Skip to main content
SpringerLink
Log in

Cloning, Overexpression, and Characterization of Halostable, Solvent-Tolerant Novel β-Endoglucanase from a Marine Bacterium Photobacterium panuliri LBS5T (DSM 27646T)

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

Abstract

A 1329 nucleotide long endoglucanase gene was amplified from marine bacterium Photobacterium panuliri strain LBS5T.The enzyme sequence was novel as protein-based similarity search revealed that it shared maximum similarity of 99 % with hypothetical protein of P. aquae and 40 % with endoglucanase of P. marinum AK15. The gene was cloned, overexpressed in Escherichia coli BL21 (DE3), and purified up to homogeneity using Ni-NTA affinity chromatography. The purified enzyme, designated as Cel8, was monomeric and has a molecular mass of 53 kDa. The enzyme was halostable and exhibited optimal carboxymethyl cellulase (CMCase) activity and stability at 2 M NaCl. Optimal activity was obtained at 40 °C and at pH 4. The enzyme exhibited remarkable stability in different organic solvents (50 %, v/v), and activity increased nearly 1.5-fold in presence of butanol, isopropanol, petroleum ether, benzene, acetone, and n-hexane. It was active in Ca2+, Ba2+, and Ni2+ and inhibited by Co2+, Cd2+, Zn2+, Cu2+, and Hg2+. Under normal physiological conditions, the enzyme has 25 % helix, 30 % sheets, and 56 % irregularities, whereas salt leads to helix to sheet transition in enzyme. Three-dimensional reconstruction analysis revealed that the enzyme has (α/β)8 structure and a TIM barrel fold-like structure at the central groove of enzyme. This is the first evidenced report on halostable, organic solvent tolerant cellulase in the marine bacterial genus Photobacterium.

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. Zhang, X. Z., & Zhang, Y. H. P. (2013). Cellulases: characteristics, sources, production, and applications. In S. T. Yang, H. A. El-Enshasy, & N. Thongchul (Eds.), Bioprocessing technologies in biorefinery for sustainable production of fuels, chemicals, and polymers. Hoboken: John Wiley & Sons, Inc.

    Google Scholar 

  2. Azevedo, H., Bishop, D., & Cavaco-Paulo, A. (2000). Effects of agitation level on the adsorption, desorption, and activities on cotton fabrics of full length and core domains of EGV (Humicola insolens) and CenA (Cellulomonas fimi). Enzyme and Microbial Technology, 27, 325–329.

    Article  CAS  Google Scholar 

  3. Murashimaa, K., Nishimuraa, T., Nakamuraa, Y., Kogaa, J., Moriyab, T., Sumidab, N., et al. (2002). Purification and characterization of new endo-1,4-β-D-glucanases from Rhizopus oryzae. Enzyme and Microbial Technology, 30, 319–326.

    Article  Google Scholar 

  4. Maki, M., Leung, K. T., & Qin, W. (2009). The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass. International Journal of Biological Sciences, 5, 500–516.

    Article  CAS  Google Scholar 

  5. Gomez, J., & Steiner, W. (2004). The biocatalytic potential of extremophiles and extremozymes. Food Technology and Biotechnology, 2, 223–235.

    Google Scholar 

  6. Marhuenda-Egea, F. C., & Bonete, M. J. (2002). Extreme halophilic enzymes in organic solvents. Current Opinion in Biotechnology, 13, 385–389.

    Article  CAS  Google Scholar 

  7. Wang, C. Y., Hsieh, Y. R., Ng, C. C., Chan, H., Lin, H. T., Tzeng, W. S., & Shyu, Y. T. (2009). Purification and characterization of a novel halostable cellulase from Salinivibrio sp. strain NTU-05. Enzyme and Microbial Technology, 44, 373–379.

    Article  CAS  Google Scholar 

  8. Simankova, M. V., Chernych, N. A., Osipov, G. A., & Zavarzin, G. A. (1993). Halocella cellulolytica gen. nov., sp. nov., a new obligately anaerobic, halophilic, cellulolytic bacterium. Systematic and Applied Microbiology, 16, 385–389.

    Article  CAS  Google Scholar 

  9. Huang, X., Shao, Z., Hong, Y., Lin, L., Li, C., Huang, F., Wang, H., & Liu, Z. (2010). Cel8H, a novel endoglucanase from the halophilic bacterium Halomonas sp. S66-4: molecular cloning, heterogonous expression, and biochemical characterization. Journal of Microbiology, 48, 318–324.

    Article  CAS  Google Scholar 

  10. Hirasawa, K., Uchimura, K., Kashiwa, M., Grant, W. D., Ito, S., Kobayashi, T., & Horikoshi, K. (2006). Salt-activated endoglucanase of a strain of alkaliphilic Bacillus agaradhaerens. Antonie van Leeuwenhoek, 89, 211–219.

