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Reaksiyon mühendisliği prensipleriyle rekombinant insan büyüme hormonu üretimi biyoproses geliştirilmesi

Bioprocess development for recombinant human growth hormone production by reaction engineering principles

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  1. Tez No: 180782
  2. Yazar: BİRGÜL ŞENTÜRK
  3. Danışmanlar: PROF. DR. TUNÇER ÖZDAMAR
  4. Tez Türü: Yüksek Lisans
  5. Konular: Kimya Mühendisliği, Chemical Engineering
  6. Anahtar Kelimeler: Belirtilmemiş.
  7. Yıl: 2006
  8. Dil: Türkçe
  9. Üniversite: Ankara Üniversitesi
  10. Enstitü: Fen Bilimleri Enstitüsü
  11. Ana Bilim Dalı: Kimya Mühendisliği Ana Bilim Dalı
  12. Bilim Dalı: Belirtilmemiş.
  13. Sayfa Sayısı: 341

Özet

ÖZETYüksek Lisans Tezi5HDNVL\RQ0KHQGLVOL÷L3UHQVLSOHUL\OH5HNRPELQDQWøQVDQ%\PH+RUPRQXhUHWLPLøoLQ%L\RSURVHV*HOLúWLULOPHVL%LUJOù(17h5.Ankara ÜniversitesiFen Bilimleri Enstitüsü.LP\D0KHQGLVOL÷L$QDELOLP'DOÕÖZDAMAR'DQÕúPDQ3URI'U+Tunçer7HUDSDWLNSURWHLQLQVDQE\PHKRUPRQXQXQK*+UHWLPLLoLQJHUoHNOHúWLULOHQYHDOWÕDúDPDGDQROXúDQ DUDúWÕUPD SURJUDPÕQÕQ LON DúDPDVÕQGD GR÷DOD |]GHú o|]QHELOLU YH EL\RORMLN RODUDN DNWLIhücre-GÕúÕ UHWLOHELOPHVL LoLQ\DSÕGD VDGHFH LQVDQGD UHWLOHELOHQ K*+¶QLQ PLNURorganizmalarcaBacillus hücre-GÕúÕHQ]LPLVHULQDONDOLSURWHD]6$3JHQLQLQsubC) pre- sinyal dizini hGH genGL]LQLQLQ |QQH 62( \|QWHPL\OH HQWHJUH HGLOHUHN ROXúWXUXODQ KLEULW JHQ |QFH S8& E.coliSODVPLGLQHNORQODQPÕú sonra da pMK4 E.coli-Bacillus shuttle plasmide sub-NORQODPD\DSÕOPÕúYHrekombinant DNA -pre(subC)::hGH::pMK4 IDUNOÕ Bacillus WUOHULQH DNWDUÕODUDN UHNRPELQDQWBacillus subtilisWUOHULHOGHHGLOPLúWLUøNLQFLDúDPDGDUHNRPELQDQWPLNURRUJDQL]PDODULOHPikro-biyolojik hava-filtreli, kesikli, VR=110cm3 oDOÕúPD KDFÕPOÕ ODERUDWXDU-ölçek biyoreaktörlerde,T=37 oC, N=200 dk-1 ve pHo LúOHWPHNRúXOODUÕQGDUHWLPRUWDPÕQDHNOHQHQSURWHD]LQKLELW|U3YH NDUERQ ND\QD÷Õ 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 XODúÕOPÕúWÕU $UDúWÕUPDQÕQ oQF DúDPDVÕQGD ODERUDWXYDU |OoHNWH=550 cm3 hDFÕPOÕPHNDQLNNDUÕúWÕUPDOÕS+VÕFDNOÕNo|]QPúEXOXQDQHQX\JXQNRúXOODUGD9RRNVLMHQ YH NDUÕúWÕUPD KÕ]Õ NRQWUROO SLORW-|OoHN NHVLNOL EL\RUHDNW|UOHUGH IDUNOÕKDYDJLULú 4o/VR=-1YH YYP YH NDUÕúWÕUPD KÕ]ODUÕQGD 1 YH GN RNVLMHQ DNWDUÕP HWNLOHUL \DQürün dD÷ÕOÕPODUÕ LOH ELUOLNWH LQFHOHQPLú RNVLMHQ DNWDUÕPÕ YH EL\RSURVHV NDUDNWHULVWLNOHUL= 0.07 kg/m3) Qo/VR= 0.5 vvm, N= 800 dk-1EHOLUOHQPLúWLU (Q \NVHN K*+ GHULúLPLQH &hGHNRúXOXQGD XODúÕOPÕúWÕU'|UGQFDúDPDGD EHOLUOHQHQHQ L\LRNVLMHQDNWDUÕPNRúXOunda hibrit geniçeren (-pre(subC)::hGH::pMK4) ve içermeyen (pMK4) plasmidli Bacillus BGSC-1A751 ileUHWLP GHQH\L JHUoHNOHúWLULOHUHN -pre(subC)::hGH KLEULW JHQLQ HWNLVL LQFHOHQPLúWLU %HúLQFLDúDPDGD EHOLUOHQHQ HQ L\L RNVLMHQ DNWDUÕP NRúXOXQGD UHWLP RUWDPÕQD IDUNOÕ ]DPDQODUGD We = 0 veVW¶WH& P0GHULúLPGHHNOHQHQVHULQDVSDUWLNDVLWO|]LQYDOLQYHJOXWDPLNDVLWLQUHWLPHHWNLVL DUDúWÕUÕOPÕúhUHWLPRUWDPÕQD D\UÕ D\UÕ HNOHQHQ VHULQ DVSDUWLN DVLW O|]LQYDOLQ YHJOXWDPLN= 0 st'te % 6.1, 34.3, 42.4 ve 48.4 , teDVLWK*+GHULúLPLQLVÕUDVÕ\ODWe VW¶WHHNOHQHQNDUÕúÕPODUÕYHO|]LQYDOLQLQLVHYHD]DOWPÕúWÕU6RQDúDPDGDLVHWHSNLPHYHPHWDEROLWWHQROXúDQ KFUH-LoL WHSNLPH VLVWHPL LoLQ PDWHPDWLN PRGHO ROXúWXUXOPXú YH UHDNVL\RQ PKHQGLVOL÷LSUHQVLSOHUL\OHWHSNLPHDNÕODUÕKHVDSODQDUDNVRQXoODU\RUXPODQPÕúWÕU2006, 314 sayfaAnahtar kelimeler : subtilis, ortamøQVDQ E\PHKRUPRQX K*+UHWLPLUHNRPELQDQWBacillusanalizi, metabolik mühendislikWDVDUÕPÕRNVLMHQDNWDUÕPÕDPLQRDVLWPHWDEROLN\Rlizi

Özet (Çeviri)

ABSTRACTMaster ThesisBioprocess development for recombinant human growth hormone productionby reaction engineering principles%LUJOù(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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