Skip to main content
SpringerLink
Log in

Efficient Purification of Nuclease P1 from Penicillium citrinum Using Polyethylene Glycol/Disodium Guanosine Monophosphate Aqueous Two-Phase System

  • Original Article
  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

Nuclease P1 (NP1) can hydrolyze nucleic acids into four 5′-mononucleotides, which are widely used in the pharmaceutical and food industries. In this paper, an aqueous two-phase system (ATPS) was developed to purify NP1 from Penicillium citrinum. Polyethylene glycol (PEG) and nucleotides salts were studied to form ATPSs, among which PEG3000/disodium guanosine monophosphate (GMPNa2) was researched, including the phase composition and pH. Using 14% (w/w) PEG3000 and 20% (w/w) GMPNa2 ATPS at pH 5.0, the best recovery and purification factor, 82.4% and 3.59, were obtained. The recovery of NP1 was 98.3% by the separation of ultrafiltration from the PEG-rich phase. The recycling use of GMPNa2 was also studied, and 95.1% of GMPNa2 in the salt-rich phase was obtained with the addition of ethanol as the solvent. These results showed that the ATPS was effective for purification of NP1.

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
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Data Availability

Not applicable.

Code Availability

Not applicable.

References

  1. Maekawa, K., Tsunasawa, S., Dibo, G., & Sakiyama, F. (1991). Primary structure of nuclease P1 from penicillium citrinum. European Journal of Biochemistry, 200, 651–661.

    Article  CAS  PubMed  Google Scholar 

  2. Tabata, N., Kazama, H., Ohgi, K., & Irie, M. (1991). Primary structure of nuclease (nuclease PA3) from penicillium sp. Agricultural and Biological Chemistry, 55, 461–469.

    CAS  PubMed  Google Scholar 

  3. Fujimoto, M., Kuninaka, A., & Yoshino, H. (1974). Purification of a nuclease from Penicillium citrinum. Agricultural and Biological Chemistry, 38, 777–783.

    Article  CAS  Google Scholar 

  4. Fujimoto, M., Kuninaka, A., & Yoshino, H. (1974). Identity of phosphodiesterase and phosphomonoesterase activities with nuclease P1 (a nuclease from Penicillium citrinum). Agricultural and Biological Chemistry, 38, 785–790.

    Article  CAS  Google Scholar 

  5. Fujimoto, M., Kuninaka, A., & Yoshino, H. (1974). Substrate specificity of nuclease P1. Agricultural and Biological Chemistry, 38, 1555–1561.

    Article  CAS  Google Scholar 

  6. Fujimoto, M., Kuninaka, A., & Yoshino, H. (1975). Some physical and chemical properties of nuclease P1. Agricultural and Biological Chemistry, 39, 1991–1997.

    CAS  Google Scholar 

  7. Lahm, A., Volbeda, A., & Suck, D. (1990). Crystallisation and preliminary crystallographic analysis of P1 nuclease from Penicillium citrinum. Journal of Molecular Biology, 215, 207–210.

    Article  CAS  PubMed  Google Scholar 

  8. Kuninaka, A. (2008). Nucleotides and related compounds. In Biotechnology (pp. 561–612). Wiley-VCH.

  9. Gerald, R., & Tilak, W. (1991). Yeast derived products. In Yeast Technology (pp. 369–412). AVI.

  10. Ying, G. Q., Shi, L. E., Yi, Y., Tang, Z. X., & Chen, J. S. (2006). Production, purification and characterization of nuclease p1 from Penicillium citrinum. Process Biochemistry, 41, 1276–1281.

    Article  CAS  Google Scholar 

  11. Vaidya, B. K., Suthar, H. K., Kasture, S., & Nene, S. (2006). Purification of potato polyphenol oxidase (PPO) by partitioning in aqueous two-phase system. Biochemical Engineering Journal, 28, 161–166.

    Article  CAS  Google Scholar 

  12. Fakhari, M. A., Rahimpour, F., & Taran, M. (2017). Response surface methodology optimization of partitioning of xylanase form Aspergillus niger by metal affinity polymer-salt aqueous two-phase systems. Journal of Chromatography, B: Analytical Technologies in the Biomedical and Life Sciences, 1063, 1–10.

    Article  CAS  PubMed  Google Scholar 

  13. Chen, X., Wang, J., & Jiao, F. (2015). Efficient enantioseparation of phenylsuccinic acid enantiomers by aqueous two-phase system-based biphasic recognition chiral extraction: Phase behaviors and distribution experiments. Process Biochemistry, 50, 1468–1478.

    Article  CAS  Google Scholar 

  14. Iqbal, M., Tao, Y., Xie, S., Zhu, Y., Chen, D., Wang, X., Huang, L., Peng, D., Sattar, A., Shabbir, M. A., Hussain, H. I., Ahmed, S., & Yuan, Z. (2016). Aqueous two-phase system (ATPS): An overview and advances in its applications. Biological Procedures Online, 18, 18.

