NL9401048A - Haloperoxidases. - Google Patents

Description

HALOPEROXIDASES

The present invention relates to haloperoxidases, in particular chloroperoxidases, in isolated form, to the genes encoding such haloperoxidases, and to various applications of the haloperoxidases, whether or not obtained by recombinant DNA techniques.

The seaweed Ascophvllum nodosum was the first species to discover a non-heme vanadium bromoperoxidase. This was followed by a large number of other seaweed species that were also found to contain these enzymes. The bromine peroxidase from A ^. nodosum has been extensively studied and characterized (1, 2). The enzyme catalyzes the formation of hypohalogen acids from the corresponding halogens. In a first step, hydrogen peroxide reacts with the enzyme to form a hydrogen peroxide-enzyme complex. After this, bromide and a proton react with the complex to form an enzyme-HOBr complex. This complex disintegrates to provide the enzyme and HOBr (3).

The known vanadium bromo peroxidases have been found to show high operational stability in aqueous and organic media. They can be stored for more than a month in organic solvents, such as acetone, methanol, ethanol and 1-propanol, without loss of activity (4). The applications or possible applications for this type of enzymes are therefore great. However, the bromoperoxidases have the drawback that for their activity bromide must be present in such potential applications.

However, this is by no means the case in all situations. Therefore, bromide will have to be added. In addition, attempts to isolate and sequence their genes for these bromo peroxidases have so far been unsuccessful. It is therefore not yet possible to obtain large amounts of recombinant bromoperoxidases for commercially viable applications.

The object of the present invention is to provide enzymes corresponding to the known bromine peroxidases, which do not depend on the presence of bromide, but on chloride, and which can be produced in large quantities by means of recombinant DNA techniques.

According to the invention it has now been found that a vanadium peroxidase, which is found, for example, in the fungus Curvularia inaeaualis. to be active can use chloride in addition to bromide. Chloride, in contrast to bromide, is very widely present in, for example, tap water, surface water and the like and does not require additional addition for the action of the enzyme in various applications. Related vanadium chloroperoxidases have now also been found in various Drechslera species. such as Drechslera biseota-ta. Drechslera fuaax. Drechslera nicotiae and Drechslera suboaoendorfii. or the Embellisia species Embellisia hyacinth i and Embellisia didvmospora. as well as in Ulocladium charta rum and Ulocladium botrvtis. The invention therefore provides a haloperoxidase in an isolated form, for example, obtainable from one of the above filamentous fungi or related species.

The above-mentioned fungal species can be obtained from the Central Bureau of Fungal Cultures in Baarn, the Netherlands via the following deposit access numbers.

The vanadium haloperoxides which can be isolated from the above fungal species are generally chloroperoxidases. Chloroperoxidases are peroxidases which have both an affinity for chloride, bromide and iodide, and can therefore use these three halides as a substrate. The terms "chloroperoxidase (n)" and "haloperoxidase (n)" can be used interchangeably in this application. However, they have the common feature that they have at least a useful affinity for chloride.

The vanadium chloroperoxidases have been found to exhibit very high thermostability and a high affinity for the substrate. For example, the Tm for the haloperoxidase of Curvularia inaeoualis is 90 ° C. The enzyme is also very stable in 40% methanol, ethanol or propanol (7). Its pH optimum is pH 5.5.

The gene encoding the haloperoxidase from Curvularia inaeoualis was isolated and fully sequenced. The isolation was as described in Example 3. In an analogous manner, the genes and sequences of the other haloperoxidases according to the invention can also be isolated and determined. The invention therefore also extends to DNA sequences encoding other vanadium haloperoxidases. The sequence of the chlorperoxidase from Curvularia inaeaualis is shown in Figure 6.

The purified enzymes of Curvularia inaeaualis and Drechslera biseotata were cleaved into peptides by proteases and CNBr. The amino acid sequence of two corresponding peptides from both fungi was determined. There appeared to be a very high degree of homology between the two species. Of the 21 amino acids, only one appeared to differ. (Asp in C. inaeaualis and Glu in D.biseptata at position 14 of the peptide).

Analogously, the genes and sequences of vanadium haloperoxidases from related fungi can be isolated, determined and expressed.

Starting from the gene sequence, whether derived from a cDNA or from a genomic clone, by recording it in a suitable expression cassette with suitable transcription initiation and termination signals, as well as a replication origin, in a suitable host, such as Aspergillus so. or Streptomvces. Bacillus. E.coli a recombinant haloperoxide can be produced. Also this recombinant enzyme, as well as biologically active derivatives thereof, are within the scope of this invention.

