Skip to main content
SpringerLink
Log in

Optimization of aqueous two-phase micellar system for partial purification of L-asparaginase from Penicillium sp. grown in wheat bran as agro-industrial residue

  • Biotechnology and Industrial Microbiology - Research Paper
  • Published:
Brazilian Journal of Microbiology Aims and scope Submit manuscript

Abstract

L-asparaginase has been used in the remission of malignant neoplasms such as acute lymphoblastic leukemia. The search for new sources of this enzyme has become attractive for therapeutics. Traditional methods for biomolecule purification involve several steps. A two-phase system may be a good strategy to anticipate one of these stages. This study aimed to produce and purify a fungal L-asparaginase through an aqueous two-phase micellar system (ATPMS) using Triton X-114. The fungus Penicillium sp.–encoded 2DSST1 was isolated from Cerrado soil. Plackett-Burman design followed by a 24 full factorial design was used to determine the best conditions to produce L-asparaginase. The evaluated variables were L-asparagine, L-proline, wheat bran, potato dextrose broth, ammonium sulfate, yeast extract, sucrose and glucose concentrations, incubation temperature, incubation period, and initial pH of the culture medium. L-asparaginase quantification was valued by the formation of β-aspartyl hydroxamate. The significant positive variables, L-asparagine, L-proline, potato dextrose broth, and sucrose concentrations, were evaluated at 2 levels (+ 1 and − 1) with triplicate of the central point. After 34 runs, maximum activity (2.33 IU/mL) was achieved at the factorial design central point. A central composite design was performed in ATPMS at two levels (+ 1 and − 1) varying Triton X-114 concentration (w/v), separation phase temperature, and crude extract concentration (w/v). The L-asparaginase partition coefficient (K) was considered the experimental design response. Out of the 16 systems that were examined, the most promising presented a purification factor of 1.4 and a yield of 100%.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  1. Lopes AM, de Oliveira-Nascimento L, Ribeiro A, Tairum CA Jr, Breyer CA, de Oliveira MA, Monteiro G, de Souza-Motta CM, de Oliveira Magalhães P, Avendaño JGF (2017) Therapeutic l-asparaginase: upstream, downstream and beyond. Crit Rev Biotechnol 37(1):82–99

    Article  CAS  PubMed  Google Scholar 

  2. Kebriaei P, Anastasi J, Larson RA (2002) Acute lymphoblastic leukaemia: diagnosis and classification. Best Pract Res Clin Haematol 15(4):597–621

    Article  CAS  PubMed  Google Scholar 

  3. Onciu M (2009) Acute lymphoblastic leukemia. Hematol Oncol Clin North Am 23(4):655–674

    Article  PubMed  Google Scholar 

  4. Moghrabi A, Levy DE, Asselin B, Barr R, Clavell L, Hurwitz C, Samson Y, Schorin M, Dalton VK, Lipshultz SE (2007) Results of the Dana-Farber Cancer Institute ALL Consortium Protocol 95-01 for children with acute lymphoblastic leukemia. Blood 109(3):896–904

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Quintanilla-Flores DL, Flores-Caballero MÁ, Rodríguez-Gutiérrez R, Tamez-Pérez HE, González-González JG (2014) Acute pancreatitis and diabetic ketoacidosis following L-asparaginase/prednisone therapy in acute lymphoblastic leukemia. Case Rep Oncol Med 2014:139169. https://doi.org/10.1155/2014/139169

  6. Egler RA, Ahuja SP, Matloub Y (2016) L-asparaginase in the treatment of patients with acute lymphoblastic leukemia. J Pharmacol Pharmacother 7(2):62–71

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Pieters R, Hunger SP, Boos J, Rizzari C, Silverman L, Baruchel A, Goekbuget N, Schrappe M, Pui CH (2011) L-asparaginase treatment in acute lymphoblastic leukemia: a focus on Erwinia asparaginase. Cancer 117(2):238–249

    Article  CAS  PubMed  Google Scholar 

  8. Torres-Obreque K, Meneguetti GP, Custódio D, Monteiro G, Pessoa-Junior A, Rangel-Yagui CO (2018) Production of a novel N-terminal PEGylated crisantaspase. Biotechnol Appl Biochem 66(3):281–289. https://doi.org/10.1002/bab.1723

  9. Chan WK, Lorenzi PL, Anishkin A, Purwaha P, Rogers DM, Sukharev S, Rempe SB, Weinstein JN (2014) The glutaminase activity of L-asparaginase is not required for anticancer activity against ASNS-negative cells. Blood 123(23):3596–3606

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Warrell RP, Chou T-C, Gordon C, Tan C, Roberts J, Sternberg SS, Philips FS, Young CW (1980) Phase I evaluation of succinylated Acinetobacter glutaminase-asparaginase in adults. Cancer Res 40(12):4546–4551

