菠萝蛋白酶的制备Preparationofbromelai.pptx

菠萝蛋白酶的制备Preparationofbromelain

目录/CONTENTS1原料与试剂准备Preparationofrawmaterialsandreagents设备与用具准备Preparationofequipmentandutensils操作方法Howitworks:23

PART01原料与试剂准备Preparationofrawmaterialsandreagents

一、原料与试剂准备原料0.1mol/LpH=7.8磷酸缓冲溶液、硫酸铵新鲜菠萝皮试剂菠萝皮磷酸缓冲溶液硫酸铵

1.Preparationofrawmaterialsandreagentsrawmaterial0.1mol/LpH=7.8Phosphatebufferedsolution,ammoniumsulfateFreshpineapplepeelreagentPineapplepeelPhosphatebufferedsolutionammoniumsulphate

PART02设备与用具准备Preparationofequipmentandutensils

透析袋磁力搅拌器榨汁机大烧杯台式天平二、设备与用具准备

DialysistubingMagneticstirrerjuicerLargebeakerBenchtopbalances2.Preparationofequipmentandutensils

PART03操作方法Howitworks:

硫酸铵盐析法提取菠萝蛋白酶操作流程去杂取汁透析降温离心加盐分别用为20%、30%、40%饱和度硫酸铵进行盐析（一）操作流程（二）操作要点称取菠萝皮材料120g，将菠萝皮在清水中洗净，沥干，切成小段后置于超高速搅拌机中，加入约60mL预冷的0.1mo1/LpH7.8PBS，持续搅拌10~15min至粉碎，搅拌完成后4层纱布过滤，滤液4°C离心（3000r/min）6min，清液备用。1.去杂取汁硫酸铵盐析法制备菠萝蛋白酶

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.加盐称取固体硫酸铵于研钵中，研磨呈粉末，缓慢分次向酶提取液中添加固体硫酸铵，边加边搅拌，使溶液最终硫酸铵饱和度为20%、40%、60%，放置于冰水混合物（4℃）30min，酶蛋白沉淀析出，观察沉淀析出速度及沉淀量。3.降温离心4.透析粗酶中加入0.1mo1/LpH7.8PBS约15mL至完全溶解，得硫酸铵酶混合液。将混合液装入2.5cm×8cm透析袋中，置于500mL烧杯中，在磁力搅拌器搅拌下用蒸馏水透析，15min换水一次，换水3～4次，直至透析完全，得菠萝蛋白酶浓缩液。处理液4°C4000rpm离心10min，收集沉淀得粗酶制品。检查SO42-是否除净方法：取2mLBaCl2溶液于试管中，滴加2～3滴透析外液，若出现白色沉淀，表示SO42-未除