According to the invention, the haloperoxides can be used for various applications.

In the presence of hydrogen peroxide or a hydrogen peroxide-generating source and chloride or other halides, hypochlorous acid (H0C1 or bleach) or other hypohalogen acids are formed (7). Since the peroxide is still present during the formation of the HOCl, singlet oxygen is also formed (5). It is known (12-15) that both the hypohalogen acids and the singlet oxygen have strong bactericidal, bleaching and oxidizing properties. The vanadium chloroperoxidases according to the invention are very stable and have a high affinity for the substrate. That is, they are able to use low concentrations of chloride and bromide. In most biological liquids, but also in food, surface water, tap water, sea water and the like, considerable concentrations of chloride are naturally present. When hydrogen peroxide is present or added to a product, a chloroperoxidase according to the invention can exert a bactericidal effect therein and inhibit the growth of gram-positive and gram-negative bacteria in foods.

The action of the enzyme is analogous to the lactoperoxidase naturally occurring in milk. However, lactoperoxidase is not stable and, moreover, cannot oxidize chloride. The action of the lactoperoxidase can be supplemented by adding the enzyme according to the invention to dairy products.

In addition, seeds and fruits can be treated with the enzyme, for example in the form of a coating applied to the product. This reduces or prevents spoilage during storage and handling. The shelf life of these products is considerably extended by the bactericidal action of the enzyme and, moreover, the current use of toxic chemicals and / or antibiotics can thereby be reduced or avoided.

Because the oxides of the invention according to the invention have a very high thermostability, they can already be added to foodstuffs before pasteurization or sterilization and thus increase the shelf life even further.

The enzyme can also find use as a bactericide in pharmaceutical or cosmetic compositions. They are also suitable for the general treatment of wounds.

Since the haloperoxidases of the invention have a high affinity for chloride, they appear to be very useful in a new enzymatic method for determining the halide concentration in aqueous solutions. The method can also be used to determine the halide concentration in body fluids, such as blood and urine. The method of the invention is very sensitive and can detect concentrations in the / molar range. The method according to the invention is preferably based on the already known monochlorodimedone determination. Monochlorodimedone reacts with the product of the enzymatic oxidation of halide to dichloro or monobromo monochlorodimedone in the presence of chloroperoxidase and to the last compound with bromoperoxidase. The reaction is followed by monitoring the absorbance at 290 nm which decreases after chlorination or bromination of monochlorodimedone.

The invention also relates to the use of haloperoxidases as an enzyme additive in detergents for bleaching stains, particularly in white tissues. In addition to the haloperoxidase, hydrogen peroxide or a hydrogen peroxide-generating source must also be present. The required chloride is sufficiently present in the tap water. The HOC1 is produced by the enzyme in only low concentrations and especially at the spots or pigments, because the enzyme is hydrophobic in nature and will preferentially adhere to the organic compounds occurring in spots and pigments. Only the organic compounds, which show a high reactivity with singlet oxygen or H0Cl, will be attacked and bleached. The fibers of the fabric are not affected. The advantage of the enzyme according to the invention is therefore that in this way no harmful amounts of H0Cl are formed and that the bleaching of the stains proceeds in a controlled manner. In addition, the bleach is only produced during washing. As a result, the use of chemical oxidants, which can be hazardous to the environment during manufacture and transport of the detergent, can be reduced or even avoided.

In nature, a number of vanadium bromoperoxidases are found on the surface of seaweeds. In the intact plant in seawater, the vanadium bromo peroxidase is accessible to added substrate and is capable of forming HOBr after addition of hydrogen peroxide. The formation of this HOBr in seawater is probably part of a defense mechanism of the plant to prevent the growth of bacteria and fungi on its surface. This anti-fouling principle used by the plant can also be imitated in anti-fouling paints for yachts and ships in both fresh and salt water. Sea water in particular contains significant amounts of hydrogen peroxide and normally 1 mM bromide is also present. Since the haloperoxidases of the invention have an affinity for chloride as well as a very high affinity for bromide, the vanadium chloroperoxidase will oxidize bromide ions to HOBr. A painted surface that contains the enzyme and has been exposed to water, especially seawater, will continuously release the bactericidal agent HOBr and prevent the growth of (micro-) organisms on the surface of ships and the like.

H0C1 is known to oxidize thiols to the corresponding sulfonic acids. Unpleasant perspiration odors are caused by a number of sulfur-containing compounds, such as mercaptans, for example. In the presence of H0Cl, the SH groups of these mercaptans will be oxidized to the less odor-emitting sulfonic acids.

