Skip to main content
SpringerLink
Log in

Cloning and characterization of a thermostable carboxylesterase from inshore hot spring thermophile Geobacillus sp. ZH1

  • Published:
Acta Oceanologica Sinica Aims and scope Submit manuscript

Abstract

The gene (741 bp) encoding carboxylesterase from the thermophilic bacterium Geobacillus sp. ZHl was cloned and overexpressed in Escherichia coli. The purified recombinant protein presented a molecular mass of about 40 kDa by SDS-PAGE analysis. Enzyme assays using p-nitrophenyl esters with different acyl chain lengths as the substrates confirmed its esterase activity, yielding highest specific activity with p-nitrophenyl acetate. Among the p-nitrophenyl esters tested, the carboxylesterase presented preference for p-nitrophenyl caprylate, but hydrolyzed p-nitrophenyl butyrate more efficiently. When p-nitrophenyl butyrate was used as a substrate, the recombinant carboxylesterase exhibited highest activity at pH 8.0 and 60°C. Almost no decrease in esterase activity was observed at 60°C for 3 h, and over 40% of activity was still maintained after incubation at 90°C for 3 h. These results indicate that Geobacillus sp. ZH1 recombinant esterase was thermostable. The enzymatic activity was inhibited by the addition of phenylmethylsulfonyl fluoride, indicating that it contains serine residue, which plays a key role in the catalytic mechanism. Except SDS and xylene, this esterase showed stability toward other tested detergents and organic solvents. Cloning, expression, and biochemical characterization of Geobacillus sp. ZH1 carboxylesterase lay a good foundation for its structural characterization and industrial application.

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

Similar content being viewed by others

References

  • Almeida R V, Alquéres S M C, Larentis A L, et al. 2006. Cloning, expression, partial characterization and structural modeling of a novel esterase from Pyrococcus furiosus. Enzyme Microbial Technol, 39(5): 1128–1136

    Article  Google Scholar 

  • Altschul S F, Madden T L, Schäffer A A, et al. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein databases search programs. Nucleic Acids Res, 25(17): 3389–3402

    Article  Google Scholar 

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

    Article  Google Scholar 

  • Arpigny J L, Jendrossek D, Jaeger K E. 1998. A novel heat-stable lipolytic enzyme from Sulfolobus acidocaldarius DSM 639 displaying similarity to polyhydroxyalkanoate depolymerases. FEMS Microbiol Lett, 167(1): 69–73

    Article  Google Scholar 

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

    Article  Google Scholar 

  • Bradford M M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein—dye binding. Anal Biochem, 72: 248–254

    Article  Google Scholar 

  • Brod F C, Vernal J, Bertoldo J B, et al. 2010. Cloning, expression, purification, and characterization of a novel esterase from Lactobacillus plantarum. Mol Biotechnol, 44(3): 242–249

    Article  Google Scholar 

  • Charbonneau D M, Meddeb-Mouelhi F, Beauregard M. 2010. A novel thermostable carboxylesterase from Geobacillus thermodenitrificans: evidence for a new carboxylesterase family. J Biochem, 148(3): 299–308

    Article  Google Scholar 

  • du Plessis E M, Berger E, Stark T, et al. 2010. Characterization of a novel thermostable esterase from Thermus scotoductus SA-01: evidence of a new family of lipolytic esterases. Curr Microbiol, 60(4): 248–253

    Article  Google Scholar 

  • Ejima K, Liu Jian, Oshima Y, et al. 2004. Molecular cloning and characterization of a thermostable carboxylesterase from an archaeon, Sulfolobus shibatae DSM5389: non-linear kinetic behavior of a hormone-sensitive lipase family enzyme. J Biosci Bioeng, 98(6): 445–451

    Google Scholar 

  • Fojan P, Jonson P H, Petersen M T, et al. 2000. What distinguishes an esterase from a lipase: a novel structural approach. Biochimie, 82(11): 1033–1041

    Article  Google Scholar 

  • Hasan F, Shah A A, Hameed A. 2006. Industrial applications of microbial lipases. Enzyme Microbial Technol, 39(2): 235–251

    Article  Google Scholar 

  • Hess M, Katzer M, Antranikian G. 2008. Extremely thermostable esterases from the thermoacidophilic euryarchaeon Picrophilus torridus. Extremophiles, 12(3): 351–364

    Article  Google Scholar 

  • Hotta Y, Ezaki S, Atomi H, et al. 2002. Extremely stable and versatile carboxylesterase from a hyperthermophilic archaeon. Appl Environ Microbiol, 68(8): 3925–3931

    Article  Google Scholar 

  • Jaeger K E, Dijkstra B W, Reetz M T. 1999. Bacterial biocatalysts: molecular biology, three-dimensional structures, and biotechnological applications of lipases. Annu Rev Microbiol, 53: 315–351

    Article  Google Scholar 

  • Jaeger K E, Reetz M T. 1998. Microbial lipases form versatile tools for biotechnology. Trends Biotechnol, 16(9): 396–403

    Article  Google Scholar 

  • Kakugawa S, Fushinobu S, Wakagi T. 2007. Characterization of a thermostable carboxylesterase from the hyperthermophilic bacterium Thermotoga maritima. Appl Microbiol Biotechnol, 74(3): 585–591

    Article  Google Scholar 

  • Kim S B, Lee W, Ryu Y W. 2008. Cloning and characterization of thermostable esterase from Archaeoglobus fulgidus. J Microbiol, 46(1): 100–107

