|
OALib Journal期刊
ISSN: 2333-9721
费用：99美元
|
|
|
|
二甲基酚橙法测定尿酸酶活性和识别尿酸酶高活性突变体
DOI: 10.3724/SP.J.1145.2013.00523, PP. 523-527
Keywords: 尿酸酶,二甲基酚橙法,突变体,高通量筛选,阳性突变体阈值,roc曲线
Abstract:
考察了采用二甲基酚橙测定过氧化氢（xylenol-orange-assay-of-hydrogen-peroxide，xoahp）法测定尿酸酶活性的可靠性以及识别高活性尿酸酶突变体的方法.将苛求芽孢杆菌（bacillusfastidious）尿酸酶4种突变体成对组合，使其催化效力比值在1.3-4.1间变化；将每种突变体表达载体转化大肠杆菌，取30个克隆分别转移到1.0ml液体培养基放大，18℃诱导表达16h，收集细胞后超声裂解得裂解液.在ph8.9的tris-hcl缓冲液中0.33mmol/l以下尿酸对xoahp法测定尿酸酶活性无干扰.sds-page分析发现不同突变体蛋白表达丰度有差异，酶活性同裂解液中总蛋白浓度正相关.receiver-operation-curve（roc）分析发现尿酸酶间催化效力比值越大，则所得曲线下面积（area-under-the-curve，auc）越接近1.00，此比值接近1.8时auc约0.95.以突变体与起始物的酶活性浓度差值超过起始物标准差1.4倍为阈值，能识别催化效力达起始物1.8倍以上的突变体.因此，用xoahp法测定裂解液中尿酸酶活性，以起始物活性均值加标准差1.4倍为阈值，有望高通量筛选尿酸酶突变体库.
| [1] | schlesingern,yasothanu,kirkpatrickp.pegloticase[j].natrevdrugdiscov,2011,10(1):17-18
|
| [2] | yangxl,yuanyh,zhancg,liaof.uricasesastherapeuticagentstotreatrefractorygout:currentstatesandfuturedirections[j].drugdevelopres,2012,73(2):66-72
|
| [3] | jackelc,hilvertd.biocatalystsbyevolution[j].curropinbiotechnol,2010,21(9):753-759
|
| [4] | hulleyme,toogoodhs,fryszkowskaa,manselld,stephensgm,gardinerjm,scruttonns.focuseddirectedevolutionofpentaerythritoltetranitratereductasebyusingautomatedanaerobickineticscreeningofsite-saturatedlibraries[j].chembiochem,2010,11(17):2433-2447
|
| [5] | mazurap,filipit,soucekp,brzobohatyb.focuseddirectedevolutionofβ-glucosidases:theoreticalversusrealeffectivenessofaminimalworkingsetupandsimplerobustscreening[j].carbohydrres,2011,346(2):238-242
|
| [6] | liaof,zhuxy,wangym,zuoyp.thecomparisonoftheestimationofenzymekineticparametersbyfittingreactioncurvetotheintegratedmichaelis-mentenrateequationsofdifferentpredictorvariables[j].jbiochembiophysmethods,2005,62(1):13-24
|
| [7] | malon,hanleyja,cerquozzis,pelletierj,nadonr.statisticalpracticeinhigh-throughputscreeningdataanalysis[j].natbiotechnol,2006,24(2):167-175
|
| [8] | zhangxd.illustrationofssmd,zscore,ssmd*,z*score,andtstatisticforhitselectioninrnaihigh-throughputscreens[j].jbiomolscreen,2011,16(7):775-85
|
| [9] | chinhnh.mechanismofinterferencebyuricacidintheglucoseoxidase/peroxidasemethodforserumglucose[j].clinchem,1974,20(4):499-501
|
| [10] | flemingjk,gadsdenrh,kabbanii.definitivecharacterizationofuricacidasaninterferentinperoxidaseindicatorreactionsandaproposedmechanismofaction[j].clinbiochem,1988,21(1):27-32
|
| [11] | 李想,冯娟,张纯,蒲军,杨晓兰,袁拥华,廖飞.反应条件对苛求芽孢杆菌胞内尿酸酶与嘌呤衍生物相互作用的影响[j].应用与环境生物学报,2011,17(1):120-123[lix,fengj,zhangc,puj,yangxl,yuanyh,liaof.effectsofreactionconditionsoninteractionsofsomepurinederivativeswiththeintracellularuricasefrombacillusfastidiosus[j].chinjapplenvironbiol,2011,17(1):120-123
|
| [12] | shermanmr,saifermg,perez-ruizf.peg-uricaseinthemanagementoftreatment-resistantgoutandhyperuricemia[j].advdrugdelivrev,2008,60(1):59-68
|
| [13] | zhaoys,yangxy,luw,liaoh,liaof.uricasebasedmethodsfordeterminationofuricacidinserum[j].microchimacta,2009,164(1):1-6
