[生物工程精品] 粗毛栓菌mnp的分离与纯化 论文

XXXA1A0A2A3A4A5A6A8A7XXX本科生毕业设计（论文）题目粗毛栓菌MNP的分离与纯化姓名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XXXA1A0A2A3A4A5A6A10A8A7A11A9A12A13A14A12A13A14A15A15A16A17A18A19A20A21A22A23A24A25A26A27A28A29A30A31A32A33A34A35A36A37A35A38A37A35QSEPHAROSEFASTFLOWA39A40A41A42A43A44A45A46A47A48A49A50A51A52A53A54A55A56A57A58A59A60A61A62A61A62A63A64A65A66A67A68A51A52A53A68A40A41A42A43A44A45ABSTRACTA69THECRUDEENZYMEFROMWHEATSTRAWSOLIDCULTUREOFTRAMETESGALLICAFRWASULTRAFILTRATED,SALTEDOUT,DIALYZEDANDAPPLIEDTOSEPHAROSEQFASTFLOWIONEXCHANGECOLUMNACOMPONENTWITHMANGANESEPEROXIDASEACTIVITYWASOBTAINEDKEYWORDSTRAMETESGALLICAFRA70MANGANESEPEROXIDASEA71IONEXCHANGECHROMATOGRAPHYA72A73A74A75A76A77A78A79A80A81A82A83A84A85A86A87A88A71A89A90A91A92A93A94A95A96A71A97A98A99A100A101A102A103A104A105A106A99A107A108A71A109A110A111A112A105A113A114A115A116A117A118A119A120A121A89A90A122A123A71A124A125A105A126A99A119A127A128A113A114A129A130A117A118A119A123A131A71A132A133A105A121A129A80A119A134A135A136A137A138A79A87A132A133A105A130A139A130A140A141A87A142A143A138A72A73A74A75A144A145A78A146A147A148A141A87A149A150A78A151A152A153A154A155A144A156A157A78A146A147A148A151A158A159A160A161A78A151A162A156A78A146A147A148A163A164A165A166A167A168A169A170A171A172A173A171A174A175A176A177A178A179A172A180A181A178A182A183A184A185A186A184A187A188A189A190A191A192A190A193A194A185A195A196A197A198A199A200A201A202A203A204A205A206A207A199A204A207A208A209A210A211A212A213A214A204A207A215A202A203A190A216A217A218A219A190A220A221A222A223A203A190A224A225A226A227A203A190A228A229A230A229A176A231A232A208A233A224A234A185A235A236A237A212A199XXXA238A239A211A240A241A242A243A179A228A229A182目录摘要1关键词1ABSTRACT1KEYWORDS1引言11材料与方法211主要仪器及试剂212菌株及培养基2121菌株2122培养基3123原酶的来源313粗酶液的制备314离子交换层析315分析方法4151锰过氧化物酶的酶活测定4152蛋白含量的测定42结果与讨论521MNP的柱层析纯化结果522蛋白含量的测定53结论5参考文献6致谢6图片目录图1粗毛栓菌（TRAMETESGALLICA）在杨木上长出的的子实体3图2牛血清蛋白含量标准曲线5XXXA244A245A246A247A248A249A250A251A252A253A254粗毛栓菌MNP的分离与纯化生物科学专业学生XXX指导教师XXX摘要A255粗酶液来自麦草粉固态培养物，经超滤浓缩、盐析、透析、QSEPHAROSEFASTFLOW阴离子交换层析后获得具有MNP活性的组分。关键词粗毛栓菌G15MNPG15离子交换层析ISOLATIONANDPURIFICATIONOFMNPINTRAMETESGALLICAFRABSTRACTA1THECRUDEENZYMEFROMWHEATSTRAWSOLIDCULTUREOFTRAMETESGALLICAFRWASULTRAFILTRATED,SALTEDOUT,DIALYZEDANDAPPLIEDTOSEPHAROSEQFASTFLOWIONEXCHANGECOLUMNACOMPONENTWITHMANGANESEPEROXIDASEACTIVITYWASOBTAINEDKEYWORDSTRAMETESGALLICAFRA0MANGANESEPEROXIDASEA2IONEXCHANGECHROMATOGRAPHY引言G11G20G12锰过氧化物酶（MNP）及G1866G1147G10995菌木G17148G13432G13512G13044G11LG76G74NOG70ELLG88LOSEG12主要G11013G13432G13512G13044G11G70ELLG88LOSEG12、G2334G13432G13512G13044G11HEG80G76G70ELLG88LOSEG12G2656木G17148G13044G11LG76G74NG76NG12组G6116G15G7171自G9994G11040G1025G5203G8879G4396在的G2499G1889G10995的G6980量G5052G3835的G10995物G17148（G69G76OG80ASS）G17176源。