Skip to main content
SpringerLink
Log in

Expression and display of a novel thermostable esterase from Clostridium thermocellum on the surface of Bacillus subtilis using the CotB anchor protein

  • Biocatalysis
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

Esterases expressed in microbial hosts are commercially valuable, but their applications are limited due to high costs of production and harsh industrial processes involved. In this study, the esterase-DSM (from Clostridium thermocellum) was expressed and successfully displayed on the spore surface, and the spore-associated esterase was confirmed by western blot analysis and activity measurements. The optimal temperature and pH of spore surface-displayed DSM was 60 and 8.5 °C, respectively. It also demonstrates a broad temperature and pH optimum in the range of 50–70, 7–9.5 °C. The spore surface-displayed esterase-DSM retained 78, 68 % of its original activity after 5 h incubation at 60 and 70 °C, respectively, which was twofold greater activity than that of the purified DSM. The recombinant spores has high activity and stability in DMSO, which was 49 % higher than the retained activity of the purified DSM in DMSO (20 % v/v), and retained 65.2 % of activity after 7 h of incubation in DMSO (20 % v/v). However, the recombinant spores could retain 77 % activity after 3 rounds of recycling. These results suggest that enzyme displayed on the surface of the Bacillus subtilis spore could serve as an effective approach for enzyme immobilization.

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
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

References

  1. Abdallah NH, Schlumpberger M, Gaffney DA, Hanrahan JP, Tobin JM, Magner E (2014) Comparison of mesoporous silicate supports for the immobilisation and activity of cytochrome c and lipase. J Mol Catal B Enzym 108:82–88

    Article  CAS  Google Scholar 

  2. Henriques AO, Moran CP (2000) Structure and assembly of the bacterial endospore coat. Methods 20:95–110

    Article  CAS  PubMed  Google Scholar 

  3. Aloni-Grinstein R, Gat O, Altboum Z, Velan B, Cohen S, Shafferman A (2005) Oral spore vaccine based on live attenuated nontoxinogenic Bacillus anthracis expressing recombinant mutant protective antigen. Infect Immun 73:4043–4053

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. Arpigny J, Jaeger K (1999) Bacterial lipolytic enzymes: classification and properties. Biochem J 343:177–183

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  5. Barbosa O, Ortiz C, Berenguer-Murcia Á, Torres R, Rodrigues RC, Fernandez-Lafuente R (2015) Strategies for the one-step immobilization–purification of enzymes as industrial biocatalysts. Biotechnol Adv. doi:10.1016/j.biotechadv.2015.03.006

  6. Chen X, Zaro JL, Shen W-C (2013) Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev 65:1357–1369

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  7. Crepin V, Faulds C, Connerton I (2004) Functional classification of the microbial feruloyl esterases. Appl Microbiol Biotechnol 63:647–652

    Article  CAS  PubMed  Google Scholar 

  8. Cutting S, Vander Horn PB (1990) Genetic analysis. In: Harwood C, Cutting S (eds) Molecular biological methods for Bacillus. Wiley, Chichester, pp 27–74

  9. Demirjian DC, Morı́s-Varas F, Cassidy CS (2001) Enzymes from extremophiles. Curr Opin Chem Biol 5:144–151

    Article  CAS  PubMed  Google Scholar 

  10. Driks A (1999) Bacillus subtilis spore coat. Microbiol Mol Biol Rev 63:1–20

    PubMed Central  CAS  PubMed  Google Scholar 

  11. Driks A, Little S (2001) Functional analysis of the Bacillus subtilis. Mol Microbiol 42:1107–1120

    Article  PubMed  Google Scholar 

  12. Duc LH, Hong HA, Fairweather N, Ricca E, Cutting SM (2003) Bacterial spores as vaccine vehicles. Infect Immun 71:2810–2818

    Article  PubMed Central  CAS  Google Scholar 

  13. Garcia-Galan C, Berenguer-Murcia Á, Fernandez-Lafuente R, Rodrigues RC (2011) Potential of different enzyme immobilization strategies to improve enzyme performance. Adv Synth Catal 353:2885–2904

