48280.土曲霉和枯草芽孢桿菌植酸酶基因的克隆及異源表達

1、中茄聾夫?qū)WHUAZIIIINGAGRICULItlRALUNI、『ERSIIY碩士學(xué)位論文MASTER’SDEGREEDISSERTATION土曲霉和桔草芽孢桿菌檀髓酶基因的克隆殛異穗衰達CLONlNG^NDHETEROLOGOUSEXPRESSIONOFPNYT扯gEGENEFROM4SPERr；tLLcSTERRHs∞H州lLLUSSLB7itJs研兜q；：伍浩走CANDIDATEWULUoFU肄斷：用健初黃提SU，。腿vIsoR：

2、PROF：zHouJUNCHU專業(yè)：蕾生特摯MAaOItIMlcRoBloLoGY研窀力舟：t生精矗目i茬FIELD：MICRoBIALGENEENGINE目UNG中國武漢二OO五≈五。土曲霉和枯草芽孢桿菌植酸酶基因的克隆及異源表達AbstractPhytateandphyticacidarethemajorphosphorusstorageformeontalnedinfoodorfeedsofplanloriginPhytasesa

3、lemyoinositolhexakisphosphatephosphohydzolasethatcatalyzethestepwiseremovalofinorganicorthophosphatefromphytateAdditionofphytaseintofeedcanresultinthefollowingadvantages：increasingtheabsorptiveefficiencyofphosphateinanim

4、albodydecreasingthephosphatepollutioninenvironment，eliminatingthechelatereactionbetweenphytateandmetalionsothatphytasecanimprovetheutilizatione餓ciencyofthefeedHencethestudyonphytase，especiallyphytaseproducedbymicroorgani

5、sm，hasbeenpaidmuchattentionbyscientistsinChinaandabroad，PhytaseproductiononalargescalearerestrictedbylowlevelofphytasegeneexpressioninwildstrainsTheobjectiveofthisstudyistoclonephytasegenefromwildstrainsandthenobtainitse

6、xpressioninheterogeneousyeastandescherichiastrainsThemainresultsofthisstudyareasthefollowing：1About14kbphyAfragmentabsentsignalsequenceandintronofAspergillusterreuswasamplifiedbyPCRwithprimersdesignedaccordingtopublished

7、phyAsequencesItwasfurtherclonedandsequencedTherecombinantexpressionplasmidpPIC9KphyAwasconstructedandtransformedintoPichiapastorisGSll5ThephytasegenewasexpressedinPichiapastorisGSll5detectedbyPeRandhydrolyticzoneonasolid

8、phytate—containingmedium乃eenzymeactivityinsupematantfromflaskfermentationafter132hoursinductionreachedto167U／m1ItsenzymeactivitywasactiveunderpH20—80anduptothethehighestunderpH55T1leoptimumtemperatureforenzymeactivitywas

9、55℃2ApairofprimersweredesignedbasedonthephyCsequenceofBacillusstubilisTheph)，CstructuregeneencodingphytasebutwithoutsignalpeptidesequenceswasobtainedbypolymerasechainreactionThenonfusionfragmentamplifiedwassubclonedintoa

10、expressionvectorpET17bwithT7promoterandthentransformedintoEcoliBL21(DE3)neehyCgeneexpressionwasdetectedbyPeRandthehydrolyticzoneobservedonasolidphytate—containingmediumKeyWords：Aspergillusterreus，BacillusstubilisPhytaseg