Skip to main content
SpringerLink
Log in

Anionic surfactant based reverse micellar extraction of l-asparaginase synthesized by Azotobacter vinelandii

  • Research Paper
  • Published:
Bioprocess and Biosystems Engineering Aims and scope Submit manuscript

Abstract

l-Asparaginase synthesized by Azotobacter vinelandii via submerged fermentation in the presence of sucrose was successfully extracted using Reverse micellar extraction. Single step enzyme purification process was developed by varying the process variables which resulted in maximum specificity and extraction of l-asparaginase. The effect of different variables, including broth pH, addition of alcohol during the forward extraction and pH of the fresh stripping aqueous phase, addition of alcohol and electrolyte during backward extraction process were studied. Lower concentration of butanol resulted in maximum activity of the enzyme during forward extraction while enzyme activity was found to increase further with the addition of higher concentrations of ammonium sulphate during backward extraction. Chromatographic analysis of l-asparaginase peak at ~7.65 min was intense for the back extracted sample confirming the maximum purity of l-asparaginase obtained. Purity of l-asparaginase was increased to about 379.68 fold.

Graphical abstract

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

Similar content being viewed by others

References

  1. Nagarethinam S, Naik AN, Udupa N, Rao VJ, Vanathi MB (2012) Microbial l-asparaginase and its future prospects. Asian J Med Res 1:159–168

    Google Scholar 

  2. Verma N, Kumar K, Kaur G, Anand S (2007) l-asparaginase: a promising chemotherapeutic agent. Crit Rev Biotechnol 27:45–62. doi:10.1080/07388550601173926

    Article  CAS  Google Scholar 

  3. Clementi A (1922) La désamidation enzymatique de l’asparagine chez les différentes espéces animales et la signification physio logique de sa presence dans l’organisme. Arch Int Physiol 19:369–398

    CAS  Google Scholar 

  4. Kidd JG (1953) Regression of transplanted lymphomas induced in vivo by means of normal guinea pig serum I. Course of transplanted cancers of various kinds in mice and rats given guinea pig serum, horse serum, or rabbit serum. J Exp Med 98:565–582

    Article  CAS  Google Scholar 

  5. Tsuji Y (1957) Studies on the amidase. IV. Supplemental studies on the amidase action of the bacteria. Japan Arch Internal Med 4:222–224

    CAS  Google Scholar 

  6. Krishnapura PR, Belur PD, Subramanya S (2016) A critical review on properties and applications of microbial l-asparaginases. CRC CR Rev Microbiol 42(5):720–737. doi:10.3109/1040841X.2015.1022505

    CAS  Google Scholar 

  7. Batool T, Makky EA, Jalal M, Yusoff MM (2016) A comprehensive review on l-asparaginase and its applications. Appl Biochem Biotechnol 178:900–923. doi:10.1007/s12010-015-1917-3

    Article  CAS  Google Scholar 

  8. Arif HM, Hussain Z (2014) Important sources and medicinal applications of l-asparaginase. Int J Pharm Sci Rev 3:35–45

    CAS  Google Scholar 

  9. Cedar H, Schwartz JH (1968) Production of l-asparaginase II by Escherichia coli. J Bacteriol 96:2043–2048

    CAS  Google Scholar 

  10. Campbell HA, Mashburn LT, Boyse EA, Old LJ (1967) Two l-asparaginases from Escherichia coli B. their separation, purification, and antitumor activity. Biochemistry 6:721–730. doi:10.1021/bi00855a011

    Article  CAS  Google Scholar 

  11. Mesas JM, Gil JA, Martín JF (1990) Characterization and partial purification of l-asparaginase from Corynebacterium glutamicum. Microbiology 136:515–519. doi:10.1099/00221287-136-3-515

    CAS  Google Scholar 

  12. Manna S, Sinha A, Sadhukhan R, Chakrabarty SL (1995) Purification, characterization and antitumor activity of l-asparaginase isolated from Pseudomonas stutzeri MB-405. Curr Microbiol 30:291–298. doi:10.1007/BF00295504