    PubMed  Google Scholar 

  11. Mazzola PG, Lopes AM, Hasmann FA, Jozala AF, Penna TC, Magalhaes PO, Rangel-Yagui CO, Pessoa A Jr (2008) Liquid–liquid extraction of biomolecules: an overview and update of the main techniques. J Chem Technol Biotechnol 83(2):143–157

    Article  CAS  Google Scholar 

  12. Nadar SS, Pawar RG, Rathod VK (2017) Recent advances in enzyme extraction strategies: a comprehensive review. Int J Biol Macromol 101:931–957

    Article  CAS  PubMed  Google Scholar 

  13. Malpiedi LP, Nerli BB, Abdala DS, de Alcântara Pessôa-Filho P, Pessoa A Jr (2014) Aqueous micellar systems containing Triton X-114 and Pichia pastoris fermentation supernatant: a novel alternative for single chain-antibody fragment purification. Sep Purif Technol 132:295–301

    Article  CAS  Google Scholar 

  14. Chuo SC, Mohd-Setapar SH, Mohamad-Aziz SN, Starov VM (2014) A new method of extraction of amoxicillin using mixed reverse micelles. Colloids Surf A Physicochem Eng Asp 460:137–144

    Article  CAS  Google Scholar 

  15. Liu Y, Sun M-H, Shao S-K, Deng G (2017) An affinity-based aqueous two-phase mixed micellar system and its purification of Yeast 3′, 5′-bisphosphate nucleotidase. J Chromatogr B 1060:215–220

    Article  CAS  Google Scholar 

  16. Liu CL, Nikas Y, Blankschtein D (1996) Novel bioseparations using two-phase aqueous micellar systems. Biotechnol Bioeng 52(2):185–192

    Article  CAS  PubMed  Google Scholar 

  17. Shiomori K, Ebuchi N, Kawano Y, Kuboi R, Komasawa I (1998) Extraction characteristic of bovine serum albumin using sodium bis (2-ethylhexyl) sulfosuccinate reverse micelles. J Ferment Bioeng 86(6):581–587

    Article  CAS  Google Scholar 

  18. Rundlett KL, Armstrong DW (1995) Effect of micelles and mixed micelles on efficiency and selectivity of antibiotic-based capillary electrophoretic enantioseparations. Anal Chem 67(13):2088–2095

    Article  CAS  PubMed  Google Scholar 

  19. Soto A, Arce A, Khoshkbarchi MK (2005) Partitioning of antibiotics in a two-liquid phase system formed by water and a room temperature ionic liquid. Sep Purif Technol 44(3):242–246

    Article  CAS  Google Scholar 

  20. Rangel-Yagui C, Pessoa-Jr A, Blankschtein D (2004) Two-phase aqueous micellar systems: an alternative method for protein purification. Braz J Chem Eng 21(4):531–544

    Article  CAS  Google Scholar 

  21. Rangel-Yagui CO, Lam H, Kamei DT, Wang DI, Pessoa A Jr, Blankschtein D (2003) Glucose-6-phosphate dehydrogenase partitioning in two-phase aqueous mixed (nonionic/cationic) micellar systems. Biotechnol Bioeng 82(4):445–456

    Article  CAS  PubMed  Google Scholar 

  22. Saitoh T, Hinze WL (1991) Concentration of hydrophobic organic compounds and extraction of protein using alkylammoniosulfate zwitterionic surfactant mediated phase separations (cloud point extractions). Anal Chem 63(21):2520–2525

    Article  CAS  PubMed  Google Scholar 

  23. Drainas C, Kinghorn J, Pateman J (1977) Aspartic hydroxamate resistance and asparaginase regulation in the fungus Aspergillus nidulans. Microbiology 98(2):493–501

    CAS  Google Scholar 

  24. Van Der Meeren P, Cocquyt J, Flores S, Demeyere H, Declercq M (2002) Quantifying wetting and wicking phenomena in cotton terry as affected by fabric conditioner treatment. Text Res J 72(5):423–428

    Article  Google Scholar 

  25. Imada A, Igarasi S, Nakahama K, Isono M (1973) Asparaginase and glutaminase activities of micro-organisms. Microbiology 76(1):85–99

    CAS  Google Scholar 

  26. Badoei-Dalfard A (2015) Purification and characterization of l-asparaginase from Pseudomonas aeruginosa strain SN004: production optimization by statistical methods. Biocatalysis Agric Biotechnol 4(3):388–397

    Article  Google Scholar 

  27. Sindhu R, Manonmani H (2018) Expression and characterization of recombinant l-asparaginase from Pseudomonas fluorescens. Protein Expr Purif 143:83–91

    Article  CAS  PubMed  Google Scholar 

  28. Souza PM, de Freitas MM, Cardoso SL, Pessoa A, Guerra ENS, Magalhaes PO (2017) Optimization and purification of L-asparaginase from fungi: a systematic review. Crit Rev Oncol Hematol 120:194–202

