产朊假丝酵母尿酸酶的纯化、特性研究及其基因扩增 (1)

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1、重庆医科大学硕士研究生学位论文PREPARATIoN、CHARACTERIZATIONANDGENICCLONINGoFTHEURlCASEFROMCAⅣD删Um坶ABSTRACTUricacidastheend—productofpurinemetabolisminhumanbodyispassedoutthroughkidney．Iftheproductionofuricacidsurpassesthatexcretedbykidney,theconcentrationofserumuricacidwillbeincreas

2、edandhyperuricemiawillOCCULT．Hyperuricemiaisassociatedwiththedevelopmentandprogressofgout，cardiovasculardiseases，tumorlysissyndromeandrenaldisease．Soitisnecessarytodevelopefficientdrugforhyperuricemia．Currently,theuricaseisconsideredtobeanidealdrugtotreathyperuricemi

3、a．Herein，theCandidautilis(C．GM．C．C．2．1008)anditsuricasewasinvestigated．1．TheincubationoftheCandidautilis(C．GM．C．C．2．1008)andpreparationandpurificationoftheuricase．Afterbeinginducedbyuricacid，Thecellswereharvestedthroughcentrifugationandbrokenwithultrasound．Thelysatei

4、sfractionedwithammoniumsulfateandtheuricasespecificactivityoftheproductreachedtomorethan0．32U／mgprotein．DuringtheDEAE—cellulosechromatography,theuricasecouldn’tbeabsorbedbutitsspecificactivityreachedto7．1U／ragaftertwiceDEAE—cellulosechromatography．2．Thecharacterizati

5、onofuricase．4重庆医科大学硕士研究生学位论文Thenativeuricasewas134．0kDbySephadexG一200gelfiltration．AftertwicechromatographyonDEAE-cellulose52，thespecificactivityofthepartlypurifieduricasewasmorethan7．0U／mgprotein．Onlysinglepolypeptideabout33．0kDwasresolvedbySDS—PAGEwiththissample．Ma

6、trix··assistedlaserdesorptionionizationtime-·of-flightMSanalysisresolveda67．7kDpolypeptideastheprincipalcomponentanda131．4kDpolypeptideofslightlylowerabundance，butnosinglepolypeptidewasdetectedwiththesamesample．TheoptimumpHforthisuricasewascloseto8．8at25+0．5℃．Thisuri

7、casewasstableat37℃for4handmorethan50％activitywasreservedatpH7．4．TheMichaelis—MentenconstantWas(32．8+3．1)IJmol／L(n=10)．Exceptthatadenosine、guanosineandinosinecouldn’tinhibittheuricase，theinhibitionconstantofOteracilpotassium、xanthine，guanine，hypoxanthine、adenineandall

8、opurinolwasindividually(1．1+0．1)“mol／L(以=3)、(4．84-0．2)gmol／L(刀=3)、(37．94-4．2)gmol／L(疗23)、(500．0-3：60．O)lmaol／L∽=3)、(748．94-82．O)lxr