SK123096A3 - Enzymatic antimicrobial compositions containing halogenperoxydase, dna sequence, preparation method for halogenperoxydase and use thereof - Google Patents

Abstract

An antimicrobial composition is provided, comprising a Vanadium haloperoxidase, a source of halide and hydrogen peroxide or a source of hydrogen peroxide. Preferably, the Vanadium haloperoxidase is a chloroperoxidase obtainable from Curvularia inaequalis.

Description

Technical field

The invention relates to enzymatic antimicrobial compositions and their use. More particularly, it relates to enzymatic antimicrobial compositions comprising a vanadium haloperoxidase, a hydrogen peroxide source, and a halide source. The invention also relates to the production of vanadium haloperoxidase by a recombinant DNA technique which can be used for enzymatic antimicrobial compositions.

BACKGROUND OF THE INVENTION

Various enzymatic antimicrobial compositions are known in the art. For example, WO-A-94/04217 discloses stabilized dentifrice compositions which are capable of forming antimicrobially effective concentrations of hypothiocyanate ions (OSCN ^- ). The compositions comprise hydrogen peroxide-forming oxidoreductase and a peroxidase enzyme capable of oxidizing thiocyanate ions, which are normally present in saliva, to antimicrobial hypotothiocyanate ions (OSCN ^- ). Suitable peroxidases include lactoperoxidase, myeloperoxidase salivary peroxidase, and chloroperoxidase.

Enzymatic antimicrobial compositions containing haloperoxidase are also described in EP-A-500 387 (Exoxemis). It is disclosed that haloperoxidases selectively bind to target microbes in the presence of peroxide and halide and inhibit their growth. Suitable haloperoxidases are mentioned in EP-A-500 387 myeloperoxidase (MPO), eosinophilic oxidase (EPO), lactoperoxidase (LPO) and chloroperoxidase (CPO). The halide to hydrogen peroxide ratio is said to be a critical factor with respect to stability and functionality of haloperoxidase. At very low ratios, hydrogen peroxide can inhibit haloperoxidase function, while at very high ratios

- 2, the halide may block the enzymatic reaction. The ratio can vary over a wide range, but is preferably greater than about 50.

For unexpected reactions of hydrogen peroxide, the actual concentration of hydrogen peroxide in antimicrobial compositions may be lower than expected. Applicants of the present invention have therefore found that it is preferable to be able to use a high initial concentration of hydrogen peroxide in an antimicrobial composition in conjunction with a conventional amount of halide.

In addition, there is a need for enzymatic antimicrobial compositions having a spectrum of antimicrobial activity that is different from the spectrum of known enzymatic antimicrobial compositions. The compositions should preferably be capable of exhibiting antimicrobial activity against difficult-to-control microorganisms, for example Streptococcus faecalis. In other circumstances, it may also be desirable to combat non-pathogenic microorganisms as they may cause spoilage of food products.

It is therefore an object of the present invention to provide an enzymatic antimicrobial composition that overcomes one or more of the above disadvantages.

We have surprisingly found that certain potent enzymatic antimicrobial compositions can be prepared using vanadium haloperoxidase.

Various haloperoxidases have been included in the above prior art for use in antimicrobial compositions, but the class of vanadium haloperoxidases has not been addressed so far.

Vanadium haloperoxidases are different from other haloperoxidases in that the prosthetic group in these enzymes has structural features similar to vanadate (vanadium V), while other haloperoxidases are heme-peroxidases.

Another object of the present invention was to clone the genetic encoding of vanadium haloperoxidase and to determine its sequence to allow its expression in other microorganisms that are more suitable for cultivation and also to increase the amount of enzyme that can be produced using the recombinant DNA technique.

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Some of the vanadium haloperoxidases are found in nature on the surface of seaweed (Vever et al., 1991). In intact plants in seawater, vanadium bromoperoxidase is achievable for added substrates and is capable of releasing HOBr upon addition of hydrogen peroxide. The role of the enzyme has not yet been determined, but it is likely that the formation of strongly oxidizing HOBr in seawater is part of the plant's defense system to prevent the growth of microorganisms or fungi on its surface.

After the discovery of non-heme vanadium bromoperoxidase from the seaweed Ascophylum nodosum, it has been shown that these enzymes also contain a large number of other seaweeds. In particular, bromoperoxidase from Ascophylum nodosum has been intensively studied and characterized (see references 2-3 for review). The prosthetic group of these enzymes has structural features similar to vanadate (vanadium V). In the catalytic mechanism that has been derived for this enzyme, hydrogen peroxide reacts with the enzyme to form a hydrogen peroxide-enzyme complex, after which it reacts with the bromide and proton complex to form the HOBr enzyme complex. It was found (De Boer et al., 1988) that this complex decomposed into enzyme and HOBr. These vanadium bromoperoxidases have also been found (De Boer et al., 1988) to have high working stability in aqueous and organic media. For example, these enzymes were stable for three weeks under conditions of use and could be stored without loss of activity for more than a month in an organic solvent such as acetone, methanol, ethanol (present up to 60% by volume) and 1-propanol. However, these enzymes have the disadvantage that bromide should be present or added for potential applications and further attempts to clone the genes encoding these bromoperoxidases from seaweed and to determine their amino acid sequence have not been successful.

