WO1995027009A1 - Antifouling paint containing haloperoxidases and method to determine halide concentrations - Google Patents

Abstract

The invention relates to applications of haloperoxidases in substantially isolated form, obtainable from a large number of filamentous fungi, in paints and in a method for determining the halide concentration in a liquid. The haloperoxidases can be enzymes isolated from the fungi or produced recombinantly. In a preferred embodiment, the invention provides a paint with growth-inhibiting properties, for instance for use on the underwater part of ships. In addition, the invention provides a test kit for determining the halide concentration in a sample.

Description

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  Antifouling paint containing haloperoxidases and method to determine halide concentrations 
The present invention relates to the use of haloperoxidases, particularly chloroperoxidases, in isolated form, whether or not obtained via recombinant DNA techniques, in a number of applications which relate to determining chloride in a solution and to different paints. 



   Haloperoxidases are enzymes which can convert halides into hypohalogenic acids. Haloperoxidases occur inter alia in a number of filamentous fungi and seaweeds. Now that such haloperoxidases have become available in isolated form, different applications have now been found to be possible. 



   A first aspect according to the invention is related to a problem which occurs with different painted surfaces, particularly with the parts of ships extending in the water, that is, the growth of unwanted organisms. Painted surfaces not exposed to water can also become for instance green on the outside through algal growth. The growth of algae and other organisms on and in painted surfaces reduces the life span of the coating of paint, inter alia because it will have to be repainted sooner from an aesthetic point of view. 



  The drawback of fouling of ships is that the resistance of the ship in the water increases. This has a negative effect on the movement of the ship, which will cost more effort or will become slower. Ships are therefore treated with growthinhibiting agents, so-called anti-foulings. Known antifoulings are however environment-unfriendly and are also not always very effective. 



   It is therefore a first object of the invention to provide a new paint, with which the above stated drawbacks can be avoided. 



   According to the invention it has now been found that the addition of a haloperoxidase to paint can reduce or even prevent the growth of unwanted organisms on a painted surface by producing disinfecting hypohalogenic acids. To this end the invention provides paints which contain at least a suitable quantity of one haloperoxidase in addition 

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 to the usual paint constituents and solvents. Understood here by "suitable quantity" is a quantity such that growth on a surface treated with the paint will be substantially prevented. Using his normal professional knowledge the skilled person will be capable of determining both the composition of the paint and the quantity of enzyme to be added. 



   In another embodiment of the invention the haloperoxidases can be used as preservative in paints. Particularly water-borne paints are subject to relatively rapid spoiling. 



  Due to its disinfecting action the enzyme can ensure the storage life of such paints during storage and when they are being used. 



   The invention is based on the conclusion that in nature a number of vanadium bromoperoxidases are found on the surface of seaweeds. In the intact plant in seawater the vanadium bromoperoxidase 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 defence mechanism of the plant to prevent the growth of bacteria and fungi on its surface. This antigrowth principle used by the plant can be imitated according to the invention in anti-fouling paints for yachts and ships in both fresh and salt water. Seawater in particular contains considerable quantities of hydrogen peroxide and 1mM of bromide is normally speaking also present.

   Since the haloperoxidases according to the invention have a high affinity for bromide in addition to an affinity for chloride, the vanadium chloroperoxidase will oxidize the bromide ions to HOBr. A painted surface which contains the enzyme and is exposed to water, particularly seawater, will continuously release the bactericidal agent HOBr and prevent the growth of (micro-) organisms on the surface of ships and the like. 



   According to a second aspect of the invention a method for determining the chloride concentration in liquids is provided. 

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   Since particular peroxidases have a high affinity for chloride, they are found to be very useful in a new enzymatic method for determining the halide concentration in aqueous solutions. The method can likewise be used for determining the halide concentration in body fluids such as blood and urine. The method according to the invention is very sensitive and can demonstrate concentrations in the  molar range. The method according to the invention is preferably based on the already known monochlorodimedone assay. Monochlorodimedone reacts with the product of the enzymatic oxidation of halide to dichloro- or monobromomonochlorodimedone in the presence of chloroperoxidase and only to the latter compound with bromoperoxidase. The reaction is monitored by measuring the absorption at 290 nm which decreases after chlorination or bromination of monochlorodimedone. 



   For the applications according to the invention all haloperoxidases can of course be used which produce (disinfecting) hypohalogenic acids when exposed to halidecontaining aqueous solutions. In a particularly advantageous embodiment of the invention however, only non-haemo vanadium haloperoxidases are used. 



   The seaweed   Ascophyllum   nodosum was the first species wherein a non-haem vanadium bromoperoxidase was discovered. 



  A large number of other seaweed species thereafter followed which were also found to contain these enzymes. The bromoperoxidase from A. nodosum has been extensively studied and characterized (1, 2). The enzyme catalyzes the formation of hypohalogenic acids from the corresponding halogens. In a first step hydrogen peroxide reacts with the enzyme and thus forms a hydrogen peroxide-enzyme complex. Bromide and a proton then react with the complex to form an enzyme-HOBr complex. This complex decomposes and thus provides the enzyme and HOBr (3). 



   The known vanadium bromoperoxidases are found to display a high operational stability in aqueous and organic media. They can be stored for over a month in organic solvents, such as acetone, methanol, ethanol, 1-propanol, 

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 without loss of activity (4). The applications or possible applications for this type of enzyme are therefore wide. 



   The bromoperoxidases have the drawback however that bromide has to be present for their activity in such potential applications. This is not however the case in a good many situations. Bromide will therefore have to be added. In addition, attempts to isolate the genes for these bromoperoxidases from seaweed and to determine their sequence have not been successful up to the present. It is therefore not yet possible to obtain recombinant bromoperoxidases in large quantities for commercially viable applications. 



   Now however, enzymes corresponding with the known bromoperoxidases have been found which are not dependent on the presence of bromide but of chloride and which can be produced in large quantities using recombinant DNA techniques. 