    Article  CAS  Google Scholar 

  14. Guzik U, Hupert-Kocurek K, Wojcieszyńska D (2014) Immobilization as a strategy for improving enzyme properties-application to oxidoreductases. Molecules 19:8995–9018

    Article  PubMed  Google Scholar 

  15. Haki G, Rakshit S (2003) Developments in industrially important thermostable enzymes: a review. Bioresour Technol 89:17–34

    Article  CAS  PubMed  Google Scholar 

  16. Han M, Enomoto K (2010) Surface display of recombinant protein on the cell surface of Bacillus subtilis by the CotB anchor protein. World J Microb Biot 27:719–726

    Article  Google Scholar 

  17. Hernandez K, Fernandez-Lafuente R (2011) Control of protein immobilization: coupling immobilization and site-directed mutagenesis to improve biocatalyst or biosensor performance. Enzyme Microb Technol 48:107–122

    Article  CAS  PubMed  Google Scholar 

  18. Hinc K, Isticato R, Dembek M, Karczewska J, Iwanicki A, Peszynska-Sularz G, De Felice M, Obuchowski M, Ricca E (2010) Expression and display of UreA of Helicobacter acinonychis on the surface of Bacillus subtilis spores. Microb Cell Fact 9:2–13

    Article  PubMed Central  PubMed  Google Scholar 

  19. Hoang TH, Hong HA, Clark GC, Titball RW, Cutting SM (2008) Recombinant Bacillus subtilis expressing the Clostridium perfringens alpha toxoid is a candidate orally delivered vaccine against necrotic enteritis. Infect Immun 76:5257–5265

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  20. Imamura D, Kuwana R, Takamatsu H, Watabe K (2010) Localization of proteins to different layers and regions of Bacillus subtilis spore coats. J Bacteriol 192:518–524

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  21. Isticato R, Cangiano G, Tran HT, Ciabattini A, Medaglini D, Oggioni MR, De Felice M, Pozzi G, Ricca E (2001) Surface display of recombinant proteins on Bacillus subtilis spores. J Bacteriol 183:6294–6301

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  22. Iyer PV, Ananthanarayan L (2008) Enzyme stability and stabilization—aqueous and non-aqueous environment. Process Biochem 43:1019–1032

    Article  CAS  Google Scholar 

  23. Kim H, Hahn M, Grabowski P, McPherson DC, Otte MM, Wang R, Ferguson CC, Eichenberger P, Driks A (2006) The Bacillus subtilis spore coat protein interaction network. Mol Microbiol 59:487–502

    Article  CAS  PubMed  Google Scholar 

  24. Kim J (2010) Functional display of target proteins on Bacillus subtilis spore and its application. J Biotechnol 150:346–347

    Article  Google Scholar 

  25. Kim J, Kim B-G (2008) Bacillus subtilis spore display system. J Biotechnol 136:S331

    Google Scholar 

  26. Kim S, Nam Y, Lee S (1997) Improvement of the production of foreign proteins using a heterologous secretion vector system in Bacillus subtilis: effects of resistance to glucose-mediated catabolite repression. Mol Cells 7:788–794

    CAS  PubMed  Google Scholar 

  27. Klibanov AM (2001) Improving enzymes by using them in organic solvents. Nature 409:241–246

    Article  CAS  PubMed  Google Scholar 

  28. Lam K, Chow K, Wong W (1998) Construction of an efficient Bacillus subtilis system for extracellular production of heterologous proteins. J Biotechnol 63:167–177

    Article  CAS  PubMed  Google Scholar 

  29. Monroe A, Setlow P (2006) Localization of the transglutaminase cross-linking sites in the Bacillus subtilis spore coat protein GerQ. J Bacteriol 188:7609–7616

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  30. Negri A, Potocki W, Iwanicki A, Obuchowski M, Hinc K (2013) Expression and display of Clostridium difficile protein FliD on the surface of Bacillus subtilis spores. J Med Microbiol 62:1379–1385

    Article  CAS  PubMed  Google Scholar 

  31. Nicholson WL, Setlow P (1990) Sporulation, germination and outgrowth. In: Harwood CR, Cutting SM (eds) Molecular biological methods for Bacillus. Wiley, Chichester, pp 391–450

