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时间:2019-01-31
《大肠杆菌植酸酶的定点突变与其热稳定性》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、万方数据2Method⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯.1O2.1Thedesignofoligonucleotideprimers⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯102.2Site—directedmutagenesisofphytaSeAPPA⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯l12.2.1111em讹ltionsPCRofpurposegene⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯..112.2.2Supe印ositionmutations⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯.122.2.3Theappraisalofr
2、ecombinantplasmid...⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯122.3Theconstnlctionofshuttleplasmid⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯142.4T}ansfomationofplasmids,expressionaIldscreellingoftherestnlcⅢngplasmidinP配厅肠p甜幻,.面s⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯142.4.1111e仃{msfo肌ationofre栅turingplaSmid⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯~162.4.2Expressiona11dscre喇ngof此sixmut觚ts⋯
3、⋯⋯⋯⋯⋯⋯⋯.163Results⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯..203.1Theap弘aisalofrecombinantplaSmid⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯.203.2Sequencingofthemutationsequences⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯203.3SDS.PAGE⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯2l4Discussion⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯..22Chapter3Theexploratio
4、nofpuri6cationmethodsofAPl?Aandthemutants⋯⋯⋯.231Materials⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯231。1Proteinpuri6cationreagents⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯231.2ProteinquantitatiVereagents⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯231.3ThereagentsofphytaseactiVit),deteminationbymolybdatebluemethod⋯231.4Instmmentsandequip
5、ments⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯.23Theexperimentalmethod⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯一242.1ThepurificationofproteinAPPAandthemutants⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯242.1.1BSAst锄dardcuⅣe⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯.242.1.2Ammoniumsulfateprecipitation⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯242.1.3Ionexchangechromato伊印hy⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯252.1.4Glucan
6、gelchromato伊印hy⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯252.2ThequaJltitatiVeanalysisofpurifiedprotein⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯252.2.1Theprep删ionofcllJdeenzyme⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯..252.2.2Thedetenninationofproteinconcentration⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯.26万方数据2.2.3SDS.PAGEofpurifiedprotein⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯262.3PhytaSeactivit),dete珊in
7、ationofpurifiedprotein⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯262.3.1Molvbdenllmbluemethodfort圭ledeteminationofphytaLses’met王lod..262.3.2ThestandardcuⅣesofmolybden啪bluemetllod⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯..272.3.3Dete锄inationofphytaseactiV时byt11ememodofmolybdateblue⋯272.4Ratioofproteinp嘶fi
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