Dissertation > Excellent graduate degree dissertation topics show

Isolation of Coenzyme B12-Producing Microorganisms and Cloning、 Expression and Application of Coenzyme B12 Biosynthetic Genes

Author: DiWeiFeng
Tutor: FangHuiYing
School: Jiangnan University
Course: Fermentation Engineering
Keywords: Glycerin Coenzyme B12 1,3 - propanediol Glycerol dehydratase Vitamin B12
CLC: TQ925
Type: Master's thesis
Year: 2011
Downloads: 23
Quote: 0
Read: Download Dissertation

Abstract


Through the reduction in glycerol, glycerol dehydratase (GDHt) a catalyst for the rate-limiting step, the catalytic process requires coenzyme B12 (adenosyl cobalamin, AdoCbl) as a coenzyme and substrate glycerol can lead AdoCbl Co-C bond is irreversible rupture, resulting in GDHt suicide inactivation. Expensive cyanocobalamin (CNCbl) added is limited fermentation of 1,3 - propanediol economic factors. To overcome the 1,3 - propanediol fermentation process GDHt exogenous added CNCbl dependence, this study focuses on the synthesis and constructed AdoCbl transformation pathways, and achieved certain results. In this study, 12 samples in the 67 single colonies were isolated, with ethanolamine as sole nitrogen source in the culture medium filter, screened strain A24. According A24 colony morphology, microscopic morphology, physiological and biochemical characteristics, combined with the 16S rRNA sequence alignment analysis identified strain A24 as Bacillus. Megaterium. To filter the resulting B. megaterium genomic DNA, obtained by PCR amplification of the coding synthesis pathway AdoCbl uroporphyrinogen key Ⅲ methyltransferase gene cobA, while the amplified from Klebsiella pneumoniae encoding glutamyl-tRNA reductase gene hemA, and successfully constructed a series hemA and cobA expression vector pUC18-tac-cobA-tac-hemA. The expression vector pUC18-tac-cobA-tac-hemA was transformed into Escherichia coli JM109 obtained recombinant E. coli JM109 (pUC18-tac-cobA-tac-hemA), after induction by IPTG fermentation broth contains a lot of porphyrins material, indicating that the correct key genes cobA with hemA expression. The expression vector pUC18-tac-cobA-tac-hemA gene containing GDHt dhaB and 1,3 - propanediol oxidoreductase gene yqhD recombinant plasmid pEtac-dhaB-tac-yqhD were transformed into E. coli JM109 obtained recombinant E . coli JM109 (pUC18-tac-cobA-tac-hemA/pEtac-dhaB-tac-yqhD), results showed that the fermentation strain E. coli JM109 (pEtac-dhaB-tac-yqhD) CNCbl the optimum dosage of 0.008 g / L, the recombinant strain E. coli JM109 (pUC18-tac-cobA-tac-hemA/pEtac-dhaB-tac-yqhD) CNCbl the optimum dosage of 0.004 g / L, and 1,3 - propanediol production unchanged , indicating key genes cobA with reduced expression of hemA exogenous recombinant add CNCbl dependence. To further study the CNCbl with the activity of AdoCbl into the process, from K. pneumoniae has been cloned cobalamin adenosyltransferase (ACA) gene btuR and reductase gene yciK, and constructed with double-promoter expression plasmid pUC18 -tac-btuR-tac-yciK, the pUC18-tac-btuR-tac-yciK transformed E. coli JM109 recombinant bacteria E. coli JM109 (pUC18-tac-btuR-tac-yciK). RT-PCR experiments and whole cell proteins by SDS-PAGE analysis showed that btuR and yciK in recombinant bacteria have got the right expression. Cobalamin adenosyl transferase activity analysis showed that the recombinant strain E. coli JM109 (pUC18-tac-btuR-tac-yciK) crude protein can be hydroxyl cobalamin (HOCbl) into AdoCbl, recombinant proteins having biological explanation activity. When the recombinant strain E. coli JM109 (pUC18-tac-btuR-tac-yciK) and the control strain E. coli JM109 (pUC18) while ethanolamine as the sole nitrogen source in culture medium for 24 h, OD600 of recombinant is 1.4 times the control bacteria, that the recombinant bacteria btuR and correct expression of yciK promoted CNCbl converted to the biologically active AdoCbl. This study is 1,3 - propanediol fermentation process GDHt resurrection studies provide new ideas.

Related Dissertations

  1. Breeding of 1, 3-propanediol-overproducing Strain by Genome Shuffling,TQ923
  2. Preparation of 1,2-propanediol from Crude Glycerol of Biodiesel Byproduct,TQ223.162
  3. High-effective Catalytic Characteristics of Glycerol Dehydratase from K.pneumoniae XJPD-Li,Q814
  4. Studies on Prefermented Frozen Dietary Fiber Dough and Its Application in Steamed Bread Making,TS213.2
  5. Research on Increasing Quality and Yield of 1,2-propylene Glycol Via Transesterification,TQ223.162
  6. Studies on Pretreatment of Wheat Straw by Glycerol,TQ645.5
  7. Add viscose fiber preparation and properties of flame retardants,TQ314.248
  8. Study on Synthesis and Application of the Internal Electron Donor 1,3-Dioldiether, Dibenzoylesters in Z-N Catalyst,TQ203.2
  9. By glycerol 1,3 - propanediol,TQ223.162
  10. Fermentation production of 1,3 - propanediol applied basic research,TQ223.162
  11. Lactobacillus diolivorans diol dehydratase and its activator function identification,Q93
  12. In Klebsiella oxytoca build independent Coenzyme B_ (12) 1,3 - propanediol pathway,Q78
  13. Catalytic Conversion of Biomass Glycerol to Acetol and Its Value-added Utilization,TQ224.2
  14. Identification of Diol Dehydratase and Its Reactivating Factor of Lactobacillus Collinoides,Q78
  15. Study of the Improved EDI Technology for 1,3-propanediol Broth Desalination,TQ923
  16. Synthesis of Polyester Elastomer and Modification of Nature Rubber,TQ334
  17. Auxiliary aids and alkaline-earth iron-based catalyst for Ag/SiO_2-ZnO vapor phase catalytic synthesis of 3 - methylindole impact,O626
  18. 1, 3-propanediol Production from Glycerol by Klebsiella Pneumoniae,TQ223.162
  19. Study on Dry Blueberry Wine and Anthocyanin Extraction from Pomace,TQ914.1
  20. The Study of Glycerol Fermentation Produced 1,3 - Propanediol,TQ223.162

CLC: > Industrial Technology > Chemical Industry > Other chemical industries > Fermentation industry > Enzyme preparation ( enzyme )
© 2012 www.DissertationTopic.Net  Mobile