Skip to main content
SpringerLink
Log in

Preparation of carboxylated magnetic particles for the efficient immobilization of C-terminally lysine-tagged Bacillus stearothermophilus aminopeptidase II

  • Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

This article reports the synthesis and use of surface-modified iron oxide particles for the simultaneous purification and immobilization of Bacillus stearothermophilus aminopeptidase II (BsAPII) tagged C-terminally with either tri- or nona-lysines (BsAPII-Lys3/9). The carboxylated magnetic particles were prepared by the simple co-precipitation of Fe3+/Fe2+ in aqueous medium and then subsequently modified with adipic acid. Transmission electron microscopy (TEM) micrographs showed that the carboxylated magnetic particles remained discrete and had no significant change in size after binding BsAPIIs. Wild-type enzyme and BsAPII-Lys3 could be purified to near homogeneity by the carboxylated magnetic particles, but it was not easy to elute the adsorbed BsAPII-Lys9 from the matrix. Free BsAPII, BsAPII-Lys3, and BsAPII-Lys9 were active in the temperature range 50–70°C and all had an optimum of 50°C, whereas the optimum temperature and thermal stability of BsAPII-Lys3 and BsAPII-Lys9 were improved as a result of immobilization. The immobilized BsAPII-Lys9 could be recycled ten times without a significant loss of the enzyme activity and had a better stability during storage than BsAPII. Owing to its high efficiency and cost-effectiveness, this magnetic adsorbent may be used as a novel purification-immobilization system for the positively charged enzymes.

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
Fig. 8

Similar content being viewed by others

References

  1. Arnau J, Lauritzen C, Petersen GE, Pedersen J (2006) Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins. Protein Expr Purif 48:1–13

    Article  CAS  PubMed  Google Scholar 

  2. Salemuddin M (1999) Bioaffinity based immobilization of enzymes. In: Fiechter A (ed) Advances in biochemical engineering/biotechnology, vol 64. Springer, Berlin, pp 203–226

    Google Scholar 

  3. Bahar T, Celebi SS (1999) Immobilization of glucoamylase on magnetic poly(styrene) particles. J Appl Polym Sci 72:69–73

    Article  CAS  Google Scholar 

  4. Koneracká M, Kopčanský P, Timko M, Ramchand CN, de Sequeira A, Trevan M (2002) Direct binding procedure of proteins and enzymes to fine magnetic particles. J Mol Catal B: Enzym 18:13–18

    Article  Google Scholar 

  5. Lei H, Wang W, Chen LL, Li XC, Yi B, Deng L (2004) The preparation and catalytically active characterization of papain immobilized on magnetic composite microspheres. Enzyme Microb Technol 35:15–21

    Article  CAS  Google Scholar 

  6. Chien LJ, Lee CK (2008) Biosilicification of dual-fusion enzyme immobilized on magnetic nanoparticle. Biotechnol Bioeng 100:223–230

    Article  CAS  PubMed  Google Scholar 

  7. Lo HF, Hu HY, Hung CP, Chen SC, Lin LL (2009) Cobalt-chelated magnetic particles for one-step purification and immobilization of His6-tagged Escherichia coli γ-glutamyltranspeptidase. Biocatal Biotransform 27:318–327

    Article  CAS  Google Scholar 

  8. Hanefeld U, Gardossi L, Magner E (2009) Understanding enzyme immobilization. Chem Soc Rev 38:453–468

    Article  CAS  PubMed  Google Scholar 

  9. Durán N, Rosa MA, D’Annibale A, Gianfreda L (2002) Applications of laccases and tyrosinases (phenoloxidase) immobilized on different supports: a review. Enzyme Microb Technol 31:907–931

    Article  Google Scholar 

  10. Girelli AM, Mattei J (2005) Application of immobilized enzyme reactor in on-line high performance liquid chromatography: a review. J Chromatogr B 819:3–16

    Article  CAS  Google Scholar 

  11. Wang G, Xu JJ, Ye LH, Zhu JJ, Chen HY (2002) Highly sensitive sensors based on the immobilization of tyrosinase in chitosan. Bioelectrochemistry 57:33–38

    Article  CAS  PubMed  Google Scholar 

  12. Petrović J, Clark RA (2005) Impact of surface immobilization and solution ionic strength on the formal potential of immobilized cytochrome. Langmuir 21:6308–6316

    Article  PubMed  Google Scholar 

  13. Goldberg AL, Cascio P, Saric T, Rock KL (2002) The importance of the proteasome and subsequent proteolytic steps in the generation of antigenic peptides. Mol Immunol 39:147–164

    Article  CAS  PubMed  Google Scholar 

  14. Terenius L, Sandin J, Sakurada T (2002) Nociceptin/orphanin FQ metabolism and bioactive metabolites. Peptides 21:919–922

    Article  Google Scholar 

  15. Pautot V, Holzer FM, Reisch B, Walling LL (1993) Leucine aminopeptidase: an inducible component of the defense response in Lycopersion esculentum (tomato). Proc Natl Acad Sci USA 90:9906–9910