    Article  PubMed  PubMed Central  Google Scholar 

  15. Hatti-Kaul, R. (2001). Aqueous two-phase systems. Molecular Biotechnology, 19, 269–277.

    Article  CAS  PubMed  Google Scholar 

  16. Li, M., Kim, J. W., & Peeples, T. L. (2002). Amylase partitioning and extractive bioconversion of starch using thermoseparating aqueous two-phase systems. Journal of Biotechnology, 93, 15–26.

    Article  CAS  PubMed  Google Scholar 

  17. Cao, X. J., Wu, X. Y., Fonseca, L. J. P., Cabral, J. M. S., & Marcos, J. C. (2004). Production of 6-aminopenicillanic acid in aqueous two-phase systems by recombinant Escherichia coli with intracellular penicillin acylase. Biotechnology Letters, 26, 97–101.

    Article  PubMed  Google Scholar 

  18. Cizova, A., Korcova, J., Farkas, P., & Bystricky, S. (2017). Efficient separation of mannan-protein mixtures by ionic liquid aqueous two-phase system, comparison with lectin affinity purification. International Journal of Biological Macromolecules, 98, 314–318.

    Article  CAS  PubMed  Google Scholar 

  19. Ufuk, G. (2000). Partitioning of bovine serum albumin in an aqueous twophase system: Optimization of partition coefficient. Journal of Chromatography B: Biomedical Sciences and Applications, 743, 259–262.

    Article  Google Scholar 

  20. Franco, T. T., Andrews, A. T., & Asenjo, J. A. (1995). Conservative chemical modification of proteins to study the effects of a single protein property on partitioning in aqueous two-phase systems. Biotechnology and Bioengineering, 49, 290–299.

    Article  Google Scholar 

  21. Fujishima, T., Uchida, K., & Yushino, H. (1972). Enzyme production by molds in sponge culture. Journal of Fermentation Technology, 50, 724–730.

    CAS  Google Scholar 

  22. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193, 265–275.

    Article  CAS  PubMed  Google Scholar 

  23. Reh, G., Nerli, B., & Pico, G. (2002). Isolation of alpha-1-antitrypsin from human plasma by partitioning in aqueous biphasis systems of polyethyleneglycol-phosphate. Journal of Chromatography B, 780, 389–396.

    Article  CAS  Google Scholar 

  24. Su, C. K., & Chiang, B. H. (2006). Partitioning and purification of lysozyme from chicken egg white using aqueous two-phase system. Process Biochemistry, 41, 257–263.

    Article  CAS  Google Scholar 

  25. Albertsson, P. A. (1986). Partition of cell particles and macromolecules (3rd ed., pp. 60–64). John Wiley and Sons.

    Google Scholar 

  26. Chen, X. C., Xu, G. M., Li, X., Li, Z. J., & Ying, H. J. (2008). Purification of an a-amylase inhibitor in a polyethylene glycol/fructose-1, 6-bisphosphate trisodium salt aqueous two-phase system. Process Biochemistry, 43, 765–768.

    Article  CAS  Google Scholar 

  27. Suppasith, K., Soottawat, B., & Wonnop, V. (2005). Partitioning and recovery of proteinase from tuna spleen by aqueous two-phase systems. Process Biochemistry, 40, 3061–3067.

    Article  Google Scholar 

  28. Isable, D. V. M., & Otero, C. (2003). Biphasic aqueous media containing polyethylene glycol for the enzymatic synthesis of oligosaccharides from lactose. Enzyme Microbial Technology, 33, 118–126.

    Article  Google Scholar 

  29. Goja, A. M., Yang, H., Cui, M., & Li, C. (2013). Aqueous two-phase extraction advances for bioseparation. Journal of Bioprocessing & Biotechniques, 4, 1–8.

    Google Scholar 

  30. Liu, Y., Guo, H. P., Gu, J. L., & Qin, W. S. (2019). Optimize purification of a cellulase from Bacillus velezensis A4 by aqueous two-phase system (ATPS) using response surface methodology. Process Biochemistry, 87, 196–203.

    Article  CAS  Google Scholar 

  31. Forciniti, D., & Hall, C. K. (1991). Protein partitioning at isoelectric point: Influence of polymer molecular weight and concentration and protein size. Biotechnology and Bioengineering, 38, 986–994.

    Article  CAS  PubMed  Google Scholar 

Download references

Funding

The authors thank the financial support by the young investigator grant program of National Natural Science Foundation of China (21606128) and the Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture (XTE1833).

Author information

Authors and Affiliations

  1. College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30, Puzhu South Road, Nanjing, 211816, China

    Xiaochun Chen, Xiaoquan Huang, Yiwen Tang & Lei Zhang

Authors
  1. Xiaochun Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiaoquan Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yiwen Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lei Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Xiaochun Chen.

Ethics declarations

Ethics Approval

Not applicable.

Conflict of Interest

The authors declare no competing interests.

Additional information

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

Chen, X., Huang, X., Tang, Y. et al. Efficient Purification of Nuclease P1 from Penicillium citrinum Using Polyethylene Glycol/Disodium Guanosine Monophosphate Aqueous Two-Phase System. Appl Biochem Biotechnol 193, 3753–3764 (2021). https://doi.org/10.1007/s12010-021-03637-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-021-03637-2

Keywords

Search

Navigation