Skip to main content
SpringerLink
Log in

Reversible Activation of Halophilic β-lactamase from Methanol-Induced Inactive Form: Contrast to Irreversible Inactivation of Non-Halophilic Counterpart

  • Published:
The Protein Journal Aims and scope Submit manuscript

Abstract

Effects of a water-miscible organic solvent, methanol, on the structure and activity of halophilic β-lactamase derived from Chromohalobacter sp.560 (HaBla), were investigated by means of circular dichroism (CD) measurement and enzymatic activity determination. Beta-lactamase activity was enhanced about 1.2-fold in the presence of 10–20% methanol. CD measurement of HaBla revealed different structures depending on the methanol concentration: native-like active form (Form I) in 10–20% methanol and methanol-induced inactive form at higher concentration (Form II in 40–60% and Form III in 75–80% methanol). Incubation of HaBla with 40% methanol led to the complete loss of activity within ~80 min accompanied by the formation of Form II, whose activity was recovered promptly up to ~80% of full activity upon dilution of the methanol concentration to 10%. In addition, when the protein concentration was sufficiently high (e.g., 0.7 mg/ml), HaBla activity of Form III in 75% methanol could be recovered in the same way (with slightly slower recovery rate), upon dilution of the methanol concentration. In contrast, non-halophilic β-lactamase from Escherichia coli K12 strain MG1655 (EcBla) was irreversibly denatured in the presence of 40% methanol. HaBla showed remarkable ability to renature from the methanol-induced inactive states.

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

Abbreviations

SDS-PAGE:

SDS-polyacrylamide gel electrophoresis

CD:

Circular dichroism

HaBla:

Halophilic β-lactamase

EcBla:

E.coli β-lactamase

References

  1. Kushner DJ (1978) Life in high salt and solute concentrations. In: Kushner DJ (ed) Microbial life in extreme environments. Academic Press, London, pp 317–368

  2. Eisenberg H, Mevarech M, Zaccai G (1992) Bichemical, structural and molecular genetic aspects of halophilism. Adv Protein Chem 43:1–62

    Article  CAS  Google Scholar 

  3. Ventosa A, Nieto JJ, Oren A (1998) Biology of moderately halophilic aerobic bacteria. Microbiol Mol Biol Rev 62:504–544

    CAS  Google Scholar 

  4. Madern D, Ebel C, Zaccai G (2000) Halophilic adaptation of enzymes. Extremophiles 4:91–98

    Article  CAS  Google Scholar 

  5. Mevarech M, Frolow F, Gloss LM (2000) Halophilic enzymes: proteins with a grain of salt. Biophys Chem 86:155–164

    Article  CAS  Google Scholar 

  6. Tokunaga H, Ishibashi M, Arakawa T, Tokunaga M (2004) Highly efficient renaturation of β-lactamase isolated from moderately halophilic bacteria. FEBS Lett 558:7–12

    Article  CAS  Google Scholar 

  7. Arai S, Yonezawa Y, Okazaki N, Matsumoto F, Shibazaki C, Shimizu R, Yamada M, Adachi M, Tamada T, Kawamoto M, Tokunaga H, Ishibashi M, Blaber M, Tokunaga M, Kuroki R (2015) Structure of a highly acidic β-lactamase from the moderate halophile Chromohalobacter sp. 560 and the discovery of a Cs(+)-selective binding site. Acta Crystallogr D 71:541–554

    Article  CAS  Google Scholar 

  8. Fukushima T, Mizuki T, Echigo A, Inoue A, Usami R (2005) Organic solvent tolerance of halophilic α-amylase from a Haloarchaeon, Haloarcula sp. strain S-1. Extremophiles 9:85–89

    Article  CAS  Google Scholar 

  9. Shafiei M, Ziaee AA, Amoozegar MA (2012) Purification and characterization of a halophilic α-amylase with increased activity in the presence of organic solvents from the moderately halophilic Nesterenkonia sp. strain F. Extremophiles 16:627–635

    Article  CAS  Google Scholar 

  10. Chang J, Lee YS, Fang SJ, Park IH, Choi YL (2013) Recombinant expression and characterization of an organic-solvent-tolerant α-amylase from Exiguobacterium sp. DAU5. Appl Biochem Biotechnol 169:1870–1883

    Article  CAS  Google Scholar 

  11. Karbalaei-Heidari HR, Shahbazi M, Absalan G (2013) Characterization of a novel organic solvent tolerant protease from a moderately halophilic bacterium and its behavior in ionic liquids. Appl Biochem Biotechnol 170:573–586

    Article  CAS  Google Scholar 

  12. Elbanna K, Ibrahim IM, Revol-Junelles AM (2015) Purification and characterization of halo-alkali-thermophilic protease from Halobacterium sp. strain HP25 isolated from raw salt, Lake Qarun, Fayoum, Egypt. Extremophiles 19:763–774

    Article  CAS  Google Scholar 

  13. Perez D, Martin S, Fernandez-Lorente G, Filice M, Guisan JM, Ventosa A, Garcia MT, Mellado E (2011) A novel halophilic lipase, LipBL, showing high efficiency in the production of eicosapentaenoic acid (EPA). PLoS ONE 6:e23325

    Article  CAS  Google Scholar 

  14. Munawar N, Engel PC (2012) Overexpression in a non-native halophilic host and biotechnological potential of NAD+-dependent glutamate dehydrogenase from Halobacterium salinarum strain NRC-36014. Extremophiles 16:463–476

