29762.高淀粉酶蛋白酶活力枯草芽孢桿菌的選育及酶基因陽性克隆的篩選

1、新疆農(nóng)業(yè)大學碩士學位論文高淀粉酶蛋白酶活力枯草芽孢桿菌的選育及酶基因陽性克隆的篩選姓名：李力申請學位級別：碩士專業(yè)：生物化學與分子生物學指導教師：羅淑萍20090501BreedingofBacillusSubtiliswithHigh—activityofAmylase，ProteaseandScreeningofPositiveClonesofEnzymeGenesAbstractToscreenBacillussubtilisst

2、rainswithhighactivityofamylaseandprotease，forthepurposeofproducinghigh—efficientmicrobialecologicalagenttopurifywaterTwostrainswithhighactivityofamylaseandproteasewereselectedfromtenstrainsofBacillussubtiliscollectedfrom

3、differentsourcesbydetectingtheactivitiesofamylaseandproteaseinfermentedliquorTheywerenamedasH001andH008andcharacterizedaccordingtomorphologycolonymorphologyandphysiologyThetwostrainsH001andH008wereprimarilyidentifiedtobe

4、Bacillussp，andtheyweresuitableforproducinghigh—efficientMicrobialecologicalagentstopurifywaterPlackettBurmandesignandBoxBehnkendesignwasemployedtooptimizeaSolidmultiplicatedmediumforBacillussubtilisstrainH001Fourimportan

5、tcomponentswerescreenedformeightrelatedfactorsTheprimefactorswereoptimizedbyresponsesurfacemethodologySOastoworkouttheiroptimumlevelResultedin15一foldincreasedlevelofbacteriagrowthmassamaximumcomparedtoinitiallevelafter24

6、hoffermentationThewaterpurificationfunctionsofthestrainBacillussubtilisH001byusing2kindsofsimulationpollutionwatersamplesWasdiscussedTheresultsindicatedthatthestraincoulddegradetheorganicmatterraidly；theCODvalueandammoni

7、aconcentrationofthewatersamplehaddroppedmoreefficientcomparewithotherfourstandardBacillussubtilisstrains，totalresidualandfilterabilityresidualdegenerationrateat144hwas633％and204％seperatelyAgenomelibraryofthestrainBacillu

8、ssubtilisH001wasestablishedby“shotgun”methodinviewofitshighamylaseandproteaseactivitythehighmolecularweightgenomeDNAwaspartiallydigestedbySau3AIrestrictionenzymePiecesof15N7KbDNAwereextracted，thefragmentswererccombinated

9、andligasedwithvectorpUC18whichweretotallydigestedwithBamHIenzyme，andhighefficiencycompetentDH5aEcolihostcellssystemwereemployedtoincreasethetransformationefficiencynearly17000transforrnantsweregotResultsofrapidScreeningo

10、ftransformantsplasmidindicatedthattherewereforeignDNAfragmentsinabout98％transformantsSevenpositivecloneswerescreenedfromlibrarybyusingallimprovedpositivetransformantscreeningmethodPlasmidsextractedformpositivetransforman

11、tswereretransformed，ThreepositivetransformantsaimgeneinsidesurelyweregotfinallytwoofthemexpressedamylaseactivitytherestoneexpressedproteaseactivityKeywords：Bacillussubtilis；amylase；protease；responsesurfacemethodology；sho