[生物工程精品] 粗毛栓菌漆酶的酶谱分析

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科学指导教师XXX职称教授2010年月日XXX教务处制目录摘要1关键词1ABSTRACT1KEYWORDS1引言11实验材料1111仪器1112试剂1113菌种2114PDA培养基2115粗酶液的制备2116等电聚焦酶谱分析2117胶体溶液的配制22实验方法2221装管2222装槽2223电泳2224剥胶2225显色3226拍照33结果与讨论3331漆酶酶谱3332讨论3参考文献3致谢4粗毛栓菌漆酶的酶谱分析生物科学专业学生XXX指导教师XXXA2A0A1A3A4A5A6A7A8A9A10A11A12A13A14A15A16A17A18A19A16A17A20A21A22A23A24A25A26A3A27A28A29A30A23A1A3A27A28A31A32A33A34A35A36A37A38A39A40A41A42A43A44A45A26A46A27A27A47A48A49A50A51A3A4A5A6A23A46A27A23A33A34A35A36A23A27A47TOMOGRAPHYFORTRAMETESGALLICA,SLACCASEABSTRACTDIAMETERGALLERYWASCULTIVATEDINWHEATPOWDERFOR30DAYSTHECRUDEENZYMEWASEXTRACTIONEDANDDIALYSISED,THENTHECRUDEENZYMEWASREACTIONWITHGUAIACOLAFTERTHEISOELECTRICFOCUSINGTOOBTAINTHETOMOGRAPHYKEYWORDSTRAMETESGALLICA,LACCASE,ISOELECTRICFOCUSINGIEFA52ZYMOGRAM引言漆酶是一种含铜的多酚氧化酶A53A54A55，最早是从日本的漆树汁中发现的，是人类最早发现的酶之一，但直到现在，人们对它的认识还不完整A53A56A55。近年来，随着漆酶G2163G14033的G17892G8505发现，G7389关漆酶的G11752G12362G2475到G7234G17953的G18337G16282。G11013G1122在G19489G16311G7420G17148G13044方G19766的G2163G14033，G1363之在制G8986G17908G13452G5049G1006，G10317G2047是G13452G8986G10995G10301G9430G11345G20058G3507G5483到G9157G1849的G11752G12362G2656G5332发A53A57A58A59A55，在G10627G1457方G19766G1075G7389G5468G3835的G9520G2159A53A60A55，G2499与G7389G8614的酚类G1328G11004，G1363含G14531氧基类的G19512G14621剂G451G11719G8845化G5049G5235G10301的G10301G17148G16311G2447G8614G5627等A53A61A55。随着G5224G11004的不G7041G2164G9157，漆酶的G11752G12362G2656G5332发G17246来G17246G2475到人们的关G8892A53A62A55。本文G4613G1209漆酶的粗酶液G17839G15904等电聚焦G8991G4462G1866G2528G5049酶谱。1实验材料111仪器G3290G11436电泳DG60G38G61G1627DG60G60G16G27G38G3423电泳仪G708G2283G1152G1857一仪器G2390G709G10639G10839管G192831G19CG80，G1881G5464G195CG80G27G161G19G1022G9915G74914G19G80G79一G1022G9915G109545G19G80G79一G1022培养G11411一G3883电G4388G3837G5191G12239液G7550G10639G10839G7846G4493G18339G109545G19G19G80G79G1016G1022G18339G12582112试剂G1005G9923G18244G14030G70829G/100MLG709G11014G2461G2464G1005G9923G18244G14030G7081G/100MLG709G1016G5627电G16311G17148AMPHOLINEG70840G451PH310G709G70850MG/MLG709G17819G11839G18252G19145G70805MG/MLG709TEMEDG7081MG/MLG709H2SO4G70804G709500MLNAOHG70804G709500MLG10768G14038G12970G70807G/100MLG709G5852G2031G7420酚G19G192G80G80OG79G18113菌种菌种是G11013XXXXXX博士提供。从山东菏泽树G7420上采集分离所G5483。经鉴G4462为粗毛栓菌TRAG80ETESGAG79G79ICA。114PDA培养基称取G2447皮马铃薯2G19G19GG18L，G2164适G18339水煮沸2G19G80IN，G11004纱布G17819滤G5483滤液，G2164G1849葡萄G129702G19G，G10768G14038粉15G，补足水至1,G19G19G19G80L，混匀，融化，分装，121灭菌2G19G80IN。此培养基主要G11004G1122粗毛栓菌的G1457存。