    Article  CAS  Google Scholar 

  15. Li X, Wang HL, Li T, Yu HY (2012) Purification and characterization of an organic solvent-tolerant alkaline cellulase from a halophilic isolate of Thalassobacillus. Biotechnol Lett 34:1531–1536

    Article  CAS  Google Scholar 

  16. Siroosi M, Amoozegar MA, Khajeh K, Fazeli M, Rezaei MH (2014) Purification and characterization of a novel extracellular halophilic and organic solvent-tolerant amylopullulanase from the haloarchaeon, Halorubrum sp. strain Ha25. Extremophiles 18:25–33

    Article  CAS  Google Scholar 

  17. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor, New York

    Google Scholar 

  18. Mizukami M, Hanagata H, Miyauchi A (2010) Brevibacillus expression system: host-vector system for efficient production of secretory proteins. Curr Pharm Biotechnol 11:251–258

    Article  CAS  Google Scholar 

  19. Onishi H, Mizukami M, Hanagata H, Tokunaga M, Arakawa T, Miyauchi A (2013) Efficient production of anti-fluorescein and anti-lysozyme as single-chain anti-body fragments (scFv) by Brevibacillus expression system. Protein Expr Purif 91:184–191

    Article  CAS  Google Scholar 

  20. Yonezawa Y, Nagayama A, Tokunaga H, Ishibashi M, Arai S, Kuroki R, Watanabe K, Arakawa T, Tokunaga M (2015) Nucleoside diphosphate kinase from psychrophilic Pseudoalteromonas sp. AS-131 isolated from Antarctic Ocean. Protein J 34:275–283

    Article  CAS  Google Scholar 

  21. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  Google Scholar 

  22. Powers RA, Shoichet BK (2002) Structure-based approach for binding site identification on AmpC β-lactamase. J Med Chem 45:3222–3234

    Article  CAS  Google Scholar 

  23. Tokunaga H, Oda Y, Yonezawa Y, Arakawa T, Tokunaga M (2006) Contribution of halophilic nucleoside diphosphate kinase sequence to the heat stability of chimeric molecule. Protein Pept Lett 13:525–530

    Article  CAS  Google Scholar 

  24. Lehrman SR, Tuls JL, Lund M (1990) Peptie α-helicity in aqueous trifluoroethanol: correlations with predicted α-helicity and the secondary structure of the corresponding regions of bovine growth hormone. Biochemistry 29:5590–5596

    Article  CAS  Google Scholar 

  25. Lee HW, Jung WK, Kim YH, Ryu BH, Kim TD, Kim J, Kim H (2016) Characterization of a novel alkaline family VIII esterase with S-enantiomer preference from a compost metagenomic library. J Microbiol Biotechnol 26:315–325

    Article  CAS  Google Scholar 

  26. Herskovits TT, Gadegbeku B, Jaillet H (1970) On the structural stability and solvent denaturation of proteins: I. Denaturation by the alcohols and glycols. J Biol Chem 245:2588–2598

    CAS  Google Scholar 

  27. Dwyer DS, Bradley RJ (2000) Chemical properties of alcohols and their protein binding sites. Cell Mol Life Sci 57:265–275

    Article  CAS  Google Scholar 

  28. Deshpande A, Nimsadkar S, Mande SC (2005) Effect of alcohols on protein hydration: crystallographic analysis of hen egg-white lysozyme in the presence of alcohols. Acta Crystallogr D 61:1005–1008

    Article  Google Scholar 

  29. Mozhaev VV (1998) Egineering stability of enzymes in systems with organic solvent. Prog Biotechnol 15:355–363

    Article  CAS  Google Scholar 

  30. Mattos C, Ringe D (2001) Proteins in organic solvents. Curr Opin Struct Biol 11:761–764

    Article  CAS  Google Scholar 

  31. Talon R, Coquelle N, Madern D, Girard E (2014) An experimental point of view on hydration/solvation in halophilic proteins. Front Microbiol 5:66

    Article  Google Scholar 

  32. Biedermannova L, Schneider B (2015) Structure of the ordered hydration of amino acids in proteins: analysis of crystal structures. Acta Crystallogr D 71:2192–2202

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors wish to acknowledge Dr. S. Arai for fruitful discussion. We also thank Drs. H. Onishi, M. Mizukami, H. Hanagata, and A. Miyauchi for helpful discussions on Brevibacillus expression.

Author information

Authors and Affiliations

  1. Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima, 890-0065, Japan

    Hiroko Tokunaga, Junpei Maeda & Masao Tokunaga

  2. Alliance Protein Laboratories, 6042 Cornerstone Court West, Suite A, San Diego, CA, 92121, USA

    Tsutomu Arakawa

Authors
  1. Hiroko Tokunaga
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Junpei Maeda
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tsutomu Arakawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Masao Tokunaga
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Masao Tokunaga.

Ethics declarations

Conflict of interest

The authors declare that they have no conflicts of interest.

Reseach Involving in Human and Animal Rights

This article does not contain any studies with human participants or animals performed by any of the authors.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Tokunaga, H., Maeda, J., Arakawa, T. et al. Reversible Activation of Halophilic β-lactamase from Methanol-Induced Inactive Form: Contrast to Irreversible Inactivation of Non-Halophilic Counterpart. Protein J 36, 228–237 (2017). https://doi.org/10.1007/s10930-017-9715-0

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10930-017-9715-0

Keywords

Search

Navigation