Transglutaminase from Streptomyces mobaraensis (MTG) is an important enzyme for numerous industrial applications. Recombinant production requires proteolytic activation of the zymogen. The study provides a convenient procedure for the preparation of the transglutaminase-activating metalloprotease (TAMP) in Escherichia coli. In contrast to wtTAMP， rTAMP exhibited the P domain of convertases as molecular mass of 55.7 kDa suggested. Protein integrity was beneficially influenced by 2-5 mM CaCl. Study of pH and temperature optima assigned rTAMP to the neutral metalloproteases， more heat-resistant than Dispase but not thermolysin. Zinc had no inhibiting effect but 3.1 μM EDTA completely reduced activity of 5 nM TAMP. MTG， exceeding concentration of rTAMP by three orders of magnitude， was largely activated within few minutes. The kinetic parameters K (1.31 ± 0.05 mM) and k (135 ± 4.3 s)， monitored by isothermal titration calorimetry (ITC)， further highlighted catalytic efficiency (103，053 M s) of rTAMP and rapid processing of MTG. ITC even revealed that inhibition of rTAMP by its intrinsic inhibitory protein SSTI was an enthalpy-driven process resulting in K of 199 ± 37.9 nM. The production procedure of rTAMP in E. coli closes the gap between production and application of recombinant MTG and may enhance relevance of MTG-mediated reactions in pharmaceutical processes.