木G17148G13044G7171G11013G14531G1005G9931G15905G10995物G2345体G7512G6116的G980G12193结G7512G3809G7446的G12447体G13605G10378物G17148，G7171G12544G21G3835G12879G3837G9994G13870G2524物G15在G6980量上G1177G8437G1122G13432G13512G13044。G11013G1122木G17148G13044G12447体结G7512的G3809G7446性G15G6164G1209G13489G3835G3822G6980G5506G10995物G993G7143G4557G1866G17839G15904G10995物G4410G19489G16311。木G17148G13044的G5506G10995物G19489G16311G1328G11004G7171G13512G6357自G9994G11040G10995物G11911G13044G5502G10627的G18337要组G6116G18108分。G19489G16311木G17148G13044的G5506G10995物主要G7171G980G1135G2499分G8864G14002G3818过氧化物酶的白G14116G11507菌G15G17837G1135酶参与G4557木G17148G13044G3835分子G13870G2524物的G7380G2033G16022G16311G2465G5224。木G17148G13044G10995物G19489G16311的酶G13007主要G2265G6336木G17148G13044过氧化物酶G11LG76G74NG76NPEROG91G76G71ASEG15G47G76PG12A3A4A5A6A7、锰过氧化物酶G11G80ANG74ANESEPEROG91G76G71ASEG15MNPG12A3A8A5A9A5A10A7G2656G9434酶（LAG70G70ASE）A3A11A7。G17837G1135酶G2499在木G17148G13044G13870G2524物G1881G5430G6116自G11013基G15G4560致键的G993G12295定G1186G13792G6183G7041木G17148G13044G3835分子A3A12A7。在G16780G3822G11507菌G1025G15MNPG7171木G17148G13044G17227G3999G19489G16311的关键酶G15G3252G1038MNPG2499G1147G10995G5390氧化态的MNA8A13G15MNA8A13G1328G1038G2499G6205G6967的氧化G17836原G1183G17148G16022G16311木G17148G13044G13870G2524物G1025的G14471G20333G10627G18108分G15G9994后在G1866G4439酶的G2339G2528G1328G11004G991G15G7380G13468G4560致G3835分子的G7041G16022A3A14A7。MNPG7171G1122G20G28G27G23G5192G11013G46G88WAHARAG12573人首先G1186黄孢原毛平革菌（PHANEROCHAETECHRYSOSPORIUM）的培养液G1025检测到的A3A15A7。随后G15G17837方面的工G1328逐渐展开，MNPG7171G980G12193依赖HA6OA6的G14002G3818酶，催化G2465G5224需要MNA6A13，MNP在G10627境保护方面具有潜在的G18337要G5224G11004价值，白G14116菌G7171已知的唯G980能把木G13044彻底G19489G16311G1038COA16G2656HA16O的G980G12879G5506G10995物。目前已G1186G3822G12193白G14116菌G1025分离纯化到该酶，如CERIPORIOPSISSUBVERMISPORA，DICHOMITUSSQUALENS，PANUSTIGRINUS，PHANEROCHAETECHRYSOSPORIUM，PHLEBIARADIATA，G1866G1025G4557黄孢原毛平革菌XXXA17A18A19A20A21A22A23A24A25A26A27（PHANEROCHAETECHRYSOSPORIUM）研究G7380G1038深入A28A29A30。G11G21G12MNP的催化机制当HA16OA16G2656二羧酸螯G2524剂（如G1005二酸盐G2656草酸盐）G4396在时，MNP能氧化MNA16A31G6116G1038MNA32A31，MNA32A31与某G1135有机酸（如草酸、G1005二酸、苹果酸、酒石酸、乳酸G12573）络G2524G5430G6116G12295定的氧化G17836原电势，后者G1328G1038G2499G6205G6967的氧化G17836原G1183G17148G17839G980步氧化各G12879酚型化G2524物，如G21，6二甲氧基酚、G20333草G1005酮G2656G14531酚G12573A28A33A34A35A33A33A35A33A16A30G113G12MNP的工业G5224G11004锰过氧化物酶（MNP）的底物专G980性弱，G2499氧化各G12879G14471G20333化G2524物，G6164G1209具有极高的工业G5224G11004潜力，如G10995物制浆、纸浆的酶法漂白、有机污染物的G19489G16311G2656G10627境的G10995物修G3809G12573。