[生物工程精品] 粗毛栓菌木聚糖酶的分离和纯化 论文正文

A1A0A2A3A4A5A6本科生毕业设计（论文）题目粗毛栓菌木聚糖酶的分离与纯化姓名XXX学号XXX系别生命科学专业生物工程指导教师XXX职称教授年月日教务处制A1A0A2A3A4A5A62中文摘要将粗毛栓菌木聚糖酶粗酶液经SEPHADEXG150分子筛层析柱，最后通过活性聚丙烯酰胺凝胶电泳进行进一步分离纯化，获得具有木聚糖酶活性的组分。关键词A7粗毛栓菌,木聚糖酶,分子筛层析，活性聚丙烯酰胺凝胶电泳ABSTRACTA8THECRUDEENZYMEFROMWHEATSTRAWCULTUREOFTRAMETESGALLICAFRWASAPPLIEDTOSEPHADEXG150GELFILTRATIONACOMPONENTWITHXYLANASEACTIVITYWASFINALLYOBTAINEDBYNATIVEPOLYACRYLAMIDEGELELECTROPHORESISKEYWORDSTRAMETESGALLICAFRA9XYLANASEA9SEPHADEXG150GELFILTRATIONA9NATIVEPAGEA10A11A12A13A14A15A163指导教师评语该生认真查阅参考文献，实验方案设计合理，在规定时间内完成了预定任务，初步掌握了从事有关研究的基本实验技能，具备了一定的独立从事科学研究的能力，达到了本科生的培养目标。该文达到了学士学位论文水平。A17A18A19A20A21A22A23A24A25A26A27A28A29A30A29A30A29A30A31A31A32A33A34A35A36A21A37A38A23A24A25A26A39A40A41A42A43A41A42A31A32A44A37A23A24A25A26A45A46A47A48A49A50A51A52A53A54A55A53A56A57A58A59A60A61A54A62A63A60A64A65A66A67A68A66A69A70A71A72A73A74A72A75A76A67A77A78A79A80A81A82A83A84A85A86A87A88A89A81A86A89A90A91A92A93A94A95A96A86A89A97A84A85A72A98A99A100A101A72A102A103A104A105A85A72A106A107A108A109A85A72A110A111A112A111A58A113A114A90A115A106A116A67A117A118A119A94A81A120A121A93A122A123A110A1114目录摘要1关键词1ABSG87G85AG70G871G46EG92G90G82G85DS1G5353G1634011G7460G7021G994方G887311G171G7460G702111G171G171G1214G313211G171G17G21G16809G2070G211G171G17G22G9354液的G18209G2058G211G171G17G23菌G12193G211G17G21实验方G8873G211G17G21G171粗酶液的G11839G18252G19145G11428析G211G17G21G17G21SEPHADEXG150分子筛层析G221G17G21G17G22木聚糖酶的活性聚丙烯酰胺凝胶电泳G221G17G21G17G23木糖标G1946G7366G13459的G8991定G231G17G21G175木聚糖酶活力的G8991定G231G17G21G17G25G15519G11345G2559G18339的G8991定G23G21G13479G7536G994分析5G21G171木糖标G1946G7366G134595G21G17G21G15519G11345G2559G18339标G1946G7366G13459G25G21G17G22粗毛栓菌木聚糖酶柱层析实验G13479G7536G25G21G17G23活性聚丙烯酰胺凝胶电泳后G7186G13479G7536G26G22G13479论G27参考文献G28G14280G16886G28G1593211木糖G8999G5242G994G1821G4506G5242G1552的关G130075G32821G171木糖标G1946G7366G13459G32825G159321G21G10287G15892G9177G15519G11345G8999G5242G994G1821G4506G5242G1552G1552的关G13007A124A124A124A1246G32821G17G21G15519G11345G2559G18339标G1946G7366G13459G25G159321G22粗毛栓菌木聚糖酶G2520步G20600的纯化G13479G7536G25G32821G17G22SEPHADEXG150柱层析G3282G16901G26G32821G17G23活性聚丙烯酰胺凝胶电泳后的G7186G14406G3282G26A120A121A93A122A123A110A1111粗毛栓菌木聚糖酶的分离和纯化生G10301G5049G12255G999G1006学生G59G59G59G6363G4560G6957G5084G59G59G59摘要将粗毛栓菌木聚糖酶粗酶液经SEPHADEXG150分子筛层析柱，最后通过活性聚丙烯酰胺凝胶电泳进行进一步分离纯化，获得具有木聚糖酶活性的组分。