[生物工程精品] 粗毛栓菌漆酶的酶谱分析1

XXX本科生毕业设计（论文）题目粗毛栓菌漆酶的酶谱分析姓名XXX学号XXX系别生命科学系专业生物工程指导教师XXX职称教授年月日XXX教务处制XXXA1A0A2A3A4A5A6摘要利用愈创木酚作为显色剂，做活性聚丙烯酰胺凝胶电泳，至少检测出粗毛栓菌一种漆酶同工酶组分。关键词粗毛栓菌,漆酶,酶谱分析ABSTRACTA7ATLEASTONELACCASEISOENZYMEINTRAMETESGALLICAWASDETECTEDOUTBYNATIVEPOLYACRYLAMIDEGELELECTROPHORESISKEYWORDSA8TRAMETESGALLICA,LACCASE,ZYMOGRAMANALYSISA9A10A11A12A13A14A15A16A17A18A19A20A21A22A23A24A25A26A27A28A29A30A31A32A33A34A35A36A37A38A39A40A41A42A38A43A44A45A46A47A48A49A50A51A43A52A53A54A55A56A52A53A57A58A59A60A38A61A62A52A53A63A64A65A56A52A53A66A35A64A65A56A67A16A17A48A68A69A70A71A72A73A74A75A76A77A78A38A32A33A79A80A81A82A25A83A84A85A86A38A87A22A88A89A90A23A91A52A56A43A92A93A94A95A96A21A22A23A24A97A98A99A100A101A102A25A97A98A47A103A104A18A23A24A105A106A30A31A107A108A43A92A56A48A109A50A110A111A93A112A105A105A106A113A114A95A115A51A43A25A116A117A47A16A17A25A107A108A118A119A23A24A25A120A63A121A122A123A124A125A85A47A126A85A127A128A129A16A95A130A47A131A132A133A134A135A136A137A138A139A140A141A142A143A144A137A145A146A147A148A149A135A150A151A137A138A139A140A152A153A154A155A156A157A158A159A160A161A162A160A163A164A165A166A167A168A161A169A170A167A171A172A173A174A175A173A176A177A178A179A180A181A179A182A183A174A184A185A186A187A188A189A190A191A192A193A194A195A196A188A193A196A197A198A199A200A201A202A203A193A196A204A191A192A179A205A206A207A208A179A209A210A211A212A192A179A213A214A215A216A192A179A217A218A219A218A165A220A221A197A222A213A223A174A224A225A226A201A188XXXA227A228A229A230A231A232A233目录摘要1关键词1ABSTRACT1KEYWORDS1引言11材料与方法211菌株212培养基2121PDA培养2122固态发酵用微量元素母液2123液体培养基213主要仪器与试剂214菌株培养方式215样品的制备3151浸提3152盐析3153透析316电泳（垂直板电泳）317染色32结果与分析321结果322分析43讨论4参考文献4致谢5XXXA227A228A229A230A231A232A233粗毛栓菌漆酶的酶谱分析生物工程专业学生XXX指导教师XXX摘要A234利用愈创木酚作为显色剂，做活性聚丙烯酰胺凝胶电泳，至少检测出粗毛栓菌一种漆酶同工酶组分。关键词A234粗毛栓菌,漆酶,酶G16901分析ZYMOGRAMANALYSISOFLACCASEINTRAMETESGALLICAFRABSTRACTA235ATLEASTONELACCASEISOENZYMEINTRAMETESGALLICAWASDETECTEDOUTBYNATIVEPOLYACRYLAMIDEGELELECTROPHORESISKEYWORDSA236TRAMETESGALLICA,LACCASE,ZYMOGRAMANALYSIS引言木G17148素G7171G12544G1120G3837G9994聚G2524G10301,木G17148素的G19489G16311G7171G11911素G5502G10627G1025的关键一G8505A237A238A239。G14270G9994G11040G1025木G17148素的G19489G16311主要G7171G17902G17819G1009G10378G11507菌,G4600G1866G7171G11345G14116G11507菌（WG75G76TEROTG73G88G81G74G76）的分G16311作用G7481G4448G6116的A237A240A239。