菠蘿蛋白酶的制備Preparationofbromelain目錄/CONTENTS1原料與試劑準備Preparationofrawmaterialsandreagents設備與用具準備Preparationofequipmentandutensils操作方法Howitworks:23PART01原料與試劑準備Preparationofrawmaterialsandreagents一、原料與試劑準備原料0.1mol/LpH=7.8磷酸緩沖溶液、硫酸銨新鮮菠蘿皮試劑菠蘿皮磷酸緩沖溶液硫酸銨1.Preparationofrawmaterialsandreagentsrawmaterial0.1mol/LpH=7.8Phosphatebufferedsolution,ammoniumsulfateFreshpineapplepeelreagentPineapplepeelPhosphatebufferedsolutionammoniumsulphatePART02設備與用具準備Preparationofequipmentandutensils透析袋磁力攪拌器榨汁機大燒杯臺式天平二、設備與用具準備DialysistubingMagneticstirrerjuicerLargebeakerBenchtopbalances2.PreparationofequipmentandutensilsPART03操作方法Howitworks:

硫酸銨鹽析法提取菠蘿蛋白酶操作流程去雜取汁透析降溫離心加鹽分別用為20%、30%、40%飽和度硫酸銨進行鹽析（一）操作流程（二）操作要點稱取菠蘿皮材料120g，將菠蘿皮在清水中洗凈，瀝干，切成小段后置于超高速攪拌機中，加入約60mL預冷的0.1mo1/LpH7.8PBS，持續(xù)攪拌10~15min至粉碎，攪拌完成后4層紗布過濾，濾液4°C離心（3000r/min）6min，清液備用。1.去雜取汁硫酸銨鹽析法制備菠蘿蛋白酶AmmoniumsulfatesaltingoutmethodtoextractbromelainRemovethemiscellaneousjuicesdialysisCooldownandcentrifugeAddsaltSaltingoutwascarriedoutwithammoniumsulfateof20%,30%and40%saturation,respectively(1)Operationalprocedures(2)Keypointsofoperation1.RemovemiscellaneousjuicesPreparationofbromelainbyammoniumsulfatesaltingoutmethodWeigh120gofpineapplepeelmaterial,washthepineapplepeelincleanwater,drainit,cutitintosmallpiecesandplaceitinanultra-high-speedmixer,addabout60mLofpre-cooled0.1mo1/LpH7.8PBS,stircontinuouslyfor10~15minuntilcrushed,filterwith4layersofgauzeafterstirring,centrifugethefiltrateat4°C(3000r/min)for6min,andsetasidetheclearsolution.2.加鹽稱取固體硫酸銨于研缽中，研磨呈粉末，緩慢分次向酶提取液中添加固體硫酸銨，邊加邊攪拌，使溶液最終硫酸銨飽和度為20%、40%、60%，放置于冰水混合物（4℃）30min，酶蛋白沉淀析出，觀察沉淀析出速度及沉淀量。3.降溫離心4.透析粗酶中加入0.1mo1/LpH7.8PBS約15mL至完全溶解，得硫酸銨酶混合液。將混合液裝入2.5cm×8cm透析袋中，置于500mL燒杯中，在磁力攪拌器攪拌下用蒸餾水透析，15min換水一次，換水3～4次，直至透析完全，得菠蘿蛋白酶濃縮液。處理液4°C4000rpm離心10min，收集沉淀得粗酶制品。檢查SO42-是否除凈方法：取2mLBaCl2溶液于試管中，滴加2～3滴透析外液，若出現白色沉淀，表示SO42-未除凈；若無沉淀出現，表示透析完全。2.AddsaltWeighthesolidammoniumsulfateinthemortar,grinditintopowder,slowlyaddsolidammoniumsulfatetotheenzymeextractinstages,stirwhileadding,makethefinalammoniumsulfatesaturationofthesolution20%,40%and60%,placeitintheice-watermixture(4°C)for30min,theenzymeproteinprecipitatesandprecipitates,andtheprecipitationspeedandprecipitationamountareobserved.3.Coolingcentrifugation4.DialysisAdd0.1mo1/LpH7.8PBSabout15mLtothecrudeenzymeuntilcompletelydissolvedtoobtainammoniumsulfatemixture.Themixturewaspackedintoa2.5cm×8cmdialysisbag,placedina500mLbeaker,dialysedwithdistilledwaterunderthestirringofamagneticstirrer,andthewaterwaschangedoncein15minand3~4timesuntilthedialysiswascompletetoobtainbromelainconcentrate.Thetreatmentsolutionwascentrifugedat4°Cat4000rpmfor10min,andtheprecipitatedcrudeenzymeproductswerecollected.CheckwhetherSO42-isremovedmethod:take2mLBaCl2solutioninthetesttube,add2~3dropsofdialysissolution,ifthereisawhiteprecipitate,itmeansthatSO42-isnotremoved;Ifnoprecipitationispresent,dialysisiscomplete.20%40%60%2.28g4.86g7.8g觀察結果1、2、3份溶液中析出沉淀的速度各不相同，硫酸鈉飽和度越高，溶液析出的菠蘿蛋白酶速度越快，析出量越多。2.28g4.86g7.8gObservationsThehigherthesaturationofsodiumsulfate,thefasterthebromelainprecipitatedinthesolution,andthemoreprecipitatedamount.2.28g4.86g7.8g5.冷凍干燥將菠蘿蛋白酶濕品放入冰箱中，于-15℃冷凍10～15h，解凍后離心（3000r/min）10min，沉淀放入裝有氯化鈣或五氧化二磷的干燥器中干燥5～10h，取出后研磨成粉末，得菠蘿蛋白酶純品，低溫（2～10℃）下保存。把上述沉析物倒入燒杯中，加0.5％硫代硫酸鈉溶液5mL，攪勻，靜置5min；加1％L-半胱氨酸溶液3mL，攪勻，離心（3500r／min）10min，棄去上清液，得菠蘿蛋白酶濕品。Theaboveprecipitateswerepouredintoabeaker,5mLof0.5%sodiumthiosulfatesolutionwasadded,stirredwell,andallowedtostandfor5min.Add3mLof1%L-cysteinesolution,stirwell,centrifuge(3500r/min)for10min,discardthesupernatant,andobtainbromelainwetproduct.5.Freeze-dryingPutthebromelainwetproductintotherefrigerator,freezeitat-15°Cfor10~15h,centrifuge(3000r/min)for10minafterthawin