羧肽酶B研究进展

PharmaceuticalBiotechnology2006,13(4):302305B3,,,,(,200237)B,BBBB;B;:Q55:A:100528915(2006)0420302204B(CarboxypeptidaseB,CPB)(EC3.4.17.2),AB(CPA/CPB),Zn,[1,2],AB,,A,B,CPB[3]B,,,B1BB(CPB),B46ku,401;CPB35ku,306B:Arg2145Asp241,CArg2145[4]Asp241AsnAla,,,,Asp241Asp2145[4]CPBArg295,,CArgCPBCPBHis269,Glu272His2196;Arg2127Glu2270;Arg2145,,Asn2144Tyr2248;Asp2255,,CPA,[5]2B2.1[6]CPB2L2,(hippuricacid)2L2(hippuryl2L2arginine):0.001mol/LL2,3ml,254nm520l,,0.001mol/L(25)40%,,10min30%100%0.36,,,0.001mol/Lunit/mgprotein2.2[7]2.2.1pH2033:2005211230:2006204202::,,,Tel:02126425225511024mol/L,pH7.98.0;,pH7.65,K0,pH8.0,k095%2.2.2L2L2,L2,,L2L2CPB2L22L2,,2.2.310,k01.5,Km3B3.1pHB[8],CPBpH8,pH12,,CPBpH5,,CPBpH53.2(60),CPB,CPBCPB,CPB[9]3.3BCPB,CPB0mol/LNaCl0.05mol/LNaClCPB,,CPB0.20.3mol/LNaCl3,CPB,,0.1mol/LNaCl36,0.1mol/LNaCl,220CPBCPB,,65%(NH4)2SO4CPB12[10]3.4B3.4.1Mn2+,Zn2+,Mg2+,Ca2+,CPBCo2+,[11,12]3.4.2Cu2+CPB,Hg2+,CPB3.5CPB10mmol/LEDTA,CPBZn2+CPB1mmol/LEDTA,01h,100%,CPBPMSF,,CPB,[11,12]pH8.0B,NaI,KSCN,NaBr,MgSO4,NaCl,KCl,NaNO3,,NaCl,KClNa2SO4pH7.2pH8.00.9mol/LNaCl(11024mol/L2L2)50%0.00125mol/LTris2HCl4mol/LNaCl(0.025mol/LTris2HCl)4BFolk[6]1960CPB,CPBCPBCPB4.1B[6]BB:16h()DEAE()Sephadex,CPB,[13],,,,,DEAE,CPBCPB4.2BB,B,B,DEAE,B,90%[16]Octyl2FF[14,15]5CPBB,,,CPBLIB(PCPB),pCPB1209bp,Blast,GenebankpCPB5,2,11LysAsn,139ArgGlu,PET224a,BL21(DE3)[14]303:B5.1PCPB,PCPB62HistagPCPB[15,16]8mol/L,6mol/L6mol/L,4mol/LSDSPCPB8mol/L,6mol/LpH12.5(1mol/L2mol/L),pH12.5,2mol/L,8mol/L20%pHPCPB8mol/LHis2tagPCPB,,8mol/LpHPCPB,pHGSSGGSHPCPBPCPB:20mmol/LTris2HClpH9.5,0.1mmol/LZnCl2,PCPB:150g/ml,1mmol/LGSH,0.5mol/L,2532h3PCPB,1h,[15]5.2PCPBPCPB,,PCPB,,BMGY,3mmol/LPMSF,28,12h0.5%,GS1152PCPB500mg/L,94%,CPB110U/mg[17]5.3CPBCPB[8,9,11,12,15][15]CPBCPBpH79,CPBpH8,pH79,35%24%CPBpH,pH78,CPB,pH9,CPB40,CPB,catfishdogfish55,,CPBCPBdogfishCPB,5025min,180%,,,26050min,40%CPBEDTA,Hg2+Mn2+,Zn2+,Mg2+,Ca2+,Zn2+dogfishCPB,Mg2+,Ca2+CPBCo2+,CPBCo2+,Co2+CPB,,CPBdog2fishCPBCo2+,3,2L22L2CPBKm,25,pH8.0,Km0.374.25,CPBKm3.550.39,2.630.54,,L2CPBCPB,20mmol/LTris,pH8.0,0.1mol/LNaCl2201mg/mlCPB6,25%CPB5.35;277nmCPB,,CPBCPB,[18],CPB,CPB[1]AvilesFXandVendrellJ,CarboxypeptidaseB.In:Hand2bookofProteolyticEnzymes,1998,133321335.[2]Gmois2RuthFX,CmopanysV,QianY,etal.CollstructureofaviancarboxypeptidaseDdomain:Aprototypefortheregulatorymetallocarboxypeptidasesubfamily[J].EMBOJ,1999,18:581725826.[3]SanSegundo,MartinezMC,VilanovaM,etal.TheseveredactivationsegmentofporeinepancreaticprocarboxypeptidaseAisapowerfulinhibitoroftheactiveenzyme[J].BiochimBiophysActa,1982,707:74280.