The known existing hem-containing chloroperoxidase from Caldariomyces fumago is less suitable for the preparation of enzymatic antimicrobial compositions, because of its inherent instability and low optimal pH of 2.75 (J.R. Kanofsky, 1984), which severely restricts its application. Similar arguments prevent the application of the enzyme myeloperoxidase (MPO) from human white blood cells, which is also capable of generating HOCI (A.R.J. Bakkenist et al., 1980).

There have also been reports (see references 8, 9) that dematic hypomycetes secrete haloperoxidases with distinct stability. In particular, it has been shown that the terrestrial fungus Curvularia-i.naequ.alis secretes vanadium chloroperoxidase (J.V.P.M. van Schijndel et al., 1993), which has a high affinity for chloride and has a pH optimum for the chlorination reaction of about pH 5.5. As with seaweed bromoperoxidase, the prosthetic group chloroperoxidase has structurally similar features to vanadate (vanadium V). In a further detailed study (J.V.P.M Van Schijndel et al. 1994), it was shown that the kinetics of the enzymatic oxidation of chloride by hydrogen peroxide is similar to that of vanadium bromoperoxidase from the seaweed A. nodosum. Three different methods have further shown that the enzyme forms oxidizing substances (HOCI) as a reaction product and is itself resistant to this product. The enzyme exhibits high thermal stability (Tm 90 ° C) and exhibits high stability to organic solvents such as 40% methanol, ethanol and propanol.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to an enzymatic antimicrobial composition comprising a vanadium haloperoxidase, a halide source and hydrogen peroxide or a source of hydrogen peroxide.

According to a second aspect, the present invention relates to the production of vanadium haloperoxidase by converting a suitable host by expressing a vector comprising the replication, transcription and termination initiation control sequences, and at least a portion of the DNA sequence encoding vanadium haloperoxidase, by culturing the host under conditions permitting structural gene expression and isolation of vanadium halide. and

DETAILED DESCRIPTION OF THE INVENTION (a) Vanadium haloperoxidase

The enzymatic antimicrobial compositions of the present invention comprise, as a first component, a vanadium haloperoxidase. The vanadium haloperoxidase may in principle be selected from the various vanadium haloperoxidases already described in the art. For example, vanadium haloperoxidase (non-hemic) produced by Curvularia inaequalis can be used, such as described in US-A-4 707 466. Alternatively, chloroperoxidase from Curvularia inaequalis (CBS 102.42) can be isolated and purified according to the method of J.V.P.M. Van Schijndel et al. (1993).

Other sources of vanadium haloperoxidases include Drechslera biseptata (CBS 371.72), Drechslera fugax (CBS 509.77), Drechslera nicotiae (CBS 655.74), Drechslera subpapendorfii (CBS 656.74), Embelisia hyacinthium (416.71), 775.71, (200.67) and Ulocladium botrytis (452.72).

Alternatively, vanadium haloperoxidase can be prepared by recombinant DNA technique by converting a suitable host by expressing a vector containing the replication, transcription and termination start sequence sequences, and a DNA sequence encoding vanadium haloperoxidase, by culturing the host under conditions that allow structural gene halide expression and vanadium peroxide isolation. This is described in detail in the examples below.

(b) Halide ion source

The second component of the sanitary compositions of the present invention is a source of halide ions. These may be any halide ions, but the source of iodide or chloride ions is preferred because it is the most efficient. Sodium chloride is the most preferred source of halide ions. The halide may be added to the enzymatic antimicrobial compositions of the present invention, or alternatively, a halide which is naturally present in tap water and which has a

- generally a concentration of the order of 2 to 5 mmol / l.

(c) Source of hydrogen peroxide.

The sanitary compositions of the invention further comprise a hydrogen peroxide source or a hydrogen peroxide generating system. Examples of suitable hydrogen peroxide generating systems are perborate or percarbonate salts, preferably sodium percarbonate or sodium perborate.

Hydrogen peroxide can also be provided by enzymatic hydrogen peroxide generation systems. The enzymatic hydrogen peroxide generating system may in principle be selected from the various enzymatic hydrogen peroxide generating systems described in the art. For example, an amine oxidase and an amine, an amino acid oxidase and an amino acid, a lactate oxidase and a lactate, a cholesterol oxidase and a cholesterol, a uric acid oxidase and a uric acid or a xanthine oxidase with xanthine may be used. However, a combination of glucose oxidase and glucose is preferred.

The amount of glucose oxidase will depend on its specific activity and the remaining catalase that may be present, but by way of example it can generally be stated that the detergent composition of the present invention will contain 10 to 1000, preferably 20 to 500 glucose oxidase units per gram or milliliter of detergent. of a mixture, the unit of enzyme activity is defined as the amount required to convert 1 pmol of substrate per minute under standard conditions.

