A heme-peptide metalloenzyme mimetic with natural peroxidase-like activity.

Mimicking enzymes with alternative molecules represents an important objective in synthetic biology, aimed to obtain new chemical entities for specific applications. This objective is hampered by the large size and complexity of enzymes. The manipulation of their structures often leads to a reduction of enzyme activity. Herein, we describe the spectroscopic and functional characterization of Fe(III)-mimochrome VI, a 3.5 kDa synthetic heme-protein model, which displays a peroxidase-like catalytic activity. By the use of hydrogen peroxide, Fe(III)-mimochrome VI efficiently catalyzes the oxidation of several substrates, with a typical Michaelis-Menten mechanism and with several multiple turnovers. The catalytic efficiency of Fe(III)-mimochrome VI in the oxidation of 2,2'-azino-di(3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) and guaiacol (k(cat)/K(m)=4417 and 870 mM(-1) s(-1), respectively) is comparable to that of native horseradish peroxidase (HRP, k(cat)/K(m)=5125 and 500 mM(-1) s(-1), respectively). Fe(III)-mimochrome VI also converts phenol to 4- and 2-nitrophenol in the presence of NO(2) (-) and H(2) O(2) in high yields. These results demonstrate that small synthetic peptides can impart high enzyme activities to metal cofactors, and anticipate the possibility of constructing new biocatalysts tailored to specific functions.

Advantages of using non-isothermal bioreactors in agricultural waste water treatment by means of immobilized urease. Study on the influence of spacer length and immobilization method.

The behavior of three different catalytic membranes, obtained by immobilizing urease on nylon sheets chemically grafted with methyl methacrylate, was studied in a bioreactor operating under isothermal and non-isothermal conditions. Membrane activation was carried out by condensation or acyl azide reaction, and spacers of different lengths, such as hexamethylendiamine or hydrazine, were used. Under isothermal conditions, the activities of the catalytic membranes and soluble urease were characterized as a function of pH, temperature, and urea concentration. Both enzyme forms showed the same optimum pH, whereas the optimum temperature was lower for the immobilized enzymes. The spacer length appeared to determine broader pH- and temperature-activity profiles for the urease derivatives. The apparent K(m) values of the insoluble urease were dependent on membrane type and were higher than those of the soluble counterpart, thus indicating an affinity loss for urea. Under non-isothermal conditions, all membranes exhibited an increase of percentage activity proportional to the applied temperature difference and decreasing with the increase of urea concentrations. A decrease of the apparent K(m) was also observed. These results suggest that substrate diffusion limitations due to the immobilization process can be overcome in the presence of temperature gradients. In addition, the remarkable reduction of the production times supports the use of non-isothermal bioreactors for the treatment of urea-polluted waste waters.