Antimicrobial effect of a modified vanadium chloroperoxidase on Enterococcus faecalis biofilms at root canal pH.

INTRODUCTION: Previous research showed an antimicrobial effect of vanadium chloroperoxidase (VCPO) on in vitro Enterococcus faecalis biofilms. The current study aimed to optimize the use of this enzyme at the root canal pH using a modified VCPO (mVCPO) that was adapted to function at a higher pH and to explore the biocompatibility of mVCPO. METHODS: The activity of the original and modified VCPO was assessed using the monochlorodimedone assay. For antimicrobial assessment, 48-hour biofilms of E. faecalis OS-16 were incubated 5 or 30 minutes with mVCPO, bromide, and hydrogen peroxide, and colony-forming units were determined. A metabolic activity assay was used to evaluate the cytotoxic effect of mVCPO on oral fibroblasts. RESULTS: Reaction products generated by mVCPO at a root canal pH of 7.7 significantly inactivated the biofilm after 5 minutes and even more after 30 minutes (Mann-Whitney U test, P < .05). The mVCPO reaction products showed less cytotoxic effects than control solutions and 0.5% sodium hypochlorite (Kruskal-Wallis test, P < .05). CONCLUSIONS: The incubation of mVCPO in the presence of its substrates with in vitro E. faecalis biofilms showed a significant antimicrobial effect at the root canal pH. Also, cytotoxicity tests showed preliminary biocompatibility. Therefore, an interappointment dressing containing mVCPO could aid in improving current endodontic treatment through continuous and local generation of antimicrobials.

Effect of vanadium chloroperoxidase on Enterococcus faecalis biofilms.

INTRODUCTION: The aim of this study was to explore the antimicrobial effect of vanadium chloroperoxidase (VCPO) reaction products on Enterococcus faecalis biofilms of 4 different strains. METHODS: Twenty-four-hour biofilms of E. faecalis strains V583, ER5/1, E2, and OS-16 were incubated in mixtures with VCPO, halide (either bromide or chloride), and hydrogen peroxide. The antibacterial efficacy was assessed by colony-forming unit counts. RESULTS: The VCPO reaction products had a similar efficacy in reducing the viability of the 4 strains of E. faecalis (94%; range, 87%-100%). Bromide as the halogen of choice was more effective on E. faecalis strains E2 and OS-16, as compared with chloride (Mann-Whitney U test; P < .05). Despite different quantities of produced biofilms by the 4 strains, VCPO treatment was similarly effective toward all strains (Kruskal-Wallis test; P < .05). CONCLUSIONS: VCPO treatment results in an antimicrobial effect toward in vitro E. faecalis biofilms and might provide an addition to current endodontic treatment, possibly as an antimicrobial dressing.

Antimicrobial activity of vanadium chloroperoxidase on planktonic Streptococcus mutans cells and Streptococcus mutans biofilms.

The aim of this study was to investigate the antimicrobial activity of vanadium chloroperoxidase (VCPO) reaction products on planktonic and biofilm cells of Streptococcus mutans C180-2. Planktonic and biofilm cells were incubated in a buffered reaction mixture containing VCPO, halide (either chloride or bromide) and hydrogen peroxide, and the killing efficacy was assessed by CFU counts. The enzymatic products formed by VCPO significantly reduced the viability of planktonic and biofilm cells compared to their negative controls and the effect on the biofilm cells was more effective than a 0.2% chlorhexidine digluconate treatment. We conclude that VCPO and its reaction products form a potent antimicrobial system against S. mutans.

Bactericidal and virucidal activity of the alkalophilic P395D/L241V/T343A mutant of vanadium chloroperoxidase.

AIMS: Vanadium chloroperoxidase and its directed evolution mutant P395D/L241V/T343A were investigated for their antibacterial and antiviral potential at slightly alkaline pH and at a H(2)O(2) concentration that is low compared to current nonenzymatic formulations. METHODS AND RESULTS: Two bacteria (the Gram-negative Pseudomonas aeruginosa and the Gram-positive Staphylococcus aureus) and two viruses (the enveloped Herpes Simplex Virus and the nonenveloped Coxsackievirus B4) were incubated with the P395D/L241V/T343A mutant, 10 mmol l(-1) H(2)O(2) and 100 mmol l(-1) Br(-) at pH 8. Strong microbial reduction was observed and bactericidal and virucidal activities of the mutant were three to six orders of magnitude higher than for the wild-type enzyme. CONCLUSIONS: The P395D/L241V/T343A mutant of vanadium chloroperoxidase has a broad antimicrobial activity at alkaline conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: For many disinfection formulations, antimicrobial activity at slightly alkaline pH values is required. To date, only the wild-type vanadium chloroperoxidase has been studied for its antibacterial activity, and only at acidic to neutral pH values. Its antiviral activity (e.g. useful for the cleaning of medical equipment) was not studied before. The observed activity for the alkalophilic P395D/L241V/T343A mutant is an important step forward in the application of this robust enzyme as a component in disinfection formulations.

Molecular structural characteristics governing biocatalytic chlorination of PAHs by chloroperoxidase from Caldariomyces fumago.