在G10627境保护G1025，工业“三废”、化G4410农药分G16311时往往G1147G10995毒性物G17148，MNP能将G1866逐步G19489G16311G1038二氧化G11911。在造纸工业G1025，MNP在纸浆的G10995物漂白方面有很G3835的G5224G11004前景。在褐煤的G10995物G19489G16311G1025，G5506G10995物G4557低阶煤具有脱硫、液化G12573能力，G2499减少能源的浪费G2656G10627境的污染，G6116G1038研究的热点，G6164G1209研究MNP具有很G18337要的价值。G11013G1122MNP底物的G3822样性及酶本身结G7512、基G3252编码、调控表达的G3809G7446性G15国G1881的研究尚处G1122G2033期阶段国G3818的研究虽G9994取得G980定G17839展G15但G4557G1122MNP的分子G10995物G4410、高效表达体G13007的G7512建G12573研究尚有待深入。本实验G7171在G4557粗毛栓菌（TGALLICA）G17839G15904麦草粉固态培养60G71，G4557MNPG17839G15904分离纯化，G1209获得具有MNP活性的组分，G5194G4557G1866G17839G15904G18108分性G17148研究，G1209G1427G1038将来有效G3332G1823G19546编码G17837G1135酶G12879的基G3252，建G12447高效表达体G13007G3892定G5529要的工G1328基G11796A28A29A30。1材料与方法11主要仪器及试剂HG61QQG1852G9213G6403G14645器（G1025国G2716G4584G9404G1008G13864电子G6228G7427开G2469有G19492G1856G2508）G727电G8903仪G39G60G60G27C型（G2283G1152G5078G1857G980仪器G2390）G727G10995化培养G12677HPSG21G240G727G7811G5242G9163G2524器G55HG2400（上G9035G8830G16211分析仪器G2390）G727G5670G8981G8905HG47G21（上G9035G8830G16211分析仪器G2390）G727G7692酸蛋白检测仪HG39G20（上G9035G8830G16211分析仪器G2390）G727自G2172G18108分G6922G19610器G37G45QG26G23G21（上G9035G2319G11004分析仪器G2390）G727阴离子交换剂QSEPHAROSEFASTFLOWG17153自PHARMACIAG1856G2508G727G13055G3818分G1821G1821G5242G16757G55G56G20G27G200（G2283G1152G7234析G17902G11004仪器有G19492G1856G2508）G727G12447G5347自G2172电热G2399力G14988G8785G9793菌G19161G47G39G61G59G230G36G44G11上G9035G11015G4445G2319G11115器G7812G2390G12。12菌株及培养基121菌株白G14116G6297子菌粗毛栓菌G13007G59G59G59G2350G3775G1122G20G28G28G26G5192G27G7388在G4677G1008G11477G14755G8913G5078G2283G18078的护G3490G3576上，G1186G15999G11745G1252的杨G7653上分离得到的。G2528G5192G4677G1008G11477G12197G4410G19510G10995物研究G6164G5506G10995物分G12879G4472的G20544G2563G7138先G10995G4557G1866G17839G15904G2033步G18504定，定G2529G1038TRAMETESGALLICAFRA36A37A38A39。研究G16789G7138该菌具有G19489G16311木G17148G13044的能力，如图G20。XXXA40A41A42A43A44A45A46A47A48A49A50A511A52A53A54A55A56TGALLICAA57A58A59A60A61A62A63A64A65A66A67A68A69A70A71122培养基G7092机盐液（G74G18G47）G29G46HA72POA73G15G20G74MG74SOA73G150G17G24G74G11G49HA73G12A72SOA73G153G74G5506量G1815G13044G8609液（2ML）G726MNSOA73G103HA72OG150G170G26G27G23G80G74G727G49ACL，0G17G20G23G80G74G727FESOA73G103G26HA72OG150G170G20G23G80G74G727COCLA72G1036HA72OG150G170G216G80G74G727G61NSOA73G103G26HA72OG150G170G170G20G23G80G74G727CG88SOA73G103G24HA720G150G170G21G20G27G23G80G74G727G46G36LG11SOA73G12A72G103G20G21HA72O0G1700G20G23G80G74G727HA74G37OA73G150G1700G20G23G80G74G727G49AA72MOOA73G103G21HA72OG150G1700G20G23G80G74G727CHA74COOCL，0G170G21G20G80G74123原酶的来源已在本实验G4472培养G1782960G3837的粗毛栓菌，G10995长G1122G15967G16025麦草粉上，G8611G15967G1881含过G200木G12591的G20130G5190杨木G19203G7423G2400G74G1209及G20G17G24G46G74G7092机盐G8712G9354液，G1861三G15967。