关键词粗毛栓菌,木聚糖酶,分子筛层析，活性聚丙烯酰胺凝胶电泳ISOLATIONANDPURIFICATIONOFXYLANASEINTRAMETESGALLICAABSTRACTTHECRUDEENZYMEFROMWHEATSTRAWCULTUREOFTRAMETESGALLICAFRWASAPPLIEDTOSEPHADEXG150GELFILTRATIONACOMPONENTWITHXYLANASEACTIVITYWASFINALLYOBTAINEDBYNATIVEPOLYACRYLAMIDEGELELECTROPHORESISKEYWORDSTRAMETESGALLICAFRA125XYLANASEA125SEPHADEXG150GELFILTRATIONA125NATIVEPAGE引言木聚糖G7171G7905G10301G13466G14002G3733G1025G1039要的G2334G13432G13512G13044成分，G7171G14270G9994G11040G1025G1177G8437G1122G13432G13512G13044的G2499G1889生的G1028G4512生G10301G17176G9316A126A127A128A129。木聚糖G19489G16311G19668要木聚糖酶G13007的G2339G2528G1328G11004，G1039要G2265G6336G1328G11004G1122G1039G19154内G18108木糖G14539键的G163G79，G23内G2011木聚糖酶G11EG81DG82G79，G23G163G39XG92G79AG81XG92G79AG81G82HG92DG85G82G79ASE，G40G38G714G22G714G21G7141G714G27G12，将木聚糖分G16311成木G4533糖G2656G4581G18339的木糖G727G1328G11004G1122木聚糖G2656G1314聚木糖G19762G17836G2419G12483的G1631，G23G714G3818G2011木聚糖酶G708EXG82G79，G23一G163G39XG92G79AG81XG92G79AG81G82HG92DMG79ASE，G40G38G714G22G714G21G7141G714G26G12，产G10301为木糖G727G1328G11004G1122G1314聚木糖G19762末G12483G163木糖G14539酶G111，G23一G163G39XG92G79G82SIDASE，G40G38G714G22G714G21G7141G714G22G26G12，释放出木糖A126A128A129。木聚糖酶在G14270G9994G11040广泛分布，海洋及陆地G13466菌、海洋藻类、真菌、酵母、瘤胃G2656反刍动G10301G13466菌、蜗G10287、甲壳动G10301、陆地G7905G10301组织G2656G2520G12193无脊椎动G10301G1025都存在。由G1122微生G10301来G9316的木聚糖G19489G16311酶普遍存在G1122G14270G9994G11040且G12193类繁多应G11004领域广泛A126A127A129。近年来，木聚糖酶在饲G7021、造纸及食品等行G1006均有广泛的应G11004，如纸浆生G10301漂G11345，功能性G1314聚木糖生产及面团改良G2070，饲G7021添加G2070等A126A127A130A129。本文G1039要对粗毛栓菌木聚糖酶进行了分离纯化以便对其酶学性质进行进一步的研究。1材料与方法A131A132A133A134A135A136A137211材料111仪器A138A139A140A141A142A143A144A145HISENSEA146A147A148A149A150A151A152A153A154A155A156A157A158A159A160A161A162A162A163A164A165A166A153A167A148A168A169A170A171A172A153A173A159A174A160A175A176A177A178A167A148A168A169A170A171A172A153A173A179A180A158A160A181A182A183A184A185A186A187A172A167A148A188A189A190A172A191A192A173A159A193A160A194A195A196A197A198A199A172A167A148A200A169A170A171A172A153A173A201A202A203A160A204A205A160A175A188A189A206A170A207A208A153A167A148A209A210A170A171A172A153A173A158A211A160A212A213A212A162A214A215A216A217A170A218A218A164A219A150A151A220A171A221A210A172A153A154A155A222A223A156A157A176A224A225A226A227A150A151A228A229A230A231A172A153A173112试剂SEPHADEXG150购G14270PHARMACIA公司G727桦木木聚糖G708BIRCHWOODXYLAN）购G14270美国SIGMA公司，其它G16809G2070均为国产分析纯。