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Improving Subtilisin Production by Engineering the Regulatory Regions of aprE in Bacillus Subtilis

Pourmohammad, Mohammad Javad | 2023

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  1. Type of Document: M.Sc. Thesis
  2. Language: Farsi
  3. Document No: 56375 (06)
  4. University: Sharif University of Technology
  5. Department: Chemical and Petroleum Engineering
  6. Advisor(s): Banaei Moghaddam, Ali Mohammad; Yaghmaei, Soheyla; Roosta Azad, Reza
  7. Abstract:
  8. Industrial enzymes can generally be classified into three groups: carbohydrases, proteases and lipases, which account for a major part of the global enzyme trade. Proteases, in particular, have always been extensively studied and investigated due to their widespread use in various industries, such as food, pharmaceutical, chemical, and agricultural sectors. Proteases are a group of proteolytic enzymes that break down proteins and peptides into their constituent amino acids by hydrolyzing peptide bonds. Among the family of proteases, alkaline serine proteases have special biocatalytic abilities in both aqueous and organic environments. Their suitable resistance in alkaline environments and high temperatures has led to wide applications in various industries, especially detergent industries. These enzymes are produced by a wide range of organisms, including plants and animals, as well as microorganisms such as fungi and bacteria. However, enzymes produced by microbial species are more important industrially than plant and animal enzymes due to their greater stability, better catalytic activity, ease of production, and optimization. Among different microbial species, Bacillus subtilis is widely used as a cell factory for microbial production of industrial enzymes, especially alkaline proteases, due to its protein secretion system and the possibility of extraction from culture media. However, it is necessary to use genetic engineering tools like CRISPR to edit the genome and modify this strain for further industrial applications. In this research, a recombinant genetic construct including the alkaline protease gene and regulatory regions such as a promoter, ribosome binding site, and signal peptide was designed and built. By replacing it in the Bacillus subtilis genome using CRISPR technology, a recombinant strain with a higher ability to produce alkaline proteases than the wild strain was created
  9. Keywords:
  10. Genome Editing ; Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)System ; Bacillus Subtilis ; Promoter ; Alkaline Protease Enzyme ; Subtilisin Enzyme ; Industrial Enzymes

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