   It has for instance been found that a vanadium peroxidase, which is found inter alia in the fungus Curvularia inaeaualis, can use chloride in addition to bromide in order to be active. In contrast to bromide, chloride is very widely present in for instance tap water, surface water and the like and does not have to be supplied additionally for the action of the enzyme in different applications. Related vanadium chloroperoxidases have now also been found in different Drechslera species, such as Drechslera biseptata, Drechslera   fuqax,   Drechslera nicotiae, and Drechslera   subpapendorfii,   or the Embellisia species Embellisia   hyacinthi   and Embellisia didymospora, as well as Ulocladium chartarum and Ulocladium botrytis. 



   Haloperoxidases in isolated or recombinant form, which can be obtained from any of the aforementioned filamentous fungi or related species, are therefore preferably used in the applications according to the invention. 



   The aforementioned fungus species can be obtained at the Centraal Bureau voor Schimmelculturen (CBS: Central Institute for Fungal Cultures) in Baarn, The Netherlands via the deposit accession numbers below. 

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 EMI5.1 
 
<tb> 
<tb> 



  FUNGUS <SEP> CBS-ACCESSION <SEP> NUMBER
<tb> Curvularia <SEP> inaequalis <SEP> 102.42
<tb> Drechslera <SEP> biseptata <SEP> 371.72
<tb> Drechslera <SEP> subpapendorfii <SEP> 656.74
<tb> Drechslera <SEP> nicotiae <SEP> 655.74
<tb> Drechslera <SEP> fugax <SEP> 509.77
<tb> Embellisia <SEP> hyacinthi <SEP> 416.71
<tb> Embellisia <SEP> didymospora <SEP> 766.79
<tb> Ulocladium <SEP> chartarum <SEP> 200.67
<tb> Ulocladium <SEP> botrytis <SEP> 452.72
<tb> 
 
The vanadium haloperoxidases, which can be isolated from the above fungus species are generally chloroperoxidases. Chloroperoxidases are peroxidases which have an affinity for both chloride and bromide and for iodide and can therefore use these three halides as substrate. The   terms "chloroperoxidase (s)" "haloperoxidase (s)" be   used interchangeably in this application.

   They have as common characteristic however that they have at least a useful affinity for chloride. 



   The vanadium chloroperoxidases are found to display a very high thermostability and a high affinity for the substrate. The Tm for the haloperoxidase of Curvularia inaequalis is for instance 90 C. The enzyme is moreover very stable in 40% methanol, ethanol or propanol (7). Its pHoptimum is   pH   5.5. 



   The gene that codes for the haloperoxidase of Curvularia   inaeaualis   has been isolated and its complete sequence determined. Example 3 indicates on the basis of Curvularia   inaeaualis   how the haloperoxidases can be isolated. The genes and sequences of the other haloperoxidases which can be used in the applications according to the invention can be isolated and determined in analogous manner. The sequence 

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 of the chloroperoxidase of Curvularia   inaeaualis   is shown in figure 6. 



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



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



   Starting from the gene sequence, either derived from a cDNA or originating from a genome clone, it is possible to produce a recombinant haloperoxidase by including the sequence in a suitable expression cassette with suitable transcription, initiation and termination signals in addition to a replication origin, in a suitable host such as   Asperaillus   sp., Saccharomvces spec. or Streptomyces, Bacillus, E. coli. This recombinant enzyme, as well as biologically active derivatives thereof, can also be used in the applications according to this invention. 



   The present invention will be further elucidated on the basis of the accompanying examples, which are however only given here by way of illustration and are not intended to limit the invention in any way. Examples 1 to 3 show the manner in which suitable haloperoxidases in isolated or recombinant form can be obtained. Example 4 shows that the haloperoxidases actually do have a bactericidal action. 



  Example 5 further explains the paint applications of the invention, while example 6 relates to the new enzymatic chloride assay. 



  EXAMPLE 1 Demonstration of extracellular chloroperoxidases in a number of fungus species. 

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  1. Material and method
Different fungi obtained from the Centraal Bureau voor Schimmelculturen in Baarn were cultured on agar plates. 



  After the growth had stopped the extracellular proteins were transferred to a nitrocellulose filter by means of blotting. 



  The filters were tested for haloperoxidase activity in an assay with ortho-dianisidine and hydrogen peroxide at different pH values and in the presence and absence of potassium bromide. 



  2. Results and discussion
Of the tested fungi Curvularia   inaeaualis,   Drechslera biseptata, Drechslera fugax, Drechslera nicotiae, Drechslera   subpapendorfii,   Embellisia hyacinthi, Embellisia didymospora, Ulocladium chartarum and Ulocladium botrytis were found to display haloperoxidase activity. 



  Chloroperoxidases were isolated from Drechslera subpapendorfii, Embellisia   didvmospora   and Ulocladium chartarum. The pH optima of the enzymes varied from pH 4.5 to pH 5.5. After addition of vanadate the enzymatic activity increases. A polyclonal antiserum that was elicited against the chloroperoxidase of Curvularia   inaecrualis   displayed cross reactivity with the enzymes from Drechslera   subpapendorfii,   Embellisia   didvmospora   and Ulocladium chartarum. 



  EXAMPLE 2 Isolation of vanadium chloroperoxidase from Drechslera biseptata. 



  1. Introduction
A large number of halogenated compounds occur in nature. They are produced by different organisms, such as marine algae, actinomycetes, lichens, fungi, bacteria and higher plants. Bromoperoxidase and chloroperoxidase are for instance involved in the production of such halogenated compounds (6). There are two groups of haloperoxidases, each of them having a different prosthetic group. The one group 

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 contains haem as prosthetic group, for instance the chloroperoxidase from the fungus   Caldariomvces   fumago. This haem protein is however not stable and its pH-optimum in the chlorination reaction lies at a low pH (8). The other group contains vanadium as prosthetic group. One such peroxidase is for instance secreted by the fungus Curvularia inaegualis (6).

   Described in this example is the isolation of a vanadium chloroperoxidase from the fungus Drechslera biseptata, which has an unusually high stability. 



