Heterologous expression and functional and structural characterization of asparaginase RmASP1 of Rhodotorula mucilaginosa

• Grant number: 17/27230-7
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: March 01, 2018
• End date: February 28, 2019
• Field of knowledge: Biological Sciences - Microbiology - Applied Microbiology
• Principal Investigator: Marcos Antonio de Oliveira
• Grantee: Vitória Isabela Montanhero Cabrera
• Host Institution: Instituto de Biociências (IB-CLP). Universidade Estadual Paulista (UNESP). Campus Experimental do Litoral Paulista. São Vicente , 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

The asparaginase (ASNases) of bacterial origin from Eschericchia coli (EcA) and Erwinia chrysantemi (ErA) are important biopharmaceuticals used in the treatment of acute lymphoblastic leukemia (ALL), a neoplasm of blood cells whose tumor cells are unable to synthesize the amino acid asparagine (Asn) and therefore necessitate of extracellular Asn for their survival. Bacterial ASNases are able to efficiently hydrolyze asparagine (Asn) to aspartic acid (Asp) and ammonia (NH3), decreasing the availability of Asn to tumor cells, which affects protein synthesis and nitrogenous bases and induces the neoplastic cells to apoptosis. Although it appears to be an alternative, human isoforms can not be used in the treatment of ALL since the Km lies in the millimolar range, while the bacterial enzymes present Km ~ 7.5 ´ 10-6 M. Commercially, international pharmaceutical industries produce ASNases of E. coli and E. chrysantemi, but this biopharmaceutical is not produced by the Brazilian pharmaceutical industry, which demonstrates the fragility of Brazil in the field of biopharmaceuticals. In addition, despite their great importance, bacterial ASNases are able to stimulate the immune system, leading from mild allergies to anaphylactic shock. One of the therapeutic possibilities is the interchangeability of EcA to ErA, respectively. However, this procedure is not possible in Brazil, since only the native form of E. coli is approved by ANVISA. In fact, even with the exchange of the drug there is still a significant portion of patients who develop allergy to existing drugs and can not effectuate treatment. In this context, the search for alternative sources of this enzyme is extremely important to mitigate the immunological effects and may also foster the development of a national biopharmaceutical that may contribute to Brazil's self-sufficiency. Studies carried out under the thematic project have revealed that an isolate from Antarctica of the yeast Rhodotorula mucilaginosa is able to produce an extracellular ASNase with high catalytic efficiency. In the present study, we identified a gene (rmasp1) whose protein product has signal peptide for export, has a moderate identity with bacterial enzymes EcA and ErA (~ 60% similarity and ~ 40% identity) and also possesses all conserved amino acids involved in the catalysis, which may represent a new alternative for the treatment of ALL. The objectives of this project are the cloning, expression and purification of the enzyme RmAsp1 and its functional and structural characterization. We believe that the results from this project are of great importance for the valorization of biodiversity and seek to strategically strengthen the study of biopharmaceuticals in Brazil (AU)