(d) Other ingredients

The enzymatically bleached detergent compositions of the present invention generally comprise from 0.01 to 50% by weight of one or more surfactants, preferably 0.1 to 5.0% by weight of one or more surfactants are wetting agents. Suitable surfactants or detergent surfactants are soap or non-soap anionic surfactants, nonionic surfactants, cationic surfactants, amphoteric or zwitterionic surfactants. Surface active system "

it usually comprises one or more anionic surfactants and one or more nonionic surfactants. The surfactant system may additionally contain amphoteric or zwitterionic substances, but this is not normally required because of their relatively high cost.

In general, the nonionic and anionic surfactants of the surfactant system may be selected from the surfactants described in Surface Active Agents Vol. 1, Schwartz & Perry, Interscience 1949 and Surface Active Agents Vol. 2, Schwartz, Perry & Berch, Interscience 1958, in the current edition of McCutcheon's Emusifiers & Detergents published by the Manufacturing Confectioners Company or in Tenside-Taschenbuch, H. Stache, 2nd Edition, Carl Hauser Verlag, 1981.

Suitable nonionic surfactants which may be used include, in particular, the reaction products of substances having a hydrophobic group and a reactive hydrogen atom, for example aliphatic alcohols, acids, amides or alkylphenols with alkylene oxides, especially ethylene oxide either alone or with propylene oxide. Specific nonionic detergent. the compounds are C8-C22 alkylphenol ethylene oxide condensates, generally 5 to 25 EO, i. 5 to 25 units of ethylene oxide per molecule and condensation products of aliphatic C8-C18 primary or secondary linear or branched alcohols with ethylene oxide, generally 5 to 40 EO.

Suitable anionic detergents which may be used are usually the water-soluble alkali metal salts of organic sulfates and sulfonates having alkyl radicals containing from about 8 to about 22 carbon atoms, the term alkyl being used to include the alkyl moiety of higher acyl radicals. Examples of suitable synthetic anionic detergents are sodium and potassium alkyl sulfates, especially sulfates obtained by suctioning higher C8-C18 alcohols made, for example, from tallow or coconut oil, sodium and potassium alkyl C8-C20 benzenesulfonates, especially sodium linear secondary alkyl benzenesulfonates; and sodium alkyl glyceryl ether sulfates, especially those ethers with higher alcohols derived from tallow or coconut oil and synthetic alcohols derived from petroleum. Preferred anionic detergents are sodium C1-pC1. alkyl benzene sulphonates and sodium C ₁₂ ^"C 18 ^a] - ^the ylsulfanyl sulfates.

Also useful are surfactants as described in EP-A-328 177 (Unilever), which exhibit sputter resistance, the alkyl polyglycoside surfactants disclosed in EP-A-070 074, and alkyl monoglycosides.

Preferred surfactant systems are mixtures of anionic surfactants with nonionic detergent active materials, especially the groups and examples of anionic and nonionic surfactants disclosed in EP-A-346 995 (Unilever). Particularly preferred is a surfactant system which is a mixture of an alkali metal salt of C 1 -C 6 primary alcohol sulfate together with 3-7 EO primary alcohol ethoxylate.

The nonionic detergent is preferably present in amounts greater than 10% by weight, for example 25 to 90% by weight of the surfactant system. For example, anionic surfactants may be present in amounts ranging from about 5% by weight to about 40% by weight of the surfactant system.

The enzymatic detergent compositions of the invention may also contain other ingredients normally used in antimicrobial compositions, such as thickening agents. Particularly useful in this regard are the surfactant combinations described in EP-A-314 232 (Unilever) which provide thickening gels upon dissolution in water.

The antimicrobial compositions of the present invention can be used to provide a hygienic effect in cleaning hard surfaces and fabric washing, but also to provide hygiene and cleaning in industrial / institutional applications such as in hospitals and for cleaning and disinfecting medical equipment. Another application is in the dairy industry to disinfect dairy equipment. Antimicrobial compositions can also be successfully used in deodorization in terms of their ability to combat odor-causing bacteria.

The antimicrobial compositions of the present invention can be used in the form of powders which have to be dissolved in water before use, but can also be formulated as liquid products or gels. In these product forms, it is important that halogenate formation does not begin prior to use of the mixture. This can be achieved! by physical separation of the enzyme and its substrate, for example by encapsulating the enzyme according to known techniques.

In use, the enzymatic antimicrobial composition is diluted 5 to 100 times by the addition of water to provide an environment having effective antimicrobial activity. This is then contacted with the surface to be disinfected and allows it to remain in contact with that surface for a time sufficient to achieve disinfection.

references

1. R. Vever, M.G.M. Tromp, B.E. Kren, A. Marj ani and M. van Tol, Bromination activity of the alga Ascophyllum nodosum: Effect on the biosphere, Envir. Sc. Techn. 25 (1991), 446-449.

2. R. Vever and K. Kustin, Vanadium: Biologically Important Element, Adv. Inorg. Chem. 35 (1990), 81-115.

3. D. Rehder, Bioinorganic Chemistry of Vanadium, Ang. Chem. Ed. Engl. 30 (1991) 148-167.

4. E. de Boer and R. Vever, Reaction mechanism of novel vanadium bromoperoxidase, steady-state kinetic analysis, J. Biol. Chem. 263 (1988), 12326-12332.

