Reaksiyon mühendisliği prensipleriyle rekombinant insan büyüme hormonu üretimi biyoproses geliştirilmesi
Bioprocess development for recombinant human growth hormone production by reaction engineering principles
- Tez No: 180782
- Danışmanlar: PROF. DR. TUNÇER ÖZDAMAR
- Tez Türü: Yüksek Lisans
- Konular: Kimya Mühendisliği, Chemical Engineering
- Anahtar Kelimeler: Belirtilmemiş.
- Yıl: 2006
- Dil: Türkçe
- Üniversite: Ankara Üniversitesi
- Enstitü: Fen Bilimleri Enstitüsü
- Ana Bilim Dalı: Kimya Mühendisliği Ana Bilim Dalı
- Bilim Dalı: Belirtilmemiş.
- Sayfa Sayısı: 341
Özet
ÖZETYüksek Lisans Tezi5HDNVL\RQ0ÂKHQGLVOL÷L3UHQVLSOHUL\OH5HNRPELQDQWøQVDQ%Â\ÂPH+RUPRQXhUHWLPLøoLQ%L\RSURVHV*HOLúWLULOPHVL%LUJÂOù(17h5.Ankara ÜniversitesiFen Bilimleri Enstitüsü.LP\D0ÂKHQGLVOL÷L$QDELOLP'DOÃÖZDAMAR'DQÃúPDQ3URI'U+Tunçer7HUDSDWLNSURWHLQLQVDQEÂ\ÂPHKRUPRQXQXQK*+ÂUHWLPLLoLQJHUoHNOHúWLULOHQYHDOWÃDúDPDGDQROXúDQ DUDúWÃUPD SURJUDPÃQÃQ LON DúDPDVÃQGD GR÷DOD |]GHú o|]ÂQHELOLU YH EL\RORMLN RODUDN DNWLIhücre-GÃúà ÂUHWLOHELOPHVL LoLQ\DSÃGD VDGHFH LQVDQGD ÂUHWLOHELOHQ K*+¶QLQ PLNURorganizmalarcaBacillus hücre-GÃúÃHQ]LPLVHULQDONDOLSURWHD]6$3JHQLQLQsubC) pre- sinyal dizini hGH genGL]LQLQLQ |QÂQH 62( \|QWHPL\OH HQWHJUH HGLOHUHN ROXúWXUXODQ KLEULW JHQ |QFH S8& E.coliSODVPLGLQHNORQODQPÃú sonra da pMK4 E.coli-Bacillus shuttle plasmide sub-NORQODPD\DSÃOPÃúYHrekombinant DNA -pre(subC)::hGH::pMK4 IDUNOà Bacillus WÂUOHULQH DNWDUÃODUDN UHNRPELQDQWBacillus subtilisWÂUOHULHOGHHGLOPLúWLUøNLQFLDúDPDGDUHNRPELQDQWPLNURRUJDQL]PDODULOHPikro-biyolojik hava-filtreli, kesikli, VR=110cm3 oDOÃúPD KDFÃPOà ODERUDWXDU-ölçek biyoreaktörlerde,T=37 oC, N=200 dk-1 ve pHo LúOHWPHNRúXOODUÃQGDÂUHWLPRUWDPÃQDHNOHQHQSURWHD]LQKLELW|UÂ3YH NDUERQ ND\QDֈ JOXNR]XQ GHULúLPOHUL EHOLUOHQPLúWLU (Q yüksek ChGH= 0.03 kg/m hGH3GHULúLPLQH Bacillus subtilis BGSC-1A751 ile CGo= 8.0 kg/m JOXNR] GHULúLPLQGH RUWDPD µlSURWHD] LQKLELW|U HNOHQHUHN XODúÃOPÃúWÃU $UDúWÃUPDQÃQ ÂoÂQF DúDPDVÃQGD ODERUDWXYDU |OoHNWH=550 cm3 hDFÃPOÃPHNDQLNNDUÃúWÃUPDOÃS+VÃFDNOÃNo|]ÂQPÂúEXOXQDQHQX\JXQNRúXOODUGD9RRNVLMHQ YH NDUÃúWÃUPD KÃ]à NRQWUROO SLORW-|OoHN NHVLNOL EL\RUHDNW|UOHUGH IDUNOÃKDYDJLULú 4o/VR=-1YH YYP YH NDUÃúWÃUPD KÃ]ODUÃQGD 1 YH GN RNVLMHQ DNWDUÃP HWNLOHUL \DQürün dD÷ÃOÃPODUà LOH ELUOLNWH LQFHOHQPLú RNVLMHQ DNWDUÃPà YH EL\RSURVHV NDUDNWHULVWLNOHUL= 0.07 kg/m3) Qo/VR= 0.5 vvm, N= 800 dk-1EHOLUOHQPLúWLU (Q \ÂNVHN K*+ GHULúLPLQH &hGHNRúXOXQGD XODúÃOPÃúWÃU'|UGÂQFÂDúDPDGD EHOLUOHQHQHQ L\LRNVLMHQDNWDUÃPNRúXOunda hibrit geniçeren (-pre(subC)::hGH::pMK4) ve içermeyen (pMK4) plasmidli Bacillus BGSC-1A751 ileÂUHWLP GHQH\L JHUoHNOHúWLULOHUHN -pre(subC)::hGH KLEULW JHQLQ HWNLVL LQFHOHQPLúWLU %HúLQFLDúDPDGD EHOLUOHQHQ HQ L\L RNVLMHQ DNWDUÃP NRúXOXQGD ÂUHWLP RUWDPÃQD IDUNOà ]DPDQODUGD We = 0 veVW¶WH& P0GHULúLPGHHNOHQHQVHULQDVSDUWLNDVLWO|]LQYDOLQYHJOXWDPLNDVLWLQÂUHWLPHHWNLVL DUDúWÃUÃOPÃúhUHWLPRUWDPÃQD D\Uà