|
| [14] | terkeltaubr.updateongout:newtherapeuticstrategiesandoptions[j].natrevrheumatol,2010,6(1):30-38
|
| [15] | richettep,bardint.gout[j].lancet,2010,375(9711):318-328
|
| [16] | bershteins,tawfikds.advancesinlaboratoryevolutionofenzymes[j].curropinchembiol,2008,12(3):151-158
|
| [17] | turnernj.directedevolutiondrivesthenextgenerationofbiocatalysts[j].natchembiol,2009,5(8):567-573
|
| [18] | zhangc,yangxl,yuanyh,puj,liaof.site-specificpegylationoftherapeuticproteinsviaoptimizationofbothaccessiblereactiveaminoacidresiduesandpegderivatives[j].biodrugs,2012,26(4):209-215
|
| [19] | jochensh,bornscheuerut.naturaldiversitytoguidefocuseddirectedevolution[j].chembiochem,2010,11(13):1861-1866
|
| [20] | fengx,sanchisj,reetzmt,rabitzh.enhancingtheefficiencyofdirectedevolutioninfocusedenzymelibrariesbytheadaptivesubstituentreorderingalgorithm[j].chemistry,2012,18(18):5646-5654
|
| [21] | zhaoys,zhao,ln,yang,gq,tao,j,liao,f.characterizationofauricasefrombacillusfastidiousa.t.c.c.26904anditsapplicationtoserumuricacidassaybyapatentedkineticuricasemethod[j].biotechnolapplbiochem,2006,45(2):75-80
|
| [22] | zhangc,yangxl,fengj,yuanyh,lix,buyq,xieyl,yuanhd,liaof.effectsofmodificationofaminogroupswithpoly(ethyleneglycol)onarecombinanturicasefrombacillusfastidiosus[j].bioscibiotechnolbiochem,2010,74(6):1298-1301
|
| [23] | fengj,lix,yangxl,zhangc,yuanyh,puj,zhaoys,xieyl,yuanhd,buyq,liaof.anewpracticalsystemforevaluatingpharmacologicalpropertiesofuricaseaspotentialdrugtohandlehyperuricemia[j].archpharmres,2010,33(11):1761-1769
|
| [24] | huangsh,wutk.modifiedcolorimetricassayforuricaseactivityandascreenformutantbacillussubtilisuricasegenesfollowingstepmutagenesis[j].eurjbiochem,2004,271(3):517-523
|
| [25] | wolffsp.ferrousionoxidationinpresenceofferricionindicatorxylenolorangeformeasurementofhydroperoxides[j].methodsenzymol,1994,233:182-189
|
| [26] | imhoffrd,powernp,borrokmj,tiptonpa.generalbasecatalysisintheurateoxidasereaction:evidenceforanovelthr-lyscatalyticdiad[j].biochemistry,2003,42(14):4094-4100
|
| [27] | 廖飞,赵运胜,杨晓兰,李想,张纯,冯娟,卜友泉,谢燕玲.一种重组苛求芽孢杆菌胞内尿酸酶及其聚乙二醇修饰与应用[p].中国发明专利,zl200910103741.1.2012-02-22
|
| [28] | bradfordmm.arapidandsensitivemethodforthequantitationofmicrogramquantitiesofproteinutilizingtheprincipleofprotein-dyebinding[j].analbiochem,1976,72(2):248-254
|
| [29] | zhaoys,yangxl,lixy,buyq,dengp,zhangc,fengj,xieyl,zhus,yuma,liaof.reversibleinactivationofanintracellularuricasefrombacillusfastidiosusviadissociationofhomotetramerintohomodimersinsolutionsoflowionicstrength[j].bioscibiotechnolbiochem,2009,73(9):2141-2144
|
| [30] | amesbn,cathcartr,schwierse,hochesteinp.uricacidprovidesanantioxidantdefenseinhumansagainstoxidant-andradical-causedagingandcancer:ahypothesis[j].procnatlacadsciusa,1981,78(11):6858-6862
|
Full-Text
|
|
Contact Us
service@oalib.com QQ:3279437679 
WhatsApp +8615387084133
|
|
