Skip to main content
SpringerLink
Log in

Purification of nattokinase by reverse micelles extraction from fermentation broth: effect of temperature and phase volume ratio

  • Original paper
  • Published:
Bioprocess and Biosystems Engineering Aims and scope Submit manuscript

Abstract

Nattokinase is a novel fibrinolytic enzyme that is considered to be a promising agent for thrombosis therapy. In this study, reverse micelles extraction was applied to purify and concentrate nattokinase from fermentation broth. The effects of temperature and phase volume ratio used for the forward and backward extraction on the extraction process were examined. The optimal temperature for forward and backward extraction were 25°C and 35°C respectively. Nattokinase became more thermosensitive during reverse micelles extraction. And it could be enriched in the stripping phase eight times during backward extraction. It was found that nattokinase could be purified by AOT reverse micelles with up to 80% activity recovery and with a purification factor of 3.9.

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

Similar content being viewed by others

References

  1. Krishna SH, Srinivas ND, Raghavarao KSMS, Karanth NG (2002) Reverse micella extraction for down stream processing of proteins/enzymes. Adv Biochem Eng Biotech 75:124-183

    Google Scholar 

  2. Chee JY, Cabral JMS, Hatton TA (1988) Recovery of an extracellular alkaline protease from whole fermentation broth using reversed micelles. Biotechnol Prog 4:218-224

    Article  Google Scholar 

  3. Chang QL, Chen JY (1995) Separation and purification of two enzymes from Bacillus subtilis using Aliquat 336 reversed micelles: study of the effect of cosolvent concentration. Chem Eng J 59:303-308

    CAS  Google Scholar 

  4. Pessoa AJ, Vitolo M. (1997) Separation of inulinase from Kluyveromyces marxianus using reversed micellar extraction. Biotechnol Tech 11:421-422

    Article  CAS  Google Scholar 

  5. Rodrigues EMG, Milagres AMF, Pessoa A Jr (1999) Xylanase recovery: effect of extraction conditions on the AOT-reversed micellar systems using experimental design. Process Biochem 34:121–125

    Article  CAS  Google Scholar 

  6. Soni K, Madamwar D (2000) Reversed micellar extraction of an extracellular acid phosphatase from fermentation broth. Process Biochem 36:311–315

    Article  CAS  Google Scholar 

  7. Zhang TX, Liu HZ, Chen JY (2001) Selective affinity separation of yeast alcohol dehydrogenase by reverse micelles with unbound triazine dye. Chinese J of Chem Eng 9:314-318

    CAS  Google Scholar 

  8. Jarudilokkul S, Paulsen E, Stuckey DC (2000) The effect of demulsifiers on lysozyme extraction from egg white using reverse micelles. Biosep 9:81-91

    Article  CAS  Google Scholar 

  9. Jarudilokkul S, Stuckey DC (2001) Continuous forward and back extraction of lysozyme from egg white using reverse micelles. Sep Sci Technol 36:657-670

    Article  CAS  Google Scholar 

  10. Jarudilokkul S, Paulsen E, Stuckey DC (2000) Lysozyme extraction from egg white using reverse micelles in a Graesser contactor: mass transfer characterization. Biotechnol Bioeng 69:618-626

    Article  PubMed  CAS  Google Scholar 

  11. Jarudilokkul S, Paulsen E, Stuckey DC (2000) Lysozyme extraction from egg white using reverse micelles in a Graesser contactor: mass transfer characterization. Biotechnol Bioeng 69:618-626

    Article  PubMed  CAS  Google Scholar 

  12. Nishiki T, Sato I, Kataoka T, Kato D (1993) Partitioning behavior and enrichment of proteins with reversed micellear extraction: 1 Forward extraction of proteins from aqueous to reversed micellar phase. Biotechnol Bioeng 42:596-600

    Article  CAS  Google Scholar 

  13. Nishiki T, Sato I, Kataoka T, Kato D (1995) Back extraction of proteins from reversed rnicellar to aqueous phase: partitioning behavior and enrichment. Chem Eng J 59:297-301