    Article  CAS  Google Scholar 

  13. del Valle EM, Freitas SC, Galán MA (2002) Separation of asparaginase and trypsin by affinity chromatography combined with batchwise adsorption and columnwise desorption. Process Biochem 38:551–557. doi:10.1016/S0032-9592(02)00167-X

    Article  Google Scholar 

  14. Kadam KL (1986) Reverse micelles as a bioseparation tool. Enzyme Microb Technol 8:266–273. doi:10.1016/0141-0229(86)90020-7

    Article  CAS  Google Scholar 

  15. Melo EP, Aires-Barros MR, Cabral JM (2001) Reverse micelles and protein biotechnology. Biotechnol Annu Rev 7:87–129. doi:10.1016/S1387-2656(01)07034-X

    Article  CAS  Google Scholar 

  16. Kilikian BV, Bastazin MR, Minami NM, Gonçalves EM, Junior AP (2000) Liquid-liquid extraction by reversed micelles in biotechnological processes. Braz J Chem Eng 17:29–38. doi:10.1590/S0104-66322000000100003

    Article  CAS  Google Scholar 

  17. Ichikawa S, Imai M, Shimizu M (1992) Solubilizing water involved in protein extraction using reversed micelles. Biotechnol Bioeng 39:20–26. doi:10.1002/bit.260390105

    Article  CAS  Google Scholar 

  18. Moore SA, Palepu RM (2007) Fluorometric investigations on the transition from reverse micelles to microemulsions in non-aqueous microemulsions. J Mol Liq 135:123–127. doi:10.1016/j.molliq.2006.11.003

    Article  CAS  Google Scholar 

  19. Perez-Gilabert M, Bru R, Sanchez-Ferrer A, García-Carmona F (1998) Hydroperoxidase activity of soybean lipoxygenase in reverse micelles. J Biotechnol 60:137–140. doi:10.1016/S0168-1656(97)00190-9

    Article  CAS  Google Scholar 

  20. Carvalho CM, Aires-Barros MR, Cabral JM (2000) Kinetics of cutinase catalyzed transesterification in AOT reversed micelles: modeling of a batch stirred tank reactor. J Biotechnol 81:1–3. doi:10.1016/S0168-1656(00)00260-1

    Article  CAS  Google Scholar 

  21. Gaikar VG, Kulkarni MS (2001) Selective reverse micellar extraction of penicillin acylase from E coli. J Chem Technol Biotechnol 76:729–736. doi:10.1002/jctb.444

    Article  CAS  Google Scholar 

  22. Motlekar NA, Bhagwat SS (2001) Activity of horseradish peroxidase in aqueous and reverse micelles and back-extraction from reverse-micellar phases. J Chem Technol Biotechnol 76:643–649. doi:10.1002/jctb.432

    Article  CAS  Google Scholar 

  23. Wu JC, He ZM, Yao CY, Yu KT (2001) Increased activity and stability of Candida rugosa lipase in reverse micelles formed by chemically modified AOT in isooctane. J Chem Technol Biotechnol 76:949–953. doi:10.1002/jctb.467

    Article  CAS  Google Scholar 

  24. Gaffar SA, Shethna YI (1977) Purification and some biological properties of Asparaginase from Azotobacter vinelandii. Appl Environ Microbiol 33:508–514

    CAS  Google Scholar 

  25. 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. doi:10.1016/0003-2697(76)90527-3

    Article  CAS  Google Scholar 

  26. Hilhorst R, Fijneman P, Heering D, Wolbert RB, Dekker M, Van’t Riet K, Bijsterbosch BH (1992) Protein extraction using reversed micelles. Pure Appl Chem 64:1765–1770. doi:10.1351/pac199264111765

    Article  CAS  Google Scholar 

  27. Krei GA, Hustedt H (1992) Extraction of enzymes by reverse micelles. Chem Eng Sci 47:99–111. doi:10.1016/0009-2509(92)80204-P

