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Author
González Camarero, Rebeca
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Abstract
Chitin is a linear polysaccharide composed of NAG (N-acetylglucosamine) monomers linked by β-1,4 bonds. It is the most abundant natural amino saccharide and the second most abundant natural biopolymer on earth after cellulase. Chitin polymers and oligomers are N-deacetylated by chitin deacetylases generating partially acetylated chitosans and chitooligosaccharides (paCOS). Chitin and its derivatives are very interesting molecules since they have a wide variety of applications in medicine and in the food, pharmaceutical and cosmetic industries, among others. Chitosan is the only natural polycationic polysaccharide, which allows it to interact with polyanionic biomolecules (membrane, nucleic acids, proteins...). The biological activity of chitosan strongly depends on the degree of acetylation (DA), degree of polymerization (DP) and pattern of acetylation (PA). Due to this, and since the different CDAs generate different PA and DA, it is of great interest to characterize different CDAs so that they can be used as catalysts in the production of chitooligosaccharides and paCOS with a specific PA and DA, so that their biological activities can be studied and used in different fields.
The main component of the bacterial cell wall is peptidoglycan, which protects the cell toward various environmental conditions. Peptidoglycan consists of NAG-NAM glycan strands covalently bound to peptide stem (stem peptide). Autolysins are involved in the continuous synthesis, hydrolysis and modification of peptidoglycan, allowing bacterial growth and division, as well as the performance of other biological functions (such as pathogenicity, cannibalism, sporulation, spore germination...). Furthermore, the peptidoglycan fragments released by these autolysins can be detected by the host's immune system during an infection, leading to an immune response. To avoid this, some pathogenic bacteria modify their peptidoglycan through different mechanisms. One of these mechanisms is carried out by peptidoglycan deacetylases that N-deacetylate the NAM or NAG residues of the peptidoglycan, avoiding its recognition by the immune system.
This work is divided into three parts. First, a bibliographic study of hydrolases and autolysins was carried out. Second, the chitin deacetylases BgCDA and SpCDA were expressed and purified. Previously, it was observed that these proteins were expressed in an insoluble way, so to carry out the purification by StrepTrap affinity chromatography, first the protein had to be denatured with urea and then refolding by dialysis so that these proteins fold in a soluble way. In addition, protein quantification and activity measurement using methyl umbelliferone acetate (AcMU) as a substrate were carried out. Finally, in order to characterize the peptidoglycan deacetylases, it is necessary to use peptidoglycan fragments of different composition. These can be obtained by digesting the peptidoglycan with different autolysins. In this third part of the work, the autolysins CwlE (DL-endopeptidase) and CwlH (N-acetylmuramyl-L-alanine amidase) from Bacillus subtilis were expressed, purified and characterized. The measurement of the enzymatic activity was carried out by detection of free amino groups using DNFB as a marker and analyzed by HPLC. These two purified autolysins can be used to generate specific peptidoglycan fragments that can act as substrates for peptidoglycan deacetylases and assist in its characterization.
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