    Article  CAS  PubMed  Google Scholar 

  16. Fitzgerald RJ, O’Cuinn G (2006) Enzymatic debittering of food protein hydrolysates. Biotechnol Adv 24:234–237

    Article  CAS  PubMed  Google Scholar 

  17. Wyvratt MJ (1988) Evolution of angiotensin-converting enzyme inhibitors. Clin Physiol Biochem 6:217–229

    CAS  PubMed  Google Scholar 

  18. Kuo LY, Hwang GY, Lai YJ, Yang SL, Lin LL (2003) Overexoression, purification, and characterization of the recombinant leucine aminopeptidase II of Bacillus stearothermophilus. Curr Microbiol 47:40–45

    Article  CAS  PubMed  Google Scholar 

  19. Kuo LY, Hwang GY, Yang SL, Hua YW, Chen W, Lin LL (2004) Inactivation of Bacillus stearothermophilus leucine aminopeptidase II by hydrogen peroxide and site-directed mutagenesis of methionine resiudes on the enzyme. Protein J 23:295–302

    Article  CAS  PubMed  Google Scholar 

  20. Hwang GY, Kuo LY, Tsai MR, Yang SL, Lin LL (2005) Histidines 345 and 378 of Bacillus stearothermophilus leucine aminopeptidase II are essential for the catalytic activity of the enzyme. Antonie van Leeuwenhoek 87:355–359

    Article  CAS  PubMed  Google Scholar 

  21. Yang SL, Chen RS, Chen W, Lin LL (2006) Identification of glutamate residues important for catalytic activity of Bacillus stearothermophilus leucine aminopeptidase II. Antonie van Leeuwenhoek 90:195–199

    Article  CAS  PubMed  Google Scholar 

  22. Wu CL, Chen YP, Yang JC, Lo HF, Lin LL (2008) Characterization of lysine-tagged Bacillus stearothermophilus leucine aminopeptidase II immobilized onto carboxylated gold nanoparticles. J Mol Catal B: Enzym 54:83–89

    Article  CAS  Google Scholar 

  23. Stoll E, Weder HG, Zuber H (1976) Aminopeptidase II from Bacillus stearothermophilus. Biochem Biophys Acta 438:212–220

    CAS  Google Scholar 

  24. Khalafalla SE, Reimers GW (1980) Preparation of dilution-stable aqueous magnetic fluids. IEEE Trans Magn 16:178–183

    Article  Google Scholar 

  25. Tsang SC, Yu CH, Gao X, Tam K (2006) Silica-encapsulated nanomagnetic particle as a new recoverable biocatalyst carrier. J Phys Chem B 110:16914–16922

    Article  CAS  PubMed  Google Scholar 

  26. Li J, Wang J, Gavalas VG, Atwood DA, Bachas LG (2003) Alumina–pepsin hybrid nanoparticles with orientation-specific enzyme coupling. Nano Lett 3:55–58

    Article  CAS  Google Scholar 

  27. Takahashi H, Li B, Sasaki T, Miyazaki C, Kajino T, Inagaki S (2000) Catalytic activity in organic solvents and stability of immobilized enzymes depend on the pore size and surface characteristics of mesoporous silica. Chem Mater 12:3301–3305

    Article  CAS  Google Scholar 

  28. Akgol S, Yalcinkaya Y, Bayramoglu A, Denizli A, Arica MY (2002) Reversible immobilization of urease onto Procion Brown MX-5BR-Ni(II) attached polyamide hollow-fibre membranes. Process Biochem 38:675–683

    Article  CAS  Google Scholar 

  29. Li GY, Huang KL, Jiang YR, Yang DL, Ding P (2008) Preparation and characterization of Saccharomyces cerevisiae alcohol dehydrogenase immobilized on magnetic nanoparticles. Int J Biol Macromol 42:405–412

    Article  CAS  PubMed  Google Scholar 

  30. Liao MH, Chen DH (2001) Immobilization of yeast alcohol dehydrogenase on magnetic nanoparticles for improving its stability. Biotechnol Lett 23:3234–3241

    Article  Google Scholar 

  31. Chen DH, Liao MH (2002) Preparation and characterization of YADH-bound magnetic nanoparticles. J Mol Catal B: Enzym 16:283–291

    Article  CAS  Google Scholar 

Download references

Acknowledgments

Financial support from the National Science Council of Taiwan (NSC 97-2628-B-415-001-MY3) is gratefully acknowledged.

Author information

Authors and Affiliations

  1. Department of Applied Chemistry, National Chiayi University, 300 University Road, Chiayi, Taiwan

    Cheng-Liang Huang, Wei-Chun Cheng, Jia-Ci Yang, Meng-Chun Chi & Long-Liu Lin

  2. Department of Food Science and Technology, Chia-Nan University of Pharmacy and Science, Tainan, Taiwan

    Jiau-Hua Chen

  3. Department of Chemistry, National Cheng Kung University, Tainan, Taiwan

    Hong-Ping Lin

Authors
  1. Cheng-Liang Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wei-Chun Cheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jia-Ci Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Meng-Chun Chi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jiau-Hua Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hong-Ping Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Long-Liu Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Long-Liu Lin.

Additional information

C.-L. Huang and W.-C. Cheng contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Huang, CL., Cheng, WC., Yang, JC. et al. Preparation of carboxylated magnetic particles for the efficient immobilization of C-terminally lysine-tagged Bacillus stearothermophilus aminopeptidase II. J Ind Microbiol Biotechnol 37, 717–725 (2010). https://doi.org/10.1007/s10295-010-0715-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10295-010-0715-8

Keywords

Search

Navigation