Production, kinetic characterization and engineering of Asparaginase 1 protein of the Saccharomyces cerevisiae for evaluate its use as biopharmaceutical

Abstract

The acute lymphoblastic leukemia (ALL) is characterized by uncontrolled production of blasts of lymphoid characteristics and by blocking of normal production of red and white cells and platelets. It is the most common neoplasm in children and teenagers, representing approximately 30% of cancer cases in this age group. The tumor cells depend of asparagines presence in circulation for protein production and cell proliferation, once this amino acid production is decreased or absent in leukemic cells. The L-Asparaginase (ASPase), enzyme obtained from Escherichia coli and Erwinia chrysanthemi, is considered an important antineoplastic in disease treatment since the 70s. The ASPase hydrolyzes asparagine resulting in aspartic acid and ammonia, blocking nourishment of tumor cells causing to cell death. However, both formulations are associated with high a rate of adverse effects, especially drug resistance caused by antibody production anti-asparaginase and severe hypersensitive, that undertakes the development of treatment. Many microorganisms can to produce asparaginase, however, the kinetic proprieties of enzymes nonbacterial has limited its use. Saccharomyces cerevisiae is an yeast of easy genetic manipulation and has the ASP1 gene responsible by production asparaginase 1 in yeast. Nevertheless, there are few studies about asparaginase produced by S. cerevisiae. The aim of present project is to evaluate the kinetic proprieties and enzyme stability of yeast and make rational mutations for improve these parameters. We will use like standard the enzymes from E. coli and E. chrysanthemi to produce an asparaginase with improved activity and less adverse effects from ASP1. Therefore, we will mutations in amino acid sequence of protein and enzymatic assays. (AU)