RESUMEN

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 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 5 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€¯0.05 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€¯37.9 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.

Asunto(s)

Proteínas Bacterianas , Escherichia coli/metabolismo , Metaloproteasas , Streptomyces/enzimología , Transglutaminasas , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Escherichia coli/genética , Metaloproteasas/genética , Metaloproteasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Transglutaminasas/genética , Transglutaminasas/metabolismo