113溶液的配制11313,5二硝基水杨酸（DNS）试剂甲液G1946确称取10GNAOH，G11004去离子水G9354G16311，定容至G2100ML，将其分为50MLG2656150ML两G18108分，在50MLNAOHG9354液G1025加入G21G苯酚，在150MLNAOHG9354液G1025加入10GG39NS，加热G9354G16311G727乙液称取G2200G酒石G18252钾钠G9354G1122500ML水G1025，加热G9354G16311。待甲乙两G12193G9354液冷却后，将二者合并、混匀，加入0G175G亚G11839G18252氢钠，搅拌、混匀，定容至1000ML。113202MOL/L醋酸钠醋酸缓冲液PH50将G2600ML的0G17G21MG82G79/L醋G18252钠G9354液G2656G2200ML的0G17G21MG82G79/L的醋G18252G9354液混合，G11004PH计调至PH5G170。11331桦木木聚糖溶液称取0G175G桦木木聚糖，加入G220MLPH5G170的醋G18252钠醋G18252缓冲液，加热、搅拌G9354G16311，冷却后G11004G2528样的缓冲液定容至50ML，G23冰箱保存。1134考马斯亮试剂称取考马斯亮蓝GG2150100MGG9354G112250MLG285的乙G18267G1025，加入10MLG275的G11979G18252，G11004去离子水G12244释到1000ML。114菌种本实验G4472保存的G10628成的具有G17751G5390G19489G16311木质G13432G13512G13044能力的菌G7678粗毛栓菌G708TRAMETESGALLICAFR），G11345G14116G6297子菌粗毛栓菌G13007的G59G59G59G2350士G11221G28G28G26年G27G7388在G4677G1008G11477G14755G8913G5078G2283G18078的G6264G3490G3576G990，从G15999G11745G1252的G7484G7653G990分离得到的。该G18338生菌G7678G1122G2528年经由G4677G1008G11477科学G19510生G10301研究A232A233A234A235A236A237A2383G6164微生G10301分类G4472的马G2563G7138G1820生对其进行了初步G18504定，定G2529为TRAMETESGALLICAA239A240A241。12试验方法121粗酶液的硫酸铵盐析G2533粗酶液G1025添加G3278G1319G11NHA240G12A242SOA240至G220G20293G2656G5242，G23过G3824，离G5527G708G25,000G85/MIG81，G23）15MIG81，G19512去G7446G15519G11345G8797G9108G10301G727G9994后将G990G9177液的G11839G18252G19145G20293G2656G5242G990调至G265，G1889G1122G23G19757G13634过G3824，离G5527G708G26,500G85/MIG81，G23）10MIG81，G5335G990G9177，得G8797G9108G13434100ML，将G8797G9108G1122G210冰箱G1025保存，备G11004。将G8797G9108G17891析G708G6142G11053G183391G211G23G46G39），G8611G19560G22G23G4579时G6454一G8437G23去离子水，G11464到G17891析G15967内酶液无G8543G11053的G11NHA240G12A242SOA240。G17891析液经聚乙二G18267G708平均分子G18339G730G210,000）G8999G13565后，G1328为G991一步进行分子筛层析的样品。122SEPHADEXG150分子筛层析16CM115CM将G3800理G3921的凝胶放在G3835G9915G7491G1025，G11004去离子水反G3809冲G8939至G1025性，G1889G1100450MMG82G79/L的醋G18252钠缓冲液G708PH5G170）冲G8939G1972G8437。将凝胶G16025G2058成1G17G25G104115G70M柱，G990样G2081G11004G990G17860缓冲液平G15925柱，过G3824。