Dissertation > Excellent graduate degree dissertation topics show

Codon Optimization and Synthesis of Nattokinase Gene and Its High-Level Expression in Pichia Pastoris

Author: LiuShuo
Tutor: DongMingSheng
School: Nanjing Agricultural College
Course: Of Food Science
Keywords: Nattokinase Pichia pastoris Codon Optimization Highly expressed
CLC: Q78
Type: PhD thesis
Year: 2007
Downloads: 631
Quote: 3
Read: Download Dissertation

Abstract


Nattokinase (Nattokinase, NK) is a serine protease produced by Bacillus subtilis natto (Bacillus subtillis var.natto) a strong thrombolytic function. The abroad studies show that enzyme can significantly dissolving vivo thrombus significantly shortened the time of the dissolution of fibrin, and with activation vein endothelial cells of tissue-type plasminogen activator (t-PA) degradation and inactivation of plasmin activator inhibitor (pAI-1) and other functions. Thrombolytic efficacy of nattokinase has been widely conclusive evidence, but have not been able to get a good promotion of the use, the application is very limited, and the reason is the lack of market supply and high prices. Expression system as a bioreactor, low-cost, large-scale land mass production nattokinase for more people, is undoubtedly one of the effective methods to solve this problem. In this study, the application of genetic engineering and protein engineering technology with independent intellectual property rights of nattokinase yield engineered bacteria, to provide a theoretical basis and conditions for nattokinase as further research and development and application of thrombolytic drugs, the main tasks include the following: 1. protoplast mutagenesis separated from our group of independent screening process producing the enzyme increased maximum natto mutagenesis strains the DU115 and wild natto BN10 genomic DNA amplification the nattokinase mature peptide gene nkD and nkB, build The recombinant expression vector pPICZα-A-nkD and pPICZα-A-NKB, the DNA sequence showed that NKD gene nkB gene, there are two nucleotide mutation occurred (A107G and C396T) AIO7G mutations result in amino acid alternative D36G (Asp → Gly). Two recombinant expression vector was Ssc Ⅰ linearization electroporation of Pichia pastoris X-33 secretion expression, separation and purification of the expressed product, the determination of enzyme activity and thermal stability testing. The results show that, nattokinase of DNK with the BNK treatment at 65 ℃ for 15 min, 20% increase in the thermal stability of the former, the activity increased by 16.6%. Comparison of the predicted structure of the of nattokinase space sites before and after the change, which can be inferred, mutation of 36 amino acid residues may be closely related to its enzyme thermal stability and activity increased. Considering select natto DU115 nattokinase gene as a material of the future research. 2 in does not change the the Nattokinase DNK on the basis of the amino acid sequence, first select the most favored codon of Pichia pastoris, and with reference to the original natto DU115 Nattokinase codon usage, use times preference codons , so as to eliminate mRNA secondary structure, designed and synthesized a the nattokinase mature peptide gene (synkD). The synthesis synkD a total length of 848 bp, was cloned into pMD18-T vector and sequencing results that are consistent with the expected results. 3 the natto kinase gene synkD of synthetic clone pPICZα-A plasmid construct the secreted recombinant yeast expression the carrier pPICZα-A-synkD transforming Pichia pastoris X-33 recombinant plasmid, the transformation after synkD expression levels than nkD 3.5 times higher. The conditions were optimized expression of recombinant proteins: proliferation when the recombinant yeast BMGY medium to OD 600 for 5 to 6, will be transferred to the bacteria the pH7.0's BMMY culture the base manipulation bacterial density OD value 1.0,28 ℃ induced 84 h, 24 h add methanol to a final concentration of 1.5%, optimized natto the kinase gene synkD shake bottle fermentation expression levels can be achieved 112μg/mL activity of reached 512 IU / mL. After ten generations passage experiments confirmed the good recombinants genetic stability. Nattokinase gene codon optimized high-level expression in Pichia pastoris shows the potential for commercial applications.

Related Dissertations

  1. Mutagenesis of a Laccase Gene from Pleurotus eryngii in Vitro and Expression in Pichia Pastoris,TQ925
  2. Expression of Glucose Oxidase Gene from Aspergillus Niger Z-25 in Pichia Pastoris,Q78
  3. Expression of a Laccase Gene from Lentinula Edodes in Pichia Pastoris and Degradation of Triarylmethane Dyes by Laccase in the Presence of Natural Mediators,TQ925
  4. Study on Bacteriostatic Mechanism of Antagonistic Yeast and Effects on the Quality of Postharvest Strawberries,S668.4
  5. Cloning and Expression of Novel β-Glucosidase Genes from Rhizopus Stolonifer Var. Reflexus,Q78
  6. Effects of Various Glutathione Synthesases on the Glutathione Production by Engineered Pichia Pastoris,TQ936.16
  7. Study on Liquid Fermentation Technology of Nattokinase,TS201.25
  8. Pichia Isolation, purification and biological activity,Q936
  9. The Effect of Codon Optimization on the Immunogenicity of Middle Hepatitis B Surface Antigen DNA Vaccine,R392
  10. Expression of Csfv Erns Protein in Pichia Pastoris and Preparation of Monoclonal Antibodies Against Erns,S852.65
  11. Cloning, Codon Optimization and Expression of Lipase Gene Penicillum Expansum,Q78
  12. The Effect of Nattokinase on Rabbit Model of Ischemic Retinopathy,R774.1
  13. Studies on the Optimum of Solid-state Fermentation Condition Using a Mixture of Bean Curd Residue and Marc and Utilization of the Fermented Materials,TS209
  14. The Optimization, Extraction and Purification of Nattokinase and Genetic Cloning,R346
  15. Cloning, Expression of Myrosinase Gene TGG5 in Arabidopsis Thaliana and Research of Myrosinase Activity,Q943.2
  16. Isolation and Expression Analysis of MiMEK2 and MiLIN45 of Plantnematode Meloidogyne Incognita,S432.45
  17. Nattokinase Gene Cloning and Expression in Pichia pastoris,Q78
  18. Establishment of Pichia Pastoris and Transgenic Medicago Sativa Expression System for Schistosoma Japonicum Antigen Sj23,Q943.2
  19. Purification of Recombined SSB Antigen Expressed in Pichia Pastoris and Establishment of Dot Immunogold Filtration Assay to Detect Anti-SSB Antibody,R446.6
  20. Construction of Novel Pichia Pastoris Cell-surface Display Systems and Its Evaluation in Non-aqueous Phase,Q78
  21. Total Gene Synthesis, Prokaryotic Expression and Structure Prediction of Lipase from Aspergillus Parasiticus,Q78

CLC: > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)
© 2012 www.DissertationTopic.Net  Mobile