Skip to main content
SpringerLink
Log in

Sequencing, cloning, and heterologous expression of cyclomaltodextrin glucanotransferase of Bacillus firmus strain 37 in Bacillus subtilis WB800

  • Research Paper
  • Published:
Bioprocess and Biosystems Engineering Aims and scope Submit manuscript

Abstract

Bacillusfirmus strain 37 produces the cyclomaltodextrin glucanotransferase (CGTase) enzyme and CGTase produces cyclodextrins (CDs) through a starch cyclization reaction. The strategy for the cloning and expression of recombinant CGTase is a potentially viable alternative for the economically viable production of CGTase for use in industrial processes. The present study used Bacillus subtilis WB800 as a bacterial expression host for the production of recombinant CGTase cloned from the CGTase gene of B. firmus strain 37. The CGTase gene was cloned in TOPO-TA® plasmid, which was transformed in Escherichia coli DH5α. The subcloning was carried out with pWB980 plasmid and transformation in B. subtilis WB800. The 2xYT medium was the most suitable for the production of recombinant CGTase. The enzymatic activity of the crude extract of the recombinant CGTase of B. subtilis WB800 was 1.33 µmol β-CD/min/mL, or 7.4 times greater than the enzymatic activity of the crude extract of CGTase obtained from the wild strain. Following purification, the recombinant CGTase exhibited an enzymatic activity of 157.78 µmol β-CD/min/mL, while the activity of the CGTase from the wild strain was 9.54 µmol β-CD/min/mL. When optimal CDs production conditions for the CGTase from B. firmus strain 37 were used, it was observed that the catalytic properties of the CGTase enzymes were equivalent. The strategy for the cloning and expression of CGTase in B. subtilis WB800 was efficient, with the production of greater quantities of CGTase than with the wild strain, offering essential data for the large-scale production of the recombinant enzyme.

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

Similar content being viewed by others

References

  1. Del Valle EMM (2004) Cyclodextrins and their uses: a review. Process Biochem 39:1033–1046

    Article  CAS  Google Scholar 

  2. Lima PSS, Lucchese AM, Araújo-Filho HG, Menezes PP, Araújo AAS, Quintans-Júnior LJ, Quintans JSS (2016) Inclusion of terpenes in cyclodextrins: preparation, characterization and pharmacological approaches. Carbohydr Polym 151:965–987

    Article  CAS  PubMed  Google Scholar 

  3. Li Y, Chen Y, Li H (2017) Recovery and purification of cholesterol from cholesterol-β-cyclodextrin inclusion complex using ultrasound-assisted extraction. Ultrason Sonochem 34:281–288

    Article  CAS  PubMed  Google Scholar 

  4. Mihailiasa M, Caldera F, Li J, Peila R, Ferri A, Trotta F (2016) Preparation of functionalized cotton fabrics by means of melatonin loaded β-cyclodextrin nanosponges. Carbohydr Polym 142:24–30

    Article  CAS  PubMed  Google Scholar 

  5. Matioli G, Zanin GM, De Moraes FF (2001) Characterization of cyclodextrin glycosyltransferase from Bacillus firmus strain nº 37. Appl Biochem Biotechnol 91(3):643–654

    Article  PubMed  Google Scholar 

  6. Mathew S, Adlercreutz P (2013) Regioselective glycosylation of hydroquinone to a-arbutin by cyclodextrin glucanotransferase from Thermoanaerobacter sp. Biochem Eng J 79:187–193

    Article  CAS  Google Scholar 

  7. Schallmey M, Singh A, Ward OP (2004) Developments in the use of Bacillus species for industrial production. Can J Microbiol 50:1–17

    Article  CAS  PubMed  Google Scholar 

  8. Wong SL, Ye R, Nathoo S (1994) Engineering and production of streptokinase in a Bacillus subtilis expression-secretion system. Appl Environ Microbiol 60:517–523

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Wong SL (1995) Advances in the use of Bacillus subtilis for the expression. and secretion of heterologous proteins. Curr Opin Biotechnol 6:517–522

    Article  CAS  PubMed  Google Scholar 

  10. Aminov RI, Golovchenko NP, Ohmiya K (1995) Expression of a celE gene from Clostridium thermocellum in Bacillus. J Ferment Bioeng 79:530–537