G990样时样品G1319G12227G993G4464过多，G13434为柱G1319G12227的15。G8939G14085G13459G17907G5242为G25G70M/H，G8611G12661G6922G19610G21ML。G7828G8991G8981出液的酶活性，合并具有G11468G2528酶活性的组分，G9994后G1889如G990G8873进行G11NHA240G12A242SOA240G11428析、离G5527G2656G17891析等步G20600A239A243A241。123木聚糖酶的活性聚丙烯酰胺凝胶电泳活性聚丙烯酰胺凝胶电泳G708G81AG87IG89EG51AGG40）A239A244A245A246A246A241G11004G1122木聚糖酶的G3250G6922G2058备及活性G7828G8991，样品为经过层析G8873分离的G18108分纯化的酶样品。G27分离胶的G2058备G708G210ML）取5G17G22MLG220凝胶G1660备液G708G2559G21G28丙烯酰胺G26561N,NG255亚甲G2464丙烯酰胺）、G21G175MLG22G170MG82G79/LG55G85ISHG38G79缓冲液G708PHG27G17G28）、0G171ML10G55G40G48G40G39、0G17G21ML10过G11839G18252G19145、11G17G28MLDHA242O，混匀，G9760入G39G60G60G2675G3423G3414G11464G7507电泳G8145的G10639G10839G7507间G19565G1025，G1363液胶的G20652G5242为G10639G10839G7507G13449G19283的G21/G22G22/G23。G990面G4565一G15192水层。G1363G10639G10839G7507在G220G5670G9213箱G1025聚合G210G220MIG81。5G8999G13565胶的G2058备G1110MLG12取1G17G26MLG220凝胶G1660备液、G21G175ML0G175MG82G79/LG55G85ISHG38G79缓冲液G708PHG25G17G26）、0G1705ML10G55G40G48G40G39、0G171ML10过G11839G18252G19145、5G17G255MLDHA247O，混匀，加G1122分离胶G990面的G12366G19565G1025。在G8999G13565胶的G990面G11053有1G21G70MG20652的G12366G19565G708G990样G11004），G1889如G990G8873进行G5670G9213聚合。进行G2058备电泳时，将酶液G994G25G104加样缓冲液G708G2559PHG25G17G26的0G17G265MG82G79/LG55G85ISHG38G79、G220G10988G8845、0G1701G9352酚蓝）混合，G990样后，G2045G11004PHG27G17G22的G55G85ISG10988G8700G18252缓冲G13007G13491G708G215MMG82L/LG55G85IS、G2150MMG82G79/LG10988G8700G18252），G1122G23进行G5670G8981G70810MA）电泳，待样品进入分离胶后，G1889将电G8981G2331至15MA，G13499G13505电泳。电泳至G9352酚蓝G6363G12046G2070离胶G5225面1G21G70M时G1584止电泳。取出并分离G10639G10839G7507，并G1363凝胶附着在一块G10639G10839G7507G990。G11004刀片G13449G2533G2011去凝胶两边的G18108分G708G8611边G134341G70M宽）。由G1122酶样品在凝胶的两边沿G3800往往略呈弓形，G993G4464G11004来进行酶的定位，故将凝胶的两边G18108分G2011G19512。G1889G11004刀片沿凝胶一侧G13449G2533G2011去宽G134341G70M的胶条，将此胶A248A249A250A251A252A253A2544条的分离胶G18108分从G990到G991G2011成G21MMG19283的胶块，放在G11468应编号的G40G51G12661G1025，捣碎胶块，G9994后G2533G8611个G40G51G12661G1025分别加入50L的1桦木木聚糖，G13634G1122G235水浴锅G1025保G92131G175H，G1889G2533G8611个G40G51G12661G1025分别滴加100L1G39NS，沸水浴煮15MIG81。观察G2520G12661G1025颜G14406变化。进行G7186G14406反应期间，G11004保鲜膜覆盖剩余胶块，并G13634G1122G23冰箱G1025暂时保存。124木糖标准曲线的测定G1946确称取干燥至G5670重的木糖标G1946品50MG，G11004蒸馏水G9354G16311，定容至50ML。