  2. Material and method 2.1. The culture of the fungus. 



   The fungus D. biseptata, strain number 371.72, was obtained from the Centraal Bureau voor Schimmelculturen (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   KH2P04,   0.64 g CuSO4.5H2O, 0.11 g FeSO4.7H2O, 0.8 g MnCl2.4H2O, 0.15 g ZnSO4.7H2O in 1 litre water. The fermentation medium consisted of 5 g casein hydrolysate (GIBCO   BRL),   3 g yeast extract and 1 g fructose in 1 litre Millipore water. The fungi were cultured in two stages. Firstly, 50 ml sterile germination medium was inoculated with a trace mass of the fungus. This culture was shaken for three days at 23 C and then transferred to a 3 litre Erlenmeyer flask containing 1 litre fermentation medium. The medium which was shaken at 23 C was collected after 14 to 17 days.

   The fungus D. biseptata 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  M phenol red and 1 mM H2O2. Conversion of the orange colour to a deep purple colour, which corresponds with the formation of bromophenol blue (4), is observed in the presence of an active haloperoxidase. The growth medium was subsequently filtered and DEAE-Sephacel was added to bind the proteins present. 

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  The column was washed with 0.2 M NaCl in 0.05 M Tris-S04 (pH 8.3) and the enzyme was eluated with 0.6 M NaCl in 0.05 M   Tris-S04   (pH 8.3). It was noted that fractions with activity contained a dark brown colorant which interfered with the quantitative assay of the chlorination activity and the protein assay. The ionic strength of the concentrated sample of the DEAE-Sephacel column was set at 2 M NaCl in 0.05 M Tris-S04 (pH 8.3) and the sample was placed on a hydrophobic interaction column Sepharose CL-4B (Pharmacia LKB Sweden). 



  The brown colorant was removed by washing the column with loading buffer and the enzyme was eluated with a gradient of 2 M to 0 M NaCl in 0.05 M   Tris-S04   (pH 8.3). The enzyme eluated at about 1.2 M NaCl. 



   The enzymatic activity of chloroperoxidase in the oxidation of Cl' to HOC1 was determined at 25 C (8) by monitoring the conversion of 50  M monochlorodimedone (E290nm   = 20.2   mM-1.cm-1) to dichlorodimedone (E290nm   = 0.2   mM-1.cm-1) in 0.1 M citrate buffers of different pH and 50   M   monochlorodimedone (Sigma). 1 unit of chloroperoxidase is defined as 1 mol monochlorodimedone chlorinated per minute in a medium with 1mM H2O2, 0.1 M citrate (pH   5.0),   50  M monochlorodimedone and 5 mM potassium chloride. H2O2 was prepared by dilution of a 30% stock solution of Perhydrol (Merck, Darmstadt, Germany). The reaction was started by adding the enzyme to the reaction medium. 



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



   SDS polyacrylamide 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. 

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   The bromoperoxidase 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, 1mM orthodianisidine and 1 mM H2O2- 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 a X-band frequency with 100 kHz magnetic field modulation. EPR-samples were prepared by reduction with sodium dithionite, whereafter the solutions were frozen in liquid nitrogen. 



   Vanadium was determined with the standard addition method using a Hitachi 180-80 Zeeman polarized spectrophotometer fitted with a Hitachi pyrolysis graphite cuvette. 



  Free and aspecifically bound vanadium were removed by centrifugation of the samples through a column of the cation exchanger Chelex-100 (Biorad) before the quantity of incorporated vanadium was determined. 



   All chemicals were of analytical quality. The water was filtered and de-ionized by carrying it through an Elgastadt B124 (Elga group) and a Milli-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 under Material and method. 



   Figure 2 shows at a number of points in time the chlorination activity of apo-chloroperoxidase which has been reactivated at a respectively low and high ionic strength by vanadate. 75 nanomolar apo-chloroperoxidase was incubated with 100  M sodium vanadate.   #--#   = 0.05 M Tris-S04 (pH 8.3) and 0.2 M   Na2S04;   0--0 = 0.05 M Tris-S04 (pH 8.3). 



   Figure 3 illustrates the thermostability of chloroperoxidase. 0.2 mg/ml enzyme was incubated for 5 minutes at 

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 different temperatures and the chlorination activity was determined. 



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



   Finally, figure 5 shows the EPR-spectra of chloroperoxidase from D. biseptata (curve a, 2.5 mg/ml) and C.   inaeaualis   (curve b, 3 mg/ml) in 50 mM Tris-S04 (pH 8.2) after reduction with sodium dithionite. The equipment was adjusted as follows: microwave power 40dB; microwave frequency 9.425 GHz; modulation frequency 0.8 mT, temperature 50 K. 



  4. Results
The yield of the enzyme was about 10 mg enzyme per litre of fermentation medium. SDS-PAGE under denaturing conditions (boiling in the presence of B-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 B-mercaptoethanol, 1 band of 66 kDa and another band with a higher molecular weight were present which both stained for activity and protein. The preparation was therefore pure but the enzyme apparently formed aggregates. A corresponding band pattern was also found for the chloroperoxidase from C. inaeaualis. 



   The chloroperoxidase from C.   inaeaualis   contains vanadium as prosthetic group and when no extra vanadium is added to the growth medium the enzyme is secreted in its apo-form. 



   The amount of activity in the different fermented materials from D. biseptata fluctuated considerably and this could be attributed to the secretion of the enzyme in its apo-form. In order to test this, different concentrations of sodium orthovanadate were added to a number of culture media at the start of the growth. After 17 days of fermentation the dry weight of the fungus was determined and the chloroperoxidase was isolated from the different culture 

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 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   M   vanadate.

   Both the dry weight of the fungal material and the quantity of protein of the purified preparation remained the same. Therefore D. biseptata likewise secretes an apo-enzyme when no extra vanadium is added to the medium. 



   Whether apo-enzyme purified from a medium without sodium orthovanadate could be reactivated was also tested. 



