Development of a purification platform of L-asparaginase II from Aliivibrio fischeri produced by Bacillus subtilis

Abstract

L-asparaginase (EC.3.5.1.1, L-asparagine amidohydrolase, L-ASNase) is an enzyme capable to hydrolyze asparagine, which makes it an important biopharmaceutical in the therapy of acute lymphoblastic leukemia (ALL), since the breakdown of this amino acid in the extracellular environment leads to the death of cancer cells that are unable to synthesize it. Even though there are several microorganisms capable to produce it, not all L-ASNases are suitable for use as biopharmaceuticals, because there is greater efficiency in the treatment of ALL and fewer side effects when the enzyme has high asparaginase activity and low glutaminase activity, characteristics that are found in L-asparaginase type II (L-ASNase II). In addition, it is commom the emergence of hypersensitivity during ALL therapy, an immune response that leads to enzyme inactivation, making it necessary to change the formulation to continue the treatment. For this reasons, the importance of new production sources for this enzyme and the development of new formulations is evident. At last, this project aims to produce L-ASNase II from Aliivibrio fischeri in modified Bacillus subtilis, using a stirred-tank bioreactor, and to establish purification techniques by evaluating the combination of low-resolution and chromatographic methods, in order to obtain an anti-leukemic biopharmaceutical to be used in the development of a new formulation suited to the needs of the market.