按G991G15932在G2520G12661G1025加入G2520G12193G16809G2070A255A109A111A0A113A1A115A2A90A53A116A83A41A117A3A4A109A17A120A5A19A42A42A6A53A42A6A83A42A6A7A42A6A8A90A6A42A124A3A9A10A17A120A52A19A42A42A6A53A42A6A83A42A6A7A42A6A8A90A6A42A127A11A12A17A120A52A19A53A6A42A90A6A8A90A6A7A90A6A83A90A6A53A90A6A42A71A13A14A130A15A17A120A52A19A90A6A41A90A6A41A90A6A41A90A6A41A90A6A41A90A6A41将G990G17860G16809G2070加完后，混匀G2520G12661，沸水浴5MIG81，冷却至G4472G9213，分别G11004去离子水将G2520G12661定容至G215ML，混匀。选G11004G1821G122551G70M的比G14406G7491在550G81MG3800比G14406，G11004G12366G11345G12661G1328对照，G8991G2520G12661G1821G4506G5242G1552，G8611G12661重G3809三G8437，求平均G1552。以木糖G8999G5242为横坐标，G1821G4506G5242G1552为G13449坐标，绘G2058标G1946G7366G13459A16A18A20。125木聚糖酶活力的测定采G11004G39NSG708DIG81IG87G85G82SAG79IG70G92G79IG70AG70ID）G8873即G22,5二硝基水G7484G18252G8873A16A21A22A23A24A20。取0G17G28ML由50MMG82G79/LNAAG70HAG70G708PHG23G175）缓冲液G18209G2058的1桦木木聚糖G1122G215ML刻G5242G16809G12661G1025，加入0G171ML适当G12244释的酶液，G1122G235水浴G1025保G9213G220MIG81后，加入G22ML1G39NSG16809G2070，混匀，在沸水浴G1025煮15MIG81，冷却至G4472G9213，补加水至G215ML，混匀，G1122550G81MG3800G8991定吸G1821G1552。以G8611分钟催化木聚糖水G16311生成1MG82G79木糖G6164G19668的酶G18339定为1个活力单位。木聚糖酶酶活力计算公式如G991木聚糖酶酶活力G708UMIG81A25A23MLA25A23）G81A/G708150G171G22G104G220）其G1025，G81为酶液G12244释倍数，A为标G1946G7366G13459G990吸G1821G1552G6164对应的木糖微克数，150G171G22为木糖分子G18339，G220为酶G1328G11004时间G708MIG81）。126蛋白含量的测定参照BG85ADFG82G85DA16A26A20G8873取一G13007列G16809G12661，按G991G15932分别加入G991列G16809G2070A248A249A250A251A252A253A2545A115A2A90A53A116A83A41A79A14A132A17A90A42A42A27A5A28A120A52A19A42A42A6A53A42A6A83A42A6A7A42A6A8A90A6A42A135A29A30A31A32A140A40A53A41A42A17A120A52A19A41A41A41A41A41A41A127A11A12A17A120A52A19A90A6A42A42A6A8A42A6A7A42A6A83A42A6A53A42A6A42将G990G17860G16809G2070加完后，混匀，G4472G9213G19757G13634G21MIG81，选G11004G1821G12255为1G70M的比G14406G7491，以G993加G10287G15892G9177G11345G15519G11345G11BSAG12的G16809G12661为对照，在5G285G81MG3800比G14406。以G10287G15892G9177G11345G15519G11345的G8999G5242为横坐标，G1821G4506G5242G1552为G13449坐标，绘G2058标G1946G7366G13459。取待G8991样品G9354液1ML，加入考马斯亮蓝GG21505ML，混匀，按G990G17860方G8873G8991定5G285G81M的G1821G4506G5242G1552。平行做G22份。比G14406后，从标G1946G7366G13459G990查出待G8991样品的G15519G11345质G8999G5242。2结果与分析21木糖标准曲线在G990G17860条件G991，得木糖标G1946品G8999G5242G994G1821G4506G5242G1552的关G13007G708G1593211）。