实验---纤维素酶活力的测定

1实验纤维素酶活力的测定（3,5-二硝基水杨酸法）一、实验目的掌握还原糖的测定原理，学习用3,5-二硝基水杨酸法测定纤维素酶活力的方法。二、实验原理纤维素酶水解纤维素，产生纤维二糖、葡萄糖等还原糖，能将3,5-二硝基水杨酸中的硝基还原成橙黄色的氨基化合物，故可利用比色法测定其还原物生成量来表示纤维素酶的活力。三、主要仪器与试剂(一)实验仪器1.25mL比色管2.722型分光光度计3.滴管4.水浴锅5.移液枪6.电炉(二)、试剂1.3,5-二硝基水杨酸显色液：称取10.0g3,5-二硝基水杨酸，溶入200mL蒸馏水中，加入20g分析纯氢氧化钠，200g酒石酸钾钠，加水至500mL，升温溶解后，加入重蒸苯酚2.0g，无水亚硫酸钠0.50g。加热搅拌，待全溶后冷却，定容至1000mL。存于棕色瓶中，放置一周后使用。2.0.1mol/LpH4.5乙酸-乙酸钠缓冲溶液。3.0.5%羧甲基纤维素钠水溶液，溶解后成胶状液，静置过夜。使用前摇匀。4.葡萄糖标准溶液：称取干燥至恒重的无水葡萄糖100mg，溶解后定容至100mL，此溶液含葡萄糖1.00mg/mL。5.纤维素酶液：将0.05g酶溶解定容至50mL，从中取出1.0mL再定容至100mL，待检测用。（用pH4.5乙酸-乙酸钠缓冲溶液配制）四、实验步骤1.标准曲线的绘制：分别吸取0.0，0.20，0.40，0.60，0.80，1.00mL葡萄糖标准液于6支25mL比色管中，均用蒸馏水稀释至1mL，加3.5-二硝基水杨酸显色剂3mL，在沸水浴中煮沸显色10min，冷却，加蒸馏水21mL，摇匀。以空白管调零，在550nm处比色。以光密度为纵坐标，以葡萄糖μg数为横坐标，绘出标准曲线。序号123456葡萄糖标液0.00.200.400.600.801.00蒸馏水1.00.800.600.400.200.03,5-二硝基水杨酸3.03.03.03.03.03.0实验操作沸水浴加热10min，冷却后，加水定容，摇匀，比色测定吸光度A550nm0.02.空白管的测定：在2支25mL试管中各加入1.0mL酶液，沸水浴5min，冷却后加3.0mL0.5%CMC-Na，与样品管同时放入50℃水浴30min。其它操作同样品管。3.样品的测定：在3支25mL试管中各加入0.5%羧甲基纤维素钠溶液3.0mL，酶液1.0mL，于50℃水浴中糖化30min，取出，立即于沸水浴中煮沸10min使酶失活，得糖化液，冷却加入3.0mL3,5-二硝基水杨酸显色液，再沸水浴10min，冷却后加水定容至25mL，混匀，以空白管调零，在550nm处测OD值，查葡萄糖标准曲线得样品的葡萄糖μg数。五、结果计算在上述条件下，1㎎酶每分钟催化纤维素水解生成1微克葡萄糖定为一个活力单位。纤维素酶活力单位（μg/mg·min）＝130)(gODN值对应的葡萄糖量2式中：N—酶液的稀释倍数，此处为10030—糖化所用时间，min1—反应酶液的mL数六、注意事项1.无论是标准液还是样品液，都要去除葡萄糖外的其他各种成分的对OD值的影响。使得到的标准曲线经过坐标原点。2.用移液管或移液枪加各试剂时，不能将移液管或移液枪头混用。各比色管中，均用蒸馏水稀释至1mL，加3.5-二硝基水杨酸显色剂3mL，在沸水浴中煮沸显色10min，冷却，加蒸馏水21mL，摇匀。DeterminationofCellulaseActivity1.PurposeofExperimentMastertheprincipleofdeterminationofreducingsugar,Learnthemethodofdeterminatingcellulaseactivityusing3,5-dinitrosalicylicacidmethod.2.ExperimentalPrincipleCellulasecanhydrolyzecellulosetoproducereducingsugarsuchascellobioseandglucose.Thosesugarscanreducenitroof3,5-dinitrosalicylicacidintoaminotoformorangeyellowaminocompounds,socolorimetricmethodcanbeusedtodeterminethereductionproductexpressedascellulaseactivity.3.TheMainInstrumentsandReagent3.1ExperimentalInstrument(1)25mLcolorimetrictubetype(2).722spectrophotometer(3)dropper(4)bath(5)pipette(6)electricfurnace3.2Reagent(1)3,5-dinitrosalicylicacidsolution:Weigh10g3,5-twonitrosalicylicacid,dissolvedin200mLofdistilledwater,adding20gsodiumhydroxideofanalyticallypure,200gsodiumpotassiumtartrate,addwaterto500mL,heatingtodissolvethosereagents.Addingredistilledphenol2.0g,sodiumsulfiteanhydrous0.50g.Heatingandstirring.Whenthereagentsarefullydissolved,cooling.Dilutewithwaterto1000mL.Storedinabrownbottle,layasideaweekbeforeuse.(2)0.1mol/LpH4.5aceticacid-sodiumacetatebuffersolution.