13粗酶液的制备培养60G3837后，培养物与过G3824G20056G1931的G2447离子G8712（G23G263）G1209G20G726G27的G8616G1375G9163G2524，G23HG5194G8611G19560G2334G4579时搅拌G980G8437，G9994后G11004G1025空G13432G13512超滤G16025置G17839G15904超滤浓缩。弃G2447滤渣，G1861获取滤液G2400G80G47G727在粗酶液G1025添加固体硫酸铵粉G7423G27G21G1823G15至30饱G2656G5242（G8611G2000G80G47加硫酸铵G206G17G23G74）G15G5194缓慢搅拌G15使硫酸铵完G1852G9354G16311，G1122G23G263G991静置过G3824离心G116000RPG80G23G263G12G20G24G80G76N，除G2447沉淀物，留取上清液将上清液硫酸铵的饱G2656G5242上调到G26G24（G8611G2000G80G47G9354液加G21G27G17G24G74粉G7423G10378硫酸铵），G1122G23G263G991静置过G3824离心G11G26G2400RPG80G23G263G12G210G80G76NG15弃G2447上清液，留取沉淀G11004适量的G14988馏G8712G9354G16311沉淀，将G9354G16311物G16025入透析G15967（G20G21G20G23G46G39A）G1881，G4557G20056G1931的G14988馏G8712透析。G8611G19560G23H换G980G8437G2447离子G8712G8712，G1122G23G263G991透析。14离子交换层析G11004阴离子交换剂QSEPHAROSEFASTFLOWG16025制G6116G980根G20G176G210G70G80柱，连接G7811G5242G9163G2524器、G5670G8981G8905、G7692酸蛋白检测仪G2656自G2172G18108分G6922G19610器。柱的G16025填G726把层析柱固定在架子上，层析柱G5529须与G3332面垂直，否则G16025好的柱床表面倾斜，影响层析分离的效果。G16025柱时，离子交换剂要分几G8437加入。如果待G12544G980G8437离子交换剂在柱G1025沉淀到十分紧密后，G1889加入G12544二批时，两者之间就会G1147G10995G7138显得节痕。在G2469G10995上述情况时，G2499在加G12544二批之前，先G11004玻璃棒轻轻搅G2172G12544G980G8437沉积的柱床的表面，使XXXA75A76A77A78A79A80A81A82A83A84A85G1866疏松G5194有G18108分的悬浮，G9994后G1889加入G12544二批悬浮液。层析柱G16025好后，G11004胶头滴管慢慢的吸取柱床上面的G9354液。上样前G20056先G11004G2447离子G8712平衡，G1889G11004G210G80G80OLG18G47的G49AG36G70HG36G70缓冲液（PHG24G17G24）平衡交换柱，平衡液体积G1038柱床体积的3G24倍，直至G8981出液的PH接G17829G24G17G24。加样G2656洗脱G726G11004G5670G8981G8905将透析后的原酶液G1209G20G170G80G47G18G80G76N的速G5242加样G1122交换柱，上样完毕后，G11004G2528样的缓冲液（即G210G80G80OLG18G47的G49AG36CHG36G70PHG24G17G24）洗柱。此时的G8981出液G11004烧杯G6922G19610。G3835约G24H后，待OG39G21G270G1821吸G6922峰回到基线，G11004G2100G80G47G49AG36G70HG36G70G11G210G80G80OLG18G47PHG24G17G24G12缓冲液G2656G11013此缓冲液配制的G2100G80G47的0G17G23G80OLG18G47的G49ACLG4557离子交换柱G17839G15904洗脱（G7811G5242G9354液G11013G7811G5242G9163G2524器获得，G49ACLG9354液的浓G5242G11013低到高）。洗脱速G5242G1038G20G170G80G47G18G80G76NG15G11004自G2172G18108分G6922G19610器G6922G19610G8981出液，G86113G80G76NG6922G19610G980管，G1861G6922G19610G20G240管。测定G8981出液的酶活性，G2524G5194具有G2528样酶活性的G8981出液。