    Article  CAS  Google Scholar 

  28. Alves JR, Fonseca LP, Ramalho MT, Cabral JM (2003) Optimisation of penicillin acylase extraction by AOT/isooctane reversed micellar systems. Biochem Eng J 15:81–86. doi:10.1016/S1369-703X(02)00181-X

    Article  CAS  Google Scholar 

  29. Lee BK, Hong DP, Lee SS, Kuboi R (2004) Analysis of protein back-extraction processes in alcohol-and carboxylic acid-mediated AOT reverse micellar systems based on structural changes of proteins and reverse micelles. Biochem Eng J 22:71–79. doi:10.1016/j.bej.2004.08.004

    Article  CAS  Google Scholar 

  30. Chang QL, Chen JY, Zhang XF, Zhao NM (1997) Effect of the cosolvent type on the extraction of α-amylase with reversed micelles: circular dichroism study. Enzyme Microb Technol 20:87–92. doi:10.1016/S0141-0229(96)00088-9

    Article  CAS  Google Scholar 

  31. Krishna SH, Srinivas ND, Raghavarao KS, Karanth NG (2002) Reverse micellar extraction for downstream processing of proteins/enzymes. History and trends in bioprocessing and biotransformation. Springer, Berlin, Heidelberg, pp 119–183. ISSN:07246145

  32. Mathew DS, Juang RS (2007) Role of alcohols in the formation of inverse microemulsions and back extraction of proteins/enzymes in a reverse micellar system. Sep Purif Technol 53:199–215. doi:10.1016/j.seppur.2006.10.001

    Article  CAS  Google Scholar 

  33. Hanstein WG (1979) Chaotropic ions and their interactions with proteins. Appl Biochem Biotechnol 4:189–206. doi:10.1007/BF02991894

    CAS  Google Scholar 

  34. Baldwin RL (1996) How Hofmeister ion interactions affect protein stability. Biophys J 71:2056. ISSN:00063495

  35. Nandini KE, Rastogi NK (2009) Reverse micellar extraction for downstream processing of lipase: effect of various parameters on extraction. Process Biochem 44:1172–1178. doi:10.1016/j.procbio.2009.06.020

    Article  CAS  Google Scholar 

  36. Gaikaiwari RP, Wagh SA, Kulkarni BD (2012) Efficient lipase purification using reverse micellar extraction. Bioresour Technol 108:224–230. doi:10.1016/j.biortech.2011.11.126

    Article  CAS  Google Scholar 

  37. Qin M, Zhao F (2003) l-Asparaginase release from Escherichia coli cells with aqueous two-phase micellar systems. Appl Biochem Biotechnol 110:11–21. doi:10.1385/ABAB:110:1:11

    Article  CAS  Google Scholar 

  38. Kumar S, Dasu VV, Pakshirajan K (2011) Purification and characterization of glutaminase-free l-asparaginase from Pectobacterium carotovorum MTCC 1428. Bioresour Technol 102:2077–2082. doi:10.1016/j.biortech.2010.07.114

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Chemical Engineering, National Institute of Technology Karnataka, Mangalore, 575025, India

    Sivananth Murugesan, Regupathi Iyyaswami, Sahana Vijay Kumar & Arathi Surendran

Authors
  1. Sivananth Murugesan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Regupathi Iyyaswami
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sahana Vijay Kumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Arathi Surendran
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Regupathi Iyyaswami.

Ethics declarations

Conflict of interest

All the authors of this manuscript express no conflict of interest towards the article submitted.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Murugesan, S., Iyyaswami, R., Kumar, S.V. et al. Anionic surfactant based reverse micellar extraction of l-asparaginase synthesized by Azotobacter vinelandii . Bioprocess Biosyst Eng 40, 1163–1171 (2017). https://doi.org/10.1007/s00449-017-1777-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00449-017-1777-z

Keywords

Search

Navigation