115粗酶液的制备菌种经麦G14621培养基培养3G19G3837后，将培养G10301取出，G2164离G4388水，浸泡4H，然后G11004G1857层纱布G17819滤，取上清液。116等电聚焦酶谱分析等电聚焦原理等电聚焦凝胶电泳是依据蛋G11345G17148分G4388的静电荷或等电点G17839G15904分离的技术，等电聚焦中，蛋G11345G17148分G4388在含G7389载体G1016G5627电G16311G17148形成的一G1022连续而稳G4462的线G5627PH梯度中电泳。载体G1016G5627电G16311G17148是G14038肪族多氨基多羧G18252，在电场中形成正极为G18252G5627，负极为碱G5627的连续的PH梯度。蛋G11345G17148分G4388在偏离G1866等电点的PH条件下带G7389电荷，因此G2499G1209在电场中G12239动；当蛋G11345G17148迁G12239至G1866等电点位置时，G1866静电荷数为零，在电场中不再G12239动，据此将蛋G11345G17148。117胶体溶液的配制G1005G9923G18244G14030贮液G70830G1005G9923G18244G14030，交联度30G709；29克G1005G9923G18244G14030G26561克G11014G2461G2464G1005G9923G18244G14030溶G1122水，G4462溶至100ML，滤G2447不G4493G10301后存G1122棕色G10954，4CG2499G1457存数月配方G708胶终浓度75G709单体2G80G79AG80PHOG79INEG195G80G79DH2O55G80G79TEMEDG27G791G19G17819G11839G18252G191455G19G79粗酶液G27G19总体G12227G27G80G79。2实验方法221装管G1820G11004G13945G11350G8939G6175，然后将G3290G11436电泳槽的G10639G10839管G8939G1940，再G1100407的G10768G14038G12970凝胶G4565G5225G1343405G2412G12871，凝G3278后将配G3921的胶液G2164G1849G17819G11839G18252G19145G11004G12239液G7550或G8892G4568器G12239G1849管G1881，液G19766G2164至管G24872MMG3800，G11004G12239液G7550G17743G2164G1849G4581G16780水水G4565，G1209G9052G19512G5379月G19766G1363胶G7621G20042G12483G5191G3386，胶管G19668G718G716G1457G9213A63A64A65A66A67，G11004G15397G13452条G2572G2447胶管上G12483水G4565。222装槽水G4565G12483G2533上，将胶管G3414直G6566G1849G3290G11436电泳槽G1881，G16855G14422G3921G2520管G20652度G16772下管G2507，G8611G6915G134341/3在槽上，2/3在槽下，G11004G717G705的G10768G14038粉煮沸后G11004G12239液G7550G3355匀G9046G6285G1122G2520G1022胶管与G3290G11436G4392连G6521G3800G19462G8502上G19766液体G9443出，上槽G2164G1849500ML04MH2SO4,下槽G2164G1849500ML04MNAOH，G9165G8821G2520管G2487G2656电极，G11004G8892G4568器G2572G2447管G2487G8680泡。223电泳G5332G2563电泳仪，G5670G2399160V聚焦23G4579时，至电G8981G17247近G1122零不再G19489G1314时G1584G8502电泳。224剥胶G6365G5132G16280方法剥胶A68A69A70，将胶条浸G1122G192G80G80OLG18LG5852G2031G7420酚中G708含2G80GG18LG38G88G54OA71A725HA73OG709。225显色将剥G3921的胶条G6930G1849配制G3921的G5852G2031G7420酚溶液中显色。226拍照G5465酶与G5225G10301G2465G5224一G8585时G19400G708G134342G163G4579时G709后G7138显G2499G16277胶条上G7389G7138显的棕色条带时G17839G15904拍照G3800理G1457存。3结果与讨论331漆酶酶谱332讨论1G451结果G726本实验G1861出现漆酶的G2528G5049酶G8616G17751G7138显的G7389G3247条，G1866中G7389G1016条显G12046漆酶的G8975G5627G5468G20652G708或G18339G5468G3835G709，G2490G3818G1016条不G3838G7138显。G17837G3247条带中的酶G7389G1861G2528的G2163G14033，G18129G14033G2656G5852G2031G7420酚G1147G10995显色G2465G5224，但G2318G2047是G17837G3247条带蛋G11345G17148的氨基G18252G13464成不G2528，G4560致PH不G2528。所G1209电泳后G1262G1584G11053在不G2528的位置。