Characterization of Erwinia chrysanthemi L-Asparaginase improved by synthetic protein evolution and optimization of production conditions

Author(s)

Débora Fernandes Custodio

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2018-05-07

Examining board members

Gisele Monteiro; Paulo Lee Ho; Ana Marisa Chudzinski Tavassi; José Andrés Yunes

• Advisor: Gisele Monteiro; Adalberto Pessoa Junior

Abstract

L-Asparaginase (L-ASNase) is a bacterial tetrameric enzyme used in chemotherapy sessions that deplete asparagine (Asn) and glutamine (Gln), transforming them into Aspartate (Asp) or glutamate (Glu), respectively, and ammonia. However, L-ASNase can induce immune response leading to the production of anti-asparaginase antibody, an important cause of drug resistance. Ideally, L-ASNase would be one with high activity, high stability and low immunogenic potential, but the L-ASNases commercially available today do not present these characteristics simultaneously. For this reason, this study used techniques of random and site-directed mutagenesis in order to create a new proteoform of E. chrysanthemi L-ASNase with improved activity and stability. In addition, culture conditions were studied in a metabolic shaker, aiming at the optimization of production conditions. A library with 1,056 clones was created, and of these clones, 19 were selected because they had activity superior or equal to the wild-type enzyme in crude protein extract. Among them, 2 mutants stood out for having different glutaminase specific activity in relation to wild-type enzyme. The 9-6D mutant also showed decreased structural disorder and immunogenic epitopes. The 9-5F mutant demonstrated a decrease in percentage of glutaminase activity when compared to the wild-type enzyme. The production study of 9-5F mutant indicated that the induction temperature followed by the inductor concentration are the most relevant parameters for the production optimization of E. chrysanthemi mutant L-ASNase. (AU)

• FAPESP's process: 15/24463-5 - Characterization of L-asparaginase of Erwinia chysanthemi improved by synthetic evolution of proteins and optimization of production conditions
• Grantee: Débora Fernandes Custodio
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)