15分析方法151锰过氧化物酶的酶活测定锰过氧化物酶（MANGANESEIIPEROXIDASES,MNPS,EC111113）活力的测定G726采G11004MICHAEL的方法。G2465G5224G9163G2524液含有2940L01MOL/L乳酸钠缓冲液（PH45）含01MMOL/LMNSO4G103H2OG265630L的01MMOL/LH2O2，30L的柱层析G6164得酶液，G2465G5224体G13007G18613ML。G2465G5224G11013加入H2O2G13792G2563G2172（G7380后G1889加）。G1209G12544G980管G1328G4557照（G3252G12544G980管几乎G7092酶活），测定G2465G5224前1MING1881在240NM处吸G1821值的增加，定义G8611分钟使1MOL/LMN2氧化G6116MN3G6164需的酶量G1328G10381个酶活力G2345位（U）。乳酸与MN3G6164G5430G6116的络G2524物的摩G4584消G1821G13007G6980G10386,500M1CM1。锰过氧化物酶酶活力G16757算G1856G5347如G991G726U/LNA106/6,500G1866G1025，NG1038酶液稀释倍G6980，AG1038G2465G5224液在1MING1881G1122240NM处吸G1821值的变化值。152蛋白含量的测定采G11004考G20544斯亮蓝G250G8616色法G726此法G1038染料结G2524法。G1209牛血清白蛋白（BSA）G1038标准液绘制标准曲线。按G991面表格G1025G6980据加入试剂G726A86A87A88A89A90A91A92A93A94A95A96A93A97A98A99A100A99A101A102012345A103A104A105BSAA94A95A106A107MLA1080102040608A101A102A109A110A106A107MLA108101010A111A112A113A107MLA108100908060402A114A115A116A117A118A119A106A12050MLA121A122A120A87A123A124A125A126A127A128A129A1305MINA131A132A133A1340A89A87A135A92A93A136A132A125A110A137595NMA138A139A140A141A99A131G2465映总体积G10383G80G47XXXA142A143A144A145A146A147A148A149A150A151A1522结果与讨论21MNP的柱层析纯化结果结果显示，在150个试管G1025，只有很少的几个试管能够测到酶活，G5194且G17837几个试管的编号G993G7171连在G980G17227的，G13792G1866他的测G993到酶活，也就G7171说，G17902过离子交换层析（QSEPHAROSEFF），没有能够得到具有锰过氧化物酶（MNP）活性的组分。22蛋白含量测定结果A153A154A155A156A157A158A159A160A161A162A163A164A165A166A167BSAA168A169A170A1711020304050A172A173A174A175A170OD595A171010302110354047205285A176A177A178A179A180A181A182A183A184A185A186A187A188A189结果测得，锰过氧化物酶的酶液G1821吸G6922值G10380G17G28G236，代入如上图G6164示的G1856G5347G1025G2499得G1866蛋白G17148含量G1038G27G24G17G21。G5194测得经过30硫酸铵盐析后G6164得沉淀的G1821吸G6922值G1038G20G17G21G286，代入G1209上G1856G5347G2499得G1866蛋白G17148含量G1038G20G206G17G26。测得经过G260硫酸铵盐析后G6164得上清的G1821吸G6922值G10380G17G26G26G27，代入G1209上G1856G5347G2499得G1866蛋白G17148含量G1038G260G17G20。3结论1粗酶液经过盐析、透析及离子交换层析G12573G980G13007列操G1328步G20600后没有获得G1114具有MNP活性的组分，G991G980步G1582实验时G5224G16280G14551操G1328G5194G17839G980步G6518G13046粗毛栓菌G1147MNP的G7477G1226，使G1866G1147G7368G3822的MNP。2经过离子交换层析G12573步G20600后，G6164得酶液G1025G7446蛋白的含量G17836G7171很G3822，锰过氧化物酶的活性G8616G17751低，G991G980步G1582实验时G5224在离子交换层析后活性电G8903回G6922G12573步G20600G1025G17839G980步G6564高酶的纯G5242。