2G451凝胶的浓度要适G4464，浓度G3838G3835G1262G1363聚G2524后形成的凝胶G4392G19565G17751G4579，G5445G2721蛋G11345G7691G2709的迁G12239，浓度G3838G4579凝胶不G7143聚G2524，本实验G20330G1820采G11004终浓度为5凝胶，结果凝胶G19602G1209聚G2524，G5445G2721剥胶。本实验最后采G11004胶终浓度为75。3G451粗酶液G2499G1209G2164G1849配制的凝胶中，粗酶液中的漆酶G2528G5049酶种类G17751多，G1000与G5225G10301G5852G2031G7420酚G2465G5224G999一G5627G5390，G2460因为酶液在不G2528的PH梯度电泳时G7693据酶不G2528的PHG1584G11053的位置是一G4462的，与G1172G1052时G1517G2164G1849酶液G7092关。4G451G5225G10301中G2164G1849铜离G4388，因为漆酶中G7389铜离G4388，与G5225G10301G2465G5224G7389关，G2164G1849G1262G2164G5567G2465G52245G451G17819G11839G18252G19145是G1664化剂，G2164G1849后胶G5468G5567G4613G1262聚G2524，因此最后G2164G1849，G2164G8617，G6683匀G5194G12447G2375装管G17902G5132化G4410聚焦的胶液，G20047在G17819G11839G18252G19145G2164G1849G2081G17839G15904G1955G2399G6289G8680G3800理。本实验不G19668要G6289G11507空G3800理G1075G2499G1209G5194G8821G7389G5445G2721。参考文献A74A75A76A77A78A79A80A81A82A83A84A85A85A84A83A86A87A88A89A89A90A91A92A89A93A94A83A84A85A95A96A84A75A84A83A74A84A76A97A98A99A100A101A102A103A104A98A105A102A106A83A107A98A108A102A100A101A103A102A83A75A95A95A109A83A107A98A105A110A101A102A108A101A108A102A105A111A112A113A106A108A112A114A101A99A103A112A103A115A106A108A112A116A111A117A118A111A114A114A111A100A105A83A119A99A114A102A103A120A99A103A117A103A116A121A122A75A109A85A123A75A95A96A84A124A83A74A125A76A126A103A108A102A120A103A112A112A111A99A100A127A122A128A111A99A114A105A119A129A122A105A101A111A117A83A75A95A95A85A83A130A131A99A113A111A101A99A103A112A103A115A132A103A112A104A98A105A112A99A114A110A108A120A100A101A102A111A101A105A100A74A133A76A83A106A134A119A110A117A105A101A101A122A84A124A135A123A95A95A96A75A85A125A83A74A109A76A97A111A117A117A128A122A119A108A114A136A105A118A83A75A95A95A135A83A137A99A100A101A103A102A121A122A130A138A105A102A138A99A105A139A111A112A113A140A104A104A117A99A114A111A101A99A103A112A103A115A119A105A113A99A111A101A105A113A118A99A116A112A114A103A117A121A101A99A114A110A121A100A101A105A141A74A133A76A83A133A126A142A130A107A134A97A137A122A143A125A123A75A143A125A96A84A85A84A83A74A143A76A126A103A117A117A111A116A133A119A122A110A98A108A101A101A117A105A139A103A102A101A98A144A118A122A145A146A147A148A149A75A95A150A150A83A118A111A114A114A111A100A105A96A141A105A113A99A111A101A105A113A151A105A101A103A131A99A115A99A114A111A101A99A103A112A103A115A128A98A105A112A103A117A99A114A97A103A141A104A103A108A112A113A100A74A133A76A83A140A104A104A117A134A112A138A99A96A102A103A112A119A99A114A102A103A120A99A103A117A122A143A109A123A125A85A124A150A96A125A85A95A75A83A7