  To this end a sample was incubated with a tenfold excess of sodium orthovanadate and the chlorination activity was measured at different time intervals. Since vanadium bromoperoxidase appears to aggregate easily at low ionic strength, the reactivation was performed in a medium which only contained 0.05 M Tris-S04 (pH 8.3) and also in one containing 0.2 M   Na2so4   in 0.05 M   Tris-S04   (pH 8.3). In figure 2 can be seen that sodium orthovanadate activates both samples. At a high salt content the chloroperoxidase is however activated much quicker, which suggests that a low ionic strength causes the formation of aggregates, wherein the reactivation speed is inhibited by sodium orthovanadate. 



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



   The effect of the chaotropic agent   guanidine-HC1   was likewise studied. Chloroperoxidase was incubated for 5 minutes in different concentrations of guanidine-HCl, whereafter the chlorination activity was measured. From the data (not shown) a G¸ of 2.7 M can be calculated, which indicates that the enzyme is not particularly stable in this denaturing agent. Conversely, the resistance to denaturation by organic solvents is considerable. Samples of the 

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 chloroperoxidase from D. biseptata, which were stored in different organic solvents such as methanol, ethanol, acetone and dioxane, remained stable for up to 6 weeks (figure   4 ) .   



   From a steady state kinetic study of the chlorination activity it was found that for other haloperoxidases a bell shaped pH-optimum was observed (not shown). The position of the pH-optimum is a function of the chloride concentration, as was also observed for the vanadium enzyme from C. inaeaualis (6). The pH-optimum shifts from pH 4.5 at 1mM Clto pH 5.5 when the chloride concentration is raised to 100 mM. Table 1 shows the Km-value for Cl- and H2O2 at different pH values. It is apparent that the affinity for both substrates is high. 



   Table 1 
Km* for chloride and hydrogen peroxide of the chloroperoxidase of D. biseptata 
 EMI13.1 
 
<tb> 
<tb> pH <SEP> Km <SEP> Cl- <SEP> (mMO <SEP> Km <SEP> H2O2 <SEP> ( M)
<tb> 4.0 <SEP> 0.18 <SEP> 27.2
<tb> 4.5 <SEP> 0.22 <SEP> 15.2
<tb> 5.0 <SEP> 0.76 <SEP> 15.4
<tb> 5.5 <SEP> 0.96 <SEP> 12.2
<tb> 6.0 <SEP> 1.99 <SEP> 4.11
<tb> 6.5 <SEP> 15.5
<tb> 7.019.6
<tb> 
 * The Km for chloride was obtained by measuring the chlorination speed at saturation levels of hydrogen peroxide and   the K   for hydrogen peroxide at saturating concentrations of chloride. The chlorination activity was determined as stated in Material and method. 

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   The EPR-spectrum of the purified enzyme was also recorded. As is the case for the other vanadium haloperoxidases (1), no EPR-signal is detectable in the oxidized enzyme. After reduction with sodium dithionite a typical vanadyl EPR-spectrum is however observed (figure 5). By way of comparison the EPR-spectrum of the haloperoxidase from C. inaeaualis is also shown. The isotropic EPR-parameters go and Ao are almost the same for both enzymes and correspond with those of the vanadium bromoperoxidases. This data suggests that the prosthetic group in these enzymes is the same. 



   The demonstration of other vanadium chloroperoxidases in normal soil fungi indicates that vanadium enzymes are widespread in nature. 



  EXAMPLE 3 Determination of the coding sequence of the CPO gene and the gene from   curvularia   inaeaualis and expression systems. 



  1. Material and method
The coding sequence of the CPO gene was determined as follows. The chloroperoxidase from C.   inaeaualis   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. On the basis of amino acid sequence 1 (see table 2) degenerated primers were designed on both sides of the coding DNA template. Using these two degenerated primers and the genome DNA of C. inaeaualis as template, the coding part of amino acid sequence 1 was amplified and cloned in a pUC18 vector. 



  The sequence of this amplified part was subsequently determined. The resulting clone was named   pCP01.   The coding sequence of amino acid sequence 2 was obtained in similar manner. The clone resulting therefrom was named pCP02. 



   On the basis of these two known sequences the new degenerated primers were designed and used in a PCR experiment with a first strand of cDNA as template. The thus 

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 obtained DNA fragment links the two already known DNA sequences. This total fragment was cloned in a pUC18 vector and the sequence thereof was determined. The resulting fragment codes for parts of the amino acid sequences 1 and 2 and also contains the region that codes for amino acid sequence 3 (see table 2). 



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



  2. Result
Shown in figure 6 is the sequence of the cDNA which codes for the chloroperoxidase from C.   inaequalis.   



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



  3. Expression of the chloroperoxidase gene
In order to express the chloroperoxidase the cDNA or a genomic fragment is cloned in known manner in an expression vector. With the resulting expression vector a suitable host cell, such as a fungus, for instance   Asperqillus   sp., Saccharomvces spec. or bacteria, for instance Streptomyces, Bacillus or E. coli, can be transformed. The culture medium is specifically adapted to the relevant host. The expressed chloroperoxidase can be recovered by known procedures, such as separation of the cells of the medium by centrifugation or filtration, precipitation of protein components in the medium by means of a salt, such as ammonium sulphate, 

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 followed by chromatographic procedures, such as ion exchange chromatography, affinity chromatography and the like. 

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   Table 2 Peptide sequences derived from vanadium chloroperoxidases 
 EMI17.1 
 
<tb> 
<tb> Sequence <SEP> Cleaving <SEP> method
<tb> C. <SEP> inaequalis
<tb> 1) <SEP> ML--LYMKPVEQPNPNPGANI <SEP> SDNAYAQLGLVLDRSVLFAa <SEP> CNBr
<tb> 2) <SEP> (S)NADETAEYDDAVRVALAVGGAQALNSa <SEP> Trypsin
<tb> 3) <SEP> (G)YHPTPGRYKFDDEP <SEP> Trypsin
<tb> 4) <SEP> IDEPEEYN <SEP> Trypsin
<tb> 5) <SEP> (D)LRQPYDPTAPIEDQPGIVRTb <SEP> Trypsin
<tb> D. <SEP> biseotata
<tb> 6) <SEP> LNGLNRDLRQPYDPTAPIEEQPGIVb <SEP> V8 <SEP> prot.
<tb> 
 a underlined sequences were used for designing the degenerated DNA primers. b homologous sequences between C.   inaequalis   and D. biseptata are printed in bold. 