5. E. de Boer, H. Plat, M.G.M. Tromp, M.C.R. Franssen, H.C. van der Plas, E.M. Meijer and H.E. Schoemaker, Vanadium bromoperoxidase: an example of oxidoreductase with high working stability in aqueous and organic media, Biotechn. Bioeng. 30 (1987), 607-610.

6. J.R. Kanofsky. Formation of singlet oxygen by chloroperoxidase - hydrogen peroxide - halide systems, J. Biol. Chem. 259 (1984), 5596-5600.

7. A.R.J. Bakkenist, J.E.G. de Boer, H. Plat and R. Vever, Halogenide complexes of myeloperoxidase and mechanism of halogenation reactions, Biochim. Biophys. Acta 613 (1980), 337-34.

8. J.C. Hunter-Cevera and L. Sotos, Searching for New Enzymes in Nature: Halogen Peroxidase Formation by Dead Valley Dematic Hypomycetes, Microb. Ecol. 12 (1986) 121-127.

9. T-N.E. Liu, T.M. Timkulu, J. Geigert, B.Volf, S.L. Neidelman, D. Silva and J.C. Hunter-Cevera, Isolation and characterization of a novel non-heme chloroperoxidase, Biochem. Biophys. Res. Commun. 142 (1987) 329-333.

10. J.V.P.M. van Schijndel, E.G.M. Vollenbroek and R. Vever, Chloroperoxidase from Curvularia inaequalis; a new vanadium enzyme, Biochim. Biophys. Acta 1161 (1993), 249-256.

11. J.V.P.M. van Schijndel, P. Barnett, J. Roels, E.G.M. Vollenbroek and R. Vever, Vanadium chloroperoxidase from the fungus Curvularia inaequalis ·, stability and steady-state kinetics, Eur. J. Biochem. 225 (1994), 151-157.

12. H. Schagger and G. von Jagow, Anal. Biochem. 166 (1987), 368-379.

13. P. Matsudaira, J. Biol. Chem. 262 (1987), 10035-10038.

14. J. Sambrook, E.F. Fritch and T. Maniatis (1989), Molecular Cloning: Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

15. R. Vever, H. Plat and E. de Boer, Biochim. Biophys. Acta 830 (1985), 181-186.

BRIEF DESCRIPTION OF THE DRAWINGS

In FIG. 1 are peptide sequences obtained from vanadium chloroperoxidase sequences.

In FIG. 2 shows a gene encoding C. inaequalis chloroperoxidase and its putative gene products.

In FIG. 3 shows the pH profile of the activity of wild-type chloroperoxidase and recombinant yeast derived chloroperoxidase.

In FIG. 4 shows an autoradiogram obtained with DNA from Curvularia inaequalis and Drechsler biseptata.

DETAILED DESCRIPTION OF THE INVENTION

The invention will be further illustrated by the following non-limiting examples.

Example 1

Minimum inhibitory concentration (MIC) of hypochlorite.

Materials:

In this example, strains of Escherichia coli NCTC 900, Pseudomonas aeruginosa ATCC 15442, Staphylococcus aureus ATCC 13565, Streptococcus faecalis NCTC 1092 and Listeria innocua ATCC 33090 serotype 6B were used. Bacteria were grown for 15-18 hours at 30 ° C in Brain Heart Infusion (BHI) broth. After cultivation, the strains were washed twice with pH 5.5 citrate buffer (20 mmol / L, Na 2 Citran-NaOH + 10 mmol / L NaCl) The bacterial suspensions were centrifuged in an Eppendorf centrifuge (14000 rpm for 5 minutes). Then, the supernatants were discarded, and then the bacterial pellet was resuspended in citrate buffer.This washing procedure was repeated with each bacterial suspension again, and the twice washed bacterial suspension was then diluted with citrate solution at pH 5.5 to obtain a suspension of approximately 10%. Bacteria and BSA solution (1% w / v in citrate solution pH 5.5) were sterilized by filtration and stored at 4 ° C. The hypochlorite solution was prepared from a stock solution ( 107000 ppm) by dilution with sterile demineralized water.

methodology:

Using a sterile technique, a suspension of 10 bacteria per ml in citrate buffer pH 5.5 was prepared. 1.9 ml aliquots were added to the sterile tubes from this suspension. Subsequently, 0.1 ml of cold hypochlorite solutions of various concentrations were added to the tubes in such a way as to obtain the desired dilution range of hypochlorite. The tubes were continuously stirred using a magnetic stirrer.

Comparative samples were also made where only sterile buffer was used instead of hypochlorite solution. The samples were incubated for exactly 5 minutes at 30 ° C. After this incubation period, 1 ml of the reaction mixture was withdrawn and added to 9 ml of cold BSA (1% w / v) solution and immediately placed on ice. This was done to stop the hypochlorite from reacting with the microorganisms. Survival as Colony Forming Units (CFU) per ml was determined by diluting the sample from the IO ^- · '' to 10 'and the application of the $ 100 μΐ samples of the various dilutions on labeled BHI-agar plates. Plates were incubated for 15-18 hours at 30 ° C. If no CFU was detectable after this incubation period, the plates were incubated again for another 24 hours at 30 ° C.

