCHARIDEHAVESHOWNWIDEAPPLICATIONVALUESINFOODINDUSTRY,COSMETICINDUSTRY,PHARMACEUTICALINDUSTRYANDOTHERSECTORSMETHODSTHEAGARASEPRODUCINGBACERIAWEREISOLATEDBYTHESPREADPLATEMETHOD,FIRSTSCREENINGBYCLEARHALOSONAGARPLATECONTAININGAGARASTHESOLECARBONSOURCE,SECONDSCREENINGOFSHAKINGCULTURETHEENZYMATICACTIVITIESWEREDETERMINEDBYIMPROVEDDNSCOLORIMETRYMETHODRESULTS18AGARDEGRADINGSTRAINSWEREISOLATEDFROMTHESEAWATER,ANDAMOREPOWERFULAGARASEPRODUCINGMARINEBACTERIUMWASOBTAINEDTHESTRAINWASDESIGNATEDASSTRAINHS0326601ANDIDENTIFIEDPRELIMINARILYASGRAMNEGATIVE,RODSHAPEDCONCLUSIONA197A198A199A200A201A202A203A204A205A206A207A208A209A210A2112007C33068A212A213A214A215A216A201A217A218A219A220A221A220A222A223A224A225A226A227A228A213A214A201A229A230A231A220A232A220A233A234A220A222A223A226A235A236TEL057188871029EMAILGQYINGZJUTEDUCNWITHREGARDTOITSHIGHGROWTHSPEED,IFFURTHERTREATEDWITHMUTAGENS,THESTRAINMIGHTBEONEWITHAHIGHENZYMICACTIVITYAFTERFERMENTATIONOPTIMIZATION,ANDITCOULDBEUSEDTOTHEFURTHERSTUDYANDAPPLIEDTOTHEPREPARATIONOFAGARASEFORAGAROOLIGOSACCHARIDEPRODUCTIONININDUSTRYKEYWORDSAGARASEPRODUCINGBACTERIAAGARASEISOLATIONSCREENING琼胶（AGAR）是一种从海洋红藻中提取得到的多糖，其在食品、医药卫生等行业已有悠久的应用历史，但由于琼胶黏度高，水溶性低，不易被吸收，因此在应用方面受到很大的限制。而琼胶寡糖（AGAROOLIGOSACCHARIDE），即琼胶多糖经水解后聚合度为210的低聚糖，又称琼胶低聚糖，主要由琼二糖的重复单位连接而成，水溶性好，有利于人体吸收，大大提高了其应用价值，琼胶寡糖具有较强的抗癌1,2、抗氧化3,4、抗炎2,5及抗病毒1,6等活性，是一种极具开发潜力的低聚糖。琼胶酶能降解琼胶生成琼胶寡糖,与琼胶的化学降解相比，琼胶酶降解琼胶则具有特异性强的优点，可选择性地酶解G2011G7041特G4462化学G19202，从而制得特G4462聚合度的寡聚糖，G5194G1000具有G2465应G7477G1226G9213G2656，降解G17819G12255易于G6523制，G7092G2115G2465应，G10627G3671G8757G7591G4581等优点，在制G3803琼胶寡糖中具有G7138G7186的优G2195。G19512了制G3803琼胶寡糖G1055G3818，琼胶酶G17836可用G1328G5049具酶、用于G3250收DNAG6122RNA、海藻多糖G13479G7512的G11752G12362等7,8。G6117G3281G4557于海藻多糖降解酶的G11752G12362G4590G3800于G17227G8505G10378G5589，G17839行的G11752G12362大多G4628限于G1147酶G14752G7678的G12591选、酶的制G3803、G13443化与性G17148G2010G7524、及酶的应用等方面，G17836G7422G16277G1863于其G6249G1849G16280G8181化发G18249生G1147的G6265G17959，G1863G19202G4613在于G13582G1059酶活力高的G14752G76789。因此G12591选高G1147琼胶酶G1147生G14752具有重要的G10714G16782G5859G1053G2656应用价值。G11458G2081，琼胶酶G1147生G14752可G1209从海水、海洋G2172G10301G13940G17959、G5225G8889等海洋G10627G3671G1209及G9258G8862、G8839G8981、G6502G8757G2487等G19482地G10627G3671中G14731得1,1012，G10990G14279从G14772G14768G7693G13007中G1075可G12591选到琼胶降解G14752G767813，但大多G6980琼胶酶G1147生G14752从海洋G10627G3671中G2010G12175得到1。G7424G4466G20576G1820后从G14323G4677、G2500G5042等地G18331G19610海水G7691，从中G2010G12175G12591选到一G7678活力较高的G14752G7678，G5194G4557其G17839行了G2033G8505G18504G4462。G7100在为琼胶酶、琼胶寡糖的G9157G1849G11752G12362G2656开发应用提G1391G3534G11796。1材料与方法11G7460G7021111海水G7691品取G14270G9005G8755G14323G4677G1888G4388G4719、G18341G3628G2656G7429家尖等藻类繁殖地G2656G2500G5042G9213岭箬横、坞沙门及三门等紫G14768养殖地海水。