    Article  CAS  Google Scholar 

  11. Wu SC, Yeung JC, Duan Y, Ye R, Szarka SJ, Habibi HR, Wong SL (2002) Functional production and characterization of a fibrin-specific single-chain antibody fragment from Bacillus subtilis: effects of molecular chaperones and a wall-bound protease on antibody fragment production. Appl Environ Microbiol 68:3261–3269

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Nakamura N, Horikoshi K (1976) Characterization and some cultural conditions of a cyclodextrin glucanotransferase from alkalophilic Bacillus sp. Agric Biol Chem 40:753–757

    Article  CAS  Google Scholar 

  13. Costa H, del Canto S, Ferrarotti S, de Jiménez Bonino MB (2009) Structure–function relationship in cyclodextrin glycosyltransferase from Bacillus circulans DF 9R. Carbohydr Res 344:74–79

    Article  CAS  PubMed  Google Scholar 

  14. Moriwaki C, Mazzer C, Pazzetto R, Matioli G (2009) Produção, purificação e aumento da performance de ciclodextrina glicosiltransferases para produção de ciclodextrinas. Quím Nova 32:2360–2366

    Article  CAS  Google Scholar 

  15. Liu YH, Lu FP, Li Y, Yin XB, Wang Y, Gao C (2008) Characterisation of mutagenised acid-resistant alphaamylase expressed in Bacillus subtilis WB600. Appl Microbiol Biotechnol 78:85–94

    Article  CAS  PubMed  Google Scholar 

  16. Sambrook J, Russell DW (2001) Molecular Cloning: A laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  17. Fenelon VC, Aguiar MF, Miyoshi JH, Martinez CO, Matioli G (2015) Ultrafiltration system for cyclodextrin production in repetitive batches by CGTase from Bacillus firmus strain 37. Bioprocess Biosyst Eng 38:1291–1301

    Article  CAS  PubMed  Google Scholar 

  18. Bradford MM (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  CAS  PubMed  Google Scholar 

  19. Tardioli PW, Zanin GM, de Moraes FF (2006) Characterization of Thermoanaerobacter cyclomaltodextrin glucanotransferase immobilized on glyoxilagarose. Enzyme Microb Technol 39:1270–1278

    Article  CAS  Google Scholar 

  20. Jemli S, Ben Messaoud E, Ben Mabrouk S, Bejar S (2008) The cyclodextrin glycosyltransferase of Paenibacillus pabuli US132 strain: molecular characterization and overproduction of the recombinant enzyme. J Biomed Biotechnol 20008:692573

    Google Scholar 

  21. Liu SL, Du K, Chen WZ, Liu G, Xing M (2012) Effective approach to greatly enhancing selective secretion and expression of three cytoplasmic enzymes in Escherichia coli through synergistic effect of EDTA and lysozyme. J Ind Microbiol Biotechnol 39:1301–1307

    Article  CAS  PubMed  Google Scholar 

  22. Solingen P van, Meijer D, van der Kleij WA, Barnett C, Bolle R, Power SD, Jones BE (2001) Cloning and expression of an endocellulase gene from a novel streptomycete isolated from an East African soda lake. Extremophiles 5:333–341

    Article  PubMed  Google Scholar 

  23. Murashima K, Chen CL, Kosugi A, Tamaru Y, Doi RH, Wong SL (2002) Heterologous production of Clostridium cellulovorans engB, using protease-deficient Bacillus subtilis, and preparation of active recombinant cellulosomes. J Bacteriol 184:76–81

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Ye R, Kim JH, Kim BG, Szarka S, Sihota E, Wong SL (1999) High level secretory production of intact, biologically active staphylokinase from Bacillus subtilis. Biotechnol Bioeng 62:87–96

    Article  CAS  PubMed  Google Scholar 

  25. Wang PZ, Doi RH (1984) Overlapping promoters transcribed by Bacillus subtilis sigma 55 and sigma 37 RNA polymerase holoenzymes during growth and stationary phases. J Biol Chem 259:8619–8625

    CAS  PubMed  Google Scholar 

  26. Wong SL (1989) Development of an inducible and enhancible expression. and secretion system in Bacillus subtilis. Gene 83:215–223

    Article  CAS  PubMed  Google Scholar 

  27. Song W, Nie Y, Mu XQ, Xu Y (2016) Enhancement of extracellular expression of Bacillus naganoensis pullulanase from recombinant Bacillus subtilis: Effects of promoter and host. Protein Expr Purif 124:23–31

