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Features of the transglutaminase-activating metalloprotease from Streptomyces mobaraensis DSM 40847 produced in Escherichia coli
Ist Teil von
Journal of biotechnology, 2018-09, Vol.281, p.115-122
Ort / Verlag
Netherlands: Elsevier B.V
Erscheinungsjahr
2018
Link zum Volltext
Quelle
Elsevier ScienceDirect Journals Complete
Beschreibungen/Notizen
•The transglutaminase-activating metalloprotease (TAMP) was produced in E. coli.•Highly purified TAMP is a thermo- and autolysis-resistant enzyme.•Transglutaminase (MTG) was rapidly activated by TAMP in low concentrations.•Strong interaction to the intrinsic inhibitory protein SSTI allows recovery of TAMP.•TAMP closes the gap between production and medicinal applications of MTG.
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 ± 0.05 mM) and kcat (135 ± 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 ± 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.