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

  • Gabriela Gregolin Gimenez
  • Hernán Costa
  • Quirino Alves de Lima Neto
  • Maria Aparecida Fernandez
  • Susana Alicia Ferrarotti
  • Graciette MatioliEmail author
Research Paper

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.

Keywords

Cloning Heterologous expression CGTase Bacillus subtilis WB800 Cyclodextrins 

Notes

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).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Gabriela Gregolin Gimenez
    • 1
  • Hernán Costa
    • 2
  • Quirino Alves de Lima Neto
    • 3
  • Maria Aparecida Fernandez
    • 3
  • Susana Alicia Ferrarotti
    • 2
  • Graciette Matioli
    • 1
    Email author
  1. 1.Departamento de FarmáciaUniversidade Estadual de Maringá (UEM)MaringáBrazil
  2. 2.Departamento de Ciencias BásicasUniversidad Nacional de LujánLujánArgentina
  3. 3.Departamento de Biologia Celular e GenéticaUniversidade Estadual de Maringá (UEM)MaringáBrazil

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