Copper incorporation into recombinant CotA laccase from Bacillus subtilis: characterization of fully copper loaded enzymes

Abstract

The copper content of recombinant CotA laccase from Bacillus subtilis produced by Escherichia coli cells is shown to be strongly dependent on the presence of copper and oxygen in the culture media. In copper-supplemented media, a switch from aerobic to microaerobic conditions leads to the synthesis of a recombinant holoenzyme, while the maintenance of aerobic conditions results in the synthesis of a copper-depleted population of proteins. Strikingly, cells grown under microaerobic conditions accumulate up to 80-fold more copper than aerobically grown cells. In vitro copper incorporation into apoenzymes was monitored by optical and electron paramagnetic resonance (EPR) spectroscopy. This analysis reveals that copper incorporation into CotA laccase is a sequential process, with the type 1 copper center being the first to be reconstituted, followed by the type 2 and the type 3 copper centers. The copper reconstitution of holoCotA derivatives depleted in vitro with EDTA results in the complete recovery of the native conformation as monitored by spectroscopic, kinetic and thermal stability analysis. However, the reconstitution of copper to apo forms produced in cultures under aerobic and copper-deficient conditions resulted in incomplete recovery of biochemical properties of the holoenzyme. EPR and resonance Raman data indicate that, presumably, folding in the presence of copper is indispensable for the correct structure of the trinuclear copper-containing site.

Effects of Environmental Factors on Soluble Expression of a Humanized Anti-TNF-α scFv Antibody in Escherichia coli

“…In another procedure, starter culture and subculture were grown at 30 °C, 120 rpm. The subculture was induced by 0.1 mM IPTG and the cultivation condition was changed to 30 rpm at 25 °C for 15 h. 27 To assess the effects of MgCl2 and sucrose on the soluble expression of GST-hD2, 0.1 and 10 mM of MgCl2 and 0.4 M sucrose in final concentration were added to each subculture, respectively. 9, 20 In this procedure, induction was achieved using 0.6 mM lactose and the cells were harvested 6 and 18 h after induction.…”

Biohydrogen for a New Generation of H2/O2 Biofuel Cells: A Sustainable Energy Perspective

“…Bacillus pumilus (Bp) [71] Bacterium BOD temp., urate, Cl À 32/391 Bacillus halodurans [53] Bacterium BOD 0.115 [c] n.d. Magnaporthe oryzae [72] Fungus BOD urea 429/664 Bacillus subtilis [318][319] Bacterium BOD temp. 124/322 Rhus vernicifera [56] Plant LAC 0.22 [b] 103/560 [320] Trametes hirsute (Th) [56] Fungus LAC 0.57 [b] 220/210 Sreptomyces coelicolor (Sc) [57] Bacterium LAC 0.47 (pH 5.5) [c] 3600/5.8 [321] E. coli [329] Bacterium CcO aa3 0.2/n.d.…”

“…The affinity for O 2 of MCOs such as BOD or LAC is low because the in vivo function is not to reduce oxygen [K m is in the order of hundreds of micromoles (see Table 1)] [56,[71][72][318][319][320][321][322] . A direct Figure 13.…”

Pushing the limits of automatic computational protein design: design, expression, and characterization of a large synthetic protein based on a fungal laccase scaffold

“…One explanation for this could be limitations in copper incorporation into the DLac protein. In addition to their catalytic significance, bound coppers have been reported to have an important structural role in multicopper oxidases (Durão et al 2006(Durão et al , 2008Sedlak and Wittung-Stafshede 2007). It seems that native glycosylations are not sufficient to enable correct refolding as the native TvL sample (that is fully glycosylated) could not be refolded from the molten globular structure.…”