康宁木霉纤维素酶发酵条件和CBH2基因表达研究

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1、河南工业大学硕士学位论文康宁木霉纤维素酶发酵条件和CBH2基因表达研究姓名：张一村申请学位级别：硕士专业：食品科学指导教师：吴兴泉20070501AbstractThecatabolismwayofcellulaseisthemajorwayofkindsofcelluloseinthenature．Trichodermaisoneofthemostefficientproducersofextracellularcellulases．ThedegradationandutilizationofabundancefibermainlyrelyonTri

2、chodermaepiphyte．SoitisnecessarytostudythecellulaseproductiveconditionofTrichoderma(TrichodermakoningiistrainsAs3．2774wit}Ihighenzymaticactivitylandenzymaticcharacteristicofcellulosesystemly．ThecelluloseproductiveconditionofTrichodermaandenzymaticcharacteristicofceUulasewerestud

3、iedinthispaper．TheregulationmechanismofgenetranslationandproteinexpressionofTrichodermakoningiistrainsCBH11wereinvestigatedbybiologyinformationmethodinthispaper．neeDNAofcellulosealzymeCBHIIgenewastransformatedinE．coliBL21(DE3)，andexpressed，andcellobiohydrolaseWasobtainedafterpur

4、ification．TheoptimumfermentationconditionsofTrichodermakoningiistrainsAs3．2774werestudiedinthispaper．includingSolidandliquidfermentation．n圮enzymaticcharacteristicofcellulasewerestudiedinthispaper．CellulaseproducedbyTrichodermakoningiiisinductiveenzyme．Theproductiverateofcellulas

5、ewouldincreasewhenthereiscelluloseinthesystem．Theproductionofcellulasewouldbesuppressedwhenthereisoligosaccharidesuchasglucose．TheinformationpeptideofcellobiohydrolaseWaspredictedbySignalPsoftwarewhichwouldlaythefoundationforstudyofregulationmechanismofgenetranslationandproteine

6、xpressionofTrichodermakoningiistrainsCBHII．111eeDNAsequenceofTrichodermakoningii曲[ai璐CBHIIWasintegratedintoPET一28“+)vector．ThentherecombinantproteinwasexpressedinE．coliBL21(DE3)．TherecombinantcellobiohydrolaseWaspurifiedwithNi-NTAAgarose．KeyWords：TrichodermakoningiiCellulaseCBHI

7、IgeneexpressionpurifysequenceanalysisII符号说明ABBREVIATIONPBSPhos：phate—bufferedSaline磷酸盐缓冲液PCRPolymeraseChanReaction多聚酶链式反应TEMED4*N—tetramethylthlyenediamine四甲基已二胺TrisThihydroxymethlaminothane三羟甲基氨基甲烷IPTGIsopropyl一B—D-Thiogalactoside异丙基一B—D-硫代半乳糖苷SDSSodDodecylSulfonate十二烷基硫酸钠PAGEP

8、01yacrylamideGelElectrophoresis聚丙烯酰胺凝胶电泳EDTAEth