Expression of Bacillus amyloliquefaciens transglutaminase in recombinant E. coli under the control of a bicistronic plasmid system in DO-stat fed-batch bioreactor cultivations

• Published in: Brazilian journal of microbiology 2021-09, Vol.52 (3), p.1225-1233
• Main Authors: Duarte, Lovaine Silva, Matte, Carla Roberta, Dall Cortivo, Paulo Roberto, Nunes, José Eduardo Sacconi, Barsé, Laisa Quadros, Bizarro, Cristiano Valim, Ayub, Marco Antônio Záchia
• Format: Article
• Language: English
• Subjects: Bacillus amyloliquefaciens; Bacillus amyloliquefaciens - enzymology; Bacillus amyloliquefaciens - genetics; Batch culture; Biomass; Biomedical and Life Sciences; Bioreactors; Biotechnology and Industrial Microbiology - Research Paper; Culture Media; E coli; Enzymatic activity; Enzyme activity; Enzymes; Escherichia coli - enzymology; Escherichia coli - genetics; Fed batch; Food Microbiology; Glucose; Life Sciences; Medical Microbiology; Microbial Ecology; Microbial Genetics and Genomics; Microbiology; Mycology; pH effects; Plasmids - genetics; Transglutaminases - biosynthesis; Transglutaminases - genetics
• ISSN: 1517-8382; 1678-4405
• DOI: 10.1007/s42770-021-00521-3

We studied the expression of Bacillus amyloliquefaciens transglutaminase cloned in Escherichia coli BL21(DE3)pLysS harboring the plasmid pBAD/3C/bTGase, a bicistronic expression system, in bioreactor cultivation. Batch and fed-batch controlled as DO-stat strategies were employed for the production of the recombinant enzyme. In 30 h-batch cultivations using Terrific broth (TB), 6 g/L of biomass and 3.12 U/mg protein of transglutaminase activity were obtained. DO-stat fed-batch cultivations under the control of oxygen concentration (DO-stat) using TB as medium but fed with glucose allowed the increment in biomass formation (17.5 g/L) and enzyme activity (6.43 U/mg protein ). DO-stat fed-batch using mineral medium (M9) and fed with glucose under the same conditions produced even higher enzymatic activity (9.14 U/mg protein ). The pH effect was investigated, and the best enzymatic activity could be observed at pH 8. In all cultivations, the bicistronic system remained stable, with 100% of plasmid-bearing cells. These results show that E. coli bearing bicistronic plasmid constructs to express recombinant TGase could be cultivated in bioreactors under DO-stat fed-batch using mineral medium and it is a promising strategy in future optimizations to produce this important enzyme.