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.Fil: Gimenez, Gabriela Gregolin. Universidad Estadual de Maringá; BrasilFil: Costa, Hernán. Universidad Nacional de Luján. Departamento de Ciencias Básicas. Area de Química Biológica; Argentina. Universidad Nacional de Luján. Instituto de Ecología y Desarrollo Sustentable. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ecología y Desarrollo Sustentable; ArgentinaFil: de Lima Neto, Quirino Alves. Universidad Estadual de Maringá; BrasilFil: Fernandez, Maria Aparecida. Universidad Estadual de Maringá; BrasilFil: Ferrarotti, Susana Alicia. Universidad Nacional de Luján. Departamento de Ciencias Básicas. Area de Química Biológica; ArgentinaFil: Matioli, Graciette. Universidad Estadual de Maringá; Brasi