112培养G3534组成G2010G12175培养G3534（）NACL25；NH4CL01；MGSO47H2O05；KCL01；FESO47H2O0002；CACL2002；NAH2PO4006；琼脂2；PH70。斜面培养G3534（）NACL25；G18249母浸出粉05；MGSO47H2O05；KCL01；FESO47H2O0002；CACL2002；NAH2PO4006；葡萄糖01；琼脂2；PH70。复G12591培养G3534（）琼脂02；G18249母浸出粉025；其他成G2010同海水G2010G12175培养G3534。G1209上培养G3534均为121、20MIN灭G14752；液体培养G3534装量为35ML/250ML摇瓶。12方法121海水G7691品的G18331G19610G18331G19610海水G7691品装于塑G7021瓶中，紫G14768等G7691品装于G7691品袋中。G18331G19610的G7691品置于4冰箱保存。122G7691品的驯化富G19610取琼胶适量，海水G7691品40ML，在酒精灯旁加G1849250ML锥形瓶中，28、200RMIN1连续振荡培养，G4462期加G1849适量蒸馏水G14279原体积，G11458测培养液G7138G7186混浊后，取培养液4ML接种于含琼胶5G、36ML该海水G7691品的三角瓶中，重复上述培养G17819G12255。123G14752G7678的G2010G12175G18331用稀释涂布平板法用G7092G14752移液管吸取海水G6122富G19610G7691品5ML，加G1849含一层玻璃珠G265645MLG7092G14752人G5049海水的三角瓶中，200RMIN1振荡30MIN，混匀制成G14752悬液，用G7092G14752人G5049海水适当稀释，移取02ML涂布于G2010G12175培养G3534平板上，28培养，直G14279平板上长出G14752落。124G14752G7678的G2033G12591观察平板上G14752落形G5589，挑取周围形成G7138G7186凹陷的G14752落，G5194在平皿G5225部G1328标记，再滴加卢戈氏碘液G7591色。保留那些平板上能G1147生透G7138圈G14752落的G4557应斜面，弃去G7591色后G7092透G7138圈G14752落的G4557应斜面，将G2081者置于28培养，G1391复G12591用。125G14752G7678的复G12591用接种G10627挑取G2033G12591G14752G7678的新鲜斜面G14752苔2G10627，接G1849复G12591摇瓶培养G3534中，28、200RMIN1发G18249培养48H。将发G18249液于3000RMIN1下G12175心10MIN后取上清液测酶活力，G12591选出G1147酶活力最高的G14752G7678G17839行G13443化。126G14752种G13443化用接种G10627挑取复G12591得到的新鲜斜面种G14752苔，用G7092G14752人G5049海水适当稀释，取02ML涂布于G2010G12175培养G3534平板上。28培养，直G14279平板上长出G14752落，将单G14752落挑G14279斜面上培养。然后G17839行复G12591，方法同124，G12591选出G1147酶活力最高的G14752G7678用于发G18249G1147酶。127琼胶酶活力的测G4462粗酶液的制G3803将发G18249液于3000RMIN1下G12175心10MIN后上清液即为粗酶液。G6925G17839的DNS比色法测G4462琼胶酶活力用PH70的NA2HPO4NAH2PO4G13543G1926液G18209制02的琼胶G5225G10301溶液20ML。加G18494MLG5465测酶液，35、150RMIN1摇G5214G2465应1H。G12447即取1MLG2465应液于G5114有G2063度的G16809管中，加G184905MLDNS溶液于G8844水G9032中G1861G99215MIN后G12447即G1931G2376G14279G4472G9213，G4462G4493G1427910ML。测520NMG8886长G3800的吸G1821度。酶活力单位G4462G1053在上述G2465应G7477G1226下，1ML酶液1MING1147生LGG17836原糖为一G1022酶活（U）。128G14752体G8999度的测G4462G18331用紫G3818可G16277G2010G1821G1821度法在620NMG8886长下测G4462培养液的吸G1821度。129G14752G7678的G2033G8505G18504G4462琼胶降解G14752G7678的G2033G8505G18504G4462G6365G2454G10043G7003G1049814G17839行。G20330G1820观察G14752落形G5589、G19248G7828观察G1022体形G5589，G19773G1860氏G7591色，再G1328G17839一G8505G18504G4462。2结果与分析21琼胶降解G14752G7678的G12591选211G14752G7678的G2033G12591在G4557G14323G4677、G2500G5042G2520地的海水G7691G17839行G7101期G12591选后发G10628，G9213岭箬横紫G14768养殖地的海水G7691中的琼胶酶G1147生G14752较多，G1000G1147酶活力G1075较高。G6937在G7424G11752G12362中G4557该海水G7691G17839行驯化富G19610，G1209琼胶为G2819一G11911G9316G17839行富G19610培养，G1209期G14731得G7368多、酶活力G7368高的琼胶降解G14752G7678。平板培养后用卢戈氏碘液G7591色，琼胶与碘G13479合而被G7591色。