Biotechnological production of L-asparaginase (ASP1) of Saccharomyces cerevisiae in a heterologous expression system Pichia pastoris.

Author(s)

Bruna de Souza Divino

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2015-11-24

Examining board members

Adalberto Pessoa Junior; Viviane Maimoni Gonçalves; Angela Faustino Jozala

• Advisor: Adalberto Pessoa Junior; Gisele Monteiro de Souza

Abstract

Used widely in the treatment of Acute lymphoblastic leukemia, L-Asparaginase is an enzyme that acts by decreasing the concentration of L-asparagine free in the plasma and thus prevents the proliferation of cancer cells. This is because such cells, in contrast to healthy cells can not synthesize L-asparagine amino acid for protein synthesis need not possess the enzyme asparagine synthetase due to a gene silencing. The L-asparaginase used in Brazil was imported and produced in bacteria, which caused problems with cost and supply, as well as cause some immune responses to users. This boosts find alternatives to domestic production of this enzyme and, if possible, less allergenic potential. For this, the production of L-asparaginase enzyme ASP1 Saccharomyces cerevisiae gene heterologous to Pichia pastoris expression system was investigated by cloning the synthetic gene (ASP1), with codons optimized for expression in P. pastoris, this study never before achieved to what is known. Furthermore, the enzyme production was performed with the aid of a factorial design in an orbital shaker taking into account the following variables: pH, temperature and concentration of inducer. The best condition found among those studied in this work was a temperature of 20 ° C, pH 6.0 and concentration of 1.5% inductor attained an activity of 6.9 U / g cell and despite the efforts, used signal peptide (S. cerevisiae alpha) was not able to promote the secretion of the enzyme into the medium extracelular.Deve be noted that the achievement of a greater number of tests using a fractional factorial design yields to find a region maximum might improve the fit of the models shown. This work was the first attempt to ASP1 gene expression of S. cerevisiae in Pichia pastoris to date, and is intended to further deepen this study. (AU)

• FAPESP's process: 13/14494-5 - Biotechnological production of L-asparaginase (ASP1) of Saccharomyces cerevisiae in heterologous expression system Pichia pastoris
• Grantee: Bruna de Souza Divino
• Support Opportunities: Scholarships in Brazil - Master