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2016353289CD-0081212*12121212121.1166222.116622Plackett-Burman、Box-BehnkenRhodotorulasp.CD-008、、SephadexG-100。pH、、pH5.34、150r/min、21.40℃6430.52U/g1.53。65%、、SephadexG-100SOD419.90U/mg9.608.2%。SDS-PAGESOD37.5ku。CD-008Q9360254-5071201603-0017-06doi:10.11882/j.issn.0254-5071.2016.03.005OptimizationoffermentationconditionsofSOD-producingRhodotorulasp.CD-008andseparationandpurificationoftheSODJINLiandou1,2,LIXiaoyan1,2*,WANGXiaohui1,2,CHINaiyu1,2,ZHANGQingfang1,2,ZHANGXujiao1,2,WANGQiang1,2(1.SchoolofLifeScienceandBiotechnology,DalianUniversity,Dalian116622,China;2.LiaoningMarineMicrobialEngineeringandTechnologyCenter,Dalian116622,China)AbstractOnthebasisofsinglefactorexperiments,theoptimumfermentationconditionsofSOD-producingRhodotorulasp.CD-008weredeter-minedbyPlackett-Burman,Box-Behnkendesignandresponsesurfaceanalysis,andtheseparationandpurificationconditionsofSODwereresearchbysaltingout,ultrafiltrationcentrifugationandSephadexG-100gelfiltrationchromatography.ResultsindicatedthatpH,rotatespeedandtempera-turehadasignificanteffectonenzymeactivity.TheoptimumfermentationconditionswaspH5.34,rotatespeed150r/minandtemperature21.40℃.Undertheconditions,theenzymeactivityinfermentationliquorwas6430.52U/gfreshcells,whichwas1.53timeshigherthanthatbeforeoptimiza-tion.Thecrudeenzymeliquidwaspurifiedbyammoniumsulfatewithdegreeofsaturation65%,ultrafiltrationcentrifugationandSephadexG-100gelfiltrationchromatography,specificenzymeactivityofSODobtainedwas419.90U/mg,purificationfoldwas9.60andproteinrecoveryratewas8.2%.SDS-PAGEelectrophoresisshowedthatmolecularweightofSODpurifiedwasabout37.5ku.KeywordsRhodotorulasp.CD-008;superoxidedismutase;fermentationconditions;separationandpurification2015-12-278632014AA0935121991-。*1972-。superoxidedismutaseSOD“”[1]。SODmRNA[2]、SODO2－·NO[3][4]SOD。SOD、、[5]。1997SODSODSODSODSOD。、、、SODSODCu/Zn-SODMn-SOD、Fe-SODStreptomycesgriseusStreptomycescoelicolorNi-SOD[6]、17··ChinaBrewing2016Vol.35No．3SerialNo．289[7-8]Co-SODSODEC-SOD。[9]SOD2075U/gZDF48[10]SOD9100U/gSn-898[11]SOD4206U/gSD06S[12]SODMarinomonassp.NJ062、NJ379、NJ522NJ548。SOD。。Plackett-BurmanP-BBox-BehnkenB-BresponsesurfacemethodologyRSMDesign-ExpertRhodotorulasp.CD-008、、SephadexG-100SOD。11.11.1.1Rhodotorulasp.CD-008123°371′E39°6972′N。1.1.22.0%0.5%1.0%2.0%pH。2.0%0.5%CuSO40.08mmol/LpH。1.1.3G-100sodiumdodecylsulfateSDS。。1.2HZP-2505417REppendorfSCIENTZ-DUV-2102CDYY-6C。1.31.3.122℃1～2d2。1.3.222℃、150r/min30h。4℃、4000r/min30min24℃、4000r/min30min。pH7.8。480W30min5s5s。4℃、10000r/min10min。