[生物工程精品] 粗毛栓菌漆酶的初步分离与纯化 本科毕业论文

本科生毕业设计（论文）题目粗毛栓菌漆酶的初步分离与纯化姓名学号系别生命科学系专业生物工程指导教师职称教授年月日教务处制A1A0A2A3A4A5A6A7A8A9A10A8A9A11A12A13A14A15A16A17A18A19A20A21A22A23A24A25A26A27A28A29A26A27A30A31A32A33A34A35A36A13A37A38A33A39A40A41A42A43A44A45A46A47A48A49A50A48A33A12A50A48A38A51A52A53A54A55A56A57A58A59A60A61A62A63A57A64A65A64A65A66A48A67A33A68A36A69A21A70A37A71A72A25A73A74A75A76A77A78A77A78A77A78A79A79A13A14A15A16A33A70A37A33A80A81A82A83A66A48ABSTRACTTRAMETESGALLICAFRWASCULTIVATEDINWHEATSTRAWPOWDERFOR25DAYSTHECRUDEENZYMEWASULTRAFILTRATED,SALTEDOUT,DIALYZEDANDAPPLIEDTOQSEPHAROSEFFIONEXCHAGECOLUMNTHEEFFLUENTWITHLACCASEACTICITYWASOBTAINEDKEYWORDSTRAMETESGALLICA,LACCASE,IONEXCHAGECHROMATOGRAPHYA84A85A86A87A88A89A90A91A92A93A94A95A96A97A98A86A99A100A101A102A103A104A90A105A106A107A108A109A110A111A112A113A114A106A115A116A117A118A119A120A121A122A123A124A117A125A126A106A127A128A129A130A123A131A132A133A134A135A136A137A138A84A107A108A139A140A106A141A142A123A143A117A137A144A145A131A132A85A146A135A136A137A140A147A106A148A149A123A84A85A86A137A150A151A152A153A154A98A90A148A149A123A146A155A146A156A89A90A157A158A154A91A92A93A94A159A160A97A161A162A163A89A90A164A165A97A166A167A168A169A170A159A171A172A97A161A162A163A166A173A174A175A176A97A166A177A171A97A161A162A163A178A179A180A181A182A183A184A185A186A187A188A186A189A190A191A192A193A194A187A195A196A193A197A198A199A200A201A199A202A203A204A205A206A207A205A208A209A200A210A211A212A213A214A215A216A217A218A219A220A221A222A214A219A222A223A224A225A226A227A228A229A219A222A230A217A218A205A231A232A233A234A205A235A236A237A238A218A205A239A240A241A242A218A205A243A244A245A244A191A246A247A223A248A239A249A200A250A251A252A227A214A253A254A255A1A0A2A3目录摘要1关键词1ABSTRACT1KEYWORDS1引言11材料与方法211材料2111菌种2112主要仪器2113主要试剂2114PDA培养基2115高产漆酶液体培养基212方法2121粗酶的制作2122粗酶的超虑浓缩3123硫酸铵盐析3124透析3125离子交换柱层析3126酶活力的测定3127蛋白含量的测定32分析和讨论321漆酶的诱导322离子交换层析对漆酶的分离与纯化3221表1洗脱组分漆酶活力测定结果4222图1粗毛栓菌漆酶的QSEPHAROSE柱层析洗脱曲线423蛋白质含量标准曲线A155A155A155A155A155A155A155A155A155A155A155A155A155A155A155A155A155A155A156231表2牛血清蛋白浓度与光密度值的关系4232图2蛋白质含量标准曲线424表3粗毛栓菌漆酶的柱层析纯化结果5参考文献5致谢6A4A5A6A7A8A9A10粗毛栓菌漆酶的初步分离与纯化生物工程G999G1006G4410生G6363导G6957G5084摘要G4570粗毛栓菌G6521种G3324含G7389G21626G14621G12893的培养基G1025培养25DG712G9036G6564G5483粗酶液G712G4584G2530G13475超G9400浓缩G451盐析和透析G712G4570透析液G990G7691G1122QSEPHAROSEG41G41层析柱G712G14731G5483G1867G7389漆酶活G5627的组分G452关键词粗毛栓菌G712漆酶G712离子交换层析SEPERATIONANDPURIFICATIONOFLACCASESINTRAMETESGALLICAFRABSTRACTTRAMETESGALLICAFRWASCULTIVATEDINWHEATSTRAWPOWDERFOR25DAYSTHECRUDEENZYMEWASULTRAFILTRATED,SALTEDOUT,DIALYZEDANDAPPLIEDTOQSEPHAROSEFFIONEXCHAGECOLUMNTHEEFFLUENTWITHLACCASEACTICITYWASOBTAINEDKEYWORDSTRAMETESGALLICA,LACCASE,IONEXCHAGECHROMATOGRAPHY引言G16780G3822G5506生物G2499对G7420质G13044G17839G15904G993G2528程度的G19489G16311G712G1866G1025G712白G14116G11507菌G708WHITEROTFUNGIG709G7380G1038G7389G6940A11A12A12A13A12A14A15G452G19512G13432G13512G13044酶和G2334G13432G13512G13044酶G1055G3818G712白G14116G11507菌G17836G14033G3827产生G1016种G14002G3818G17819G8699化物酶G712G2375G7420质G13044G17819G8699化物酶G708LIGNINPEROXIDASEG712LIPG709A11A12A16A13A12A17A15和G19204G17819G8699化物酶G708MANGANESEDEPENDENTPEROXIDASE,MNPG709A11A12A18A15G712G16780G3822白G14116G11507菌G17836G2528G7114产生G980种含G19120的G18222G8699化酶G708COPPERCONTAININGPHENOLOXIDASEG709G2375漆酶G708LACCASEG709G452漆酶G7171G980种含G19120的G3822G18222G8699化酶G712G7380G7101G7171G1186G7097G7424的漆G7653G8725G1025G2469G10628的G712G7171G1166G12879G7380G7101G2469G10628的酶G1055G980G712G1306G11464G2052G10628G3324G712G1166G1216对G4439的G16760G16794G17836G993G4448G6984A11A14A15G452G17829G5192G7481G712G19555G11540漆酶G2163G14033的G17892G8505G2469G10628G712G7389关漆酶的G11752G12362G2475G2052G7234G17953的G18337G16282G452G11013G1122G3324G19489G16311G7420质G13044方G19766的G2163G14033G712G1363G1055G3324制G8986G17908G13452工G1006G712G10317G2047G7171G13452G8986生物G9430白G20058G3507G5483G2052G9157G1849的G11752G12362和G5332G2469A11A16A13A17A15G712G3324G10627G1457方G19766G1075G7389G5468G3835的G9520力A11A18A15G712漆酶G2499与G7389G8614的G18222G12879作G11004G712G1363含苯G8699基G12879的G19512G14621剂G451石油化工废物的物质G16311去G8614G5627等A11A19A15G452G19555G11540应G11004的G993断加G9157G712漆酶的G11752G12362和G5332G2469越G7481越G2475G2052G1166G1216的关注G452G7424文就以漆酶的分离G451纯化G17839G15904初G8505G11752G12362G4521材料与方法11材料111菌种A20A21A22A23A24A25A26菌种TRAMETESGALLICG41RG7171G11013G6957授G6564供G452该菌种系G6957授1997G51928月G1186山东菏泽护城河堤的杨G7653G990首先G2469G10628并采集分离所G5483G452G13475初G8505鉴定G1038粗毛栓菌TRAMETESGALLICAFRA11A12A15G452112主要仪器A157A27A28A29A22A30A31A32A164A33A34A35A52A36A37A125A38A39A40A41A42A43A44A45A46A47A48A29A49A50A51A53A54A55A29A56A31A22A57A58A59A60A186A61A62A52A188A63A37A190A64A65A66A67A41A68A69A70A71A72A61A186A62A52A196A73A198A74A75A76A77A49A50A78A79A28A29A80A81A71A72A205A82A83A84A85A86A87A62A88A89A90A91A92A52A188A63A93A215A94A95A88A89A90A91A71A72A217A186A52A96A37A37A218A76A97A98A29A49A50A99A100A101A102A28A29A31A186A164A52A92A222A103A104A49A50A99A100A101A102A28A29A31A105A106A92A37A37A36