Skip to main content
SpringerLink
Log in

Isolation, Expression and Characterization of the Thermophilic Recombinant Esterase from Geobacillus thermodenitrificans PS01

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

Abstract

Esterases are widely used in the food industry. Here, a new thermophilic bacterium, Geobacillus thermodenitrificans PS01, was isolated and the esterase-encoding gene est1 was cloned, sequenced, and recombinant expressed in Escherichia coli Tuner (DE3). The highest activity of recombinant Est1 was detected at pH 8.0, and 40 °C and the extreme stability was observed at pH 6–9 over 30 days at 4 °C. In particular, Est1 can hydrolyze short- to medium-chain (C2–C10) triglycerides and p-nitrophenyl esters (C2–C12) and was not inhibited by most metal ions. Kinetic parameters of p-nitrophenyl butyrate hydrolysis under optimal conditions were determined: Km, 22.76 μM; kcat, 10,415 s−1; and kcat/Km, 457.53 μM−1 s−1. The outstanding specification of Est1 indicates its potential for use in industrial applications.

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

    Article  CAS  Google Scholar 

  2. Bornemann, S., Cassells, J., Dordick, J., & Hacking, A. (1992). The use of enzymes to regioselectively deacylate sucrose esters. Biocatalysis, 7, 1–12.

    Article  CAS  Google Scholar 

  3. Bornscheuer, U. T. (2002). Microbial carboxyl esterases: classification, properties and application in biocatalysis. FEMS Microbiology Reviews, 26(1), 73–81.

    Article  CAS  Google Scholar 

  4. Charbonneau, D. M., Meddeb-Mouelhi, F., & Beauregard, M. (2010). A novel thermostable carboxylesterase from Geobacillus thermodenitrificans: Evidence for a new carboxylesterase family. Journal of Biochemistry, 148(3), 299–308.

    Article  CAS  Google Scholar 

  5. Elleuche, S., Schäfers, C., Blank, S., Schröder, C., & Antranikian, G. (2015). Exploration of extremophiles for high temperature biotechnological processes. Current Opinion in Microbiology, 25, 113–119.

    Article  CAS  Google Scholar 

  6. Margolin, A. L. (1993). Enzymes in the synthesis of chiral drugs. Enzyme and Microbial Technology, 15, 266–280.

    Article  CAS  Google Scholar 

  7. Mohr, P., Rösslein, L., & Tamma, C. (1989). Kinetic resolution of racemic β,γ-epoxy esters with pig liver esterase (PLE, e.c. 3.1.1.1.). Tetrahedron Letters, 30, 2513–2516.

    Article  CAS  Google Scholar 

  8. Montoro-García, S., Martínez-Martínez, I., Navarro-Fernández, J., Takami, H., García-Carmona, F., & Sánchez-Ferrer, Á. (2009). Characterization of a novel thermostable carboxylesterase from Geobacillus kaustophilus HTA426 shows the existence of a new carboxylesterase family. Journal of Bacteriology, 191(9), 3076–3085.

    Article  Google Scholar 

  9. Morana, A., Di Prizito, N., Aurilia, V., Rossi, M., & Cannio, R. (2002). A carboxylesterase from the hyperthermophilic archaeon Sulfolobus solfataricus: cloning of the gene, characterization of the protein. Gene, 283(1-2), 107–115.

    Article  CAS  Google Scholar 

  10. Park, Y.-J., Choi, S. Y., & Lee, H.-B. (2006). A carboxylesterase from the thermoacidophilic archaeon Sulfolobus solfataricus P1; purification, characterization, and expression. Biochimica et Biophysica Acta, 1760(5), 820–828.

    Article  CAS  Google Scholar 

  11. Wood, A. N. P., Fernandez-Lafuente, R., & Cowan, D. A. (1995). Purification and partial characterization of a novel thermophilic carboxylesterase with high mesophilic specific activity. Enzyme and Microbial Technology, 17(9), 816–825.

    Article  CAS  Google Scholar 

  12. Marilley, L., & Casey, M. G. (2004). Flavours of cheese products: metabolic pathways, analytical tools and identification of producing strains. International Journal of Food Microbiology, 90(2), 139–159.

    Article  CAS  Google Scholar 

  13. Marten, B., Pfeuffer, M., & Schrezenmeir, J. (2006). Medium-chain triglycerides. International Dairy Journal, 16, 1374–1382.

    Article  CAS  Google Scholar 

  14. Waché, Y., Husson, F., Feron, G., & Belin, J.-M. (2006). Yeast as an efficient biocatalyst for the production of lipid-derived flavours and fragrances. Antonie Van Leeuwenhoek, 89(3-4), 405–416.

