Loading...

Creating Random Mutations on The Human Transglutaminase 2 Gene and Lysozyme Gene to Increase The Solubility of the Enzymes in order to Perform Promiscuous Organic Reactions Using The Recombinant Enzymes

Moghaddasi, Ahmad | 2023

93 Viewed
  1. Type of Document: M.Sc. Thesis
  2. Language: Farsi
  3. Document No: 55973 (03)
  4. University: Sharif University of Technology
  5. Department: Chemistry
  6. Advisor(s): Kalhor, Hamid Reza
  7. Abstract:
  8. Transglutaminase 2 enzyme is expressed in various parts of the human body and is present in almost all tissue cells. The function of this enzyme is multiple and it catalyzes different reactions depending on where it is located in the cell. The most important activities of this enzyme are: isopeptide bond formation between the side chains of two proteins (transamidation reaction), diamidation reaction (hydrolysis of amide bond), esterification, kinase activity. The hen egg white lysozyme protein is another natural antimicrobial protein that has been extensively studied. The muramidase activity of egg white lysozyme against highly gram-positive bacteria is well known, and this property has caused lysozyme protein to be widely used as food additives to destroy gram-positive bacteria in food. Since these enzymes are insoluble, their recombinant expression in the bacterial cell causes the formation of protein masses. In this case, it becomes impossible to check the activity and structure of the enzymes in vivo conditions. Investigations in vivo conditions require expression of the recombinant protein in the host cell in a soluble form. In addition to the medical field, these enzymes can also be used in industry (dairy and meat). For this reason, changing the protein sequence is essential to achieve high solubility. In order to improve these abilities, the current project tries to achieve a structure with high solubility by using a random mutagenesis technique on the human transglutaminase 2 enzyme gene and the hen egg white lysozyme enzyme gene. The human transglutaminase 2 enzyme gene has been cloned by the subcloning technique next to the beta-galactosidase protein gene. In this structure, the two subunits of the beta-galactosidase protein and the breakdown of the soluble species of transglutaminase 2 can be selected. After transglutaminase 2 gene mutation using polymerase chain reaction technique, several mutated species were investigated for expression of transglutaminase 2 protein. None of the species expressed the desired protein. Sequencing of the mutated transglutaminase 2 gene showed that stop codons were created by applying forward mutations on the gene, and this prevents the expression of the desired protein. In order to compare the effect of gene sequence length in this technique, hen egg white lysozyme enzyme gene was used and all the steps performed for transglutaminase 2 gene were used for lysozyme enzyme gene
  9. Keywords:
  10. Lysozyime Protein ; Polymerase Chain Reaction (PCR) ; Mutation Metries ; In-Vivo Body ; Hen Egg White Lysozyme Enzyme ; Mutagenesis ; Transglutaminase 2 Enzyme

 Digital Object List

 Bookmark

No TOC