    Article  Google Scholar 

  • Laemmli U K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259): 680–685

    Article  Google Scholar 

  • Levisson M, van der Oost J, Kengen S W. 2007. Characterization and structural modeling of a new type of thermostable esterase from Thermotoga maritima. FEBS J, 274(11): 2832–2842

    Article  Google Scholar 

  • Manco G, Adinolfi E, Pisani F M, et al. 1998. Overexpression and properties of a new thermophilic and thermostable esterase from Bacillus acidocaldarius with sequence similarity to hormone-sensitive lipase subfamily. Biochem J, 332(Pt 1): 203–212

    Google Scholar 

  • Manco G, Giosuè E, D’Auria S, et al. 2000. Cloning, overexpression, and properties of a new thermophilic and thermostable esterase with sequence similarity to hormone-sensitive lipase subfamily from the archaeon Archaeoglobus fulgidus. Arch Biochem Biophys, 373(1): 182–192

    Article  Google Scholar 

  • Morana A, Di Prizito N, Aurilia V, et al. 2002. A carboxylesterase from the hyperthermophilic archaeon Sulfolobus solfataricus: cloning of the gene, characterization of the protein. Gene, 283(1–2): 107–115

    Article  Google Scholar 

  • Ollis D L, Cheah E, Cygler M, et al. 1992. The alpha/beta hydrolase fold. Prot Eng, 5(3): 197–211

    Article  Google Scholar 

  • Oppenheimer C H, ZoBell C E. 1952. The growth and viability of sixty-three species of marine bacteria as influenced by hydrostatic pressure. J Mar Res, 11(1): 10–18

    Google Scholar 

  • Park Y J, Choi S Y, Lee H B. 2006. A carboxylesterase from the thermoacidophilic archaeon Sulfolobus solfataricus P1; purification, characterization, and expression. Biochim Biophys Acta, 1760(5): 820–828

    Article  Google Scholar 

  • Sobek H, Görisch H. 1988. Purification and characterization of a heat-stable esterase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. Biochem J, 250(2): 453–458

    Google Scholar 

  • Soliman N A, Knoll M, Abdel-Fattah Y R, et al. 2007. Molecular cloning and characterization of thermostable esterase and lipase from Geobacillus thermoleovorans YN isolated from desert soil in Egypt. Process Biochem, 42(7): 1090–1100

    Article  Google Scholar 

  • Stok J E, Huang Huazhang, Jones P D, et al. 2004. Identification, expression, and purification of a pyrethroid-hydrolyzing carboxylesterase from mouse liver microsomes. J Biol Chem, 279(28): 29863–29869

    Article  Google Scholar 

  • Sun Lei, Levisson M, Hendriks S, et al. 2007. Crystallization and preliminary crystallographic analysis of an esterase with a novel domain from the hyperthermophile Thermotoga maritima. Acta Crystallogr Sect F Struct Biol Cryst Commun, 63(Pt 9): 777–779

    Article  Google Scholar 

  • Suzuki Y, Miyamoto K, Ohta H. 2004. A novel thermostable esterase from the thermoacidophilic archaeon Sulfolobus tokodaii strain 7. FEMS Microbiol Lett, 236(1): 97–102

    Article  Google Scholar 

  • Tekedar H C, Şanlı-Mohamed G. 2011. Molecular cloning, over expression and characterization of thermoalkalophilic esterases isolated from Geobacillus sp. Extremophiles, 15(2): 203–211

    Article  Google Scholar 

  • Verger R. 1997. Interfacial activation of lipases: fact and artifacts. Trends Biotechnol, 15(1): 32–38

    Article  Google Scholar 

  • Vieille C, Zeikus G J. 2001. Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol Mol Biol Rev, 65(1): 1–43

    Article  Google Scholar 

  • Wang Baijing, Lu Dongmei, Gao Renjun, et al. 2004. A novel phospholipase A2/esterase from hyperthermophilic archaeon Aeropyrum pernix K1. Protein Expr Purif, 35(2): 199–205

    Article  Google Scholar 

  • Zhang Jian, Liu Jingfang, Zhou Jian, et al. 2003. Thermostable esterase from Thermoanaerobacter tengcongensis: high-level expression, purification and characterization. Biotechnol Lett, 25(17): 1463–1467

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. School of Biotechnology Engineering, Jimei University, Xiamen, 361021, China

    Yanbing Zhu, Guangming Liu, Jingwen Liu, Xiaoming Bai, Rong Guan & Huinong Cai

  2. Research Center of Food Microbiology and Enzyme Engineering Technology (Jimei University), Fujian Province University, Xiamen, 361021, China

    Yanbing Zhu & Huinong Cai

  3. Research Center of Food Biotechnology of Xiamen City, Xiamen, 361021, China

    Yanbing Zhu & Huinong Cai

  4. Xiamen Medical College, Xiamen, 361008, China

    Hebin Li

Authors
  1. Yanbing Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Guangming Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hebin Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jingwen Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xiaoming Bai
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Rong Guan
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Huinong Cai
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Huinong Cai.

Additional information

Foundation item: Scientific Research Fund of Fujian Provincial Education Department, China under contact No. JA11153; the Natural Science Foundation of Fujian Province, China under contact Nos 2010J06012 and 2010J01261; the Foundation for Innovative Research Team of Jimei University, China under contact No. 2010A005.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zhu, Y., Liu, G., Li, H. et al. Cloning and characterization of a thermostable carboxylesterase from inshore hot spring thermophile Geobacillus sp. ZH1. Acta Oceanol. Sin. 31, 117–126 (2012). https://doi.org/10.1007/s13131-012-0258-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13131-012-0258-0

Key words

Search

Navigation