G19512G13432G13512素酶G2656G2334G13432G13512素酶G1055G3818，G11345G14116G11507菌G17836G14033G3827G1147G10995G1016种G14002G3818G17819G8699G2282G10301酶，G2375木G17148素G17819G8699G2282G10301酶（G79G76G74G81G76G81G83EROG91G76DASE，G47G76P）A237A241A242A239G2656G19204G17819G8699G2282G10301酶（G80AG81G74AG81ESEDEG83EG81DEG81TG83EROG91G76DASE,G48G81P），G16780G3822G11345G14116G11507菌G17836同G7114G1147G10995一种G2559G19120的酚G8699G2282酶（COG83G83ERCOG81TAG76G81G76G81G74G83G75EG81OG79OG91G76DASE）G2375漆酶（G79ACCASE）A237A240A239。漆酶G6365G1866主要G7481G9316分为漆G7653漆酶G2656G11507菌漆酶G1016G3835G12879,G11752G12362G15932G7138,漆酶G2499作用的G5225G10301G14551G3272G5203G8879。粗毛栓菌G7171一种G19489G16311木G17148G13432G13512素G14033G2159G5390，G1147木G17148G13432G13512素G19489G16311酶酶G13007G21796G1852的G11345G14116G6297G4388菌，G3324G6117G3281G5203G8879分G5079，主要G10995G19283G1122G7484木G990，G7171G7484木的主要G10995G10301G19489G16311G13785。G4466G20576G15932G7138G16825菌G1075G14033G10995G19283G1122G1904作G10301G12236G7450G990，G3926G3324G21626G14621G990G10995G192836G19G3837G2530，G14033G3827G9052G13803G21626G14621G102566G8的G13432G13512素，72G8的G2334G13432G13512素G26567G19G8的木G17148素，G16840G7138G16825菌G1867G7389G5468G5390的G19489G16311木G17148G13432G13512素的G14033G2159A237A240A239。G6164G1209G17839一G8505G11752G12362G2656分析TGALLICAG1147木G17148G13432G13512素G19489G16311酶G4600G1866G7171G1147G10995木G17148素G19489G16311酶活G2159发酵G7477G1226G7171G5468G7389G5529要的。G7424论文工作的一G20045G18337要G1881G4493G7171G11752G12362粗毛栓菌G18338G10995菌株G1147G10995漆酶的培养方法G1209及酶G16901分析，G1209便为将G7481G7389效的克隆编码这些酶G12879的基因奠定G5529要的工作基础A237A240A239。众G6164周知，酶G16901技术G7171用G7481鉴别酶的种G12879、分析酶G13007组G6116G2656检G20576酶活性的G4466G20576技术。活性聚丙烯酰胺凝胶电泳技术常用G7481G17839行酶的检测G2656定位。利用愈创木酚作为显色剂，对粗毛栓菌的漆酶酶G16901G17839行初G8505分析A243A244A245。XXXA246A247A248A249A250A251A2521材料与方法11菌株G11345G14116G6297G4388菌G13007XXXG11221997年8月G3324山东省菏泽市北郊的护城堤G990，从被砍伐的G7484G7653G990分离得到的。G16825G18338G10995菌株G1122同年经由山东省科学院G10995G10301G11752G12362G6164微G10995G10301分G12879室的马启G7138先G10995对G1866G17839行了初G8505鉴定，定为TRAMETESGALLICAFRA243A253A245。12培养基121PDA培养基称取去皮马铃薯2G19G19G74，加适量水煮沸2G19G80G76G81，用纱G5079G17819滤得滤液，加入葡萄糖（或蔗糖）2G19G74，琼脂粉15G74，补足水至1,G19G19G19G80G47，混匀，融G2282，分装，121灭菌2G19G80G76G81。此培养基主要用G1122粗毛栓菌的保存。122固态发酵用微量元素母液（MG/L）CACG79A2532HA253O,7G19G19G48G81SOA244HA253O,5G19G19FESOA2447HA253O,5G19G19CG88SOA2445HA253O,6G19G19ZG81SOA2447HA253O,5G19HA254BOA254,2G19G19NAA253G48OOA2442HA253O,2G19G19COCG79A2536HA253O,1G19G19。