Departamento de Farmácia, Universidade Estadual de Maringá (UEM), Av. Colombo, 5790, Maringá, PR, CEP 87020-900, Brazil.
Departamento de Ciencias Básicas, Universidad Nacional de Luján, Rutas 5 y 7 (6700), Luján, Buenos Aires, Argentina.
Bioprocess Biosyst Eng. 2019 Apr;42(4):621-629. doi: 10.1007/s00449-018-02068-4. Epub 2019 Jan 3.
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.
坚硬芽孢杆菌 37 株产生环麦芽寡糖葡聚糖转移酶（CGTase）酶，CGTase 通过淀粉环化反应产生环糊精（CDs）。克隆和表达重组 CGTase 的策略是一种具有经济可行性的替代方法，可用于工业生产中具有经济可行性的 CGTase 生产。本研究使用枯草芽孢杆菌 WB800 作为细菌表达宿主，用于克隆来自坚硬芽孢杆菌 37 株的 CGTase 基因的重组 CGTase 的生产。CGTase 基因克隆在 TOPO-TA 质粒中，该质粒转化大肠杆菌 DH5α。亚克隆在 pWB980 质粒上进行，并转化枯草芽孢杆菌 WB800。2xYT 培养基最适合重组 CGTase 的生产。枯草芽孢杆菌 WB800 重组 CGTase 粗提物的酶活为 1.33µmolβ-CD/min/mL，比野生型 CGTase 粗提物的酶活高 7.4 倍。经纯化后，重组 CGTase 的酶活为 157.78µmolβ-CD/min/mL，而野生型 CGTase 的酶活为 9.54µmolβ-CD/min/mL。当使用坚硬芽孢杆菌 37 株 CGTase 的最佳 CDs 生产条件时，观察到 CGTase 酶的催化特性相当。在枯草芽孢杆菌 WB800 中克隆和表达 CGTase 的策略是有效的，产生的 CGTase 量比野生型更多，为重组酶的大规模生产提供了必要的数据。
