Vanadium containing bromoperoxidase: An example of an oxidoreductase with high operational stability in aqueous and organic media

Abstract

The conversion is described of phenolsulphonephtalein (phenol red) to 3,3',5,5'-tetrabromophenolsulphonephthalein (bromophenol blue) by bromoper-oxidase from the brown alga Ascophyllum nodosum. This reaction provides a convenient assay for the detection of bromoperoxidase activity in vitro. Bromoperoxidase was shown to be stable under turnover conditions for three weeks at room temperature, catalyzing the bromination of phenol red into bromophenol blue. When stored at room temperature in organic sol vents such as acetone, methanol, ethanol [present up to 60% (v/v)], and 1-propanol [40% (v/v)], bromoperoxidase was stable for more than one month. As far as we know this is the first example of an oxidoreductase which displays such great stability. This enhances the applicability of the enzyme in organic synthesis.

Vanadium haloperoxidases: From the discovery 30 years ago to X-ray crystallographic and V K-edge absorption spectroscopic studies

“…Standard assays for the determination of VHPO activity are therefore based on the reactivity of this mixture with commercial chemicals. Phenol red and o-dianisidine were first reported as chemical probes for the detection of VHPO-catalyzed halogenation, but are restricted to qualitative studies [45][46][47]. The monochlorodimedone (MCD) halogenation provides a simple assay to determine steady-state kinetic parameters of VCPO and VBPO at acidic pH by monitoring the loss of absorbance of MCD at 290 nm [48][49][50].…”

“…VHPO enzymes are also extremely thermostable, resistant to high concentration of organic solvents such as methanol, ethanol, 1-propanol, acetone [45] and dioxane and still active in concentrated solutions of guanidine hydrochloride or SDS [61,63].…”

“…In view of the still increasing power of computational approaches such as the ADF Density Functional Theory package [145], it is also tempting to generate computational structures which can be validated/adjusted in both the EXAFS and XANES part of the X-ray absorption spectrum. and dioxidovanadium (V) stabilized by organic molecules [45,152]. However, recent studies [56] have shown that the influence of the protein stabilization of the active center goes beyond the first coordination sphere.…”

Laboratory-evolved Vanadium Chloroperoxidase Exhibits 100-Fold Higher Halogenating Activity at Alkaline pH

“…The heme peroxidases have a significant disadvantage of rapid inactivation during turnover because of an oxidative reaction of the heme group with the peroxide substrate and the formed hypohalous acids. Vanadium haloperoxidases (VHPOs), 2 which contain vanadate (VO 4 3Ϫ ) as a prosthetic group, do not suffer from this disadvantage (9); in addition they are much more resistant toward heat, detergent, and solvent denaturation (10). The major drawback of all haloperoxidases including the stable VHPOs is that they are mainly active at mildly acidic pH values, whereas for many applications activity at mildly alkaline pH values is required.…”

Isolation, Characterization, and Primary Structure of the Vanadium Chloroperoxidase from the Fungus Embellisia didymospora

“…The DEAE was loaded on to a 3-cm diameter column and subsequently washed with 10 column volumes of 0.1 M NaCl in 50 mM Tris/SO 4 , pH 8.0. Column elution was carried out using 0.25 M NaCl in 50 mM Tris/SO 4 , pH 8.0, and vanadium chloroperoxidase (vCPO) 1 active fractions, assessed using a phenol red qualitative assay (22), were pooled, and NaCl was added to a final concentration of 2 M. A 5-ml prepacked, buffer washed (2 M NaCl in 50 mM Tris/SO 4 , pH 8.0), phenyl-Sepharose Cl-4B (Amersham Pharmacia Biotech) column was used to bind the vCPO. This was then washed with 10 column volumes of 0.6 M NaCl in 50 mM Tris/SO 4 and eluted with 50 mM NaCl in 50 mM Tris/SO 4 , pH 8.0. vCPO active fractions were pooled, dialyzed overnight against 50 mM Tris/SO 4 , pH 8.0.…”