A106A101A102A41A68A107A108A49A50A198A107A41A68A28A29A80A81A71A72A35A167A217A52A62A228A109A110A38A111A112A28A49A50A39A40A57A28A113A31A186A33A52A188A151A75A114A115A116A117A28A29A49A50A99A100A101A102A28A29A31A238A217A118A52A239A36A52A92A39A40A170A101A119A120A29A49A50A54A121A101A102A28A29A31113主要试剂G11752G12362G1025所G11004的主要试剂G7389QSEPHAROSEG41ASTG41LOWG451MNSOA17HA14OG451硫酸铵G451酵母G12893和G5506量元G13044G451乳酸钠乳酸缓冲液；025R250考马斯亮蓝染液及脱色液A11A122A15G452114PDA培养基称取去皮马铃薯40GG712加适量水煮沸20MING712G11004纱布G17819G9400G5483G9400液G712加G1849葡萄糖4GG712琼脂G128933GG712补足水至200MLG712混匀G712融化G712分装G712121灭菌20MING452此培养基主要G11004G1122粗毛栓菌的G1457存G452115高产漆酶液体培养基KHA14POA171GG712MGSOA177HA14O05GG712酒石酸铵2GG712蛋白胨30GG712酵母G128931GG712G5506量元G13044母液2MLG712加去离子水至1000MLG452取500ML加去离子水至1LG712分装G3324500ML的三角烧瓶G1025,共5瓶；另500ML分装G1849500ML的三角烧瓶G1025装2瓶G452121灭菌20MIN备G11004G45212方法121粗酶液的制备菌种G3324高产漆酶液体培养基培养25天G2530G712G4570培养物取出G712G11004G1016层纱布G17819G9400G712取G990清液G452122粗酶的超滤浓缩G4570粗酶液G11013超G9400浓缩装置处理G712G5483G2052浓度较高的酶液G452123硫酸铵盐析首先G3324粗酶液G1025添加固体G708NHA123G709A124SOA123G12893末至30饱和度G712离心G7087200R/MING71215MING712A126A127A128A129A130A131A1324A133CG709G19512去部分杂蛋白沉淀物G712收集G990清液；然G2530G4570G708NHA123G709A124SOA123饱和度G990调G205275G712G11224静置G17819G3824G452124透析离心G7087200R/MING71220MING7124G709G712G5335去G990清液G712G11004适量去离子水G9354G16311沉淀G712G4570G9354G16311物装G1849透析G15967G708G6142G11053分子量G10381214G78DAG709G1881对去离子水G17839G15904透析G7122G4579G7114G7368换G980G8437去离子水G712G7368换34G8437G452125离子交换柱层析首先G11004G19464离子交换剂SEPHAROSEQG41ASTG41LOW装制G6116G980G769316G10420CM柱G712G17842G6521G7692酸蛋白G13055G3818G7828测仪和G14270G2172部分收集器G452G990G7691G2081G20056先G11004300MLG70810MMOL/LG709G18271酸钠缓冲液G708PH55G709G5191G15925交换柱G712G990G7691G2530G11004含G73891MOL/LNACL的缓冲液G708PH45G709G17839G15904G7811度洗脱G712收集G7389漆酶活力的组分G452126酶活力的测定3ML的G2465应液G1025含G7389G11004005MOL/L的G18271酸缓冲液G708PH40G709G18209制的05MMOL/LAG37G55S20G173L酶液G712G112220G991G2465应3MING712测420NM处的G2572光值G452127蛋白含量的测定参G10043G37RADG73ORDA134A135A136A137法取G980系G2027试G12661G712分G2047加G18490G71202,04G71206G71208G71210ML的标准牛血清白蛋白G9354液100G173G/MLG712然G2530G2533G8611试G12661G1025分G2047加G1849考马斯亮蓝G42250G708001G7095MLG712G7380G2530G11004水补G1817至6MLG712混匀G712G4472G9213静置2MING712G17885G11004光程G10381CM的G8616色G7491G712以G993加牛血清白蛋白G37SA的试G12661G1038对G10043G712G3324595NM处G8616色G452以牛血清白蛋白的浓度G1038G8190G3364标G712光密度值G1038G13449G3364标G712G13484制标准曲线G452取G5465测G7691G2709G9354液1MLG712加G1849考马斯亮蓝G422505MLG712混匀G712