D\Uà HNOHQHQ VHULQ DVSDUWLN DVLW O|]LQYDOLQ YHJOXWDPLN= 0 st'te % 6.1, 34.3, 42.4 ve 48.4 , teDVLWK*+GHULúLPLQLVÃUDVÃ\ODWe VW¶WHHNOHQHQNDUÃúÃPODUÃYHO|]LQYDOLQLQLVHYHD]DOWPÃúWÃU6RQDúDPDGDLVHWHSNLPHYHPHWDEROLWWHQROXúDQ KÂFUH-LoL WHSNLPH VLVWHPL LoLQ PDWHPDWLN PRGHO ROXúWXUXOPXú YH UHDNVL\RQ PÂKHQGLVOL÷LSUHQVLSOHUL\OHWHSNLPHDNÃODUÃKHVDSODQDUDNVRQXoODU\RUXPODQPÃúWÃU2006, 314 sayfaAnahtar kelimeler : subtilis, ortamøQVDQ EÂ\ÂPHKRUPRQX K*+ÂUHWLPLUHNRPELQDQWBacillusanalizi, metabolik mühendislikWDVDUÃPÃRNVLMHQDNWDUÃPÃDPLQRDVLWPHWDEROLN\Rlizi
Özet (Çeviri)
ABSTRACTMaster ThesisBioprocess development for recombinant human growth hormone productionby reaction engineering principles%LUJÂOù(17h5.Ankara UniversityGraduate School of Natural and Applied SciencesDepartment of Chemical EngineeringSupervisor : Prof. Dr. H. Tunçer ÖZDAMARMetabolic engineering in Bacillus subtilis for extracellular human growth hormone (hGH) synthesissystem design is conceptually based on construction of a hybrid gene of two DNA fragments, i.e. signalDNA sequence of a B.subtilis extracellular enzyme gene and the DNA encoding human therapeuticprotein. Complementary oligodeoxyribo-nucleotide (oligo) primers and the polymerase chain reaction(PCR) are used to generate two DNA fragments having overlapping ends. The two DNA fragmentshaving overlapping ends are combined to form the hybrid gene in a subsequent `fusion? reaction inwhich the overlapping ends anneal, allowing the 3' overlap of each strand to serve as a primer for the 3'extension of the complementary strand. Extracellular protease (subC) pre- signal DNA sequence of B.licheniformis chromosomal DNA and plasmid carrying hGH cDNA were used as templates in PCRs,respectively, for the amplification of subC signal DNA sequences and hGH mature peptide sequence.Extension of the overlaps by DNA polymerase has yielded a recombinant hybrid-gene. Hybrid genefragment was cloned into pUC19, and then sub-cloned into pMK4 E.coli- Bacillus shuttle plasmid.Thus, the new expression vector was transferred into host B. subtilis strains. Effects of oxygen-transferon product and by-product distributions in extracellular rhGH production by recombinant B. subtiliscarrying -pre(subC)::hGH::pMK4 were investigated in 1.0dm3 batch-bioreactor-systems at fourdifferent conditions with the parameters, air-inlet rate of QO/VR =0.5 vvm, and agitation rates of N= 500(low:LOT), 700 (medium:MOT1), 800 (medium:MOT2) min-1 and, at QO/VR=0.7 vvm, N=800 min-1(high:HOT), on a glucose-based defined-medium. Cell, rhGH, amino and organic acid concentrations,yield values, KLa, and oxygen-uptake and oxygen-ransfer rates were determined throughout thebioprocess. Considering the cell and rhGH concentration profiles, the bioprocess was divided into fiveperiods. Period I (0
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