A3311A34A35A36A37A38A39A43A37A44A45A46A47A124A3A48A49A50MG/ML020406081A96A51A54A55A17OD550A1901010280046406220747以木糖G2559G18339为横坐标，G1821G4506G5242G1552为G13449坐标，绘G2058出标G1946G7366G13459G708G32821G171）。计算出标G1946G7366G13459方G12255为A0G17G2715X0G170G23G26G708RA2470G17G28G28G23）G13479G7536G15932G7138，在00G17G27MG/ML范围内木糖G8999G5242G994G1821G4506G5242G1552G13459性关G13007良G3921。A56A57A58A59A60A61A62A63A64A65A6622蛋白含量标准曲线在G990G17860条件G991，得G10287G15892G9177G15519G11345G8999G5242G994G1821G4506G5242G1552的关G13007G708G159321G21）A67A68A69A70A72A73A74A75A76A77A74A78A78A80A81A82BSAA146A84A85G/MLA8620406080100A87A88A84A89A85OD595A8602360469067607880906A91A92A93A94A95A97A98A99A100A93A94A93A101A102A102A103A104A92A93A94A105A105A101A95A93A93A94A98A97A93A93A94A106A93A94A101A93A94A107A93A94A95A97A97A94A106A108A110A112A114A114A118A118MG/MLA119A121A122A123A125A126OD550A128A129A131A133A134A136A137A138A139A141A142A143A144A1456以G10287G15892G9177G11345G15519G11345G8999G5242为横坐标，G1821G4506G5242G1552为G13449坐标绘G2058出标G1946G7366G13459G708G32821G17G21）。得到标G1946G7366G13459的G3250归方G12255为A0G1700G27G215X0G1711G26G11RA1470G17G28G261G12，G13479G7536G15932G7138，G10287G15892G9177G11345G15519G11345G18339在0G17G211MG/ML范围内G994G1821G4506G5242G1552G13459性关G13007良G3921G708G32821G17G21）。A148A149A150A151A152A153A154A155A156A157A158A15923粗毛栓菌木聚糖酶柱层析实验结果木聚糖酶粗酶液经过G11428析离G5527，分子筛层析等纯化步G20600G13479G7536见G1593213。A16013A161A162A163A164A165A166A167A168A169A170A171A172A173A174A175A176TABLE13SUMMARYOFTHEPURIFICATIONOFXYLANASEINTRAMETESGALLICAA177A178A179A180A181A182A183/UA181A184A185/MGA186A182A183A187U/MGA188A189A190A191A177A178A192A193PURIFICATIONTOTALTOTALSPECIFICRECOVERYPURIFICATIONSTEPACTIVITYPROTEINACTIVITYRATEFOLDA194A195A196A197A198454A199105100A1991044541001NH42SO4FRACTIONATIONA200A201A202A203A204286A199104225A1991021271163281SEPHADEXG150MOLECULARSIEVINGA194A195A196A197A198248A1991033596689660051519NH42SO4FRACTIONATIONSEPHADEXG150分子筛层析G8939G14085G7366G13459见G328213。