大肠杆菌植酸酶的定点突变与其热稳定性

《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