Skip to main content
SpringerLink
Log in

Gene Cloning and Characterization of the Geobacillus thermoleovorans CCR11 Carboxylesterase CaesCCR11, a New Member of Family XV

  • Original Paper
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

A gene encoding a carboxylesterase produced by Geobacillus thermoleovoras CCR11 was cloned in the pET-3b cloning vector, sequenced and expressed in Escherichia coli BL21(DE3). Gene sequence analysis revealed an open reading frame of 750 bp that encodes a polypeptide of 250 amino acid residues (27.3 kDa) named CaesCCR11. The enzyme showed its maximum activity at 50 °C and pH 5–8, with preference for C4 substrates, confirming its esterase nature. It displayed good resistance to temperature, pH, and the presence of organic solvents and detergents, that makes this enzyme biotechnologically applicable in the industries such as fine and oleo-chemicals, cosmetics, pharmaceuticals, organic synthesis, biodiesel production, detergents, and food industries. A 3D model of CaesCCR11 was predicted using the Bacillus sp. monoacyl glycerol lipase bMGL H-257 structure as template (PBD code 3RM3, 99 % residue identity with CaesCCR11). Based on its canonical α/β hydrolase fold composed of 7 β-strands and 6 α-helices, the α/β architecture of the cap domain, the GLSTG pentapeptide, and the formation of distinctive salt bridges, we are proposing CaesCCR11 as a new member of family XV of lipolytic 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

Similar content being viewed by others

References

  1. Sharma, R., Chisti, Y., & Banerjee, U. C. (2001). Production, purification, characterization, and applications of lipases. Biotechnology Advances, 19, 627–662.

    Article  CAS  Google Scholar 

  2. Gupta, R., Kumari, A., Syal, P., & Singh, Y. (2015). Molecular and functional diversity of yeast and fungal lipases: their role in biotechnology and cellular physiology. Progress in Lipid Research, 57, 40–54.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. Soliman, N. A., Knoll, M., Abdel-Fattah, Y. R., Schmid, R. D., & Lange, S. (2007). Molecular cloning and characterization of thermostable esterase and lipase from Geobacillus thermoleovorans YN isolated from desert soil in Egypt. Process Biochemistry, 42, 1090–1100.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. Rengachari, S., Bezerra, G. A., Riegler-Berket, L., Gruber, C. C., Sturm, C., Taschler, U., et al. (2012). The structure of monoacylglycerol lipase from Bacillus sp. H-257 reveals unexpected conservation of the cap architecture between bacterial and human enzymes. Biochimica et Biophysica Acta, 1821, 1012–1021.

    Article  CAS  Google Scholar 

  9. Charbonneau, D. M., & Beauregard, M. (2013). Role of key salt bridges in thermostability of G. thermodenitrificans EstGtA2: distinctive patterns within the new bacterial lipolytic enzyme family XV. PLoS One, 10(8), e0136940. doi:10.1371/journal.pone.0136940.

    Article  Google Scholar 

  10. Rao, L., Xue, Y., Zheng, Y., Lu, J. R., & Ma, Y. (2013). A novel alkaliphilic Bacillus 324 esterase belongs to the 13th bacterial lipolytic enzyme family. PLoS One, 8, e60645. doi:10.1371/journal.pone.0060645.

    Article  CAS  Google Scholar 

  11. Castro-Ochoa, L. D., Rodríguez-Gómez, C., Valerio-Alfaro, G., & Oliart-Ros, R. (2005). Screening, purification and characterization of the thermoalkalophilic lipase produced by Bacillus thermoleovorans CCR11. Enz. Microb. Technol., 37, 648–654.

    Article  CAS  Google Scholar 

  12. Quintana-Castro, R., Díaz, P., Valerio-Alfaro, G., García, H. S., & Oliart-Ros, R. (2009). Gene cloning, expression and characterization of the Geobacillus thermoleovorans CCR11 thermoalkaliphilic lipase. Molecular Biotechnology, 42, 75–83.

    Article  CAS  Google Scholar 

  13. Sánchez-Otero, M. G., Ruiz-López, I. I., Avila-Nieto, D. E., & Oliart-Ros, R. M. (2011). Significant improvement of Geobacillus thermoleovorans CCR11 thermoalkalophilic lipase production using response surface methodology. New Biotechnology, 28, 761–766.