The haloperoxidases according to the invention can therefore be used, for example, in deodorants as an odor suppressant. Hydrogen peroxide can be added to the deodorant. However, the bacteria present can themselves also be used as a hydrogen peroxide-generating source. Since the enzymes of the invention are very stable, they can be formulated in liquid form or as liquids containing alcohols or detergents.

The present invention will be further elucidated with reference to the accompanying examples, which are, however, given herein by way of illustration only and are not intended to limit the invention in any way.

EXAMPLE 1

Detection of extracellular chloroperoxidases in a number of fungal species.

1. Material and method

Several fungi obtained from the Central Bureau of Fungal Cultures in Baarn were grown on agar plates. After growth was completed, the extracellular proteins were blotted into a nitrocellulose filter. The filters were tested for haloperoxidase activity in an orthodianisidine and hydrogen peroxide assay at various pH values and in the presence and absence of potassium bromide.

2. Results and discussion

Curvularia inae-aualis was found to be one of the fungi tested. Drechslera biseotata. Drechslera fuaax, Drechslera nicotiae. Drechslera suboapendorfii. Embellisia hvacinthi. Embellisia didymospora. Ulocladium chartarum and Ulocladium botrvtis exhibit haloperoxidase activity. Chloroperoxidases were isolated from Prechslera subpaoendorf ii. Embellisia didvmospora and Ulocladium chartarum. The pH optima of the enzymes ranged from pH 4.5 to pH 5.5. The enzymatic activity increases after addition of vanadate. A polyclonal antiserum raised against the chloroperoxidase from Curvularia inaeoualis showed cross-reactivity with the enzymes from Drechslera subpapendorfii. Embellisia didvmospora and Ulocladium chartarum.

EXAMPLE 2

Isolation of vanadium chloroperoxidase from Drechslera bisep-tata.

1 Introduction

A large number of halogenated compounds occur in nature. They are produced by various organisms, such as marine algae, actinomycetes, lichens, fungi, bacteria and higher plants. For example, broor peroxidase and chloroperoxidase are involved in the production of such halogenated compounds (6). There are two groups of haloperoxidases, each of which has a different prosthetic group. One group contains heme as a prosthetic group, for example the chlorperoxidase from the fungus Caldariomvces fumago. However, this heme protein is not stable and its pH optimum in the chlorination reaction is at a low pH (8). The other group contains vanadium as a prosthetic group. One such peroxidase is secreted, for example, by the fungus Curvularia inaequalis (6). This example describes the isolation of a vanadium chloroperoxidase from the fungus Drechslera biseptata, which has an unusually high stability.

2. Material and method 2.1. Growing the fungus

The fungus D. biseptata. strain number 371.72, was obtained from the Central Bureau of Mold Cultures (CBS, Baarn, the Netherlands). The germination medium consisted of 15 g fructose, 3 g yeast extract (GIBCO BRL) and 1 ml micro-element solution (0.8 g KH2PO4, 0.64 g CuS04.5 HzO, 0.11 g FeS04.7 H2O, 0.8 g MnCl2.4 H20, 0.15 g ZnS04.7 H20 in 1 liter of water The fermentation broth consisted of 5 g of casein hydrolyzate (GIBCO BRL), 3 g of yeast extract and 1 g of fructose in 1 liter of millipore water The fungi were grown in two phases First, 50 ml of sterile germination medium was inoculated with a spore mass of the fungus This culture was shaken for three days at 23 ° C and then transferred to a 3 liter conical flask containing 1 liter of fermentation medium The medium shaken at 23 ° C was collected after 14 to 17. The fungus D. biseotata secreted a haloperoxidase into the medium.

2.2. Activity determination

To determine the activity of the secreted peroxidase, a qualitative assay was used containing 0.1 M potassium phosphate (pH 6.5), 0.1 M KBr, 40 μΜ phenol red and 1 mM H 2 O 2. Conversion of the orange color to a deep purple color, which corresponds to the formation of bromophenol blue (4), is observed in the presence of an active haloperoxidase. The growth medium was then filtered and DEAE-Sephacel was added to bind the proteins present. The column was washed with 0.2 M NaCl in 0.05 M Tris-SO4 (pH 8.3) and the enzyme eluted with 0.6 M NaCl in 0.05 M Tris-SO4 (pH 8.3). It was noted that fractions with activity contained a dark brown dye that interfered with the quantification of the chlorination activity and the protein determination. Therefore, the ionic strength of the concentrated sample from the DEAE-Sepha cell column was adjusted to 2 M NaCl in 0.05 M Tris-SO4 (pH 8.3) and the sample to a hydrophobic interaction column Sepharose CL-4B (Pharmacia LKB Sweden). The brown dye was removed by washing the column with loading buffer and the enzyme eluted with a gradient from 2 M to 0 M NaCl in 0.05 M Tris-SO4 (pH 8.3). The enzyme eluted at about 1.2 M NaCl.