Definition: the minimum inhibitor concentration (MlC-value) is defined herein as the hypochlorite concentration, which under the experimental conditions used results in a log reduction of the colony forming units of the particular microorganism tested at least 6.

The results are shown in Table I. The values found are in the same range as reported in the literature.

Table I. MIC values for various microorganisms

microorganisms MIC values in ppm Escherichia coli 2-3 Staphylococcus aureus 2-3 Listeria innocua 2-3 Pseudomonas aeruginosa 2 - 3 Streptococcus faecalis 2-3

Example 2

Minimum inhibitory concentration of hypochlorite and hypochlorite generated enzymatically by vanadium chloroperoxidase (V-CPO) from Curvularia inaequalis.

In this example, the killing effect of hypochlorite is compared with the killing effect of hypochlorite generated enzymatically by vanadium chloroperoxidase (V-CPO) from Curvularia inaequalis. A micropump was used to allow comparison. This was done to adjust the hypochlorite concentration during the experiment to follow the same profile as in the situation where hypochlorite is generated enzymatically. V-CPO activity was also determined very carefully under the conditions of the experiment, in order to know the amount of hypochlorite present at each point of the experiment.

Materials:

In this example, strains of Escherichia coli NCTC 900, Pseudomonas aeruginosa ATCC 15442, Staphylococcus aureus ATCC 13565, Streptococcus faecalis NCTC 1092 and Listeria innocua ATCC 33090 serotype 6B were used. The bacteria were cultured for 15-18 hours at 30 ° C in Brain Heart Infusion broth (BHI). After cultivation, the strains were washed twice with citrate buffer pH 5.5 (20 mmol / l, Na 2 Citran-NaOH + 10 mmol / l NaCl). The bacterial suspensions were centrifuged in an Eppendorf centrifuge (14,000 rpm for 5 minutes). Then, the supernatants were discarded and then the bacterial pellet was resuspended in citrate buffer. This washing procedure was then repeated with each bacterial suspension again. The twice washed bacterial suspension was then diluted with citrate solution at pH 5.5 to obtain a suspension of approximately 10 bacteria per ml. During the washing steps, cells were kept on ice. Buffers and BSA-solution (1% w / v in citrate solution pH 5.5) were sterilized by filtration and stored at 4 ° C. The hypochlorite solution was prepared from the stock solution (107000 ppm) by dilution with sterile demineralized water. H ₂ O ₂ solutions were prepared from a 30% stock solution by dilution with sterile demineralized water. Casitone was purchased from Difco. The chloroperoxidase from Curvularia inaequalis was purified according to van Schijndel et al. (1993). Chloroperoxidase activity in the conversion of the C although determined spectrophotometrically in 20 mmol / 1 citrate buffer pH 5.5, 10 mmol / ml of NaCl, 100 g mol / l H ₂ 0 _2, 50 mmol / 1 monochlorodimedone at 30 C, by following the conversion of monochlorodimedone (e (290 nm) = 20.2 mmole .1.cm ^{-1 -1)} to dichlorodimedone (e (290 nm) = 0.2 mM ^-1 .1.cm ^-1). A chloroperoxidase unit is defined as the amount of enzyme that converts 1 pmol of monochlorodisone (Sigma) per minute.

methodology;

Using a sterile technique, a suspension of about 10 bacteria per ml in citrate buffer pH 5.5 was prepared. From this suspension, 1.8 ml aliquots were added to sterile tubes. Subsequently, 0.2 ml of V-CPO solutions of various concentrations were added to the reaction vials (calculations see below) in such a way as to obtain the desired dilution range of V-CPO. A comparative sample was also prepared which contained 0.2 ml of sterile buffer instead of 0.2 ml of V-CPO. The reaction vials were incubated at 30 ° C. Then 0.5 ml of H ₂ O ₂ stock solution was added to initiate the V-CPO reaction. The stock solution concentration used depended on the hypochlorite concentration and was chosen in such a way that the final concentration of H ₂ O ₂ (at the end of the flow) was a five-fold molar excess compared to the final concentration of hypochlorite. The samples were incubated for exactly 5 minutes at 30 ° C. After this incubation period, 1 ml of the reaction mixture was withdrawn and added to 9 ml of cold BSA (1% w / v) solution and immediately placed on ice. This was done to stop the hypochlorite from reacting with the microorganisms. Survival as Colony Forming Units (CFU) per ml was determined by diluting the sample 101-10 ^- 5 and plating 100 μΐ samples of the various dilutions on labeled BHI-agar plates. Plates were incubated for 15-18 hours at 30 ° C. If after this incubation period no CFUs were detectable, the plates were incubated again for another 24 hours at 30 ° C.

The MIC value obtained was compared to the MIC value obtained when equal amounts of hypochlorite were added by a micropump. It was done as follows:

Using a sterile technique, a suspension of approximately 10 bacteria per ml in citrate buffer pH 5.5 was prepared.