    Article  Google Scholar 

  15. Nazina, T. N., Tourova, T. P., Poltaraus, A. B., Novikova, E. V., Grigoryan, A. A., Ivanova, A. E., Lysenko, A. M., Petrunyaka, V. V., Osipov, G. A., Belyaev, S. S., & Ivanov, M. V. (2001). Taxonomic study of aerobic thermophilic bacilli: descriptions of Geobacillus subterraneus gen. nov., sp. nov. and Geobacillus uzenensis sp. nov. from petroleum reservoirs and transfer of Bacillus stearothermophilus, Bacillus thermocatenulatus, Bacillus thermoleovorans, Bacillus kaustophilus, Bacillus thermodenitrificans to Geobacillus as the new combinations G. stearothermophilus, G. th. International Journal of Systematic and Evolutionary Microbiology, 51, 433–446.

    Article  CAS  Google Scholar 

  16. Anand, A., Kumar, V., & Satyanarayana, T. (2013). Characteristics of thermostable endoxylanase and β-xylosidase of the extremely thermophilic bacterium Geobacillus thermodenitrificans TSAA1 and its applicability in generating xylooligosaccharides and xylose from agro-residues. Extremophiles, 17, 357–366.

    Article  CAS  Google Scholar 

  17. McMullan, G., Christie, J. M., Rahman, T. J., Banat, I. M., Ternan, N. G., & Marchant, R. (2004). Habitat, applications and genomics of the aerobic, thermophilic genus Geobacillus. Biochemical Society Transactions, 32(Pt 2), 214–217.

    Article  CAS  Google Scholar 

  18. Ratnadewi, A. A. I., Fanani, M., Kurniasih, S. D., Sakka, M., Wasito, E. B., Sakka, K., Nurachman, Z., & Puspaningsih, N. N. T. (2013). β-D-Xylosidase from Geobacillus thermoleovorans IT-08: biochemical characterization and bioinformatics of the enzyme. Applied Biochemistry and Biotechnology, 170(8), 1950–1964.

    Article  CAS  Google Scholar 

  19. Kim, S.-B., Lee, Y.-M., Park, S.-W., Kim, J.-H., Song, S.-H. and Lee, K.-P. (2012) Recombinant GRAS strains expressing thermophilic arabinose isomerase as an active form and method of preparing food grade tagatose by using the same. Google Patents. United States Patent 8137946.

  20. Olempska-Beer, Z. S., Merker, R. I., Ditto, M. D., & DiNovi, M. J. (2006). Food-processing enzymes from recombinant microorganisms—a review. Regulatory Toxicology and Pharmacology, 45(2), 144–158.

    Article  CAS  Google Scholar 

  21. Kim, E. K., Jang, W. H., Ko, J. H., Kang, J. S., Noh, M. J., & Yoo, O. J. (2001). Lipase and its modulator from Pseudomonas sp. strain KFCC 10818: proline-to-glutamine substitution at position 112 induces formation of enzymatically active lipase in the absence of the modulator. Journal of Bacteriology, 183(20), 5937–5941.

    Article  CAS  Google Scholar 

  22. Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution, 39, 783–791.

    Article  Google Scholar 

  23. Saitou, N., & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4(4), 406–425.

    CAS  PubMed  Google Scholar 

  24. Tamura, K., Stecher, G., Peterson, D., Filipski, A., & Kumar, S. (2013). MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution, 30(12), 2725–2729.

    Article  CAS  Google Scholar 

  25. Chuang, H.-H., Chen, P.-T., Wang, W.-N., Chen, Y.-T., & Shaw, J.-F. (2011). Functional proteomic analysis of rice bran esterases/lipases and characterization of a novel recombinant esterase. Journal of Agricultural and Food Chemistry, 59(5), 2019–2025.

    Article  CAS  Google Scholar 

  26. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.

    Article  CAS  Google Scholar 

  27. Ewis, H. E., Abdelal, A. T., & Lu, C.-D. (2004). Molecular cloning and characterization of two thermostable carboxyl esterases from Geobacillus stearothermophilus. Gene, 329, 187–195.

    Article  CAS  Google Scholar 

  28. Lee, L.-C., Yen, C.-C., Malmis, C. C., Chen, L.-F., Chen, J.-C., Lee, G.-C., & Shaw, J.-F. (2011). Characterization of codon-optimized recombinant Candida rugosa lipase 5 (LIP5). Journal of Agricultural and Food Chemistry, 59(19), 10693–10698.

    Article  CAS  Google Scholar 

  29. Feng, L., Wang, W., Cheng, J., Ren, Y., Zhao, G., Gao, C., Tang, Y., Liu, X., Han, W., Peng, X., Liu, R., & Wang, L. (2007). Genome and proteome of long-chain alkane degrading Geobacillus thermodenitrificans NG80-2 isolated from a deep-subsurface oil reservoir. Proceedings of the National Academy of Sciences of the United States of America, 104(13), 5602–5607.

