A.P.M. Tavares

Claudia G Silva

Joaquim Luís Faria

M.G. Freire

R.A.M. Barros

Raquel O. Cristóvão

S.A.C. Carabineiro

V.C. Santos-Ebinuma

Repositório Aberto da Universidade do Porto

Carbon xerogels as a purification platform for L-asparaginase purificationCláudia G. Silvaa,b,*, Rita A.M. Barros a,b, Raquel O. Cristóvão a,b, Teresa P. Marramaque a,b, Sónia A.C. Carabineiro c, Mara G. Freired, Joaquim L. Fariaa,b, Valéria C. Santos-Ebinumae, Ana P. M. Tavaresd, a) LSRE-LCM – Laboratory of Separation and Reaction Engineering - Laboratory of Catalysis and Materials, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal; b) ALiCE – Associate Laboratory in Chemical Engineering, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal; c) LAQV-REQUIMTE, NOVA School of Science and Technology, 2829-516 Caparica, Portugal; d) CICECO-Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal; e) Department of Engineering Bioprocess and Biotechnology, School of Pharmaceutical Sciences, UNESP-University Estadual Paulista, Araraquara, Brazil*cgsilva@fe.up.ptL-asparaginase (ASNase, EC 3.5.1.1) is an enzymatic biopharmaceutical used as chemotherapeutic agent for acute lymphoblastic leukaemia (ALL) treatment [1]. Since patients with ALL cannot synthesize L-asparagine due to the absence of asparagine synthetase, the starvation of serum asparagine by ASNase leads to selective death of tumour cells. Current techniques for therapeutic enzyme purification are focused on the use of chromatography, which is highly costly and time-consuming [2]. Compared to conventional approaches, the use of nanomaterials in medical applications presents massive advantages due to their peculiar properties and biocompatibility. Carbon xerogels (CXs) are known for their unique properties such as high surface area, customizable porosity and high adsorption capacities [3]. This work aims the development of an efficient and fast strategy for cost-effective ASNase purification. For that, pristine and functionalized CXs were studied as a purification platform for ASNase from a cell extract of Bacillus subtilis. Different operating conditions were studied during cell extract adsorption onto CXs, such as cell extract concentration (1-15 mg mL⁻¹), materials functionalization and mass (12, 18 and 24 mg), and net adsorption volume (1.5, 2 and 15 mL tubes). SDS-PAGE analysis was carried out to complement the results.The most promising results were obtained during the adsorption of 3 mg mL⁻¹ of cell extract onto pristine CXs in 15 mL tubes, attaining a 1.63-fold increase in the ASNase specific activity in the supernatant through adsorption of unwanted proteins onto the nanostructured carbon materials while keeping ASNase free in solution. SDS-PAGE analysis confirmed that these conditions seem to be the ideal relationship between cell extract concentration/material type and mass/net adsorption volume. The adsorption of cell extract onto functionalized CXs proved to be unfavourable, as the purity of the enzyme decreased in most cases.The results obtained in this work revealed a promising and straightforward alternative to traditional ASNase purification processes through carbon xerogels nanomaterials. This method allows the removal of most impurities from a cell extract of B. subtillis containing ASNase, increasing the purity of the enzyme and decreasing the number of steps in the enzyme purification process.This work was financially supported by: LA/P/0045/2020 (ALiCE) and UIDB/50020/2020 - UIDP/50020/2020 (LSRE-LCM) funded by national funds through FCT/MCTES (PIDDAC), and POCI-01-0145-FEDER-031268 funded by FEDER, through COMPETE2020-Programa Operacional Competitividade e Internacionalização (POCI), and by national funds (OE), through FCT/MCTES. This work was also developed within the scope of the project CICECO-Aveiro Institute of Materials, UIDB/50011/2020, UIDP/50011/2020 & LA/P/0006/2020, financed by national funds through the FCT/MEC (PIDDAC). Ana P. M. Tavares acknowledges FCT for the research contract CEEC CEECIND/2020/01867.[1] Lopes A.M.; Oliveira-Nascimento L.d.; Ribeiro, A., et al., Crit Rev Biotechnol, 2017, 37, 82-99.[2] Tundisi L.L.; Coêlho D.F.; Zanchetta B., Separ & Purif Ver, 2017, 46, 35-43.[3] Carabineiro S.A.C.; Thavorn-Amornsri T.; Pereira M.F.R.; Figueiredo J.L., Water Research, 2011, 45, 4583-4591.

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Carbon xerogels as a purification platform for L-asparaginase purification

Carbon xerogels as a purification platform for L-asparaginase purification

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Repositório Aberto da Universidade do Porto