    Article  Google Scholar 

  14. Schmidt-Dannert, C., Rúa, L., Atomi, H., & Schmid, R. (1996). Thermoalkalophilic lipase of Bacillus thermocatenulatus. I. Molecular cloning, nucleotide sequence, purification and some properties. Biochimica et Biophysica Acta, 1301, 105–114.

    Article  Google Scholar 

  15. 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 

  16. Prim, N., Sánchez, M. M., Ruiz, C., Pastor, F. I. J., & Díaz, P. (2003). Use of methylumbeliferyl-derivative substrates for lipase activity characterization. Journal of Molecular Catalysis B: Enzymatic, 22, 339–346.

    Article  CAS  Google Scholar 

  17. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193, 265–275.

    CAS  Google Scholar 

  18. Nawani, N., Dosanjh, S., & Kaur, J. (1998). A novel thermostable lipase from a thermophilic Bacillus sp.: characterization and esterification studies. Biotechnology Letters, 20, 997–1000.

    Article  CAS  Google Scholar 

  19. Ausubel, F., Brent, R. R., Kingstone, M. D., Seidman, S. J., & Struhl, K. (Eds.) (1997). Short protocols in molecular biology. A compendium of methods from current protocols in molecular biology. Vol. I Preparation and analysis of DNA and enzymatic manipulation of DNA (pp. 2-1–3-50) New York: Wiley.

  20. Maniatis, T., Fritsch, E., & Sambrook, J. (1982). Molecular Cloning: A laboratory manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.

    Google Scholar 

  21. Grossman, T. H., Kawasaki, E. S., Punreddy, S. R., & Osburne, M. S. (1998). Spontaneous cAMP-dependent derepression of gene expression in stationary phase plays a role in recombinant expression instability. Gene, 209, 95–103.

    Article  CAS  Google Scholar 

  22. Kalendar, R., Lee, D., & Schulman, A. H. (2009). FastPCR Software for PCR primer and probe design and repeat search genes: Focus on bioinformatics. Genes, Genomes and Genomics, 3(1), 1–14.

    Google Scholar 

  23. Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M. R., Appel, R. D., & Bairoch, A. (2005). Protein identification and analysis tools on the ExPASy Server. In J. M. Walker (Ed.), The proteomics protocols handbook (pp. 571–607). Totowa: Humana Press.

    Chapter  Google Scholar 

  24. Rost, R., Yachdav, G., & Liu, J. (2004). The predict protein server. Nucleic Acids Research, 32(Web Server issue), W321–W326.

    Article  CAS  Google Scholar 

  25. Thompson, J. D., Higgins, D. G., & Gibson, T. J. (1994). CLUSTAL-W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position, specific gap penalties and weight matrix choice. Nucleic Acids Research, 22, 4673–4680.

    Article  CAS  Google Scholar 

  26. Tamura, K., Stecher, G., Peterson, D., Filipski, A., & Kumar, S. (2013). MEGA6: Molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution, 30, 2725–2729.

    Article  CAS  Google Scholar 

  27. Robert, X., & Gouet, P. (2014). Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Research, 42(Web Server issue), W320–W324.

    Article  CAS  Google Scholar 

  28. Källberg, M., Wang, H., Wang, S., Peng, J., Wang, Z., Lu, H., & Xu, J. (2012). Template-based protein structure modeling using the RaptorX web server. Nature Protocols, 7, 1511–1522.

    Article  Google Scholar 

  29. Constantini, S., Colonna, G., & Facchiano, A. M. (2008). ESBRI: A web server for evaluating salt bridges in proteins. Bioinformation, 3, 137–138.

    Article  Google Scholar 

  30. Takami, H., Takaki, Y., Chee, G. J., Nishi, S., Shimamura, S., Suzuki, H., et al. (2004). Thermoadaptation trait revealed by the genome sequence of thermophilic Geobacillus kaustophilus. Nucleic Acids Research, 32, 6292–6303.

    Article  CAS  Google Scholar 

  31. Rúa, M. L., Schmidt-Dannert, C., Wahl, S., Sprauer, A., & Schmid, R. D. (1997). Thermoalkalophilic lipase of Bacillus thermocatenulatus large-scale production, purification and properties: aggregation behavior and its effect on activity. Journal of Biotechnology, 56, 89–102.

