Skip to main content

Advertisement

SpringerLink
Log in

Characterization of Organic Solvent-Tolerant Lipolytic Enzyme from Marinobacter lipolyticus Isolated from the Antarctic Ocean

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

Abstract

The Antarctic marine environment provides a good source of novel lipolytic enzymes that possess beneficial properties, i.e., resistance to extreme physical and chemical conditions. We found a lipolytic Escherichia coli colony that was transformed using genomic DNA from Marinobacter lipolyticus 27-A9 isolated from the Antarctic Ross Sea. DNA sequence analysis revealed an open reading frame of lipolytic enzyme gene. The gene translates a protein (LipA9) of 404 amino acids with molecular mass of 45,247 Da. Recombinant LipA9 was expressed in E. coli BL21 (DE3) cells and purified by anion exchange and gel filtration chromatography. The kcat/Km of LipA9 was 175 s−1 μM−1, and the optimum temperature and pH were 70 °C and pH 8.0, respectively. LipA9 had quite high organic solvent stability; it was stable toward several common organic solvents up to 50% concentration. Substrate specificity studies showed that LipA9 preferred a short acyl chain length of p-nitrophenyl ester and triglyceride. Sequence analysis showed that LipA9 contained catalytic Ser72 and Lys75 in S-x-x-K motif, like family VIII esterases. Homology modeling and site-directed mutagenesis studies revealed that Tyr141 and Tyr188 residues were located near the conserved motif and played an important role in catalytic activity.

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

Similar content being viewed by others

References

  1. Arpigny, K. L., & Jaeger, K. E. (1999). Bacterial lipolytic enzymes: classification and properties. Biochemical Journal, 343(1), 177–183.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Jaeger, K. E., & Eggert, T. (2002). Lipases for biotechnology. Current Opinion in Biotechnology, 13(4), 390–397.

    Article  CAS  PubMed  Google Scholar 

  3. Perez, D., Martin, S., Fernandez-Lorente, F., Filice, M., Guisan, J. M., Ventosa, A., Garcia, M. T., & Mellado, E. (2011). A novel halophilic lipase, LipBL, showing high efficiency in the production of eicosapentaenoic acid (EPA). PLoS One, 6(8), e23325. https://doi.org/10.1371/journal.pone.0023325.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Dror, A., Shemesh, E., Dayan, N., & Fishman, A. (2014). Protein engineering by random mutagenesis and structure-guided consensus of Geobacillus stearothermophilus lipase T6 for enhanced stability in methanol. Applied and Environmental Microbiology, 80(4), 1515–1527.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Niehaus, F., Bertoldo, C., Kahler, M., & Antranikian, G. (1999). Extermophiles as a source of novel enzymes for industrial application. Applied Microbiology and Biotechnology, 51(6), 711–729.

    Article  CAS  PubMed  Google Scholar 

  6. Karan, R., Capes, M. D., & DasSarma, S. (2012). Function and biotechnology of extremophilic enzymes in low water activity. Aquatic Biosystems, 8(1), 4. https://doi.org/10.1186/2046-9063-8-4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Mateo, C., Palomo, J. M., Fernandez-Lorente, G., Guisan, J. M., & Fernandez-Lafuente, R. (2007). Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme and Microbial Technology, 40(6), 1451–1463.

    Article  CAS  Google Scholar 

  8. Han, J. Y., & Kim, H. K. (2011). Transesterification using the cross-linked enzyme aggregate of Photobacterium lipolyticum lipase M37. Journal of Microbiology and Biotechnology, 21(11), 1159–1165.

    Article  CAS  PubMed  Google Scholar 

  9. Sheldon, R. A. (2007). Enzyme immobilization: the quest for optimum performance. Advanced Synthesis & Catalysis, 349(8-9), 1289–1307.

    Article  CAS  Google Scholar 

  10. Bornscheuer, U. T. (2002). Microbial carboxylesterases: classification, properties and application in biocatalysis. FEMS Micobiology Reviews, 26(1), 73–81.

    Article  CAS  Google Scholar 

  11. Petersen, E. I., Valinger, G., Solkner, B., Stubenrauch, G., & Schwab, H. (2011). A novel esterase from Burkholderia gladioli which shows high deacetylation activity on cephalosporins is related to beta-lactamases and DD-peptidases. Journal of Biotechnology, 89, 11–25.