The enzymatic activity of chloroperoxidase in the oxidation of Cl 'to H0Cl was determined at 25 ° C (8) by following the conversion of 50 μl monochlorodimedone (6290nm = »20.2 mM'1.cm'1) to dichlorodimedone ( € 290rm = 0.2 mM '1.cm'1) in 0.1 M citrate buffers of various pH and 50 μl monochlorodimedone (Sigma). 1 unit of chloroperoxidase is defined as 1 μπιοί monochlorodimedone chlorinated per minute in a medium containing 1 mM H 2 O 2, 0.1 M citrate (pH 5.0), 50 μΜ monochlorodimedone and 5 mM potassium chloride. H 2 O 2 was prepared by diluting a 30% stock solution of Perhydrol (Merck, Darmstadt, Germany). The reaction was started by adding the enzyme to the reaction medium.

2.3. Other provisions

The Bradford method (Anal. Biochem. 72, 248-254 (1976)) with bovine serum albumin as standard was used for the protein determination.

SDS polyacrylamine gel electrophoresis was performed with 10% gels as described by Laemmli (Nature 227, 680-685 (1970)). Standard proteins (low molecular weight 14.4-94 kDa, Pharmacia, LKB, Sweden) were used for the molecular weight determination.

The protein staining was performed with Coomassie Brilliant Blue G250.

Bromine peroxidase activity in SDS-PAGE gels was determined by immersing the gels in a solution of 0.1 M potassium phosphate (pH 6.5), 0.1 M potassium bromide, 1 mM orthodianisidine and 1 mM H 2 O 2. When the peroxidase is present, a brown precipitate is formed in the gel.

The optical measurements were performed on a Cary-17 spectrophotometer.

EPR spectra (EPR = Electron Paramagnetic Resonance) were recorded on a Bruker ECS-106 spectrometer. The instrument was used at an X-band frequency with 100 kHz magnetic field modulation. EPR samples were prepared by reduction with sodium dithionite and the solutions were frozen in liquid nitrogen.

Vanadium was determined by the standard addition method using a Hitachi 180-80 Zeeman polarized spectrophotometer equipped with a Hitachi pyrolysis graphite cuvette. Free and nonspecifically bound vanadium were removed by centrifugation of the samples through a column of the cation exchanger Chelex-100 (Biorad) before determining the amount of built-in vanadium.

All chemicals were of analytical grade. The water was filtered and deionized by passing it through an Elgastadt B124 (Elga group) and a Mill-Q (Millipore Corporation) water purification system.

3. Figures

Figure 1 shows the total number of units of chloroperoxidase isolated from media containing different concentrations of vanadate. The activity was determined as described in Material and Method.

Figure 2 shows the chlorinating activity at a number of times of apo-chloroperoxidase reactivated by vanadate at a low and high ionic strength, respectively. 75 nanomolar apo-chloroperoxidase was incubated with 100 µl sodium vanadate. □ - □ = 0.05 M Tris-SO4 (pH 8.3) and 0.2 M Na2 SO4; ♦ - ♦ = 0.05 M Tris-SO4 (pH 8.3).

Figure 3 illustrates the thermostability of chloroperoxidase. 0.2 mg / ml enzyme was incubated at different temperatures for 5 minutes and the chlorinating activity determined.

Figure 4 illustrates the stability of the enzyme in organic solvents. The chloroperoxidase was stored in organic solvents and samples were taken to determine the cholerating activity.

Finally, in Figure 5, the EPR spectra of chloroperoxidase from D. biseotata (curve a, 2.5 mg / ml) and C. inaeaualis (curve b, 3 mg / ml) are shown in 50 mM Tris-SO4 (pH 8.2 ) after reduction with sodium dithionite. The equipment was set as follows: microwave power 40 dB; microwave frequency 9.425 GHz; modulation frequency 0.8 mT, temperature 50 K.

4. Results

The enzyme yield was about 10 mg of enzyme per liter of fermentation broth. SDS-PAGE under denaturing conditions (boiling in the presence of fi-mercaptoethanol) of the chloroperoxidase preparation showed one important band at 66 kDa (not shown). When the chloroperoxidase sample was not boiled in the buffer with β-mercaptoethanol, 1 band of 66 kDa and another higher molecular weight band were present, both staining for activity and protein. The preparation was therefore pure, but apparently the enzyme formed aggregates. A similar banding pattern was also found for the chloroperoxidase from C. inaeoualis.