G1147琼胶酶的G14752G7678，由于其G14752落周围琼胶被降解形成琼胶寡糖，而琼胶寡糖具有G17836原性，不能G11540色，因此其G14752落周围可形成透G7138圈，G17837G7691G1427可G1209很好地从海水G7691中G2010G12175G12591选到琼胶降解G14752。观察培养G3534平板可发G10628G14752落G6164在的培养G3534G2588G18352G5225形凹陷，G5194G1000有较大的透G7138圈，G12591选的平板透G7138圈G16277G32821。G18331用此法，G2010G12175到了18G7678琼胶降解G14752，G2010G2047测G4462其G14752落直G5464G2656透G7138圈直G5464，G13479G7536G16277G159321。图1琼胶酶G12591选平板经卢戈氏碘液G7591色后的G10043G10267FIG1PHOTOGRAPHOFTHEAGARPLATESTAINEDBYLUGOLSFORAGAROLYTICSTRAINSSCREENING表118G7678琼胶降解G14752G14752落直G5464G2656透G7138圈直G5464TAB1DIAMETEROFCOLONYANDHALOFROM18STRAINSSTRAINSDIAMETEROFCOLONIESDC/CMDIAMETEROFINNERCLEARHALOSDH/CMHCVALUEDH/DC102140438204720230046620263020204282119401620360222250186039020976052410181943701200304253380240048420179021603981843100198041220811101000286286012005402083852130118024620851402140536250515018804882596160830201624291702000452226018021005102429由G159321可G1209G11487出，6、14、16G2507G14752G7678G6164G1147的透G7138圈较大，但由于即G1363同一G1022平皿上长出的G14752落，其大G4579G1075不一，G13466G14002生G10301量G1075有G6164不同，透G7138圈的大G4579G5194不能G2465G7156G14752G7678G1147酶的比活性，因此G2010G2047测G4462G4439G1216的G14752落直G5464，但发G10628G1783718G7678G14752G7678的HC值（DH/DC）G1075相G5058不大。为了G4470观地G2465G7156G14752体的生长G2656G1147酶能力，将此18G7678G14752G7678G17839行G17839一G8505的G12591选。212G14752G7678的复G12591利用平板G2033G12591G2494能G4462性地挑选出G1147酶G14752G7678，而G1209摇瓶培养形G5347G17839行的复G12591则可G1209G4462量地比较G2520G14752G7678G1147酶活力及其G14752体生长G5785G1929。复G12591培养G3534G1185G1209琼胶G1328为G2819一G11911G9316，培养48H后G2010G2047测G4462发G18249液的琼胶酶活力G2656G14752体G8999度。G13479G7536G16277G159322。表2复G12591G2520G14752G7678G1147酶活力的比较TAB2COMPARISIONOFAGARASEACTIVITYPRODUCEDBYDIFFERENTSTRAINSSTRAINSENZYMEACTIVITY/UCONCENTRATIONOFSTRAINSOD62019381964242510863920161245090813544307196120911907509144483591469949006181054615721160208071238706931350911341448115341565807621611250285175271012186581639由G159322可G16277，6G265616G2507G14752G7678的酶活较高，G2010G2047为1209UG26561125U，选G2081者G1328为高G1147G14752G7678，G13546G2507为HS03266。在此值得一提的是，16G2507G14752G7678的透G7138圈直G5464为2016CM，其HC值为2429CM，而6G2507G14752G7678透G7138圈G2656HC值G2520为1018CMG26561943CM。可G1627716G2507G14752G7678的透G7138圈G2656HC值均比6G2507大，而G2081者的酶活G2376不G3926后者，其他G14752G7678的G5785G1929G1075与此类G1296，G17837G4613G16840G7138在G16792价G14752G7678G1147酶活性高低G7114，应G1209摇瓶复G12591G6980G6466为G1946，而G2033G12591中透G7138圈G2656HC值的大G4579G1177G1328G2454G13783。G17837与G8760海G197509G6363出的“透G7138圈的大G4579与G14752G7678G1147酶活性的高低有一G4462G8503相G1863”G5194不一G14280。G1863于透G7138圈G2656酶活G1055G19400的G1863G13007，有G5465G17839一G8505G11752G12362。