G6365G990G17860方法测定595NM的光密度值G452G5191G15904G15823G1233G452G8616色G2530G712G1186标准曲线G990G7609出G5465测G7691G2709的蛋白质浓度G4522结果与讨论21漆酶的诱导G1038G14731G5483较高产量的漆酶G712G7424文采G11004G1114高产漆酶天然培养基G712G13475培养G712该菌G3324生G19283G7411G19400G712G1262分G16311产生G3835量的G1025G19400产物G712G17837G1135产物G2460G6116G1114漆酶的诱导物A134A138A137,G3252此G712漆酶活力G7389G7509G3835的G6564高G71222离子交换柱层析对漆酶的分离与纯化离子交换柱剂G15441然G14033对漆酶G17839G15904静G8502G2572G19480G712G1306G11013G1122G2469酵液G1025存G3324G3835量的色G13044G712G1363洗脱G991G7481的漆酶液G2588G9157G21656G16100色液体G712G5445G2721G1114漆酶的活G5627G712G1038G1114G5483G2052G7368G1038纯化的漆酶G712G11004QSEPHAROSEG2499G14731G5483较G3921的分离G6940果G712收集G7389酶活G5627的部分G712G2375G5483部分纯化的组分A134A139A137G452221洗脱组分漆酶活力测定结果,见表1。A126A127A128A129A130A131A132漆酶酶活力G16757G12651G1856G5347G56/LG32NG104G141AG10410A140/23,300G104323,300G1038G3324420NM处的G6717G4584G9052光系G6980,G141AG1207表G2572光值,NG1207表G12244G18334G1505G6980G712NG32250表1洗脱组分漆酶活力测定结果G708G172G32420；20G709A244A141A142A237A143A144A145A146A147A148A149A150A250A152A153A154A244A141A142A237A143A144A145A146A147A148A149A150A250A152A153A154A158A168A168A159A168A160A161A252A162A163A165A165A166A159A169A158A163A168A159A163A168A161A252A162A158A161A171A158A159A158A158A161A168A159A163A161A163A252A162A158A171A165A254A159A171A158A158A168A159A161A169A171A252A162A171A165A254A171A159A166A158A254A168A159A254A166A254A252A162A163A254A160A163A158A159A169A158A166A161A159A254A171A169A252A162A171A168A168A166A158A159A160A158A160A161A159A168A171A168A252A162A160A160A165A166A161A159A171A158A169A163A159A160A168A165A252A162A166A163A169A165A163A159A171A158A171A168A159A165A169A163A252A162A158A163A161A166A166A159A254A158A165A168A159A160A161A169A252A162A161A168A163A171A161A159A254A254A168A168A159A254A165A160A252A162A163A166A165A160A166A159A169A254A163A168A159A158A169A158A252A162A163A161A168A168A160A159A254A254A161A168A159A161A171A161A252A162A165A168A169A169A159A158A254A158A168A159A161A158A169A252A162A169A160A161A171A159A171A254A254A168A159A163A171A254A252A162A166A165A161A161A159A169A254A166A168A159A163A171A254A252A162A166A165A161A161A159A169222以洗脱体积为横坐标，酶活力值做纵坐标。所得洗脱曲线见图1。A172A173A174A174A175A176A177A178A179A180A181A175A176A178A179A180A181A175A182A182A183A183A184A185A187A189A191A192A193A194A185A184A185A191A192A185A195A197A199A195A197A195G11013图G2499G11705G1867G7389较G3835漆酶活G5627的组分主要集G1025G33241395MLG20522025MLG1055G19400G45223蛋白质含量标准曲线231牛血清蛋白浓度与光密度值的关系,见表2。