[4]CollM,GuaschA,AvilesFX,etal.Three2dimensionalstructureofporcineprocarboxypeptidaseB:astructuralbasisofitsinactivity[J].EMBOJ,1991,10:129.[5]BurgosFJ,SalvaM.VillegasV,etal.Analysisoftheacti2vationprocessofporcineprocarboxypeptidaseBanddetermi2nationofthesequeneceofitsactivationsegment[J].Bio2chemistry,1991,30:408224089.[6]FolkJE,PiezKA,CarrollWA,etal.CarboxypeptidaseB,403134IV.Purificationandcharaterizationoftheporcineenzyme[J].JBiolChem,1960,235(8):227222277.[7]FolkJE,WolffEC,SchirmerEW,etal.Thekineticsofcar2boxypeptidaseBactivity..Effectsofalcoholonthepepti2daseandesteraseactivities;kineticmodels[J].JBiolChem,1962,237(10):310523109.[8]GraemeB,RynoJN,KojiM,etal.Ostrich(Struthiocame2lus)CarboxypeptidaseB:Purification,KineticPropertiesandCharacterizationofthePancreaticEnzyme[J].IntJBio2chem,1996,28(5):5212529.[9]HidekiKishimura,KenjiHayashi.Isolationandcharacteris2ticsofcarboxypeptidaseBfromthepyloriccecaofthestarfishAsteriasamurensis[J].ComparativeBiochemistryandPhysiologyPartB,2002,133:1832189.[10],,,.B[J].,2002,23(4):1852186.[11]YoshinakaR,SatoM,MorishitaJ,etal.PurificationandsomepropertiesoftwocarboxypeptidaseBfromthecatfishpancreas[J].NippoSuisanGakkaishi,1984b,50:172321727.[12]HajjiouM,SmineA,GuerardE,etal.PurificationandsomepropertiesofacarboxypeptidaseBfromdogfishScylio2chinuscamicula.Comp[J].BiochemPhysiol,1995,110B:7912798.[13],,,.B[J].,2002,16217.[14]LiSX,ZhangY,TianLP,etal.Cloning,ExpressionofANewProcarboxypeptidaseBGeneinEscherichiacoliandPur2ificationofRecombinationCarboxypeptidaseB[J].ProteinandPeptideLetters,2003,10(6):1210.[15]ZhangXY,LiSX,YuanQS.Therenaturationofpro2car2boxypeptidaseBbyureagradientgelfiltrationandsomepropertiesofrecombinantcarboxypeptidaseB[J].ProteinandPeptideLetters,2005,12(7),6712676.[16],.B[J].,2005,18(2):1462148.[17],,,.B[J].,2005,29(3):2442247.[18],,,.BC[J].(),2004,30(30):10213.AduancesinCarboxypeptidaseBLISu2xia,ZHANGXiao2yan,ZHANGYing2xin,YANGZi,YUANQin2sheng(StateKeyLaboratoryofBioreactorEngineering,EastChinaUniversityofScienceandTechnology,Shanghai200237,China)AbstractCarboxypeptidaseBisanecessaryenzymeespeciallyintheprocessingofrecombinantinsulinandisoftenwidelyusedinthesequencingofproteins.Inthisarticle,thestructure,methodofactivitydetermination,characteristics,stability,thestoragecondition,theresolutionways,theproductionandpropertiesofrecombinantcarboxypeptidaseBwereallsummarized.KeywordsCarboxypeptidaseB,RecombinantCarboxypeptidaseB,characteristic200610322006104TNF200610520061062