resumo
L-asparaginase (ASNase, EC 3.5.1.1) is an enzyme with wide applications in the pharmaceutical sector and food processing industries. It is mainly used as a biotherapeutic for treating Acute Lymphoblastic Leukemia (ALL) and to reduce acrylamide formation in starchy compounds. Despite its relevance, current purification methods for microbial enzymes involve complex and expensive techniques. To overcome this drawback, Carbon Xerogels (CXs) were here investigated as novel adsorbents to be applied in an one-step ASNase purification process, using a flow-through-like setup, from a cell extract of genetically engineered Bacillus subtilis. Different operating conditions were studied for optimizing the adsorption onto CXs, including total protein concentration (3-15 mg/ mL), CXs amount (12, 18 and 24 mg), and adsorption volume of cell extract (1.5, 2.0 and 15 mL). Ultimately, CXs were packed into a column to evaluate the feasibility of semi-continuous ASNase purification. CXs have high affinity for other proteins present in the cell extract, while leaving ASNase in the supernatant or eluted sample. Purification folds of 2.5 and 3.8 for ASNase were obtained in batch and semi-continuous experiments, respectively, revealing the potential of CXs as novel adsorbent materials for ASNase purification directly from a complex matrix.
palavras-chave
CHROMATOGRAPHY; GLUTAMINASE; PROTEINS; RAMAN; SIZE
categoria
Engineering
autores
Cristóvao, RO; Barros, RAM; Marramaque, TP; Aguiar, GG; Almeida, MR; Carabineiro, SAC; de Paiva, GB; Pedrolli, DB; Freire, MG; Faria, JL; Santos-Ebinuma, VC; Tavares, APM; Silva, CG
nossos autores
Ana Mafalda Rodrigues Almeida Rocha
Investigador
Ana Paula Mora Tavares
Professor Auxiliar
Mara Guadalupe Freire Martins
Professor CatedráticoProjectos
CICECO - Aveiro Institute of Materials (UIDB/50011/2020)
CICECO - Aveiro Institute of Materials (UIDP/50011/2020)
Associated Laboratory CICECO-Aveiro Institute of Materials (LA/P/0006/2020)
Collaboratory for Emerging Technologies, CoLab (EMERGING TECHNOLOGIES)
agradecimentos
This work was supported by national funds through FCT/MCTES (PIDDAC): LSRE-LCM, UIDB/50020/2020 (DOI: 10.54499/UIDB/50020/2020) and UIDP/50020/2020 (DOI: 10.54499/UIDP/50020/2020); and ALiCE, LA/P/0045/2020 (DOI: 10.54499/LA/P/0045/2020). It was also supported by POCI-01-0145-FEDER-031268 funded by FEDER, through COMPETE2020-Programa Operacional Competitividade e Internacionalizacao (POCI), and by national funds (OE), through FCT/MCTES. This work was developed partly within the scope of the project CICECO-Aveiro Institute of Materials, UIDB/50011/2020 (DOI10.54499/UIDB/50011/2020), UIDP/50011/2020 (DOI 10.54499/UIDP/50011/2020) & LA/P/0006/2020 (DOI 10.54499/LA/P/0006/2020), financed by national funds through the FCT/MCTES (PIDDAC). This work was also partially supported by the Associate Laboratory for Green Chemistry-LAQV, financed by national funds from FCT/MCTES (DOIs: 10.54499/LA/P/0008/2020, 10.54499/UIDP/50006/2020, 10.54499/UIDB/50006/2020). Valeria C. Santos-Ebinuma and Gabriela B. de Paiva acknowledges FAPESP (Grant no. 2018/06908-8, 2021/01284-9, 2021/06686-8). Ana P.M. Tavares and Sonia A.C. Carabineiro acknowledge FCT for the research contracts CEEC-IND/2020/01867 (doi: 10.54499/2020.01867.CEECIND/CP1589/CT0013) and CEECINST/00102/2018 (DOI: 10.54499/CEE-CINST/00102/2018/CP1567/CT0026), respectively. R.A.M. Barros ac-knowledges FCT for her PhD grant 2022.12055.BD. Valeria C. Santos- Ebinuma thanks the National Council of Scientific and Technological Development, Brazil (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico - CNPq) - grant no. 312463/2021-9. Danielle B. Pedrolli acknowledges CNPq grant 305324/2023.