The chloroperoxidase from C. inaeoualis contains vanadium as a prosthetic group and when no additional vanadium is added to the growth medium, the enzyme is excreted in its apo form.

The amount of activity in the various fermentations of D1 biseptata fluctuated considerably and this could be due to the secretion of the enzyme in its apo form. To test this, different concentrations of sodium orthovanadate were added to a number of culture media at the beginning of growth. After 17 days of fermentation, the dry weight of the fungus was determined and the chloroperoxidase was isolated from the different culture media. The samples containing activity were concentrated and the protein content and activity determined. The number of isolated units was a function of the concentration of vanadate present in the medium (see Figure 1). A constant activity level was observed when the medium contained more than 10 μΜ vanadate. Both the dry weight of the fungal material and the amount of protein of the purified preparation remained the same. Therefore, D. biseptata also secretes an apoenzyme when no additional vanadium is added to the medium.

It was also tested whether apoenzyme purified from a medium without sodium orthovanadate could be reactivated. To this end, a sample was incubated with a 10-fold excess of sodium orthovanadate and the chlorinating activity was measured at different time intervals. Since vanadium bromo peroxidase seems to aggregate easily at low ionic strength, the reactivation was performed in a medium containing only 0.05 M Tris-SO4 (pH 8.3) and also one containing 0.2 M Na2 SO4 in 0.05 M Tris -SO 4 (pH 8.3). Figure 2 shows that sodium orthovanadate activates both samples. However, at a high salt content, the chloroperoxidase is activated much faster, suggesting that low ionic strength causes the formation of aggregates, in which the reactivation rate is inhibited by sodium orthovanadate.

Since vanadium bromoperoxidase enzymes are relatively stable (4), stability experiments were performed. Figure 3a shows the thermostability of the chloroperoxidase from D. biseptata. From this figure, a midpoint temperature of 82.5 ° C can be calculated, demonstrating that this enzyme exhibits very high thermostability.

The effect of the chaotropic agent guanidine-HCl has also been studied. Chloroperoxidase was incubated in different concentrations of guanidine-HCl for 5 minutes, after which the chlorinating activity was measured. From the data (not shown) a G1 / 2 of 2.7 M can be calculated, demonstrating that the enzyme is not particularly stable in this denaturing agent. In contrast, the resistance to denaturation by organic solvents is considerable. Samples of the chloroperoxidase from D. biseptata, which were stored in various organic solvents, such as methanol, ethanol, acetone and dioxane, remained stable for up to 6 weeks (Figure 4).

A steady-state kinetic study of the chlorinating activity shows that for other haloperoxidases a beiform pH optimum was observed (not shown). The position of the pH optimum is a function of the chloride concentration, as is also observed for the vanadium enzyme from C1. inaequalis (6). The pH optimum shifts from pH 4.5 at 1 mM Cl 'to pH 5.5 when the chloride concentration is increased to 100 mM. Table 1 shows the waarde ,,, value for Cl 'and H 2 O 2 at different pH values. It is clear that the affinity for both substrates is very high.

Table 1 K * * for chloride and hydrogen peroxide of the chloroperoxidase from D. biseptata

* The K2 for chloride was obtained by measuring the rate of chlorination at saturation levels of hydrogen peroxide and K2 for hydrogen peroxide at saturating concentrations of chloride. The chlorination activity was determined as set forth in Material and Method.

The EPR spectrum of the purified enzyme was also included. As with the other vanadium haloperoxidases (1), no EPR signal is detectable in the oxidized enzyme. However, after reduction with sodium dithionite, a typical vanadyl EPR spectrum is observed (Figure 5). For comparison, the EPR spectrum of the haloperoxidase from Cj_ inaeoualis is also shown. The isotropic EPR parameters g0 and A0 are almost the same for both enzymes and are similar to those of the vanadium bromoperoxidases. These data suggest that the prosthetic group in these enzymes is the same.

Detection of other vanadium chloroperoxidase in common soil fungi indicates that vanadium enzymes are widely distributed in nature.

EXAMPLE 3

Determination of the coding sequence of the CPO gene and the gene from Curvularia inaecrualis and expression systems.

