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Diversity and Synthetic applications of subtilases

Meekels, L.K.M. (2011) Diversity and Synthetic applications of subtilases. Bachelor's Thesis, Biology.

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Abstract

Subtilases are proteinases which catalyze the hydrolysis of peptide bonds in peptides and proteins. Their catalytic triad consist of serine (Ser), histidine (His) and aspartic acid (Asp). Next to the catalytic triad, subtilases contain a pre-pro peptide sequence. The pre-sequence acts as a signal peptide, resulting in translocation through the cell membrane. The pro-sequence acts as an intramolecular chaperone for guiding of correct folding and as a protease self-inhibitor. Siezen et al. (1997) introduced six groups within the subtilases family; subtilisin, thermitase, kexin, lantibiotic, proteinase K and pyrolysin family. To see whether other enzymes, with known crystal structure, have similar sequences to subtilisin BPN’, blast searches have been performed. These showed that several differences in residues in the S4 binding pocket are present and the differences do not initially lead to inactivation or a decrease in activation of the enzyme. The S1 binding pocket was a conserved region for the 10 homolog sequences. Subtilases can be used for several synthetic applications. This report only focuses on the application of subtilases in peptide synthesis. The use of subtilases in the peptide synthesis has the advantage that no side-chain protection is necessary, however, the use of subtilases for peptide synthesis did not come without problems and optimization is necessary. To improve the substrate specificity, enzymes are modified by introducing different residues in the substrate binding pockets. Next to the genetic modifications of the enzymes, also medium engineering is a possibility. Peptide synthesis can be performed in different kind of types of media, aqueous-organic mixtures, neat organic solvents and nonconventional media. Using aqueous-organic mixtures gives the opportunity that hydrophobic reactants can be used, also the hydrolysis reactions can be reduced. To further reduce hydrolysis, neat organic solvents are used but this leads to a dramatically decrease of the catalytic activity of the enzyme. Nonconventional media look promising, but the equipment is too expensive. The use of different enzymes such as sortase A, which is a cysteine peptidase, or enzymes which are organic-solvent tolerant, such as the metalloprotease from P. aeruginosa, look promising for the peptide synthesis in organic solvents. To further improve enzymatic peptide synthesis more investigation needs to be done on the effects on substrate specificity and activity when mutations are made within the substrate binding pockets of the enzyme.

Item Type: Thesis (Bachelor's Thesis)
Degree programme: Biology
Thesis type: Bachelor's Thesis
Language: English
Date Deposited: 15 Feb 2018 07:45
Last Modified: 15 Feb 2018 07:45
URI: http://fse.studenttheses.ub.rug.nl/id/eprint/9554

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