    Article  CAS  Google Scholar 

  11. Aygan, A., & Arikan, B. (2008). A new halo-alkaliphilic, thermostable endoglucanase from moderately halophilic Bacillus sp. C14 isolated from Van Soda Lake. International Journal of Agriculture and Biology, 10, 369–374.

    CAS  Google Scholar 

  12. Li, X., Wang, H. L., Li, T., & Yu, H. Y. (2012). Purification and characterization of an organic solvent-tolerant alkaline cellulase from a halophilic isolate of Thalassobacillus. Biotechnology Letters, 34, 1531–1536.

    Article  CAS  Google Scholar 

  13. Deep, K., Poddar, A., & Das, S. K. (2014). Photobacterium panuliri sp. nov., an alkalitolerant marine bacterium isolated from eggs of spiny lobster, Panulirus penicillatus from Andaman Sea. Current Microbiology, 69, 660–668.

    Article  CAS  Google Scholar 

  14. Tabor, S. (1990). Expression using the T7 RNA polymerase/promoter system. In F. A. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, & K. Struhl (Eds.), Current protocols in molecular biology (pp. 16.2.1–16.2.11). NY: Greene Publishing and Wiley-Interscience.

    Google Scholar 

  15. Boyd, J., Oza, M. N., & Murphy, J. R. (1990). Molecular cloning and DNA sequence analysis of a diphtheria tox iron-dependent regulatory element (dtxR) from Corynebacterium diphtheriae. Proceedings of the National Academy of Sciences of the United States of America, 87, 5968–5972.

    Article  CAS  Google Scholar 

  16. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., et al. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research, 25, 3389–3402.

    Article  CAS  Google Scholar 

  17. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.

    Article  CAS  Google Scholar 

  18. Bernfeld, P. (1955). Amylases α and β. Method Enzymol, 1, 149–158.

    Article  CAS  Google Scholar 

  19. Li, X., & Yu, H. Y. (2012). Purification and characterization of an organic-solvent-tolerant cellulase from a halotolerant isolate, Bacillus sp. L1. Journal of Industrial Microbiology and Biotechnology, 39, 1117–1124.

    Article  CAS  Google Scholar 

  20. Bailey, T. L., Bodén, M., Buske, F. A., Frith, M., Grant, C. E., et al. (2009). MEME SUITE: tools for motif discovery and searching. Nucleic Acids Research, 37, W202–W208.

    Article  CAS  Google Scholar 

  21. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., & Kumar, S. (2011). MEGA 5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 10, 2731–2739.

    Article  Google Scholar 

  22. Saitou, N., & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4, 406–42526.

    CAS  Google Scholar 

  23. Ciria, R., Abreu-Goodger, C., Morett, E., & Merino, E. (2004). GeConT: gene context analysis. Bioinformatics, 14, 2307–2308.

    Article  Google Scholar 

  24. Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N., & Sternberg, M. J. (2015). The Phyre2 web portal for protein modelling, prediction and analysis. Nature Protocols, 10, 845–858.

    Article  CAS  Google Scholar 

  25. Seeliger, D., & de Groot, B. L. (2010). Ligand docking and binding site analysis with PyMOL and Autodock/Vina. Journal of Computer-Aided Molecular Design, 24, 417–422.

    Article  CAS  Google Scholar 

  26. Roy, A., Yang, J., & Zhang, Y. (2012). COFACTOR: An accurate comparative algorithm for structure-based protein function annotation. Nucleic Acids Research, 40, W471–W477.

    Article  CAS  Google Scholar 

  27. Wiedemann, C., Bellstedt, P., & Görlach, M. (2013). CAPITO—a web server based analysis and plotting tool for circular dichroism data. Bioinformatics, 29, 1750–1757.

    Article  CAS  Google Scholar 

  28. Bohm, G., Muhr, R., & Jaenicke, R. (1992). CDNN: quantitative analysis of protein far UV circular dichroism spectra by neural networks. Protein Engineering, 5, 191–195.

    Article  CAS  Google Scholar 

  29. Baneyx, F. (1999). Recombinant protein expression in Escherichia coli. Current Opinion in Biotechnology, 10, 411–421.

    Article  CAS  Google Scholar 

  30. Klinke, H. B., Thomsen, A. B., & Ahring, B. K. (2004). Inhibition of ethanol producing yeast and bacteria by degradation products produced during pre-treatment of biomass. Applied Microbiology and Biotechnology, 66, 10–26.

    Article  CAS  Google Scholar 

  31. Park, J. I., Steen, E. J., Burd, H., Evans, S. S., Redding-Johnson, A. M., Batth, T., et al. (2012). A thermophilic ionic liquid-tolerant cellulase cocktail for the production of cellulosic biofuels. PLoS ONE, 7, e37010.