1. Material and method

The coding sequence of the CPO gene was determined as follows. The chloroperoxidase from C. inaeoualis was cleaved with protease or CNBr to obtain peptides. The amino acid sequence of these peptides was determined using a gas phase sequencer. The resulting sequences are shown in Table 2. Based on amino acid sequence 1 (see Table 2), degenerate primers were designed on both sides of the coding DNA template. Using these two degenerate primers and the genome DNA of C. inaeoualis as template, the coding part of amino acid sequence 1 was amplified and cloned into a pUC18 vector. The amplified portion was then sequenced. The resulting clone was designated pCPO1. Similarly, the coding sequence of amino acid sequence 2 was obtained. The resulting clone was designated pCP02.

Based on these two known sequences, new degenerate primers were designed and used in a PCR experiment with a first strand cDNA as template. The DNA fragment obtained in this way links the two already known DNA sequences. This total fragment was cloned into a pUC18 vector and sequenced.

The resulting fragment encodes portions of amino acid sequences 1 and 2 and also contains the region encoding amino acid sequence 3 (see Table 2).

In order to obtain the 5 'end of the cDNA encoding chloroperoxidase, the 5'RACE kit from Clontech Laboratories (USA) was used. Sequence was determined from one of the resulting clones (pCP04). The 3 'end of the cDNA was obtained in a PCR using cDNA as template. The primers used herein were based on the known DNA sequence and on the NotI-d (T) 18 bifunctional primer from a Pharmacia first strand synthesis kit. The resulting 1.4 kb fragment was cloned into pUC18. DNA sequencing confirmed that this fragment encodes the C-terminal part of the CPO.

2. Result

Figure 6 shows the sequence of the cDNA encoding the chlorina oxidase from C.inaeaualis.

The chloroperoxidase apoprotein can be reactivated by the addition of vanadate (see example 2) and therefore it is likely that no other genes are involved in the incorporation of the prosthetic group into these enzymes.

3. Expression of the chloroperoxidase gene

To express the chloroperoxidase, the cDNA or a genomic fragment is cloned in a known manner in an expression vector. With the resulting expression vector, a suitable host cell, such as a fungus, for example, Aspergillus so .. or bacteria, for example, Streotomvces. Bacillus or E. coli. Are transformed. The culture medium is tuned to the respective host. The expressed chlorperoxidase can be recovered from this medium by known procedures such as the separation of the cells from the medium by centrifugation or filtration, precipitation of protein components in the medium by a salt, such as ammonium sulfate, followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography and the like.

Table 2

Peptide sequences derived from vanadium chloroperoxidases

8 underlined sequences were used to design degenerate DNA primers. b homologous sequences between C. inaeaualis and D. bisepta-ta are in bold.

EXAMPLE 4

Determination of the chloride concentration in a liquid.

1 Introduction

Chloride concentrations in natural and wastewater are usually determined by volumetric methods with silver nitrate or mercury (II) nitrate or spectrophotometrically using mercury (II) thiocyanate and iron (III) ions. This example illustrates the use of a new enzymatic method for determining total halide (excluding fluoride) and chloride in aqueous solutions. The process is based on the specific oxidation of halides to hypohalogen acids, which oxidation is catalyzed by chlorine and bromine oxidases. The hypohalogenic acid is scavenged by mono-chlorodimedone. The quantitative halogenation of monochloro-dimedone is determined spectrophotometrically.

Haloperoxidases form a class of enzymes capable of oxidizing halides (X '= Cl', Br 'or I') in the presence of hydrogen peroxide to the corresponding hypohalogen acids according to the reaction: H202 + X '+ H + - * H20 + HOX ( 1)

When a suitable nucleophilic acceptor is present, a reaction with HOX will occur and a halogenated compound is formed.

Haloperoxidase can be divided into chlorine, bromine and iodine peroxidases, according to the most electro-negative element that can be oxidized by these enzymes. For example, chloroperoxidases are capable of oxidizing Cl ', Br * and I', while bromoperoxidase oxidizes only Br 'and I' and iodine peroxidases only iodide.

In this example, two vanadium-containing enzymes, namely the chlorine peroxidase from the fungus C. inaecrualis and the bromine peroxidase from the lichen X ^ parina (9), are used to determine total halide (Cl ', Br', I ') concentrations . The data will demonstrate that the enzymatic determination of halide concentrations is easy to perform and provides reliable quantitative results.

