产朊假丝酵母尿酸酶的纯化、特性研究及其基因扩增

《产朊假丝酵母尿酸酶的纯化、特性研究及其基因扩增》由会员上传分享，免费在线阅读，更多相关内容在学术论文-天天文库。

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

2、lbeincreasedandhyperuricemiawillOCCULT．Hyperuricemiaisassociatedwiththedevelopmentandprogressofgout，cardiovasculardiseases，tumorlysissyndromeandrenaldisease．Soitisnecessarytodevelopefficientdrugforhyperuricemia．Currently,theuricaseisconsideredtobeanidealdru

3、gtotreathyperuricemia．Herein，theCandidautilis(C．GM．C．C．2．1008)anditsuricasewasinvestigated．1．TheincubationoftheCandidautilis(C．GM．C．C．2．1008)andpreparationandpurificationoftheuricase．Afterbeinginducedbyuricacid，Thecellswereharvestedthroughcentrifugationandb

4、rokenwithultrasound．Thelysateisfractionedwithammoniumsulfateandtheuricasespecificactivityoftheproductreachedtomorethan0．32U／mgprotein．DuringtheDEAE—cellulosechromatography,theuricasecouldn’tbeabsorbedbutitsspecificactivityreachedto7．1U／ragaftertwiceDEAE—cel

5、lulosechromatography．2．Thecharacterizationofuricase．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．Onlysinglepolypeptid

6、eabout33．0kDwasresolvedbySDS—PAGEwiththissample．Matrix··assistedlaserdesorptionionizationtime-·of-flightMSanalysisresolveda67．7kDpolypeptideastheprincipalcomponentanda131．4kDpolypeptideofslightlylowerabundance，butnosinglepolypeptidewasdetectedwiththesamesam

7、ple．TheoptimumpHforthisuricasewascloseto8．8at25+0．5℃．Thisuricasewasstableat37℃for4handmorethan50％activitywasreservedatpH7．4．TheMichaelis—MentenconstantWas(32．8+3．1)IJmol／L(n=10)．Exceptthatadenosine、guanosineandinosinecouldn’tinhibittheuricase，theinhibitionc

8、onstantofOteracilpotassium、xanthine，guanine，hypoxanthine、adenineandallopurinolwasindividually(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