Sequencing, cloning, and heterologous expression of cyclomaltodextrin glucanotransferase of Bacillus firmus strain 37 in Bacillus subtilis WB800
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.
KeywordsCloning Heterologous expression CGTase Bacillus subtilis WB800 Cyclodextrins
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).
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.
- 8.Wong SL, Ye R, Nathoo S (1994) Engineering and production of streptokinase in a Bacillus subtilis expression-secretion system. Appl Environ Microbiol 60:517–523Google 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 68:3261–3269CrossRefGoogle Scholar
- 16.Sambrook J, Russell DW (2001) Molecular Cloning: A laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New YorkGoogle 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 20008:692573Google 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 259:8619–8625Google 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 12:753–759Google 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 3:78–85Google Scholar