(3)0.5%CMC-Naliquid:Weigh0.50gsodiumcarboxymethylcellulose,dissolvedinwatertomakeacolloidalsolution,standingforanight.Shakewellbeforeusing.(4)1.0mg/mLglucosestandardsolution:weigh100mganhydrousglucosedriedtoconstantweight,dissolveinwaterto100mL.(5)Cellulaseliquid:0.05genzymedissolvein50mLpH4.5aceticacid-sodiumacetatebuffersolution,thensuck1.0mLtoa100mLvolumetricflask，dilutewiththebuffersolutiontoscale.4.ExperimentalSteps4.1StandardCurveDrawing:Adding0.0,0.20,0.40,0.60,0.80,1.00mLglucosestandardsolutionin6colorimetrictubeof25mL,respectively.Ineverycolorimetrictube,addingdistilledwateruntil1.0mL,thenadding3.5-dinitrosalicylicacidsolution3.0mL,boiledinboilingwaterbath10minforcolordeveloping,thencooling,add21mLofdistilledwater,shaking.Setemptytubezero,determineODvalueat3130...).(.cosvalueODtorelatedgAmonteGluN550nm.Usingtheopticaldensityasordinate,glucoseμgnumberasabscissa,todrawthestandardcurve.SerialNumber123456glucosestandardsolution(mL)0.00.200.400.600.801.00Distilledwater(mL)1.00.800.600.400.200.03,5-dinitrosalicylicacid(mL)3.03.03.03.03.03.0ExperimentOperationHeating10mininboilingwaterbath,dilutewithwatertoscaleaftercooling,shake,colorimetricdeterminationAbsorbanceA550nm0.04.2DeterminationofBlank:Adding1.0mLenzymeliquidineachofthe2tubesof25mL.putinboilingwaterbath5min,aftercoolingadd3.0mL0.5%CMC-Naliquid,thenputin50℃waterbathfor30min.Thesubsequentoperationissameassample.4.3.DeterminationofSample:Ineachof325mLtubesadding0.5%CMC-Naliquid3.0mL,cellulaseliquid1.0mL,putat50℃waterbathexactly30minforglycosylation.Thenremoveimmediatelytoputinboilingwaterbathfor10mintoinactivatetheenzyme.Afterthesaccharificationliquidcooling,add3.0mL3,5-dinitrosalicylicacidsolution,thenputagaininboilingwaterbath10mintodevelopcolor,thencooling,dilutewithwaterto25mL,mixing.Setemptytubezero,determineODvalueat550nm.Checkonglucosestandardcurvetogetglucoseμgnumberofsamples5.ResultsCalculationUndertheaboveconditions,1mgofcellulasecatalyzeshydrolysisofcellulosetoform1microgramglucosewithin1minuteisdefinedasaunitofactivity.ActivityofCellulaseUnits(μg/mg·min)=N—enzymeliquid,hereis10030—mashingtimeused,min1—mLnumberofenzymeliquid6.Notice(1)whetherstandardorsamplesolution,theotheringredientsexceptglucoseshouldberemovedtoelimilatetheinfluenceeffectly.Thestandardcurveobtainedshouldgothroughtheoriginofcoordinates.(2)Whenusingpipettestoaddreagents,don’tmixpipetteswithpipettetips.实验食品中黄酮含量的测定-可见分光光度法一、实验目的掌握可见分光光度法测定食品中总黄酮含量的方法。二、实验原理黄酮类化合物中的酚羟基能与Al3+的碱性溶液中生成红色络合物，其颜色深浅与黄4酮含量成正比，在510nm波长处有最大吸收，故可比色测定。三、适用范围适