    Article  PubMed  Google Scholar 

  29. Baskar G, Renganathan S (2012) Optimization of L-asparaginase production by Aspergillus terreus MTCC 1782 using response surface methodology and artificial neural network-linked genetic algorithm. Asia Pac J Chem Eng 7(2):212–220

    Article  CAS  Google Scholar 

  30. Dias FF, Sato HH (2016) Sequential optimization strategy for maximum l-asparaginase production from Aspergillus oryzae CCT 3940. Biocatalysis Agric Biotechnol 6:33–39

    Article  Google Scholar 

  31. Farag AM, Hassan SW, Beltagy EA, El-Shenawy MA (2015) Optimization of production of anti-tumor l-asparaginase by free and immobilized marine Aspergillus terreus. Egypt J Aquat Res 41(4):295–302

    Article  Google Scholar 

  32. Gurunathan B, Sahadevan R (2012) Optimization of culture conditions and bench-scale production of L-asparaginase by submerged fermentation of Aspergillus terreus MTCC 1782. J Microbiol Biotechnol 22(7):923–929

    Article  CAS  PubMed  Google Scholar 

  33. Huang L, Liu Y, Sun Y, Yan Q, Jiang Z (2014) Biochemical characterization of a novel L-Asparaginase with low glutaminase activity from Rhizomucor miehei and its application in food safety and leukemia treatment. Appl Environ Microbiol 80(5):1561–1569

    Article  PubMed  PubMed Central  Google Scholar 

  34. Duarte AWF, Lopes AM, Molino JVD, Pessoa A, Sette LD (2015) Liquid–liquid extraction of lipase produced by psychrotrophic yeast Leucosporidium scottii L117 using aqueous two-phase systems. Sep Purif Technol 156:215–225

    Article  CAS  Google Scholar 

  35. Spir LG, Ataide JA, De Lencastre Novaes LC, De Borba GD, Moriel P, Silveira E, Pessoa A Jr, Tambourgi EB, Mazzola PG (2015) Application of an aqueous two-phase micellar system to extract bromelain from pineapple (Ananas comosus) peel waste and analysis of bromelain stability in cosmetic formulations. Biotechnol Prog 31(4):937–945

    Article  CAS  PubMed  Google Scholar 

  36. Jaramillo PMD, Gomes HAR, de Siqueira FG, Homem-de-Mello M, Ferreira Filho EX, Magalhães PO (2013) Liquid–liquid extraction of pectinase produced by Aspergillus oryzae using aqueous two-phase micellar system. Sep Purif Technol 120:452–457

    Article  CAS  Google Scholar 

  37. de Freitas MM, Souza PM, Cruvinel K, Barros T, Santos SN, Long PF, Pessoa A, Magalhães PO (2019) Interferences that impact measuring optimal l-asparaginase activity and consequent errors interpreting these data. Appl Microbiol Biotechnol 103(13):5161–5166

    Article  PubMed  Google Scholar 

  38. Drainas D, Drainas C (1985) A conductimetric method for assaying asparaginase activity in Aspergillus nidulans. Eur J Biochem 151(3):591–593

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was carried out with the financial support of the State Funding Agency of Distrito Federal (FAPDF), National Council for Scientific and Technological Development (CNPq), Coordination of Superior Level Staff Improvement (CAPES), and University of Brasilia.

Funding

This work was supported by the State Funding Agency of Distrito Federal (FAPDF) – process number 193.001.661/2017 and the São Paulo Research Fundation (FAPESP) – process number: 13/08617-7.

Author information

Authors and Affiliations

  1. Laboratory of Natural Products, Department of Pharmacy, Health Sciences School, University of Brasilia, Brasilia, Brazil

    Samuel L. Cardoso, Marcela M. de Freitas, Paula M. de Souza, Mauricio Homem-de-Mello, Dâmaris Silveira, Yris Maria Fonseca-Bazzo & Pérola O. Magalhães

  2. Institute of Biology, University of Brasília, Brasilia, Brazil

    Edivaldo X. Filho

  3. Department of Biochemical and Pharmaceutical Technology, University of São Paulo, São Paulo, Brazil

    Adalberto P. Junior

Authors
  1. Samuel L. Cardoso
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marcela M. de Freitas
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Paula M. de Souza
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mauricio Homem-de-Mello
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Dâmaris Silveira
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yris Maria Fonseca-Bazzo
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Edivaldo X. Filho
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Adalberto P. Junior
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Pérola O. Magalhães
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Pérola O. Magalhães.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Responsible Editor: Solange I. Mussatto

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Cardoso, S.L., de Freitas, M.M., de Souza, P.M. et al. Optimization of aqueous two-phase micellar system for partial purification of L-asparaginase from Penicillium sp. grown in wheat bran as agro-industrial residue. Braz J Microbiol 51, 979–988 (2020). https://doi.org/10.1007/s42770-020-00269-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s42770-020-00269-2

Keywords

Search

Navigation