Kinetic and stability studies on the chloroperoxidase complexes in presence of tert-butyl hydroperoxide.

The inactivation of native chloroperoxidase (CPO) from Caldariomyces fumago in the presence of tert-butyl hydroperoxide (tert-BuOOH) was investigated. A kinetic analysis was made and the inactivation constants (V(3) and K(3)) were evaluated. In prolonged times, uni-exponential equation describes the enzyme time course inactivation. A method based on the rate of inactivation of the enzyme in the presence of the inactivating molecule tert-BuOOH was also performed. A second group of inactivation constants (j(3) and K) was obtained, which is sufficiently close to the first two, thus verifying that the decreasing of enzyme absorbance corresponds to the decay of activity.

High-pressure-assisted reconstitution of recombinant chloroperoxidase.

An expression vector containing a T7 promoter and an OmpA signal sequence followed by the DNA sequence of mature chloroperoxidase from the fungus Caldariomyces fumago has been transformed into Escherichia coli. This construct gave high-level expression of apochloroperoxidase when induced with isopropyl thiogalactopyranoside. The nonglycosylated apoenzyme was secreted into periplasmic space. The recombinant apochloroperoxidase was expressed at a level representing about 2% of the total cellular protein. Before conversion to holoenzyme, the apochloroperoxidase was denatured in 8 M urea and partially purified by DEAE chromatography. Maximum yields of holoenzyme were obtained when the denatured apochloroperoxidase, dissolved in a refolding buffer containing iron protoporphyrin IX, calcium ions, and oxidized glutathione, was subjected to high pressure (207 MPa) at -12 degrees C and then allowed to refold at atmospheric pressure and room temperature. The recombinant holoenzyme was characterized by absorption and CD spectroscopy and tested for halogenation and peroxidation activity. The yield of active holochloroperoxidase was about 5% when high-pressure treatment was used as part of the reconstitution process. In the absence of pressure treatment, holoenzyme was formed at about the 1% level. The holochloroperoxidase preparations which resulted from high-pressure treatment showed, upon return to atmospheric pressure, a considerably higher content of native-like secondary structure compared to the nonpressurized preparations. These experiments show that active recombinant chloroperoxidase molecules can be produced, and prove that glycosylation is not a mandatory requirement for chloroperoxidase refolding.