Pseudomonas putida S1海藻糖合成酶基因在大腸桿菌中的表達(dá)及其條件研究.pdf

1、江南大學(xué)碩士學(xué)位論文PseudomonasputidaS1海藻糖合成酶基因在大腸桿菌中的表達(dá)及其條件研究姓名：姚林申請學(xué)位級別：碩士專業(yè)：生物化工指導(dǎo)教師：毛忠貴段作營20080601AbstractAbstractTrehaloseisakindofnon—redoingdisaccharideandiswidespreadinnatureBecausetrehalosehasspecialphysical，chemicalprope

2、rtiesandbiologicalfunction，itcouldbewidelyusedinmolecularbiology，food，cosmetic，pharmaceuticalindustryandagricultureetcTrehalosesynthaseCallconvertmaltoseintotrehalosebyintramoleculartransglucosylationItisoneofthemostimpo

3、rtantwaysaboutthesynthesisoftrehaloseT論SgeneencodingtrehalosesynthaseweresuccessfullyamplifiedfromPseudomonasputidaS1byPeR111eamplifiedfragmentwerelinkedtopMD19TvectorbyTAcloningmethod，andthentransformedthecompetentcells

4、EcoliJM109Thetmnsformantswerescreenedonplatewhichhaveamplicillin／Xgal—IPTGandgettherecombinantplasmidpMDTS2Afteridentifyingtherecombinantplasmidbyrestrictionenzymedigestanalysisandsequencing，usingrestrictionendonucleases

5、BamHIand月砌dIIIdigestbotllpMDTS2andPQE30TObjectgenefragmentwererecollectedandlinkedtoeachotherbyT4DNAligaseIdentifiedbyenzymedigest，therecombinantplasmidpQE—TS2wereconstructed111ecompetentcellsEcoliM15weretransformedwithp

6、lasmidpQETS2andinducedbyIPTGDetectedbySDSPAGE，thetrehalosesynthasegenewasexpressedinEcolianditsmolecularweightwereabout77kDabutthemwereexistedintheformofinclusionbodyinthehosteellsTheconditionsforEcoliM15(pQETS2)cellgrow

7、thandexpressioninshakeflaskwereoptimizedTheoptimizedconditionisthatwhenthecellsgrewintoOD600≈06，IPTGwasaddedtothefinalconcentrationof001mmol／Landlastfor20hoursat20℃Theexpressionoftrehalosesynthasecouldbeashighas186％ofthe

8、totalsolubleproteinsTheactivityoftrehalosesynthaseinthefermentationbrothwasdetectedas19U／mLwhichwasabout50foldhigherthanthatoforiginalstrainnleoptimizedmediumsuitableforhighdensityfermentationiscomposedofglucose10g／L，(NH

9、4)28043g／L，peptone15∥L，citricacid159／L，KH2P041259／L，MgS04‘7H20lg／L，traceelementssolution(TES)5mL／LTheeffectofdissolvedoxygen(DO)concentrationandthefeedingofcarbonandnitrogensourceforthegrowthofEcoliM15(pQETS2)werestudied

10、ata5LfermentorWhentheDOwascontrolledabove30％，carbonandnitrogensourcewasfeededatlogphase，thedrycellweightreached172egLatlastTheexpressionleveloftrehalosesynthasereached102％ofthetotalsolubleproteinwhichwas69timesthanthat、Ⅳ

11、itllflaskfermentationinunitvolumeRecombinanttrehalosesynthasecouldconvertsubstratemaltoseintotrehaloseinonestepTheinfluencesofbufferpH，temperature，reactiontime，substrateconcentration，theamountofenzymeontheconversionofthe

12、maltosewerestudiedItsoptimumpHandoptimumtemperaturewerepH85and20“C，respectivelyTheenzymewasstableunder30℃andbetweenpH75topH90ItshouldbestoredatlowtemperaturesThereactingconditionsoftrehalosesynthasewereresearchedthrought

13、heorthogonaltest。neresultsshowedthat：theoptimumconditionsforconversionofmaltosebytrehalosesynthasewere：pH85，temperature10℃，theamountofenzyme6U／gmaltose，thelastingtimeofreaction36hoursUndertheseconditionsthedegreeofconver