Characterization of L-asparaginase of Erwinia chysanthemi improved by synthetic evolution of proteins and optimization of production conditions

• Grant number: 15/24463-5
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: March 01, 2016
• End date: September 11, 2016
• Field of knowledge: Health Sciences - Pharmacy
• Principal Investigator: Gisele Monteiro
• Grantee: Débora Fernandes Custodio
• Host Institution: Faculdade de Ciências Farmacêuticas (FCF). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 13/08617-7 - Production of extracellular L-asparaginase: from bioprospecting to the engineering of an antileukemic biopharmaceutical, AP.TEM

Abstract

L-Asparaginase (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, ASNase can induce immune response leading to the production of anti-asparaginase antibody, the main cause of drug resistance. Ideally ASNase would be one with high activity, high stability and low allergenic potential, but trade today ASNases obtained by Escherichia coli or Erwinia chrysanthemi do not meet these characteristics simultaneously. For this reason, this study uses techniques of random mutagenesis and site-directed in order to create a new isoform of E. chrysanthemi ASNase with improved activity and stability in human serum. Moreover, with factorial design aid will be studied in metabolic shaker, the mutant strain that express the best recombinant protein of interest, aiming to optimize the production process. It is a master's project conversion direct doctorate, since it was already obtained a significant number of promising mutants. By the time the specific activity of the enzyme standard was established as 455 U/mg and a library of 1,056 mutant was created; these thirty clones were selected for presenting activity 80 to 200% of the activity value obtained for the standard enzyme. Of the thirty four mutants showed no mutation seven mutants showed only silent mutations and nineteen mutants (64%) have at least one mutation in the protein. These 19 mutants will have their resulting proteins analyzed for kinetic parameters and cytotoxicity and the most promising will be selected to study the best conditions and production optimization. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

WLODARCZYK, SAMARINA R.; CUSTODIO, DEBORA; PESSOA JR, ADALBERTO; MONTEIRO, GISELE. Influence and effect of osmolytes in biopharmaceutical formulations. EUROPEAN JOURNAL OF PHARMACEUTICS AND BIOPHARMACEUTICS, v. 131, p. 92-98, OCT 2018. (15/24463-5, 13/08617-7, 13/24024-6, 15/07749-2)

Academic Publications

(References retrieved automatically from State of São Paulo Research Institutions)

CUSTODIO, Débora Fernandes. Characterization of Erwinia chrysanthemi L-Asparaginase improved by synthetic protein evolution and optimization of production conditions. 2018. Doctoral Thesis - Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ) São Paulo.