使用G7114，取2G80G47G16825母液加入1,G19G19G19G80G47水G1025，再补加KHA253POA2441G74，G48G74SOA2447HA253OG195G74，G14270G9994G83H值A243A254A245。123液体培养基KHA253POA244，G48G74SOA2447HA253O，酒石酸铵，葡萄糖，微量元素母液，酵母粉，硫酸G19204。此培养基作为粗毛栓菌漆酶G1147酶培养基A243A244A245。13主要仪器和试剂TH5G19G19梯度混G2524器伤害沪西分析仪器厂；QSEG83G75AROSEFF交换柱；XWTSG4579G3423G2500式G16772G5417仪G990G9035G14270G2172G2282仪G15932G989厂；HD1G7692酸G15519G11345检测仪G990G9035沪西分析仪器厂；BG45QG18108分G6922G19610器G990G9035G2319用分析仪器厂；G47DZX4G19AG44G3423G12447式G14270G2172电G9921G2399G2159G14988G8785灭菌器G990G9035G11015G4445G2319G11115器G7812厂；G6403G14645培养G12677HZQX1G19G19G2656G10995G2282培养G12677HPS16G19（G2716G4584G9404东G13864电G4388技术G1856G2508）；离G5527G7438（G990G9035G4445G1153科学仪器厂）；电泳仪DG60G60G8028CG3423（北G1152市G1857一仪器厂）；酸度仪；G9900G12677G12573A243A255A245。G6164用到的试剂主要G7389G726愈创木酚、TG40G48G40D、G7692G21656素、TRG76S、盐酸、G8700基G1069酸、G9352酚G1860、G10988G8845、丙烯酰胺、N,NG255G1134G11014G2464丙烯酰胺G12573,KHA253POA244，G48G74SOA2447HA253O，酒石酸铵，葡萄糖，微量元素母液，酵母粉，G48G81SOA244HA253O。14菌株培养方式KHA253POA244G191G8，G48G74SOA2447HA253OG19G195G8，酒石酸铵G192G8，葡萄糖2G8，微量元素母液2G48G47G18G47，酵母粉G19G19G195G8，硫酸G192042G19G48G42G6365G8616G1375G18209G136342G19G19G19G80G47，分8G10954装,G8611G1095425G19G80G47。G1122121灭菌3G19G19G80G76G81，G1931G2376至3G19G5050G2503G7114，G8611G15967G6521入G134345G19G74活G22826D（26）的TGALLICA菌G1009体（G6521种G7114用G6521种G19142）。27G8985G4630G19757G13634培养4周。15样品的制备XXXA246A247A248A249A250A251A252151浸提培养28DG2530，将培养G10301取出，G6365料G29水1G297加去离G4388水，G11224浸提4G75；用6G4630纱G5079G17819滤，G5335滤G9207；取G990G9177液（G2375得G2419酶液）A243A1A245。152盐析G20330先G3324G2419酶液G1025G9167加固体（NHA244）A253SOA244粉G7423至3G19G8G20293G2656度，G6617G6304G3355匀，G19757G13634G17819G3824，离G5527（6G19G19G19RG18G80G76G81，15G80G76G81，4）G19512去G18108分G7446G15519G11345G8797G9108G10301；G9994G2530将（NHA244）A253SOA244G20293G2656度G990G16855到75G8，G11224G19757G136343G75；离G5527（75G19G19RG18G80G76G81，2G19G80G76G81，4），G5335去G990G9177液。153透析用适量G14988G20323水G20056G19314G75G9354G16311G8797G9108，将G9354G16311G10301G8892入透析G15967（G6142G11053分G4388量为1214G78DA）G1881对G14988G20323水G17839行透析。G861123G75换一G8437水,此G17819G12255为2DA243A0A245。16电泳（垂直板电泳）活性聚丙烯酰胺凝胶电泳（G81ATG76G89EPAG42G40）A243A0A245。样品分别为G18039些经G17819透析G2530的酶液G2656浸提G2530的G2419酶液。8G8分离胶的制备（2G19G80G47）G726取53G80G473G19G8凝胶G1660备液（G255929G8丙烯酰胺G26561G8N,NG255G1134G11014G2464丙烯酰胺）、25G80G473G19G80OG79G18G47RG76SHCG79G13543G1926液（G83H89）、G191G80G471G19G8TG40G48G40D、G192G80G47G19G195G8G7692G21656素、119G80G47DHA253O，混匀，G9760入DG60CZ28AG3423垂直板电泳G8145的G10639G10839板G19400G19565G1025，使液胶的G20652度为G10639G10839板G13449G19283的2G1833G184。