    Article  CAS  Google Scholar 

  32. Zhang, T., Datta, S., Eichler, J., Ivanova, N., Axen, S. D., et al. (2011). Identification of a haloalkaliphilic and thermostable cellulase with improved ionic liquid tolerance. Green Chemistry, 13, 2083–2090.

    Article  CAS  Google Scholar 

  33. Trincone, A. (2011). Marine biocatalysts: enzymatic features and applications. Marine Drugs, 9, 478–499.

    Article  CAS  Google Scholar 

  34. Zhang, G., Li, S., Xue, Y., Mao, L., & Ma, Y. (2012). Effects of salts on activity of halophilic cellulase with glucomannanase activity isolated from alkaliphilic and halophilic Bacillus sp. BG-CS10. Extremophiles, 16, 35–43.

    Article  CAS  Google Scholar 

  35. Gaur Voget, S., Steele, H. L., & Streit, W. R. (2006). Characterization of a metagenome-derived halotolerant cellulase. Journal of Biotechnology, 126, 26–36.

    Article  Google Scholar 

  36. Doukyu, N., & Ogino, H. (2010). Organic solvent-tolerant enzymes. Biochemical Engineering Journal, 48, 270–282.

    Article  CAS  Google Scholar 

  37. Gaur, R., & Tiwari, S. (2015). Isolation, production, purification and characterization of an organic-solvent-thermostable alkalophilic cellulase from Bacillus vallismortis RG-07. BMC Biotechnology, 15, 19.

    Article  Google Scholar 

  38. Trivedi, N., Gupta, V., Kumar, M., Kumari, P., Reddy, C. R., & Jha, B. (2011). Solvent tolerant marine bacterium Bacillus aquimaris secreting organic solvent stable alkaline cellulase. Chemosphere, 83, 706–712.

    Article  CAS  Google Scholar 

  39. Hu, Y., Zhang, G. M., Li, A. Y., Chen, J., & L. X., M. (2008). Cloning and enzymatic characterization of a xylanase gene from a soil derived metagenomic library with an efficient approach. Applied Microbiology and Biotechnology, 80, 823–830.

    Article  CAS  Google Scholar 

  40. Singh, J., Batra, N., & Sobti, R. C. (2004). Purification and characterization of alkaline cellulase produced by a novel isolate, Bacillus sphaericus JS1. Journal of Industrial Microbiology and Biotechnology, 31, 51–56.

    Article  CAS  Google Scholar 

  41. Coolbear, T., Whittaker, J. M., & Daniel, R. M. (1992). The effect of metal ions on the activity and thermostability of the extracellular proteinase from a thermophilic Bacillus, strain EA.1. Biochemical Journal , 287, 367–374.

    Article  CAS  Google Scholar 

  42. Sierecka, J. K. (1989). Purification and partial characterization of a neutral protease from a virulent strain of Bacillus cereus. The International Journal of Biochemistry & Cell Biology , 30, 579–595.

    Article  Google Scholar 

  43. Yang, J., Roy, A., & Zhang, Y. (2013). BioLiP: a semi-manually curated database for biologically relevant ligand-protein interactions. Nucleic Acids Research, 41, D1096–D1103.

    Article  CAS  Google Scholar 

  44. Kelly, S. M., Jess, T. J., & Price, N. C. (2005). How to study proteins by circular dichroism. Biochimica et Biophysica Acta, 1751, 119–139.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We are grateful to the Central Instrumentation Facilities at Bose Institute, Kolkata, for CD spectroscopic analysis. This work was supported in part by the funding received from Ministry of Earth Sciences, Government of India (MoES/11-MRDF/1/59/P/08), and Department of Biotechnology, Government of India (D.O. No. BT/PR7661/AAQ/3/629/2013) to SKD. The authors, KD and AP, acknowledge the University Grant Commission (UGC) and Council of Scientific and Industrial Research (CSIR), Government of India, New Delhi, respectively, for providing the research fellowship.

Author information

Authors and Affiliations

  1. Department of Biotechnology, Institute of Life Sciences, Nalco Square, Bhubaneswar, 751 023, India

    Kamal Deep, Abhijit Poddar & Subrata K. Das

Authors
  1. Kamal Deep
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Abhijit Poddar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Subrata K. Das
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Subrata K. Das.

Ethics declarations

Conflict of Interest

No conflict of interest exists.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

ESM 1

(DOCX 2763 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Deep, K., Poddar, A. & Das, S.K. Cloning, Overexpression, and Characterization of Halostable, Solvent-Tolerant Novel β-Endoglucanase from a Marine Bacterium Photobacterium panuliri LBS5T (DSM 27646T). Appl Biochem Biotechnol 178, 695–709 (2016). https://doi.org/10.1007/s12010-015-1903-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-015-1903-9

Keywords

Search

Navigation