Production and characterization of Bacillus subtilis CwlH autolysin


Bierkens Limia, Carlos


Bacteria are microorganisms that possess a cell wall. The cell wall in bacteria is of great importance, since it gives them resistance to different adverse conditions and provides them with the necessary rigidity to maintain their shape. The cell wall in bacteria is formed by a peptidoglycan network, which is formed by the chain linkage of two sugars and a tetrapeptide. The sugars that form it are N-acetylglucosamine and N-acetylmuramic acid. The tetrapeptide, also known as "stem peptide", forms the amide bonds between the N-acetylmuramic acid bases, thus conferring greater stability to the mesh. This peptidoglycan that forms the cell wall is extremely dynamic, as it must be able to be modified throughout the life of the bacterium. For this purpose, there are a number of proteins that bacteria use to modify the cell wall. These proteins are known as autolysins, of which there are many types, and they allow the cell wall to be shaped by breaking the bonds that form its components. In this project we use a specific autolysin from Bacillus subtilis to generate a fragmented peptidoglycan substrate, where we are able to separate the glycan chain from the stem peptide. For this purpose, CwlH autolysin is expressed in a protease-deficient E. coli strain to increase the stability of the protein produced. Once expression and small-scale production is obtained, the enzyme activity is characterized, and the process is scaled up. For scale-up, a 10 L fermenter is used where a fed-batch protocol is proposed.



Carnicer Heras, Marc


IQS SE - Undergraduate Program in Biotechnology