A205A206A207A208A207A207A209A210A211A212A213A207A208A214A214A215A216A217A218A206A207A208A219A215A214A211A207A207A208A211A214A210A207A220A207A221A207A209A207A214A207A207A222A223A224A225A226A225A227A228A229UG/MLA230A231A232A233A234A235OD595A236A237A238A239A240A241A242A2437A244A245A246A247SEPHADEXG150A248A249A250A244A251FIG13ELUTIONCURVEOFTHESEPHADEXG150CHROMATOGRAPHY24活性聚丙烯酰胺凝胶电泳后显色结果按照1G171G17G23G1025G6164G17860方G8873得到的G13479G7536如G32821G17G23G6164G12046A252A253A254A255A45A0A1A2A3A4A5A6A7A8A9A10A11A12A13A252从G7186G14406G13479G7536G990G2499以看出，1号到G25号的G40G51G12661的颜G14406比其它G2520G12661的颜G14406深，即分离胶G990从负极到正极第G211G21MM的位G13634G990有酶存在G727而第G210到第G21G27G12661的颜G14406也比其他它的G2520G12661的颜G14406深，G2499能G7171G9352酚蓝G6363G12046G2070的干扰。3讨论本G16809验从粗毛栓菌的木聚糖酶粗酶液G1025,经过G11428析，离G5527G2656分子筛层析等操G1328后,G13479G7536得到一G12193木聚糖酶的混合组分，木聚糖酶粗酶液得到初步分离G2656纯化。首G1820经过G11839G18252G19145G11428析得到的组分经SEPHADEXG150分子筛层析，此阶段酶的纯化倍数G7171G21G17G271，G3250G6922率G7171G25G17G22G727G1889经过G11428析后，酶的纯化倍数G2656G3250G6922率分别G717115G171G28G26560G1705。得到的组分通过活性聚丙烯酰胺凝胶电泳G2499以G11004G1122进一步的分离纯化G2656G3250G6922。经SEPHADEXG150分子筛层析后得到的酶液纯化倍数得到很G3835的提G20652，但G3250G6922率很G1314，酶的损失很G3835，并且纯G5242G2656比活力均G1314G1122范美霞A14A15A16A17的G11468应G13479G7536，其G2419因G2499能G7171G990样G18339G17751G3835，造成样品分布过广且未充分G3250G6922。另G3818，分离纯化步G20600过G1122单一，G1363该实验未能达到理想的纯化效G7536。0100200300400500600A18A19A20A21A22A21A23A21A21A23A24A25A23A25A21A23A26A21A23A20A23A23A22A19A24A21A21A24A24A21A24A19A21A27A28A29A30A31MLA32A33A34A35UA36000501015020250303504A37A38A39A40A41A42A43A44A46A47A48A49A50A51A52A538参考文献A54A55A56A57A58A59A60A61A62A62A63A60A64A65A66A67A68A69A70A71A72A73A74A75A76A77A78A79A80A60A81A76A82A83A84A85A86A87A88A89A61A90A91A55A92A93A94A55A92A88A60A54A61A56A95A96A60A61A62A62A63A60A97A98A99A100A101A68A69A70A94A67A68A69A70A102A100A101A68A69A70A94A103A104A70A105A106A70A78A107A108A109A110A71A111A70A84A112A113A114A115A116A117A118A84A119A120A121A122A86A93A94A93A60A54A87A56A123A124A60A61A62A62A125A60A126A127A128A97A98A99A67A68A69A70A78A129A108A102A70A84A130A131A132A133A60A134A135A81A76A118A84A136A120A121A122A86A61A94A61A60A54A137A56A138A138A138A115A139A140A141A115A142A143A144A60A61A62A62A61A60A145A146A147A148A149A150A151A152A67A131A153A154A155A78A156A157A132A133A86A64A65A66A84A158A159A86A61A61A89A55A90A91A61A137A94A61A125A60A54A93A56A160A161A162A115A163A164A165A115A166A167A168A169A170A171A172A173A174A175A176A177A178A179A180A181A182A183A184A185A186A186A187A188A189A190A