Based on some fundamental quantum chemical descriptors computed by PM3 Hamiltonian, a quantitative structure-property relationship (QSPR) for specific activity of 17 polycyclic aromatic hydrocarbons (PAHs) of biocatalytic chlorination by chloroperoxidase (CPO) from Caldariomyces fumago was developed using partial least squares (PLS) regression. The model can be used to estimate biocatalytic chlorination reaction rates of PAHs. The main factors affecting specific activity of PAHs of biocatalytic chlorination by CPO from Caldariomyces fumago are absolute hardness, dipole moment, absolute electronegativity, and molecular bulkness of the PAH molecules. The biocatalytic chlorination reaction rates of PAHs with large values of absolute hardness, absolute electronegativity, and molecular bulkness tend to be slow. Increasing dipole moment of PAHs leads to increase the specific activity.

Cl K-edge X-ray spectroscopic investigation of enzymatic formation of organochlorines in weathering plant material.

The contribution of halocarbons from plant weathering to the total organohalogen budget of terrestrial systems is gaining recognition. To evaluate the formation of such halocarbons, speciation of chlorine in Sequoia sempervirens (redwood) needles was examined in the presence of an external chloroperoxidase (CPO) enzyme using Cl K-edge X-ray absorption spectroscopy. The Cl forms in fresh and naturally weathered needles and in model laboratory reactions were compared. To provide a straightforward analogue to the enzymatic chlorination in plants, chlorination reactions were conducted for phenol, a common moiety of plant macromolecules. Plant material chlorination was also examined in the presence of hypochlorite in an ancillary mechanistic investigation. The dominant form of Cl in fresh, unreacted plant material was found to be inorganic Cl-, which was partially converted to organochlorine in the presence of CPO. Chlorination is affected by the nature of reactant (CPO, H2O2) addition, reaction time, and temperature. The organochlorines produced in these laboratory investigations closely resemble those produced during the natural weathering of redwood needles. A striking consistency in chlorine speciation observed among the various sample types suggests that (i) CPO produced by terrestrial organisms could play a vital role in the generation of organochlorines associated with the degradation of plant material and (ii) initial targets of enzymatic chlorination might include lignin-like macromolecules rich in aromatic character and hydroxyl groups. These findings lend further credibility to a significant biogenic contribution to the global organohalogen burden by elucidating a probable route of enzymatic chlorination of natural organic matter in terrestrial systems.

Antifungal and peroxidative activities of nonheme chloroperoxidase in relation to transgenic plant protection.

Nonheme chloroperoxidase (CPO-P) of Pseudomonas pyrrocinia catalyzes the oxidation of alkyl acids to peracids by hydrogen peroxide. Alkyl peracids possess potent antifungal activity as found with peracetate: 50% killing (LD(50)) of Aspergillus flavus occurred at 25 microM compared to 3.0 mM for the hydrogen peroxide substrate. To evaluate whether CPO-P could protect plants from fungal infection, tobacco was transformed with a gene for CPO-P from P. pyrrocinia and assayed for antifungal activity. Leaf extracts from transformed plants inhibited growth of A. flavus by up to 100%, and levels of inhibition were quantitatively correlated to the amounts of CPO-P activity expressed in leaves. To clarify if the peroxidative activity of CPO-P could be the basis for the increased resistance, the antifungal activity of the purified enzyme was investigated. The LD(50) of hydrogen peroxide combined with CPO-P occurred at 2.0 mM against A. flavus. Because this value was too small to account for the enhanced antifungal activity of transgenic plants, the kinetics of the enzyme reaction was examined and it was found that the concentration of hydrogen peroxide needed for enzyme saturation (K(m) = 5.9 mM) was already lethal. Thus, the peroxidative activity of CPO-P is not the basis for antifungal activity or enhanced resistance in transgenic plants expressing the gene.

Effects of chloroperoxidase and hydrogen peroxide on the viabilities of Aspergillus flavus conidiospores.

The effects of chloroperoxidase [EC 1.1.1.10] and hydrogen peroxide on the viabilities of quiescent and germinating conidiospores of an aflatoxigenic fungus, Aspergillus flavus, were determined. Hydrogen peroxide was found moderately lethal and chloroperoxidase produced a 30-fold increase in the lethality of hydrogen peroxide to germinating conidia, which were 75-fold more susceptible to chloroperoxidase than were quiescent conidia. According to infrared examinations of fungal corpses, mortality occurred by oxidation rather than peroxidative chlorination.

Oxygenation of organosulfur compounds by peroxidases: evidence of an electron transfer mechanism for lactoperoxidase.

The oxygenation of benzyl methylsulfide, thioanisole, and thiobenzamide to the respective sulfoxides was found to be catalyzed by chloroperoxidase, lactoperoxidase, and horseradish peroxidase. The activities of lactoperoxidase and horseradish peroxidase were similarly low toward benzyl methylsulfide and thioanisole but lactoperoxidase efficiently catalyzed the oxygenation of thiobenzamide while horseradish peroxidase showed low activity. Chloroperoxidase had high reactivity toward all three substrates tested in halide-independent reactions and only small differences in the rates of enzymatic sulfoxidation were observed. The logarithm of lactoperoxidase activity was found to linearly correlate with the voltammetric peak potentials for oxidation of the three substrates tested. The results of this study are consistent with a one-electron transfer mechanism for lactoperoxidase-mediated sulfoxidation.