2. Material and method

The halide content of the aqueous solutions was determined using the monochlorodimedone assay (8). Monochlorodimedone reacts with the product of the enzymatic oxidation of halide to dichloro or monobromochloro dimonone in the presence of chloroperoxidase and to the last compound with bromoperoxidase. The reaction was followed by monitoring the absorbance at 290 nm which decreases after chlorination or bromination of monochlorodimedone. At pH 3.6, the extinction coefficient at 290 nm for monochlorodimedone is 15.09 mM'1 cm -1, while the extinction coefficient at the same wavelength for dichlorodimedone and for monobromochlorodimedone are both 0.1 mM * 1 cm -1.

A 50 μΜ concentration of monochlorodimedone was used in the spectrophotometric assay. A partial decrease in absorption was observed after addition of hydrogen peroxide and enzymes to a solution containing less than 50 μΜ halide. The difference between the initial and final absorption gives a value for the amount of halide present.

Addition of chloroperoxidase to the assay will give the total amount of halide present in the test mixture, while addition of bromoperoxidase will give a value for the halides except chloride. When the absorbance values of the test with chloroperoxidase and those with bromoperoxidase are subtracted from each other, the difference gives the amount of chloride present in the solution.

All halide assays were performed in 0.1 M citrate buffer (pH 3.6), 1 mM hydrogen peroxide, 50 μΜ monochlorodimedone and 0.29 μΜ chloroperoxidase or 0.1 μΜ bromo peroxidase in a 2.5 ml quartz cuvette using a Cary 17 spectrophotometer. All buffers and solutions were prepared with water that had been filtered and deionized through an Elgastad B12H (Elga group) and a MilliQ (Millipo-re) water purification system. All reagents were of analytical grade.

3. Results

Figure 7 shows the decrease in absorption observed after conversion of monochlorodimedone by the bromine peroxidase from Xi. parietina or by the chloroperoxidase from Ci. inaeaualis shown. The reaction mixture contains 0.1 M

citrate buffer (pH 3.6), 1 mM hydrogen peroxide, 25 μΜ chloride and 15 μΜ bromide (final concentration of halides: 40 μΜ). Graph A shows the absorbance decrease in the presence of 0.1 μΜ bromine peroxide, while Graph B shows the absorbance decrease i in the presence of 0.3 μΜ chlorine peroxide.

Figure 8 shows the relationship between the absorbance change at 290 nm and the chloride concentration. Chloride concentration varies between 4 μΜ and 32 μΜ. The enzyme concentration is 0.29 μΜ. Values shown are mean values from three experiments. The relationship between the chloride concentration and the decrease in absorption appears to be linear.

Table 3 shows the result of an experiment in which mixtures of bromide and chloride were analyzed for bromide and chloride content using the enzymatic assay of the invention. A comparison of the originally present concentrations of bromide and chloride with the result of the enzymatic determination shows that the enzymatic method measures both halides separately and accurately and is therefore able to fully convert the halides present into hypohalogen acid. The method is reliable because the deviation of the measurements is small.

The chloride content of a number of water samples is measured with the enzymatic method according to the invention. The results are shown in Table 4. It is clear that the determined values correspond to the given specifications and that accurate data can be obtained since the deviation in the measurements is small) (4%).

Table 3

Determination of a Chloride / Bromide Mixture by the Method of the Invention.

The total concentration of halides is 24 µM.

*: Relative standard deviation (%). n = 3

Table 4

Chloride content of a number of water samples. The concentration of chloroperoxidase was 0.29 / xM, bromoperoxidase was 0.1 μΚ.

*: Relative standard deviation (%). n = 3.

a: as specified on the bottle b! the value depends on the source, as specified by the Amsterdam Water Supply Company.

4. Discussion

The results show that the new enzymatic method for the quantitative measurement of total halide (excluding fluoride) and chloride is simple and straightforward. The monochlorodimedone and its brominated and chlorinated derivatives have known extinction coefficients and can therefore be used as an internal standard for the halide determination. The calibration curve shows a linear relationship between the chloride content and the absorption changes in the concentration range between 1 and 35 μΜ. The method according to the invention is particularly sensitive. The concentration range used in the enzymatic assay of this example is tenfold lower than in the known method of Sagara et al. (Anal. Chim. Acta 270, 217 (1992)). The method of Fajans (Z. anorg. Allgem. Chem 137, 221 (1924)) is only accurate when the solutions contain more than 5 mM chloride. This is about a thousand times more concentrated than the solutions that can be determined by the enzymatic method according to the invention. The method described is also more sensitive than that using ion-selective electrodes, where chloride concentrations below 10 μ 10 cannot be measured (10).

The measured bromide / chloride ratios are in accordance with the ratio of the actual values (see Table 3). The results demonstrate that the combination of the two vanadium-containing enzymes used in this assay provide reliable results not only in terms of the halide content, but also in the nature of the halide. It is even possible, when using a high affinity iodide peroxidase for iodide, to separately analyze the content of each halide in any given mixture.