From this suspension, 1.8 ml aliquots were added to sterile tubes, which were stirred continuously with a magnetic stirrer during the experiment. The reaction vials were incubated at 30 ° C. Then, 0.2 ml of a stock solution of H ₂ 0 ₂ The concentration of the stock solution of H ₂ 0 ₂ depended on the hypochlorite concentration produced, and was chosen such that the final concentration of H ₂ 0 ₂ (at the end of the flux) was a fivefold molar excess compared with a final hypochlorite concentration. The applied flow of 0.5 ml of hypochlorite solutions of a known concentration for 5 min (flow: 0.1 ml per min) at 30 ^e C did a series of experiments each with different concentrations of hypochlorite solutions such that the desired range of hypochlorite end concentrations (calculations see below). After an incubation period of 5 minutes, 1 ml of the reaction mixture was collected and added to 9 ml of cold BSA (1% w / v) solution and immediately placed on ice. This was done to stop the hypochlorite from reacting with the microorganisms. Survival as the number of colony forming units (CFU) per ml was determined by diluting the sample from 10 µL to 10 µL and loading 100 µL samples of various dilutions onto labeled BHI-agar plates. Plates were incubated for 15-18 hours at 30 ° C. If no CFU was detectable after this incubation period, the plates were incubated again for another 24 hours at 30 ° C.

The similarity of the hypochlorite profiles obtained in the above-described experiments with V-CPO and hypochlorite from the stock solution was confirmed using the same experimental setup (as for comparative experiments without added microorganisms) coupled to a spectrophotometer in which hypochlorite concentrations could be monitored at 290 nm. using the conversion of monochlorodisone (e (290 nm) = 20.2 mmol "1.1.cm" l) to dichlorodisone (e (290 nm) = 0.2 mmol "- * - 1. cm · · ·)

calculations:

The final concentration of hypochlorite generated by V-CPO was calculated as follows:

With 0.01 U per ml of V-CPO, 0.01 mmol per ml per minute of hypochlorite is formed, which is equivalent to 0.05 μιηοΐ hypochlorite per ml in 5 minutes due to the effect of dilution with a flow of 0.5 ml added to the initial volume of 2.0 ml, the final concentration after 5 minutes is (2.0 / 2.5) * 0.05 pmol per ml = 0.04 gmol hypochlorite per ml after 5 minutes. This concentration could then be expressed as 0.04 mmol of hypochlorite per ml = 0.04 * 52.5 ppm of hypochlorite = 2.10 ppm of hypochlorite. Other end concentrations of hypochlorite were obtained by increasing or decreasing the V-CPO concentration. The final concentration of hypochlorite added was calculated as follows:

Since a 0.5 ml flow is added to a reaction volume of 2.0 ml, the final volume is 2.5 ml, which corresponds to a five-fold dilution. To obtain a final concentration of 2.10 ppm, a stock solution of 10.50 ppm was used. Other end concentrations of hypochlorite were obtained by increasing or decreasing the concentration of the hypochlorite stock solution.

Definition: the minimum inhibitor concentration (MlC-value) is defined herein as the hypochlorite concentration, which under the experimental conditions used results in a log reduction of the colony forming units of the particular microorganism tested at least 6.

The results are shown in Table II. The lethal effect of various end concentrations of hypochlorite formed by either V-CPO or at the end of the hypochlorite flow was compared. As can be concluded from Table II, the hypochlorite formed enzymatically by V-CPO has always provided complete growth inhibition at concentrations of hypochlorite below 0.4 ppm for all test organisms except Staphylococcus aureus, where 2 ppm of hypochlorite is required to obtain the same growth inhibition. This shows that vanadium containing haloperoxidase is a more effective hygiene component than could only be expected from its hypochlorite-forming capacity. It is also evident that V-CPO provides the killing of all tested microorganisms, both pathogenic and non-pathogenic, whereas the hemoperoxide peroxidase is claimed to provide effective killing of only pathogenic bacteria (see EP-A-500 387).

Table II. MIC values for various microorganisms

microorganisms MIC values with hypochlorite (ppm) MIC values with V-CPO (ppm) Escherichia coli 2 0.4 Staphylococcus aureus 2 - 3 1.6 Listeria innocua 2 0.4 Pseudomonas aeruginosa 2 0.4 Streptococcus faecalis 2 0.4

Example 3

Lethal effects of hypochlorite and hypochlorite generated enzymatically by V-CPO in the presence of protein hydrolyzate

In obtaining hygiene, it is known that in situations that occur in practice, an overdose of hypochlorite is needed, since this reactive molecule will not only react with microorganisms, but also with other substances present. Therefore, it is important to test the behavior of vanadium-containing haloperoxidase in a fluid, such as a protein hydrolyzate.