  EXAMPLE 4 Assay of the chloride concentration in a liquid. 



  1. Introduction 
Chloride concentrations in natural and waste water are usually determined by means of volumetric methods with    silver nitrate or mercury (II) or spectrophotometrically using mercury (II) and iron(III)   ions. Illustrated in this example is the application of a new enzymatic method for determining total halide (with the exception of fluoride) and chloride in aqueous solutions. 



  The method is based on the specific oxidation of halides to hypohalogenic acids, which oxidation is catalyzed by chloro- and bromoperoxidases. The hypohalogenic acid is captured by 

 <Desc/Clms Page number 18> 

 monochlorodimedone. The quantitative halogenation of monochlorodimedone is determined spectrophotometrically. 



   Haloperoxidases form a class of enzymes which are capable of oxidizing halides (X' - Cl-, Br' or I-) in the presence of hydrogen peroxide to the corresponding hypohalogenic acids in accordance with the reaction: H2O2 + X- +   It -+   H2O + HOX   (1)   When a suitable nucleophilic acceptor is present, a reaction will occur with HOX and a halogenated compound will be formed. 



   Haloperoxidases can be subdivided into chloro-, bromoand iodoperoxidases in accordance with the most electronegative element that can be oxidized by these enzymes. 



  Chloroperoxidases are thus capable of oxidizing Cl-, Br' and I', while bromoperoxidases only oxidize Br' and I' and iodoperoxidases only iodide. 



   In this example two vanadium-containing enzymes, namely the chloroperoxidase of the fungus C.   inaequalis   and the bromoperoxidase from the lichen X. parietina (9) are used to determine total halide (Cl-, Br- I-) concentrations. It will be found from the data that the enzymatic assay of halide concentrations is easy to perform and gives 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 monobromo-monochlorodimedone in the presence of chloroperoxidase and to only the latter compound with bromoperoxidase. The reaction was monitored by measuring the absorption 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 coefficients at the same wavelength for di- 

 <Desc/Clms Page number 19> 

 chlorodimedone and for monobromo-monochlorodimedone are both 0.1 mM-1 cm-1. In the spectrophotometric assay a 50  M concentration of monochlorodimedone was used.

   After adding hydrogen peroxide and enzymes to a solution containing less than 50  M halide a partial absorption decrease was observed. The difference between the initial and final absorption indicates a value for the quantity of halide present. 



   Addition of chloroperoxidase to the assay will give the total quantity of halide present in the test mixture, while addition of bromoperoxidase gives a value for the halides with the exception of chloride. When the absorption values of the test with chloroperoxidase and that with bromoperoxidase are subtracted from one another, the difference gives the quantity 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   M   monochlorodimedone and 0.29   M   chloroperoxidase or 0.1   M   bromoperoxidase in a 2.5 ml quartz cuvette using a Cary 17 spectrophotometer. All buffers and solutions were prepared with water which had been filtered and de-ionized by an Elgastadt B12H (Elga group) and a Milli-Q (Millipore) water purification system. All reagents were of analytical grade. 



  3. Results
Figure 7 shows the decrease in absorption which is observed after conversion of monochlorodimedone by the bromoperoxidase from X. parietina or by the chloroperoxidase from C.   inaeaualis.   The reaction mixture contains 0.1 M citrate buffer (pH 3.6), 1 mM hydrogen peroxide, 25  M chloride and 15  M bromide (final concentration of halides: 40   M).   Graph A shows the absorption decrease in the presence of 0.1   M   bromoperoxidase, while graph B shows the absorption decrease in the presence of 0.3   M   chloroperoxidase. 



   Figure 8 shows the relation between the absorption change at 290 nm and the chloride concentration. The 

 <Desc/Clms Page number 20> 

 chloride concentration varies between 4   M   and 32  M. The enzyme concentration is 0.29   M.   The values shown are average values of three experiments. The relation between the chloride concentration and the absorption decrease is found to be linear. 



   In table 3 is shown the result of an experiment in which mixtures of bromide and chloride were analysed for bromide and chloride content using the enzymatic assay according to the invention. A comparison of the initially present concentrations of bromide and chloride with the result of the enzymatic assay shows that the enzymatic method measures both halides separately and accurately and is therefore capable of fully converting the halides which are present into hypohalogenic acid. The method is reliable because the measurements only deviate slightly. 



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



   Table 3 
Assay of a mixture of chloride and bromide by the method of the invention. 



   The total concentration of halides is 24   M.   
 EMI20.1 
 
<tb> 
<tb> 



  Mol <SEP> ratio <SEP> chlo- <SEP> Determined <SEP> value <SEP> Determined <SEP> ratio
<tb> ride <SEP> and <SEP> bromide <SEP> ( M) <SEP> of <SEP> the <SEP> total <SEP> of <SEP> chloride <SEP> and
<tb> concentration <SEP> bromide
<tb> 0.25 <SEP> 24.2 <SEP> (3.5) <SEP> * <SEP> 0.22 <SEP> (3.0)*
<tb> 0.67 <SEP> 23.8 <SEP> (3.1)* <SEP> 0.70 <SEP> (2.8) <SEP> * <SEP> 
<tb> 4 <SEP> 24.3 <SEP> (2.8) <SEP> * <SEP> 4.2 <SEP> (3.2)*
<tb> 
 *: Relative standard deviation (%). n=3. 