使G10639G10839板G173291G19G19G10938G9795G8885G1343415CG80，G1821G10043聚G25242G193G19G80G76G81。5G8G8999G13565胶的制备1G19G80G47G726取17G80G473G19G8凝胶G1660备液、25G80G47G195G80OG79G18G47TRG76SHCG79G13543G1926液（G83H67）、G19G195G80G471G19G8TG40G48G40D、G191G80G47G19G195G8G7692G21656素、565G80G47DHA253O，混匀，加G1122分离胶G990G19766的G12366G19565G1025，加G990G7815G4388。再G3926G990法G17839行G1821G10043聚G2524。G17839行制备电泳G7114，将酶液与6G104加样G13543G1926液（G2559G83H67的G1975G80OG79G18G47TRG76SHCG79、3G19G8G10988G8845、G19G195G8G9352酚G15025）混G2524，G990样G2530，利用G83H83的TRG76SG10988G8700酸G13543G1926G13007统（25G80G80OG79G18G47TRG76S、25G19G80G80OG79G18G47G10988G8700酸），G17839行恒流（1G19G80A）电泳，待样品G17839入分离胶G2530，再将电流升至15G80A，继续电泳。G990样前，若样品G8999度太低,G2499用聚G1069G1120醇2G19G19G19G19G17839行适当G8999G13565A243A2A245。17染色漆酶G1867G7389G5203G8879的G5225G10301专一性A243A3A245，G1375G3926，2,6G1120G11014基2,6DG48O）、愈创木酚G2656ABTSG12573都曾分别被用G1122漆酶活性的检测。G7424G4466G20576为了检测漆酶活性，电泳G2530,放G3324G5225G10301胶G99045水浴恒温,放少G16780醋酸醋酸钠G13543G1926液G11222G19G19G80G47,加入125A30G47愈创木酚,放G332437恒温G12677G18811G75G2530拍G10043。2结果与分析21结果G3324分离胶G990分别G14033G3827看到显色带（图1）为经G17819透析G2530的酶液。由G1122加样量G17819G3835导致酶G16901未G4448G1852分开，不易识别漆酶同工酶。XXXA246A247A248A249A250A251A252A31A32A33A34A4A5A37A6A7A8A9A42A10A11A12A46A1322分析G7424G4466G20576用愈创木酚作G5225G10301，对漆酶G17839行了酶G16901分析A243A254A245。从图1G2499G1209看出，对应G1122漆酶活性的显色带呈现弥散形分G5079图1，G15932G7138粗毛栓菌G3324G7424G4466G20576培养G7477G1226下G1147G10995了G3835量的漆酶同工酶A243A14A15A245。3讨论愈创木酚作G5225G10301，G3324G3835量漆酶存G3324G7114，做初G8505的酶G16901分析，G2499G1209得到G9177晰的显色带。G1866现象G8616较G7138显，酶G16901呈现G9177晰的红棕色，易G1122识别。已经G7389G11752G12362G13785初G8505分离并纯G2282了G1147G14270G16825菌株的至少14种漆酶同工酶，它们的G12573电点分G5079G112227至57G1055G19400A16A17A18。参考文献1XXX2G19G192A48A19A20A21A22A23A24A25A26A27A28A29A35A36A38A39A40A41A43A48A62A44A45A47A49A50A47A51A52A532XXXA69A70A54A55A69A56A57A582G19G192A48A19A20A21A22A23A24A25A26A27A28A29A35A36A38A39A40A41A43A48A75A59A60A47A61A63A4822（1）G7262426A483A79A64A652G19G196A82A22A66A67A68A71A22A72A38A73A74A88A89A76A77A78A67A47A80A28A41A43A93A81A83A84A84A854A96A86,A98A87,A100A90A91,A103A92A942G19G194（G193A95A106A97A99A101A102A104A105A107A108A109A110A111A112JA113A114A115A116A117A116A1185A119A120A121A122A123A124A125A122A126A127A128A122A123A129A1301991A131A132A133A116A134