    Article  CAS  Google Scholar 

  30. Lenfant, N., Hotelier, T., Velluet, E., Bourne, Y., Marchot, P., & Chatonnet, A. (2013). ESTHER, the database of the α/β-hydrolase fold superfamily of proteins: tools to explore diversity of functions. Nucleic Acids Research, 41, D423–D429 Database issue.

    Article  CAS  Google Scholar 

  31. Liu, P., Ewis, H. E., Tai, P. C., Lu, C.-D., & Weber, I. T. (2007). Crystal structure of the Geobacillus stearothermophilus carboxylesterase Est55 and its activation of prodrug CPT-11. Journal of Molecular Biology, 367(1), 212–223.

    Article  CAS  Google Scholar 

  32. Abeijón Mukdsi, M. C., Medina, R. B., Katz, M. B., Pivotto, R., Gatti, P., & González, S. N. (2009). Contribution of lactic acid bacteria esterases to the release of fatty acids in miniature ewe’s milk cheese models. Journal of Agricultural and Food Chemistry, 57, 1036–1044.

    Article  Google Scholar 

  33. Amaki, Y., Edgard, E. T., Ueda, S., Ohmiya, K., & Yamane, T. (1992). Purification and properties of a thermostable esterase of Bacillus stearothermophilus produced by recombinant Bacillus brevis. Bioscience, Biotechnology, and Biochemistry, 56, 238–241.

    Article  CAS  Google Scholar 

  34. Esteban-Torres, M., Barcenilla, J. M., Mancheño, J. M., De las Rivas, B., & Muñoz, R. (2014). Characterization of a versatile arylesterase from Lactobacillus plantarum active on wine esters. Journal of Agricultural and Food Chemistry, 62(22), 5118–5125.

    Article  CAS  Google Scholar 

  35. Singh, T., Drake, M., & Cadwallader, K. (2003). Flavor of cheddar cheese: a chemical and sensory perspective. Comprehensive Reviews in Food Science and Food Safety, 2, 166–189.

    Article  Google Scholar 

  36. Russell, W. R., Hoyles, L., Flint, H. J., & Dumas, M.-E. (2013). Colonic bacterial metabolites and human health. Current Opinion in Microbiology, 16, 246–254.

    Article  CAS  Google Scholar 

  37. Lin, H. V., Frassetto, A., Kowalik Jr., E. J., Nawrocki, A. R., Lu, M. M., Kosinski, J. R., Hubert, J. A., Szeto, D., Yao, X., Forrest, G., & Marsh, D. J. (2012). Butyrate and propionate protect against diet-induced obesity and regulate gut hormones via free fatty acid receptor 3-independent mechanisms. PLoS One, 7, e35240.

    Article  CAS  Google Scholar 

  38. Fung, K. Y. C., Cosgrove, L., Lockett, T., Head, R., & Topping, D. L. (2012). A review of the potential mechanisms for the lowering of colorectal oncogenesis by butyrate. The British Journal of Nutrition, 108(5), 820–831.

    Article  CAS  Google Scholar 

  39. Yang, Z., Zhang, Y., Shen, T., Xie, Y., Mao, Y., & Ji, C. (2013). Cloning, expression and biochemical characterization of a novel, moderately thermostable GDSL family esterase from Geobacillus thermodenitrificans T2. Journal of Bioscience and Bioengineering, 115(2), 133–137.

    Article  CAS  Google Scholar 

Download references

Funding

This work was supported by the Ministry of Science and Technology, Taiwan (grant no. MOST 107-2621-M-218-001-, MOST 104-2313-B-005-007-MY2, MOST 106-2313-B-005-035-MY2).

Author information

Authors and Affiliations

  1. Department of Biotechnology and Food Technology, Southern Taiwan University of Science and Technology, Tainan, 71005, Taiwan

    Po-Ting Chen

  2. Department of Food Science and Biotechnology, National Chung Hsing University, Taichung, 40227, Taiwan

    Cheng-Huan Liu & Fang-Yu Hsu

  3. Institute of Genomics and Bioinformatics, National Chung Hsing University, Taichung, 40227, Taiwan

    Yu-Ting Chen

  4. Department of Biological Science & Technology, I-Shou University, Kaohsiung City, 84001, Taiwan

    Jei-Fu Shaw

Authors
  1. Po-Ting Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Cheng-Huan Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yu-Ting Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Fang-Yu Hsu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jei-Fu Shaw
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Po-Ting Chen or Jei-Fu Shaw.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chen, PT., Liu, CH., Chen, YT. et al. Isolation, Expression and Characterization of the Thermophilic Recombinant Esterase from Geobacillus thermodenitrificans PS01. Appl Biochem Biotechnol 191, 112–124 (2020). https://doi.org/10.1007/s12010-020-03225-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-020-03225-w

Keywords

Search

Navigation