 <Desc/Clms Page number 21> 

 



   Table 4 
Chloride content of a number of water samples The concentration of chloroperoxidase was 0.29   M,   bromoperoxidase was 0.1   M.   
 EMI21.1 
 
<tb> 
<tb> 



  Sample <SEP> Measured <SEP> chloride <SEP> Reference <SEP> values
<tb> content <SEP> (mM) <SEP> (mM)
<tb> Spa <SEP> mineral <SEP> water <SEP> 0.15 <SEP> (3.7)* <SEP> 0.16a
<tb> Amsterdam <SEP> tap <SEP> 3.1 <SEP> (4.0)* <SEP> 2.1-4.2b
<tb> water
<tb> Amsterdam <SEP> canal <SEP> 6.5 <SEP> (2.5)*
<tb> water
<tb> 
 *: Relative standard deviation (%). n=3. a: as specified on the bottle b: the value is dependent upon the source, as specified by the Amsterdam Water Board. 



  4. Discussion
The results show that the new enzymatic method for the quantitative measurement of total halide (with the exception of fluoride) and chloride is simple and clear. The monochlorodimedone and its brominated and chlorinated derivatives have known extinction coefficients and can therefore be used as internal standard for the halide assay. 



  The calibration curve shows a linear relation between the chloride content and the absorption changes in the concentration range between 1 and 35   M.   The method according to the invention is exceptionally sensitive. The concentration range used in the enzymatic assay as according to this example is ten times 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 

 <Desc/Clms Page number 22> 

 only accurate when the solutions contain more than 5 mM chloride. This is about a thousand times more concentrated than the solutions which can be determined by the enzymatic method according to the invention. The described method is also more sensitive than that making use of ion-selective electrodes, wherein chloride concentrations of less than 10  M cannot be measured (10). 



   The measured bromide/chloride ratios correspond with the ratio of the actual values (see table 3). The results show that the combination of the two vanadium-containing enzymes used in this test provides reliable results, not only in respect of the halide content but also concerning the nature of the halide. When a iodoperoxidase with a high affinity for iodide is used it is even possible to analyze separately the content of any halide in any given mixture. 



  EXAMPLE 5 The demonstration of the anti-bacterial action of vanadium chloroperoxidase 1. Introduction
With the object of testing the anti-bacterial action of the chloroperoxidase from Curvularia inaeaualis, 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 in a culture medium (10 g yeast extract, 16 g trypton and 5 g NaCl per litre demineralized water), were washed with a physiological saline solution followed by a washing step in 0.1 M sodium acetate (pH 5.0). 1 ml of this bacteria 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  M chloroperoxidase and a concentration of hydrogen peroxide of 0,0.01 mM, 0.05 mM to 0.10 mM. As a control the bacteria suspension was also 

 <Desc/Clms Page number 23> 

 incubated in a sterile medium to which no chloroperoxidase (CPO) was added.

   After incubation for one hour samples of 50  l were taken and subsequently diluted 102x, 104x and 106X in a physiological saline solution. 100  l was taken from each of these dilutions and these were placed on agar plates (2% agar in culture medium). After overnight incubation at 37 C the number of bacteria per plate were counted. 



  3. Results and discussion
Table 5 shows that an incubation in the presence of only hydrogen peroxide also has a bactericidal action. The effect is however increased by adding the chloroperoxidase and surviving bacteria are no longer found at incubation in 0.05 mM hydrogen peroxide. 



   As is seen from the experiment in which no chloride is added (table 6), the bactericidal action of chloroperoxidase is here also observed. In view of the high affinity for chloride, the enzyme presumably uses the traces of chloride from the buffer system. The greatly declining number of bacteria in the incubation without chloroperoxidase can be the result of very unfavourable conditions (the absence of chloride) during the incubation. 



   Table 5
Number of surviving bacteria (cells per plate) after incubation with chloroperoxidase in 10 mM Cl' 
 EMI23.1 
 
<tb> 
<tb> H2O2 <SEP> (mM) <SEP> no <SEP> CPO* <SEP> 0.05 <SEP>  M <SEP> CPO*
<tb> 0 <SEP> 46 <SEP> 104 <SEP> 50 <SEP> 104
<tb> 0.01 <SEP> 9 <SEP> 104 <SEP> 1.2 <SEP> 104
<tb> 0.05 <SEP> 6 <SEP> 104 <SEP> 0
<tb> 0 <SEP> .10 <SEP> 4 <SEP> 104 <SEP> 0
<tb> 
 * CPO = chloroperoxidase 

 <Desc/Clms Page number 24> 

 
Table 6 Number of surviving bacteria (cells per plate) after incubation with chloroperoxidase in presence of extra chloride in the incubation medium 
 EMI24.1 
 
<tb> 
<tb> H2O2 <SEP> (mM) <SEP> no <SEP> CPO* <SEP> 0.05 <SEP>  M <SEP> CPO*
<tb> 0 <SEP> 2 <SEP> .

   <SEP> 7 <SEP> 0 <SEP> x <SEP> 104 <SEP> 4 <SEP> x <SEP> 104
<tb> 0.01 <SEP> 0.43 <SEP> x <SEP> 104 <SEP> 0
<tb> 0.05 <SEP> 0.08 <SEP> x <SEP> 104 <SEP> 0
<tb> 0.10 <SEP> 0.08 <SEP> x <SEP> 104 <SEP> 0
<tb> 
 * CPO = chloroperoxidase 
The results of this experiment therefore provide proof of the bactericidal action of the chloroperoxidase. 



  EXAMPLE 6 1. Introduction
This example demonstrates the formation of HOBr by (recombinant) chloroperoxidase from the fungus Curvularia   inaeaualis   immobilized in a paint or polymer system. Based on the bactericidal action demonstrated in example 4, it is possible to extrapolate to a growth-inhibiting action of the paint. 



  2. Materials and method
In order to determine the activity of peroxidase immobilized in a paint, a qualitative assay was used based on the bromination of 40  M phenol red to (tetra)bromophenol blue. Conversion of the red colour to a deep purple colour will be observed if the peroxidase system produces HOBr. 

 <Desc/Clms Page number 25> 

 



  This assay enables the formation of HOBr to be demonstrated in a simple visual manner. 



   Used in the experiments was a recombinant vanadium chloroperoxidase which had been expressed in a yeast strain. 