A190A191A192A193A192A194A194A194A195A196A192A193A192A192A192A197A198A199A191A192A193A200A201A197A192A192A193A194A192A193A202A192A193A197A192A193A203A192A193A204A192A193A205A194A192A202A192A197A192A203A192A204A192A206A207A208A209A210A211A212A213A214A215A216A217A218A219A220A221A222A223A222A224XXXA142A143A144A145A146A147A148A149A150A151A152参考文献A225A226A227A228A229A230A231A231A232A233A234A228A235A229A236A237A238A239A226A240A241A242A239A243A244A245A246A247A246A248A249A250A246A235A231A249A231A236A248A251A249A245A249A248A250A230A245A246A252A249A250A246A235A231A235A247A249A231A230A253A250A245A249A248A230A229A229A244A229A249A245A237A231A254A255A93A236A230A73A230A231A236A230A231A250A73A230A245A235A253A246A236A249A0A230A247A245A235A1A250A251A230A229A246A2A231A246A231A93A236A230A2A245A249A236A246A231A2A3A249A0A246A236A246A235A1A4A248A230A250A230A0A234A243A251A249A231A248A230A245A235A248A251A249A230A250A230A248A251A245A4A0A235A0A73A235A245A246A244A1A239A85A245A248A251A239A5A235A246A248A251A230A1A239A5A246A235A73A251A4A0A234A6A7A6A8A9A6A240A93A9A7A226A239A225A6A227A243A230A245A246A230A80A238A234A10A251A230A231A2A11A234A228A235A229A236A237A238A239A226A240A240A12A239A243A244A245A246A247A246A248A249A250A246A235A231A249A231A236A248A251A249A245A249A248A250A230A245A246A252A249A250A246A235A231A235A247A250A13A235A1A249A231A2A249A231A230A0A230A73A230A245A235A253A246A236A249A0A230A246A0A235A252A4A1A230A0A247A245A235A1A250A251A230A13A251A246A250A230A93A245A235A250A3A249A0A246A236A246A235A1A4A248A230A250A230A11A246A248A251A235A1A246A250A244A0A0A83A244A249A229A230A231A0A239A5A246A235A248A251A246A1A246A248A249A230A250A5A246A235A73A251A4A0A246A248A249A85A248A250A249A234A226A6A240A67A8A226A9A240A93A226A7A241A239A225A9A227A233A246A245A70A14A233A249A231A236A15A16A80A249A245A245A230A229A229A239A226A240A241A67A239A17A231A252A4A1A249A250A246A248A18A19A235A1A3A244A0A250A246A235A231A20A8A250A251A230A1A246A248A245A235A3A246A249A229A236A230A2A245A249A236A249A250A246A235A231A235A247A229A246A2A231A246A231A239A85A231A231A244A15A230A66A237A246A248A245A235A3A246A235A229A239A7A226A8A7A12A242A93A242A79A242A239A225A7A227A238A249A250A249A70A70A249A85A239A226A240A240A7A239A16A246A2A231A246A231A235A229A4A250A246A248A230A231A252A4A1A230A0A247A245A235A1A0A230A229A230A248A250A230A236A13A251A246A250A230A93A245A235A250A247A244A231A2A246A8A73A245A235A236A244A248A250A246A235A231A249A231A236A245A235A229A230A246A231A229A246A2A231A246A231A236A230A2A245A249A236A249A250A246A235A231A239A80A17A237A10A237A246A248A245A235A3A246A235A229A15A230A66A239A226A9A8A226A6A242A93A9A242A239A225A242A227A123A123A123A239A124A21A22A23A24A25A26A27A28A29A30A31A32A33A34A35A36A37A38A141A39A40A239A144A41A42A43A234A6A79A79A242A239A225A226A227A123A123A123A44A6A79A79A7A239A124A21A22A23A25A26A27A28A29A30A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