213G14752种G13443化从G14270然G10627G3671中的G5506生G10301G7691品，经G17819一G8437G2010G12175形成单G14752落，G17728接斜面培养得到的G14752G7678，因G14085G12175了G14270然G10627G3671G7477G1226，在G1268G1207培养中G13466G14002发生性G10378G15940G17876的G20069G10587G3698加，直接G1363用G5460G5460存在酶活G5625G2107下降，G10990G14279G1019G3845酶活，G19668要经G17819多G8437G13443化培养G6177能G17810到G12295G4462的G17963G1268。G4466G20576G4557复G12591得到的高G1147琼胶降解G14752G7678HS03266G17839行G16809管斜面活化G2656平板再G8437G13443化，G1209G12591选出G1147酶活力较高G1000G12295G4462的G14752G7678。经G17819G13443化培养，得到高G1147G14752G7678HS0326601，其酶活为2078U，较G13443化G2081提高了72，G1000酶活G12295G4462。22G14752G7678HS0326601的G2033G8505G18504G4462G14752G7678HS0326601在G2010G12175培养G3534平板上培养，34DG3837可G1209形成G14752落，G14752落大G4579中等。G14752落形G5589特G5461为G14752落G2588G3290形，G9141G21656色，G17805G13548G6984G21796，G15932面G9299G9082，中G3842凹陷，不透G7138（G32822）。斜面培养21HG5050G2503G14752苔G19150G9397斜面，G14752苔G2588G9141G21656色，G2414度中等（G32823）。G19248G7828观察G14752G7678的G1022体形G5589G2656G19773G1860氏G7591色G13479G7536G15932G7138，G14752G7678HS0326601为G19773G1860氏G19464性G13466G14752，G14752体G2588G11713G7450G10378，G16277G32824。图2琼胶降解G14752的平板G13443化G3282FIG2CLONESOFAGAROLYTICBACTERIAINPLATECULTIVATION图3琼胶降解G14752的斜面培养FIG3TUBECULTUREOFAGAROLYTICBACTERIA图4G7186G5506G19248观察的G14752体（G6930大1000G1505）FIG4THESHAPEOFSTRAINOBSERVEDBYMICROSCOPY（1000）3讨论31在G4557紫G14768养殖地的海水G7691、紫G14768G7691及土G7691G17839行G7101期G12591选后发G10628，海水G7691中G12591选到的琼胶降解G14752G7678较多，G1000G1147酶活力G7138G7186高于后两者。G6937G7424G4466G20576G4557海水G7691G17839行驯化富G19610，G1209期G14731得G7368多、G1147酶活力G7368高的琼胶降解G14752G7678。32G7424G4466G20576中发G10628，透G7138圈G2656HC值均较大的G14752G7678，其酶活不一G4462较高，G16840G7138在G16792价G14752G7678G1147酶活性高低G7114，G6117G1216应G1209摇瓶复G12591G6980G6466为G1946，而G2033G12591中透G7138圈G2656HC值的大G4579G1177G1328G2454G13783。33G14752G7678HS0326601在G2010G12175培养G3534平板上生长较慢，一般G19668要34DG5050G2503G6177能长成大G4579适宜的单G14752落。而斜面培养G7114生长则较快，培养21HG5050G2503即可长出较G2414的G14752苔。G18504于该G14752G7678生长速度快，通G17819G4557其G17839一G8505的诱变G2656发G18249G7477G1226优化，有望成为高G1147琼胶酶G1147生G14752，从而为琼胶酶、琼胶寡糖的G9157G1849G11752G12362G2656开发应用奠G4462G3534G11796。此G3818，G4466G20576中G17836发G10628此类G14752G7678G4557G10627G3671变化较为敏感，G17963G1268性能不够G12295G4462，多G8437G1268G1207后G1147酶活力有G6164下降。因此，在后续G4466G20576中，G19512了G4557G14752种G17839行保藏G1055G3818，G17836G19668G4557G14752G7678G17839行G4462期活化。G11458G2081G8503在G4557此G14752G7678G17839行G17839一G8505的G18504G4462，同G7114，为提高其G1147酶活力，G4557该G14752G7678的诱变选育G2656发G18249G7477G1226优化G1075G8503在G17839行G1055中。REFERENCES1WANGJX,MOUHJ,JIANGXLBIOLOGICALACTIVITIESOFANEUTRALWATERSOLUBLEAGARPOLYSACCHARIDEPREPAREDBYAGARASEDEGRADATIONJHIGHTECHLETT,2005,1144154202ENOKIT,SAGAWAH,TOMINAGAT,ETALDRUGS,FOODSORDRINKSWITHTHEUSEOFALGAEDERIVEDPHYSIOLOGICALLYACTIVESUBSTANCESUS,6475990P200211053XUECH,XUQ,ZHAOX,ETALRADICALSCAVENGINGABILITYOFAGAROLIGOMERJJFISHCHINA237A238A239A240A241A242