  32. Pan ZY, Yang ZM, Pan L, Zheng SP, Han SY, Lin Y (2014) Displaying Candida antarctica lipase B on the cell surface of Aspergillus niger as a potential food-grade whole-cell catalyst. J Ind Microbiol Biotechnol 41:711–720

    Article  CAS  PubMed  Google Scholar 

  33. Potot S, Serra CR, Henriques AO, Schyns G (2010) Display of recombinant proteins on Bacillus subtilis spores, using a coat-associated enzyme as the carrier. Appl Environ Microbiol 76:5926–5933

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  34. Ricca E, Cutting SM (2003) Emerging applications of bacterial spores in nanobiotechnology. J Nanobiotechnol. doi:10.1186/1477-3155-1-6

  35. Sanbrook J, Fritsch E, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

    Google Scholar 

  36. Shao H, Xu L, Yan Y (2013) Isolation and characterization of a thermostable esterase from a metagenomic library. J Ind Microbiol Biotechnol 40:1211–1222

    Article  CAS  PubMed  Google Scholar 

  37. Stepankova V, Bidmanova S, Koudelakova T, Prokop Z, Chaloupkova R, Damborsky J (2013) Strategies for stabilization of enzymes in organic solvents. ACS Catal 3:2823–2836

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  39. Turner P, Mamo G, Karlsson EN (2007) Potential and utilization of thermophiles and thermostable enzymes in biorefining. Microb Cell Fact 6:1–23

    Article  Google Scholar 

  40. Vafiadi C, Topakas E, Biely P, Christakopoulos P (2009) Purification, characterization and mass spectrometric sequencing of a thermophilic glucuronoyl esterase from Sporotrichum thermophile. FEMS Microbiol Lett 296:178–184

    Article  CAS  PubMed  Google Scholar 

  41. Wang N, Chang C, Yao Q, Li G, Qin L, Chen L, Chen K (2011) Display of Bombyx mori alcohol dehydrogenases on the Bacillus subtilis spore surface to enhance enzymatic activity under adverse conditions. PLoS One 6:e21454

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  42. Wang W, Xu Y, Qin X, Lan D, Yang B, Wang Y (2014) Immobilization of lipase SMG1 and its application in synthesis of partial glycerides. Eur J Lipid Sci Technol 116:1063–1069

    Article  CAS  Google Scholar 

  43. Wriggers W, Chakravarty S, Jennings PA (2005) Control of protein functional dynamics by peptide linkers. Biopolymers 80:736–746

    Article  CAS  PubMed  Google Scholar 

  44. Xu X, Gao C, Zhang X, Che B, Ma C, Qiu J, Tao F, Xu P (2011) Production of N-acetyl-d-neuraminic acid by use of an efficient spore surface display system. Appl Environ Microbiol 77:3197–3201

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  45. Zhang B, Weng Y, Xu H, Mao Z (2012) Enzyme immobilization for biodiesel production. Appl Microbiol Biotechnol 93:61–70

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by the Open Funding Project of National Key Laboratory of Biochemical Engineering, the National Key Basic Research Program of China (973 Program, No. 2011CBA00800), and the Key Agriculture Support Project of Jiangsu Province, China (No. BE2013400).

Author information

Authors and Affiliations

  1. Institute of Life Sciences, Jiangsu University, Zhenjiang, 212013, Jiangsu Province, China

    Huayou Chen, Tianxi Zhang, Jinru Jia, Rui Tian, Zhong Ni, Zhi Chen, Keping Chen & Shengli Yang

  2. National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 10090, China

    Huayou Chen

  3. Division of Biological Sciences, University of California at San Diego, La Jolla, CA, 92093-0116, USA

    Huayou Chen & Ake Vastermark

Authors
  1. Huayou Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tianxi Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jinru Jia
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ake Vastermark
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Rui Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Zhong Ni
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Zhi Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Keping Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Shengli Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Huayou Chen.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chen, H., Zhang, T., Jia, J. et al. Expression and display of a novel thermostable esterase from Clostridium thermocellum on the surface of Bacillus subtilis using the CotB anchor protein. J Ind Microbiol Biotechnol 42, 1439–1448 (2015). https://doi.org/10.1007/s10295-015-1676-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10295-015-1676-8

Keywords

Search

Navigation