    Article  Google Scholar 

  32. Salameh, M. A., & Wiegel, J. (2010). Effects of detergents on activity, thermostability and aggregation of two alkalithermophilic lipases from Thermosyntropha lipolytica. Open Biochemistry Journal, 4, 22–28.

    Article  CAS  Google Scholar 

  33. Schlieben, N. H., Niefind, K., & Schomburg, D. (2004). Expression, purification and aggregation studies of His-tagged thermoalkalophilic lipase from Bacillus thermocatenolatus. Protein Expression and Purification, 34, 103–110.

    Article  CAS  Google Scholar 

  34. Imamura, S., & Kitaura, S. (2000). Purification and characterization of a monoacylglycerol lipase from the moderately thermophilic Bacillus sp. H-257. Journal of Biochemistry, 127, 419–425.

    Article  CAS  Google Scholar 

  35. Kitaura, S., Suzuki, K., & Imamura, S. (2001). Monoacylglycerol lipase from moderately thermophilic Bacillus sp. strain H-257: molecular cloning, sequencing, and expression in Escherichia coli of the gene. Journal of Biochemistry, 129, 397–402.

    Article  CAS  Google Scholar 

  36. Zhu, Y., Lid, J., Caib, H., Nib, H., Xiaob, A., & Hou, L. (2013). Characterization of a new and thermostable esterase from a metagenomic library. Microb. Res., 168, 589–597.

    Article  CAS  Google Scholar 

  37. Ceroni, A., Passerini, A., Vullo, A., & Frasconi, P. (2006). DISULFIND: A disulfide bonding state and cysteine connectivity prediction server. Nucleic Acids Research, 34(1), W177–W181.

    Article  CAS  Google Scholar 

  38. Boonmak, C., Takahasi, Y., & Morikawa, M. (2013). Draft genome sequence of Geobacillus thermoleovorans strain B23. Genome Announcements, 1(6), e00944-13. doi:10.1128/genomeA.00944-13.

    Article  Google Scholar 

  39. Rengachari, S., Aschauer, P., Schittmayer, M., Mayer, N., Gruber, K., Breinbauer, R., et al. (2013). Conformational plasticity and ligand binding of bacterial monoacylglycerol lipase. Journal of Biological Chemistry, 288, 31093–31104.

    Article  CAS  Google Scholar 

  40. Lee, D. W., Kim, H. W., Lee, K. W., Kim, B. C., Choe, E. A., Lee, H. S., et al. (2001). Purification and characterization of two distinct thermostable lipases from the gram-positive thermophilic bacterium Bacillus thermoleovorans ID-1. Enzyme and Microbial Technology, 29, 363–371.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

M.Sc. Graciela Espinosa Luna and M.Sc. Rodrigo E. Matus Toledo acknowledge their scholarships from the National Council for Science and Technology (Conacyt). This work was supported by Grants No. 105636 from Conacyt, 362.06-P from the General Direction of Superior Technological Education (DGEST-SEP) and PROMEP.

Author information

Authors and Affiliations

  1. Unidad de Investigación y Desarrollo en Alimentos, Instituto Tecnológico de Veracruz, Miguel Ángel de Quevedo 2779, Col. Formando Hogar, 91897, Veracruz, Mexico

    Graciela Espinosa-Luna, Rodrigo Eloir Matus-Toledo & Rosa María Oliart-Ros

  2. Facultad de Bioanálisis, Universidad Veracruzana. Carmen Serdán esq. Iturbide, C.P. 91700, Veracruz, Veracruz, Mexico

    María Guadalupe Sánchez-Otero & Rodolfo Quintana-Castro

Authors
  1. Graciela Espinosa-Luna
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. María Guadalupe Sánchez-Otero
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rodolfo Quintana-Castro
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rodrigo Eloir Matus-Toledo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Rosa María Oliart-Ros
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Rosa María Oliart-Ros.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Espinosa-Luna, G., Sánchez-Otero, M.G., Quintana-Castro, R. et al. Gene Cloning and Characterization of the Geobacillus thermoleovorans CCR11 Carboxylesterase CaesCCR11, a New Member of Family XV. Mol Biotechnol 58, 37–46 (2016). https://doi.org/10.1007/s12033-015-9901-2

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-015-9901-2

Keywords

Search

Navigation