Extracellular production of active-form Streptomyces mobaraensis transglutaminase in Bacillus subtilis

Transglutaminase (TG) from Streptomyces mobaraensis has been widely used in the food industry. It is secreted naturally as an inactive zymogen, which is then activated by the removal of the N-terminal pro-peptide. In this study, the mtg gene from S. mobaraensis was expressed in a food-grade strain of bacterium, Bacillus subtilis. When its native signal peptide was replaced by signal peptide SacB (SP_sacB) and the pro-peptide was replaced by that derived from S. hygroscopicus, an extracellular activity of 16.1 U/mg was observed. A modified Saccharomyces cerevisiae vacuolar ATPase subunit (VMA) intein was introduced into the zymogen to simplify its activation process by controlling temperature. When the cleavage site in the C-terminal of VMA was placed between the pro-peptide and core domain, the activation process was carried out at 18 °C. Promoter replacement further increased the enzymatic activity. Finally, the extracellular enzymatic activity reached 2.6 U/mg under the control of the constitutive promoter P_yvyD. This is the first report on the extracellular production of active-form Streptomyces TG in B. subtilis without splicing with the cleavage enzyme.