  The enzymatic and catalytic properties (inter alia the pH optimum and the specific activity of about 20 Units/mg) of this enzyme are identical to those of the original fungal enzyme in its purest form. The original enzyme can of course also be used for the described experiments. 



   Three test systems were used as model for the growthinhibiting effect of the paints. 



  Test system 1
2 grams acrylic latex (Sikkens) was mixed with 100  l (2mg/ml) chloroperoxidase and this was applied to a piece of wood of 2x2 cm. The paint then contained 0.1 mg enzyme per gram latex. The piece of wood was subsequently dried in air for several hours. In a subsequent experiment a 10 x larger quantity of enzyme was mixed with the paint to 1 mg enzyme per gram latex and dried for the same length of time. The drying process immobilizes the chloroperoxidase. 



   The pieces of wood were placed in 2 ml medium and carefully shaken with a table shaker to limit diffusion limitation of substrates and products. 



  Test system 2
A small quantity (about 2 ml) chlorinated rubber antifouling 2000 (AKZO) was mixed as well as possible with 0.5 ml chloroperoxidase (2.1 mg/ml). The enzyme preparation dissolved in buffer mixes very badly with the paint. This paint was also applied to a piece of wood and dried for several hours. 



  Test system 3
To increase the accessibility of the enzyme, a matrix was also used of 20% polyacrylamide in which the chloroperoxidase, in a final concentration of 0.1 mg enzyme per ml acrylamide, was immobilized by causing it to 

 <Desc/Clms Page number 26> 

 copolymerize during the polymerization process. For a description of this procedure see Baily J. E. and Ollis, D. F. in Biochemical Engineering Fundamentals. McGraw-Hill Book Company, second edition, pp. 180-202. 



   The matrix was prepared by mixing 10 ml acrylamide/ bisacrylamide with 0.46 ml chloroperoxidase (2.1 mg/ml). The pores in the matrix system allow easy access of substrates and draining of the formed products, while the enzyme however remains bound to the matrix and thus cannot diffuse therefrom. 



   To test the formation of HOBr, pieces of gel with a surface area of about 0.5 by 0.5 cm (about 2 mm thick) were cut out and tested in media of 2 ml which were also shaken. 



   The pieces of wood obtained according to the three test systems were held in a number of media for some time. Used as media were: Medium A:
100 mM KBr
0.1 M phosphate buffer (pH 6.5)   4 0    M phenol red Medium B: natural seawater with 40   M   phenol red 3. Results
When the piece of treated wood according to test system 1 is placed in medium A and 1mM of hydrogen peroxide is subsequently added, a blue coloration of the phenol red occurs within 30 minutes as a result of the bromination reactions by HOBr. 



   When the piece of wood with the higher enzyme concentration is placed in this same medium, an accelerated blue coloration occurs, as expected. 



   With test system 2 in the same medium only a very slow reaction is measured and the conversion of the phenol red to 

 <Desc/Clms Page number 27> 

 bromophenol blue is only measurable after a few days. The slow reaction is probably caused by the poor mixing of this paint with the chloroperoxidase dissolved in water and a small inclusion of the enzyme in the paint. This observation does however show that the enzyme is apparently resistant to the solvent used in the conventional growth-inhibiting agents. It has already been established that the enzyme retains its activity in organic solvents mixable with water (7) . 



   The piece of painted wood from test system 1 (1 mg enzyme per ml latex) was also tested in more natural conditions and placed in 2 ml medium B, to which 1 mM hydrogen peroxide was also added. Here also bromination of phenol red occurs, although the full conversion takes a number of hours. These experiments show that the enzyme is accessible for its substrates (hydrogen peroxide and bromide) via pores in the latex paint and is capable of continuous formation of HOBr. 



   The polyacrylamide gels in which the enzyme is immobilized also show that HOBr is formed in seawater after addition of hydrogen peroxide. When the seawater (pH about 7.8) is first brought to pH 6.8 with citric acid, a bromination reaction occurs which is complete in about 15 minutes. This is probably the result of the fact that the pH optimum of the chloroperoxidase in the bromination reaction lies at pH 4-6. 



   The concentration of hydrogen peroxide does not influence the speed of HOBr formation. A test shows that this speed is not affected if the concentration of hydrogen peroxide is reduced to 200  M. 



   In all cases a blue coloration of the gel occurs first which spreads further into the medium. To demonstrate that the bromination reactions are not caused by the enzyme leaking out of the matrix, a piece of gel was incubated in 2 ml 1mM Br- in 0.1 M citrate buffer (pH 6.5) for 3 x 24 hours. After each incubation of 24 hours the medium was changed. When hydrogen peroxide was added to the piece of gel in fresh medium, a rapid bromination reaction once again 

 <Desc/Clms Page number 28> 

 occurred which proceeded as rapidly as initially prior to incubation. This data indicates that the enzyme remains immobilized and remains active for a minimum of three days. 



  4. Discussion
The above described experiments demonstrate that it is possible to immobilize the vanadium chloroperoxidase and that such an enzyme added to paint or a polymer is also capable of formation of HOBr in seawater. The naturally present Br' (1 mM) is herein oxidized by the enzyme to HOBr in the presence of hydrogen peroxide. It is possible that the enzyme also uses the Cl- from the seawater, although the Km of the enzyme for bromide is much smaller than for chloride and this means that the chloroperoxidase will preferentially oxidize bromide in a mixture of the two halides. 



   As demonstrated in example 4, HOBr and HOC1 have a strong biocidal action. It will therefore be possible to apply the enzyme system as growth-inhibiting agent in paints. 



   The second substrate for the enzyme is hydrogen peroxide. It is known from the literature that seawater contains sufficient hydrogen peroxide (11). The concentration amounts to approximately 1  M and is a result of biological activity and photochemical reactions in seawater under the influence of sunlight. The enzyme is certainly capable of using this low concentration of hydrogen peroxide as oxidizing agent for bromide. The data of Van Schijndel et al. (7) show that the Km for hydrogen peroxide amounts to about 10   M   at pH 5.0 and at higher pH values such as in seawater this Km will decrease still further. This means that very low concentrations of hydrogen peroxide can also be used by the enzyme. The biological activity of adhering micro-organisms can also result in a number of cases in a locally increased hydrogen peroxide concentration.

