Features of the transglutaminase-activating metalloprotease from Streptomyces mobaraensis DSM 40847 produced in Escherichia coli

J Biotechnol. 2018 Sep 10;281:115-122. doi: 10.1016/j.jbiotec.2018.07.004. Epub 2018 Jul 4.

Abstract

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.

Keywords: Streptomyces mobaraensis; TAMP features; Transglutaminase processing; Transglutaminase-activating metalloprotease; Transglutaminase-activating metalloprotease inhibitor.

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins* / genetics
  • Bacterial Proteins* / metabolism
  • Base Sequence
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Metalloproteases* / genetics
  • Metalloproteases* / metabolism
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Analysis, DNA
  • Streptomyces / enzymology*
  • Transglutaminases* / genetics
  • Transglutaminases* / metabolism

Substances

  • Bacterial Proteins
  • Recombinant Proteins
  • Transglutaminases
  • Metalloproteases