表2牛血清蛋白浓度与光密度值值的关系A200A201A202A203A204A150A206A212A207A213A208A154A161A168A254A168A160A168A171A168A163A168A168A209A210A204A146A150A211A214A216A219A216A154A168A159A161A160A166A168A159A254A171A158A168A159A169A160A166A168A159A165A158A161A163A159A168A158A160232蛋白质含量标准曲线，见图2。G5483G2052标准曲线的G3250G5414方程G1038AG320009955G91G1400989G712结果表G7138G712牛血清白蛋白量G332401MG/MLG14551G3272G1881与光密度值线G5627关系G14403G3921G452A220A221A220A220A220A220A223A220A220A220A220A224A220A220A220A220A225A220A220A220A220A226A220A220A220A220A227A220A220A220A220A229A220A220A220A220A230A220A220A220A220A231A220A220A220A220A221A224A226A221A225A220A221A225A225A221A225A231A221A226A224A221A226A230A221A227A223A221A227A229A221A229A221A221A229A227A221A230A220A221A230A226A221A230A231A221A231A225A221A231A230A223A220A224A223A221A227A223A224A220A223A225A224A223A226A229A223A229A220A223A230A225A223A231A229A224A221A221A224A223A225A224A224A230A224A226A221A224A227A226A225A220A226A225A221A231A232A233A234A235A236A240A241A242A243A245A246A247A248A249A251A253A255A1A0A1A1A2A2A3A3A4A5A1A0A1A2A6A2A1A0A11A1A0A7A1A0A6A13A13A0A8A9A10A12A14A15A16A17A18A19A20A21A22A23A24A25A25A26A27A26A28A29A30A31A32A33A34A35A36A30A31A32A33A35A3624粗毛栓菌漆酶的柱层析纯化结果，见表3。表3粗毛栓菌漆酶的纯化结果SUMMARYOFTHEPURIFICATIONOFLACCASEINTRAMETESGALLICAFRA37A184A38A39A40A41A42A43A252A40A234A44A43A213A45A46A41A42A147A252A43A213A45A149A47A241A48A49A37A184A50A247A187A51A58A52A53A52A54A57A55A52A211A56A217A211A55A57A59A217A211A55A57A59A60A61A62A54A52A53A52A54A63A62A54A211A64A62A58A65A187A51A58A52A53A52A54A57A55A52A211A56A66A55A62A61A57A54A55A52A64A52A55A65A61A58A211A55A62A52A56A57A54A55A52A64A52A55A65A58A57A55A62A53A211A59A67A68A41A69A188A70A71A188A72A73A71A74A74A75A76A73A74A76A74A74A73A74A77A75A188A71A71A188A197A58A51A67A62A62A56A78A65A213A62A79A80A81A82A83A84A85A188A86A75A86A73A75A70A74A71A73A70A86A72A72A73A76A77A87A188A71A88A188A188A73A76A77A73A71A60A62A61A89A57A58A211A66A62A90A91A53A57A66A55A53A59A211A92参考文献G621G64G7429G19530G712G17225G8716G144712002,粗毛栓菌漆酶的分离纯化及部分G5627质G11752G12362G712G8506G8733G3835G4410G4410G6265G708理G4410G10268G709209212G622G64CHISTROPHERFTHURSTOR1994,THESTRUTUREANDFUNCTIONOFFUNGALLACCASEJMICROBIOLOGY,1401926G623G64BOURBONNAISR,PAICEMG,ETAL1990,OXIDATIONOFNONPHENICSUBSTRATESJFEMSLETT,26799103G624G64G2283G1152G3835G4410生物系生物化G4410G6957G11752G44721991生物化G4410G4466G20576G6363导G625G64G10591G12180G3867G712G12218G9125G4207G712高天G5947G712G20080G2333G25431999基G11796生物化G4410G4466G20576G708第二G10268G709G626G64卢圣栋主编G10628G1207分子生物G4410G4