1.3.3SODSOD[13-14]。1mL25℃50%SODU/g。1.3.4100r/min、120r/min、140r/min、160r/min、180r/min、70mL/250mL、80mL/250mL、90mL/250mL、100mL/250mL、110mL/250mL、1%、2%、3%、4%、5%、18℃、20℃、22℃、24℃、26℃、pH4.5、5.5、6.5、7.5、8.5100%。SOD。1.3.51Plackett-Burman512N=12Design-Expert。2Plackett-Burman1。2Plackett-Burman。3Box-BehnkenPlackett-Burman3Design-ExpertBox-Behnken332。A/℃B/%C/r·min-1D/mL·250mL-1EpH-11202424130150801004.56.51Plackett-BurmanTable1FactorsandlevelsofPlackett-BurmanexperimentsResearchReport18··20163532891.3.6SODCD-008SOD65%4℃12h0.05mol/LpH7.810ku4℃4000r/min30minpH7.80.05mol/L2mLSODpH7.80.05mol/LSephadexG-1001.6cm×100cm2mLSOD。1.3.71[15]y=0.0092x+0.0204R2=0.9946。2SODLAEMMLIUK[16]8%5%sodiumdodecylsulfatepolyacrylamidegelelectrophore-sisSDS-PAGE30minMarkerThermo26616SOD。22.1、、、pHSOD1。2Box-BehnkenTable2FactorsandlevelsofBox-Behnkenexperiments-101X1pHX2/r·min-1X3/℃5.0130205.5140226.0150241(a)、(b)、(c)、(d)、pH(e)SODFig.1Effectsofrotatespeed(a),liquidvolume(b),inoculum(c),temperature(d),pH(e)onSODrelativeenzymeactivity1SOD、、、pH。SOD140r/min、90mL/250mL、3%、22℃、pH5.5。2.22.2.1Plackett-BurmanSOD5Rhodotorulasp.CD-008SODYN=12P-B3。3A、B、C、D、E-414.3、263.8、-448.4、-370.1、521.0PSODE＞C=A＞D＞B19··ChinaBrewing2016Vol.35No．3SerialNo．289pH＞=＞＞pH、。。2.2.2Plackett-Burman3pH、4。43pH5.5、140r/min22℃。2.2.3Box-BehnkenRhodotorulasp.CD-008Box-BehnkenBox-Behnken56。Design-Expert8.6.0.15SODYY=6216.06-258.10X1+132.90X2+41.43X3-337.36X12-65.73X22-579.62X32+152.54X1X2+2.97X1X3-404.93X2X36X1X3X1X3X22SODP＜0.05SODP＜0.001pHpH。R2=0.9974R2Adj=0.99280.72%。P＜0.0001＜0.0010.0973＞0.05Rhodotorulasp.CD-008。pH、Rhodotorulasp.CD-008SOD2。23[17-18]tPABCDE-414.3263.8-448.4-370.1521.0-6.143.91-6.64-5.487.720.0090.0300.0070.0120.005352413Plackett-BurmanTable3SignificanceanalysisofPlackett-BurmanexperimentspH/r·min-1/℃/U·g-112344.55.56.57.5130140150160232221206002.566189.305584.274972.864Table4Designandresultsofthesteepestascentpathexperiments5Box-BehnkenTable5DesignandresultsoftheBox-BehnkenexperimentsX1X2X3/U·g-11234567891011121314151-110000-100011-1-11000100-1-1-11-101001101000-11-10-10-15779.205627.195158.946219.205330.966230.196198.786054.385001.315689.626062.365291.124965.726029.725545.876Tabble6VarianceanalysisofregressionequationP＜0.05P＜0.001。FPX1X2X3X1X2X1X3X2X3X12X22X32111111111953214R2=0.99745.33E+051.41E+0513732.3993073.8135.46.56E+054.20E+0515953.381.24E+062.99E+067704.347196.22508.113.00E+065.33E+0591.7113732.3993073.8135.46.56E+054.20E+0515953.381.24E+063.33E+051540.872398.74254.06R2Adj=0.9928345.8591.718.9160.40.023425.65272.7310.35804.05＜0.00019.44＜