    Article  Google Scholar 

  12. Rashamuse, K., Magomani, V., Ronneburg, T., & Brady, D. (2009). A novel family VIII carboxylesterase derived from a leachate metagenome library exhibits promiscuous β-lactamase activity on nitrocefin. Applied Microbiology and Biotechnology, 83(3), 491–500.

    Article  CAS  PubMed  Google Scholar 

  13. Cao, J., Dang, G., Li, T., Yue, Z., Li, N., Liu, Y., Liu, S., & Chen, L. (2015). Identification and characterization of lipase activity and immunogenicity of LipL from Mycobacterium tuberculosis. PLoS One, 10(9), e0138151. https://doi.org/10.1371/journal.pone.0138151.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Nishizawa, M., Shimizu, M., Ohkawa, H., & Kanaoka, M. (1995). Steroselective production of (+)-trans-chrysanthemic acid by a microbial esterase: cloning, nucleotide sequence, and overexpression of the esterase gene of Arthrobacter globiformis in Escherichia coli. Applied and Environmental Microbiology, 61(9), 3208–3215.

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Jeon, J. H., Kim, S. J., Lee, H. S., Cha, S. S., Lee, J. H., Yoon, S. H., Koo, B. S., Lee, C. M., Choi, S. H., Lee, S. H., Kang, S. G., & Lee, J. H. (2011). Novel metagenome-derived carboxylesterase that hydrolyzes β-lactam antibiotics. Applied and Environmental Microbiology, 77(21), 7830–7836.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Cornell, W. D., Cieplak, P., Bayly, C. I., Gould, I. R., Merz, K. M., Ferguson, D. M., Spellmeyer, D. C., Fox, T., Caldwell, J. W., & Kollman, P. A. (1995). Second generation force field for the simulation of proteins, nucleic acids, and organic molecules. Journal of the American Chemical Society, 117(19), 5179–5197.

    Article  CAS  Google Scholar 

  17. Tischer, W., & Kasche, V. (1999). Immobilized enzymes: crystals or carriers? Trends in Biotechnology, 17(8), 326–335.

    Article  CAS  PubMed  Google Scholar 

  18. Jaeger, K. E., Dijkstra, B. E., & Reetz, M. T. (1999). Bacterial biocatalysts: molecular biology, three-dimensional structures, and biotechnological applications of lipases. Annual Review of Microbiology, 53(1), 315–351.

    Article  CAS  PubMed  Google Scholar 

  19. Kulakova, L., Galkin, A., Nakayama, T., Nishino, T., & Esaki, N. (2004). Cold-active esterase from Psychrobacter sp. Ant300: gene cloning, characterization, and the effects of Gly–>Pro substitution near the active site on its catalytic activity and stability. Biochimica et Biophysica Acta, 1696(1), 59–65.

    Article  CAS  PubMed  Google Scholar 

  20. Velasco-Lozano, S., Lopez-Gallego, F., Mateos-Diaz, J. C., & Favela-Torres, E. (2015). Cross-linked enzyme aggregated (CLEA) in enzyme improvement-a review. Biocatalysis, 1, 166–177.

    Google Scholar 

  21. Sangeetha, K., & Abraham, T. E. (2008). Preparation and characterization of cross-linked enzyme aggregates (CLEA) of substilisin for controlled release applications. International Journal of Biological Macromolecules, 43(3), 314–319.

    Article  CAS  PubMed  Google Scholar 

  22. Kumar, A., Dhar, K., Kanwar, S. S., & Arora, P. K. (2016). Lipase catalysis in organic solvents: advantages and applications. Biological Procedures Online, 18(1). https://doi.org/10.1186/s12575-016-0033-2.

  23. Kamal, M. Z., Yedavalli, P., Deshmukh, M. V., & Rao, N. M. (2013). Lipase in aqueous-polar organic solvents: activity, structure and stability. Protein Science, 22(7), 904–915.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Gorman, L. A., & Dordick, J. S. (1992). Organic solvents strip water off enzymes. Biotechnology and Bioengineering, 39(4), 392–397.

    Article  CAS  PubMed  Google Scholar 

  25. Elend, C., Schmeisser, C., Leggewie, C., Babiak, P., Carballeira, J. D., Steele, H. L., Reymond, J. L., Jaeger, K. E., & Streit, W. R. (2006). Isolation and biochemical characterization of two novel metagenome derived esterases. Applied and Environmental Microbiology, 72(5), 3637–3645.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Sharma, S., & Kanwar, S. S. (2014). Organic solvent tolerant lipases and applications. Scientific World Journal. https://doi.org/10.1155/2014/625258.