   The hydrogen peroxide will be converted effectively into the biocidal HOBr by the enzyme present on and in the painted surface to be adhered to. 

 <Desc/Clms Page number 29> 

 



   This example demonstrates that it is possible to include a haloperoxidase or a mixture of a number of haloperoxidases in a paint without the enzyme losing its activity. 

 <Desc/Clms Page number 30> 

 



  REFERENCE LIST 1. R. Wever en K. Kustin. Vanadium: a biologically rele- vant element. Adv. Inorg. Chem. 35 (1990) 81-115. 



  2. D. Rehder. The bioinorganic chemistry of vanadium. Ang. 



   Chem. Ed. Engl. 30 (1991) 148-167. 



  3. E. de Boer en R. Wever. The reaction mechanism of the novel vanadium bromoperoxidase, a steady-state kinetic analysis. J. Biol. Chem. 263 (1988) 12326-12332. 



  4. E. de Boer, H. Plat, M. G.M. Tromp, M. C.R. Franssen,
H. C. van der Plas, E. M. Meijer en H. E. Schoemaker. 



   Vanadium-containing bromoperoxidase: an example of an oxidoreductase with high operational stability in aqueous and organic media. Biotechn. Bioeng. 30 (1987)
607-610. 



  5. J. R. Kanofsky. Singlet oxygen production by chlorope- roxidase-hydrogen peroxide-halide systems. J. Biol. 



   Chem. 259 (1984) 5596-5600. 



  6. J. W.P.M. van Schijndel, E. G.M. Vollenbroek en R. Wever. 



   The chloroperoxidase from the fungus Curvularia inae- qualis; a novel vanadium enzyme. Biochim. Biophys. Acta
1161, 249-256. 



  7. J.W.P.M. van Schijndel, P. Barnett, J. Roelse, E.G.M. 



   Vollenbroek en R. Wever. The stability and steady-state kinetics of vanadium chloroperoxidase from the fungus
Curvularia inaequalis. Europ. J. Biochem. 225, 151-157 (1994). 



  8. L. P. Hager, D. R. Morris, F. S. Brown en H. Eberwein. 



   Chloroperoxidase II utilization of halogen anions. J. 



   Biol. Chem. 241 (1966) 1769-1777. 

 <Desc/Clms Page number 31> 

 



   9. H. Plat, B. E. Krenn en R. Wever. The bromoperoxidase from the lichen Xanthoria   parietina   is a novel vanadium enzyme. Biochem. J. , 248 (1987) 277-279. 



  10. Ingold Laboratory Electrode Guide (1993). Ion selective and gas sensitive electrodes p. 13. 



  11. Petasne, R. G., Zika, R. G. fate of superoxide in coastal seawater Nature 325 (1987), 516-518.

Claims

CLAIMS 1. Paint comprising the usual paint constituents and solvents in addition to a suitable concentration of one or more haloperoxidases.

2. Paint as claimed in claim 1, characterized in that the haloperoxidase can be obtained from a fungus chosen from the group consisting of Curvularia inaeaualis, Drechslera biseptata, Drechslera fuqax, Drechslera nicotiae, Drechslera subpapendorfii, Embellisia hyacinthi, Embellisia didymospora, Ulocladium chartarum, Ulocladium botrvtis.

3. Paint as claimed in claim 1 or 2, characterized in that the enzyme is the chloroperoxidase from Curvularia inaeaualis.

4. Paint as claimed in claim 1,2 or 3, characterized in that the enzyme is a recombinant enzyme.

5. Paint as claimed in claim 4, characterized in that the DNA sequence which codes for the recombinant enzyme comprises at least a part of the sequence according to figure 6 or modified versions thereof.

6. Paint as claimed in any of the foregoing claims, characterized in that it is a growth-inhibiting paint.

7. Haloperoxidase in substantially isolated form obtainable from a fungus chosen from the group consisting of Curvularia inaeaualis, Drechslera biseptata, Drechslera fugax, Drechslera nicotiae, Drechslera subpapendorfii, Embellisia hvacinthi, Embellisia didymospora, Ulocladium chartarum, Ulocladium botrytis for use in a paint as claimed in any of the claims 1-6.

8. Haloperoxidase for use as preservative in paint.

9. Use of haloperoxidase obtainable from a fungus chosen from the group consisting of Curvularia inaequalis, Drechslera biseptata, Drechslera fugax, Drechslera nicotiae, Drechslera subpapendorfii, Embellisia hyacinthi, Embellisia <Desc/Clms Page number 33> didvmospora, Ulocladium chartarum, Ulocladium botrytis in paint.

10. Use of haloperoxidase as claimed in claim 9, characterized in that the paint is a growth-inhibiting agent, for instance for ships.

11. Method for determining the halide concentration in a liquid, comprising of adding to the liquid for testing a hydrogen peroxide and one or more haloperoxidases, monitoring the oxidation reaction by means of an indicator system and determining the halide concentration on the basis of the indicator system.

12. Method as claimed in claim 11, characterized in that the indicator system is formed by monochlorodimedone.

13. Haloperoxidase in substantially isolated form obtainable from a fungus chosen from the group consisting of Curvularia inaequalis, Drechslera biseptata, Drechslera fuaax, Drechslera nicotiae, Drechslera subpapendorfii, Embellisia hyacinthi, Embellisia didymospora, Ulocladium chartarum, Ulocladium botrvtis for use in a method for determining the halide concentration in a sample.

14. Use of haloperoxidases in a method for determining the halide concentration in a sample.

15. Test kit for determining the halide concentration in a sample, comprising at least one haloperoxidase, a source of hydrogen peroxide and an indicator system for monitoring the oxidation reaction.

16. Test kit as claimed in claim 15, characterized in that the source of hydrogen peroxide is pure hydrogen peroxide or a source generating hydrogen peroxide and the indicator system is monochlorodimedone.