    CAS  Google Scholar 

  14. Regalado C, Asenjo JA, Pyle D Leo (1994) Protein extraction by reverse micelles: studies on the recovery of horseradish peroxidase. Biotechnol Bioeng 44:674-681

    Article  CAS  Google Scholar 

  15. Larsson KM, Adlercreutz P, Mattiasson B (1990) Enzymatic catalysis in microemulsions:enzyme reuse and product recovery. Biotechnol Bioeng 36:135-141

    Article  CAS  Google Scholar 

  16. Huang SY, Chang HL (1995) Separation and purification of horseradish peroxidase from Armoracia rusticana root using reversed micellar extraction. Bioseparation 5:225

    CAS  Google Scholar 

  17. Kommareddi NS, John VT, Waguespack YY, McPherson GL (1993) Temperature and gas pressure induced microstructural changes in AOT water-in-oil microemulsions: characterization through electron paramagnetic resonance spectroscopy. J Phys Chem 97:5752-5761

    Article  CAS  Google Scholar 

  18. Sumi H, Hamado H, Tsushima H, Mihara H, Murica H (1987) A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto: a typical and popular soybean food in the Japanese diet. Experimentia 43:1110-1111

    Article  CAS  Google Scholar 

  19. Fujita M, Hong K, Ito Y, Fuji R, Kariya K, Nishimuro S (1995) Thrombolytic effect of nattokinase on a chemically induced thrombosis model in rat. Biol Pharm Bul1 18:1387-1391

    CAS  Google Scholar 

  20. Sumi H, Hamada H, Nakanishi K, Hiratani H (1990) Enhancement of the fibrinolytic activity in plasma by oral administration of nattokinase. Acta Haemotol 84:139-143

    CAS  Google Scholar 

  21. Fujita M, Hong K, Ito Y, Misawa S, Takeuchi N, Kariya K, Nishimuro S (1995) Transport of nattokinase across the rat intestinal tract. Biol Pharm Bull 18:1194-1196

    PubMed  CAS  Google Scholar 

  22. Fujita M, Nomura K, Hong K, Ito Y, Asada A, Nishimuro S (1993) Purification and characterization of a strong fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto, a popular soybean fermented food in Japan. Biochem Bioph Res Co 197:1340-1347

    Article  CAS  Google Scholar 

  23. Liu JG, Xing JM, Shen R, Yang CL, Liu HZ (2004) Reverse micellar extraction of nattokinase from fermentation broth. Biochem Eng J 21:273-278

    Article  CAS  Google Scholar 

  24. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254

    Article  PubMed  CAS  Google Scholar 

  25. Ansen ML (1939) The estimation of pepsin, trypsin, papain, and cathepsin with hemoglobin. J Gen Physiol 22:78-79

    Google Scholar 

  26. Dekker M (1990) Enzyme recovery using reversed micelles, PhD Thesis, Agricultural University of Wageningen, The Netherlands

  27. Yu YC, Chu Y, Ji JY (2003) Study of the factors affecting the forward and back extraction of yeast-lipase and its activity by reverse micelles. J Coll Interface Sci 267:60-64

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors acknowledge the financial support from the National Natural Science Foundation of China (No.20221603).

Author information

Authors and Affiliations

  1. College of Bioscience and Bioengineering, Hebei University of Science and Technology, 050018, Shijiazhuang, Hebei Province, China

    Jun-Guo Liu & Tian-Shi Chang

  2. Laboratory of Separation Science and Engineering, Institute of Process Engineering, Chinese Academy of Sciences, 100080, Beijing, China

    Jian-Min Xing & Hui-Zhou Liu

Authors
  1. Jun-Guo Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jian-Min Xing
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tian-Shi Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hui-Zhou Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jun-Guo Liu.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Liu, JG., Xing, JM., Chang, TS. et al. Purification of nattokinase by reverse micelles extraction from fermentation broth: effect of temperature and phase volume ratio. Bioprocess Biosyst Eng 28, 267–273 (2006). https://doi.org/10.1007/s00449-005-0032-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00449-005-0032-1

Keywords

Search

Navigation