  27. Dachuria, V., Boyinenia, J., Choia, S., Chung, H. S., Jang, S. H., & Lee, C. W. (2016). Organic solvent-tolerant, cold-adapted lipases PML and LipS exhibit increased conformational flexibility in polar organic solvents. Journal of Molecular Catalysis B Enzymatics, 131, 73–78.

    Article  CAS  Google Scholar 

  28. Perez, D., Kovacic, F., Wilhelm, S., Jaegaer, K. E., Garcia, M. T., Ventosa, A., & Mellado, E. (2012). Identification of amino acids involved in the hydrolytic activity of lipase LipBL from Marinobacter lipolyticus. Microbiology, 158(Pt_8), 2192–2203.

    Article  CAS  PubMed  Google Scholar 

  29. Wagner, U. G., Petersen, E. I., Schwab, H., & Kratky, C. (2002). EstB from Burkholderia gladioli: a novel esterase with a beta-lactamase fold reveals steric factors to discriminate between esterolytic and bata-lactam cleaving activity. Protein Science, 11(3), 467–478.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Stryer, L., Berg, J. M., and Tymoczko, J. L. (2002). Catalytic strategies. Biochemistry (5th ed.). San Francisco: W.H. Freeman. ISBN 0-7167-4955-6.

  31. Wiederstein, M., & Sippl, M. J. (2007). ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins. Nucleic Acids Research, 35(Web Server), W407–W410.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Lüthy, R., Bowie, J. U., & Eisenberg, D. (1992). Assessment of protein models with three-dimensional profiles. Nature, 356(6364), 83–85.

    Article  PubMed  Google Scholar 

  33. Colovos, C., & Yeates, T. O. (1993). Verification of protein structures: patterns of nonbonded atomic interactions. Protein Science, 2(9), 1511–1519.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Rouached, H., Berthomieu, P., Kassis, E. E., Cathala, N., Catherinot, V., Labesse, G., Davidian, J.-C., & Fourcroy, P. (2005). Structural and functional analysis of the C-terminal STAS (sulfate transporter and anti-sigma antagonist) domain of the Arabidopsis thaliana sulfate transporter SULTR1.2. The Journal of Biological Chemistry, 280(16), 15976–15983.

    Article  CAS  PubMed  Google Scholar 

  35. Oefner, C., D’Arcy, A., Daly, J. J., Gubemator, K., Charnas, R. L., Heinze, I., Hubschwerlen, C., & Winkler, F. K. (1990). Refined crystal structure of beta-lactamase from Citrobacter freundii indicates a mechanism for beta-lactam hydrolysis. Nature, 343(6255), 284–288.

    Article  CAS  PubMed  Google Scholar 

  36. Thangavelu, K., Pan, C. Q., Karlberg, T., Balaji, G., Uttamchandani, M., Schuler, H., Low, B. C., & Sivaraman, J. (2012). Structural basis for the allosteric inhibitory mechanism of human kidney-type glutaminase (KGA) and its regulation by Raf-Mek-Erk signaling in cancer cell metabolism. Proceedings of National Academy of Sciences USA, 109(20), 7705–7710.

    Article  Google Scholar 

Download references

Funding

This work was supported by the Research Fund 2017 of The Catholic University of Korea. This work was also supported by Korea Polar Research Institute (PE18100).

Author information

Authors and Affiliations

  1. Division of Biotechnology, The Catholic University of Korea, Bucheon, 420-743, Republic of Korea

    Se Hyeon Park, Soo-jin Kim & Hyung Kwoun Kim

  2. Department of Chemistry, Center for NanoBio Applied Technology, Institute of Basic Sciences, Sungshin Women’s University, Seoul, 136-742, Republic of Korea

    Seongsoon Park

Authors
  1. Se Hyeon Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Soo-jin Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Seongsoon Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hyung Kwoun Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hyung Kwoun Kim.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflict of interest.

Electronic Supplementary Material

ESM 1

(DOCX 657 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Park, S.H., Kim, Sj., Park, S. et al. Characterization of Organic Solvent-Tolerant Lipolytic Enzyme from Marinobacter lipolyticus Isolated from the Antarctic Ocean. Appl Biochem Biotechnol 187, 1046–1060 (2019). https://doi.org/10.1007/s12010-018-2865-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-018-2865-5

Keywords

Search

Navigation