EXAMPLE 5

Demonstrating the antibacterial activity of vanadium chloroperoxidase 1. Introduction

For the purpose of testing the antibacterial activity of the chloroperoxidase according to the invention, E. coli bacteria (HB101) were exposed to a combination of chloroperoxidase, hydrogen peroxide and chloride in 100 mM NaAc (pH 5.0).

2. Material and method

The E. coli cells grown up in a culture medium (10 g yeast extract, 16 g tryptone and 5 g NaCl per liter demineralised water) were washed with physiological saline followed by a washing step in 0.1 M sodium acetate (pH 5.0). 1 ml of this bacterial suspension was taken and added to incubation media containing 0.1 M sodium acetate (pH 5.0), 0 or 10 mM NaCl, and 0.05 μl chloroperoxidase and a hydrogen peroxide concentration of 0.01 mM, 0.05 mM to 0.10 mM. For control, the bacterial suspension was also incubated in a sterile medium to which no chloroperoxidase (CPO) had been added. After an hour incubation, samples of 50 μΐ were taken which were diluted 102x, 104x and 106x successively in physiological saline. 100 µl of each of these dilutions were taken and placed on agar plates (2% agar in culture medium). After overnight incubation at 37 ° C, the number of bacteria per plate was counted.

3. Results and discussion

Table 5 shows that incubation in the presence of hydrogen peroxide alone also has a bactericidal effect. However, the effect is enhanced by adding the chloroperoxidase and no surviving bacteria are found when incubated in 0.05 mM hydrogen peroxide.

As can be seen from the experiment in which no chloride was added (Table 6), the bactericidal activity by chloroperoxidase is also observed here. Given the high affinity for chloride, the enzyme presumably uses the trace amounts of chloride from the buffer system. The strongly decreasing number of bacteria in the incubation without chlorine peroxidase can be traced back to very unfavorable conditions (the lack of chloride) during the incubation.

The results of this experiment thus provide evidence for the bactericidal activity of the chloroperoxidase.

Table 5

Number of surviving bacteria (cells per plate) after incubation with chloroperoxidase in 10 mM Cl '

CPO = chloroperoxidase

Table 6

Number of surviving bacteria (cells per plate) after incubation with chloroperoxidase in the absence of extra chloride in the incubation medium

CPO = chloroperoxidase

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Claims (17)

1. Haloperoxidase obtainable in substantially isolated form from a fungus selected from the group consisting of Curvularia inaequalis, Drechslera bisoetata. Drechslera f uaax. Drechslera nicotiae. Drechslera suboaoen-dorfii. Embellisia hvacinthi. Embellisia didvmospora. Ulo-cladium chartarum. Ulocladium botrvtis. DNA sequence encoding a haloperoxidase comprising at least part of the sequence of Figure 6 or modified versions thereof. 3. Recombinant protein with haloperoxidase activity, obtainable by expressing at least part of the sequence of Figure 6 in a suitable host. 4. Haloperoxidase for use in anti-fouling agents for vessels, for example. 5. Haloperoxidase for use as an odor suppressant in a deodorant. 6. Haloperoxidase for use as a detergent additive. 7. Haloperoxidase for use in a method of determining the halide concentration in a sample. 8. Haloperoxidase for use as a bactericide. 9. Haloperoxidase for use in food preservation. 10. Haloperoxidase for use as a disinfectant in pharmaceutical compositions. Detergent, in particular for bleaching white fabric stains, comprising one or more suitable detergents, one or more haloperoxidases and hydrogen peroxide. 12. Deodorant, comprising one or more haloperoxidases and hydrogen peroxide or a hydrogen peroxide generating source. Anti-fouling paint, comprising the usual paint ingredients and solvents, as well as one or more haloperoxidases. Expression vector, comprising a replication origin, suitable transcription initiation and termination signals, and at least part of the sequence of Figure 6, or modified versions thereof. A method of producing a recombinant haloperoxidase by transforming a host with an expression vector of claim 11, culturing the vector under conditions permitting expression of the haloperoxidase, and isolating the haloperoxidase from the culture medium. A method for determining the halide concentration in a liquid, comprising adding a hydrogen peroxide and one or more haloperoxidases to the liquid to be investigated, monitoring the oxidation reaction by means of an indicator system, and using the indicator system to determine the halide concentration. A method according to claim 16, characterized in that the indicator system is mono-chlorodimedone.