    Article  CAS  PubMed  Google Scholar 

  28. Wu XC, Lee W, Tran L, Wong SL (1991) Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracelular proteases. J Bacteriol 173:4952–4958

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Qi Q, Zimmermann W (2005) Cyclodextrin glucanotransferase: from gene to applications. Appl Microbiol Biotechnol 66:475–485

    Article  CAS  PubMed  Google Scholar 

  30. Schmid G (1989) Cyclodextrin glycosyltransferase production: yield enhancement by overexpression of cloned genes. Trends Biotechnol 7:244–248

    Article  CAS  Google Scholar 

  31. Lee K-W, Shin H-D, Lee Y-H (2002) Extracellular Overproduction of β-Cyclodextrin Glucanotransferase in a Recombinant E. coli Using Secretive Expression System. J Microbiol Biotechnol 12:753–759

    Google Scholar 

  32. Do EJ, Shin HD, Kim C, Lee YH (1993) Selection and characterization of catabolite repression resistant mutant of Bacillus firmus var. alkalophilus producing cyclodextrin glucanotransferase. J Microbiol Biotechnol 3:78–85

    CAS  Google Scholar 

  33. Paloheimo M, Haglund D, Aho S, Korhola M (1992) Production of cyclomaltodextrin glucanotransferase of Bacillus circulans var. alkalophilus ATCC21783 in B. subtilis. Appl Microbiol Biotechnol 36:584–591

    Article  CAS  PubMed  Google Scholar 

  34. Li Z, Li B, Gu Z, Du G, Wu J, Chen J (2010) Extracellular expression and biochemical characterization of alpha-cyclodextrin glycosyltransferase from Paenibacillus macerans. Carbohydr Res 345:886–892

    Article  CAS  PubMed  Google Scholar 

  35. Liu Y, Lu F, Chen G, Snyder CL, Sun J, Li Y, Wang J, Xiao J (2010) High-level expression, purification and characterization of a recombinant medium-temperature α-amylase from Bacillus subtilis. Biotechnol Lett 32:119–124

    Article  CAS  PubMed  Google Scholar 

  36. Chen J, Chen X, Dai J, Xie G, Yan L, Lu L, Chen J (2015) Cloning, enhanced expression and characterization of an α-amylase gene from a wild strain in B. subtilis WB800. Int J Biol Macromol 80:200–207

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We would like to thank Dr. Sui Lam Wong for generously providing the WB800 strain of B. subtilis and the pWB980 plasmid. We would also like to thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES-Mincyt) program for financially supporting this study, and the Agencia Nacional de Promoción Científica y Tecnológica (PICT 2016-0240).

Funding

This study was funded in part by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES-Mincyt 244/14).

Author information

Authors and Affiliations

  1. Departamento de Farmácia, Universidade Estadual de Maringá (UEM), Av. Colombo, 5790, Maringá, PR, CEP 87020-900, Brazil

    Gabriela Gregolin Gimenez & Graciette Matioli

  2. Departamento de Ciencias Básicas, Universidad Nacional de Luján, Rutas 5 y 7 (6700), Luján, Buenos Aires, Argentina

    Hernán Costa & Susana Alicia Ferrarotti

  3. Departamento de Biologia Celular e Genética, Universidade Estadual de Maringá (UEM), Av. Colombo, 5790, Maringá, PR, CEP 87020-900, Brazil

    Quirino Alves de Lima Neto & Maria Aparecida Fernandez

Authors
  1. Gabriela Gregolin Gimenez
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hernán Costa
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Quirino Alves de Lima Neto
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Maria Aparecida Fernandez
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Susana Alicia Ferrarotti
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Graciette Matioli
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Graciette Matioli.

Ethics declarations

Conflict of interest

The authors declare that they have no competing interests.

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

Gimenez, G.G., Costa, H., de Lima Neto, Q.A. et al. Sequencing, cloning, and heterologous expression of cyclomaltodextrin glucanotransferase of Bacillus firmus strain 37 in Bacillus subtilis WB800. Bioprocess Biosyst Eng 42, 621–629 (2019). https://doi.org/10.1007/s00449-018-02068-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00449-018-02068-4

Keywords

Search

Navigation