RESUMO
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 55.7â¯kDa suggested. Protein integrity was beneficially influenced by 2-5â¯mM CaCl2. 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 5â¯nM TAMP. MTG, exceeding concentration of rTAMP by three orders of magnitude, was largely activated within few minutes. The kinetic parameters KM (1.31⯱â¯0.05â¯mM) and kcat (135⯱â¯4.3 s-1), monitored by isothermal titration calorimetry (ITC), further highlighted catalytic efficiency (103,053 M-1 s-1) 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 Kd of 199⯱â¯37.9â¯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.