Materials:

In this example, a strain of Escherichia coli NCTC 900 was used. The bacteria were cultured for 15-18 hours at 30 ° C in a Brain Heart Infusion (BHI) broth environment. After cultivation, the strains were washed twice with citrate buffer pH 5.5 (20 mmol / l, NajCitran-NaOH + 10 mmol / l NaCl). The bacterial suspensions were centrifuged in an Eppendorf centrifuge (14000 rpm for 5 minutes). Then, the supernatants were discarded and then the bacterial pellet was resuspended in citrate buffer. This washing procedure was then repeated with each bacterial suspension again. The twice washed bacterial suspension was then diluted with citrate solution at pH

Ο

5.5 to give a suspension of approximately 10 ° bacteria per ml. During the washing steps, cells were kept on ice. Buffers and BSA-solution (1% v / v in citrate buffer pH 5.5) were filter-sterilized and stored at 4 ^e C. The hypochlorite solutions were made from a stock solution (107,000 ppm) by dilution with sterile demineralised water. H 2 O 2 solutions were prepared from a 30% stock solution by dilution with sterile demineralized water. Casitone was purchased from Difco. Chloroperoxidase from Curvularia inaequalis was purified according to van Schijndel et al. (1993).

methodology:

Using a sterile technique, a suspension was prepared

Q approximately 10 ° bacteria per ml in citrate buffer pH 5.5. 1.3 ml aliquots of this suspension were added to sterile tubes and mixed continuously during the experiment. 0.5 ml of 0.5 mg / ml casitone (in citrate buffer pH 5.5) of the solution, respectively, was added. 0.5 ml citrate buffer pH 5.5. Subsequently, 0.2 ml of V-CPO solutions of various concentrations (calculations see Example 2) were added to the reaction vials in such a way that a final concentration of 3.2 ppm of hypochlorite or 6.5 ppm of hypochlorite was reached. A comparative sample was also prepared which contained 0.2 ml of sterile buffer instead of 0.2 ml of V-CPO solution. The reaction vials were incubated at 30 ° C. Then 0.5 ml Η ₂ 0 _{2 of the} solution was added (the concentration was chosen to give a five-fold molar excess of hydrogen peroxide to the end of the flow) to start the V-CPO reaction. The samples were incubated for exactly 5 minutes at 30 ° C. After this incubation period, 1 ml of the reaction mixture was withdrawn and added to 9 ml of cold BSA (1% v / v) solution and immediately placed on ice. This was done to stop the hypochlorite from reacting with the microorganisms. Survival as Colony Forming Units (CFU) per ml was determined by diluting the sample to 10 "L and 10" ⁶ _and plating 100 μΐ samples of the various dilutions on labeled BHI-agar plates. Plates were incubated for 15-18 hours at 30 ° C. If no CFU was detectable after this incubation period, the plates were incubated again for another hour at 30 ° C.

They obtained the lethal efficacy and compared to the lethal efficacy obtained when equal amounts of hypochlorite were added by a micropump. It was done as follows:

Using a sterile technique, a suspension of approximately 10 ⁸ bacteria per ml in citrate buffer pH 5.5 was prepared. From this suspension, 1.3 ml aliquots were added to sterile tubes, which were stirred continuously with a magnetic stirrer during the experiment. 0.5 ml of a 0.5 mg / ml casitone solution (in citrate buffer pH 5.5), respectively, was added. 0.5 ml citrate buffer pH 5.5. Subsequently, 0.2 ml of sterile citrate pH buffer was added to the reaction vials

5.5. The reaction vials were incubated at 30 C. C. Then a flow of 0.5 ml of hypochlorite was applied for 5 minutes, giving a final concentration of 3.2 ppm of hypochlorite, respectively. 6.5 ppm of hypochlorite (calculations see Example 2) (flow: 0.1 ml per minute) at 30 C. C. After an incubation period of 5 minutes, 1 ml of the reaction mixture was collected and added to 9 ml of cold BSA (1% w / v) solution and immediately placed on ice. This was done to stop the hypochlorite from reacting with the microorganisms. Survival as the number of colony forming units (CFU) per ml was determined by diluting the sample from 10 to 106 and loading 100 μΐ samples of various dilutions on labeled BHI-agar plates. Plates were incubated for 15-18 hours at 30 ° C. If no CFU was detectable after this incubation period, the plates were incubated again for another 24 hours at 30 C. The results are shown in Table III.

- 20 Table III

Effect of casitone solution on the lethal efficacy of HCl-generated V-CPO.

concentric log log log log tration reduction reduction reduction reduction casitone with 3.2 ppm with 3.2 ppm with 6.5 ppm with 6.5 ppm (Mg / ml) hypochlorite hypochlorite hypochlorite hypochlorite gen. V-CPO gen. V-CPO 0.0 8.0 8.2 8.0 8.2 (Total = (Total = (= Total (Total = slaughter) slaughter) slaughter) slaughter) 0.1 5.0 0.5 8.0 (Total = slaughter) 1.0

Example 4

Comparison between the lethal efficacy of vanadium halo peroxide oxidase (V-CPO) and haem-containing haloperoxidase

Materials:

In this example, strains of Escherichia coli NCTC 900, Streptococcus faecalis NCTC 1092 and Listeria innocua ATCC 33090 serotype 6B were used. The bacteria were cultured for 15-18 hours at 30 ° C in Brain Heart Infusion broth (BHI). After cultivation, the strains were washed twice with citrate buffer pH 5.5 (20 mmol / l, Na 2 Citran-NaOH + 10 mmol / l NaCl). The bacterial suspensions were centrifuged in an Eppendorf centrifuge (14000 rpm for 5 minutes). Then, the supernatants were discarded and then the bacterial pellet was resuspended in citrate buffer. This washing procedure was then repeated with each bacterial suspension again. The twice washed bacterial suspension was then diluted with citrate solution at pH 5.5 to obtain a suspension of approximately n bacteria per ml. During the washing steps, cells were kept on ice. Buffers and BSA-solution (1% w / v in citrate solution pH 5.5) were sterilized by filtration and stored at 4 ° C. The chloroperoxidase from Curvularia inaequalis was purified by van

- 2ί Schijndela et al. (1993). Chloroperoxidase activity in the conversion of Cl to HOCI was determined spectrophotometrically in 20 mM citrate buffer pH 5.5, 10 mM NaCl, 100 gmol / L

H 2 O 2, 50 mmol / l monochlorodisone at 30 ° C by monitoring the conversion of monochlorodisone (e (290 nm) = 20.2 mmol ..cm to dichlorodimedone (e (290 nm) = 0.2 mmol ^. 1.c.cm ^-1 ). A unit of chloroperoxidase is defined as the amount of enzyme that converts 1 gmol of monochlorodisone (Sigma) per minute The same assay (and activity definition) was used for both enzymes so that both enzymes could be dosed with the same activity.

methodology:

Using a sterile technique, a suspension of n

approximately 10 bacteria per ml in 20 mM citrate pH buffer

5.5, 10 mmol / l NaCl. 1.9 ml aliquots were added to the sterile tubes from this suspension. Subsequently, 0.1 ml stock solutions of vanadium-containing chloroperoxidase and hem-containing chloroperoxidase were added to the reaction vials in such a way as to obtain the desired dilution range for both enzymes. A comparative sample was also prepared which contained 0.2 ml of sterile buffer instead of 0.2 ml of enzyme stock solution. Then 0.5 ml of H 2 O 2 stock solution of 25 mmol / l was added. The samples were incubated for exactly 5 minutes at 30 ° C. After this incubation period, 1 ml of the reaction mixture was withdrawn and added to 9 ml of cold BSA (1% w / v) solution and immediately placed on ice. Survival as the number of colony forming units (CFU) per ml was determined by diluting the sample from 10 µL to 10 -5 _and loading 100 µL samples of various dilutions on labeled BHI-agar plates. Plates were incubated for 15-18 hours at 30 ^° C. If no CFU was detectable after this incubation period, the plates were incubated again for another 24 hours at 30 ° C.

The results are shown in Table IV. The data clearly show that vanadium-containing chloroperoxidase provides a far more effective hygiene system than hem-containing chloroperoxidase, even when dosed with the same activity.

Table IV

10 mmol / 1 Načl vanadium chloroperoxidase (U per ml) hemic chloroperoxidase (U per ml) log reduction E. coli 0,000 0.0 0.00156 4.0 0.0030 5.0 0.0060 7.0 * 0,025 7.0 * 0,012 7.0 * 0,050 7.0 * 0.10 7.0 * 0,000 0.0 0.00156 0.1 0.0030 0.1 0.0060 0.1 0,012 0.2 0,012 1.0 0,050 1.2 0.10 1.2 L. innocua 0,000 0.0 0.00156 7.0 * 0.0030 7.0 * 0.0060 7.0 * 0,025 7.0 * 0,012 7.0 * 0,050 7.0 * 0.10 7.0 * 0,000 0.0 0.00156 0.0 0.0030 0.0 0.0060 0.0 0,012 0.0 0,012 0.0 0,050 0.0 0.10 0.0

Table IV - continued

10 mmol / 1 Načl vanadium chloroperoxidase (U per ml) hemic chloroperoxidase (U per ml) log reduction S.faecalis 0,000 0.0 0.00156 5.0 0.0030 5.0 0.0060 7.0 * 0,025 7.0 * 0,012 7.0 * 0,050 7.0 * 0.10 7.0 * 0,000 0.0 0.00156 0.0 0.0030 0.1 0.0060 0.1 0,012 0.1 0,012 0.1 0,050 0.1 0.10 0.9

* = complete killing

Example 5

Effect of various sources of hydrogen peroxide on the inhibitory effects of V-CPO

In the previous examples, hydrogen peroxide, which is one of the substrates in the V-CPO reaction, was added from a stock solution. In this example, the effects of using other sources of hydrogen peroxide are described. Oxidases were tested at 30 ° C in a Biological Oxygen Monitor YSI Model 5300 (oxygen chamber model 5301), which monitors oxygen consumption, using citrate buffer at pH 5.5 (20 mmol / l, Na 2 Citran-NaOH + 10 mmol). NaCl). The buffer was saturated with air at 30 ° C. Glucose 15 gl ^- * (final concentration) was used as substrate for glucose oxidase. Escherichia coli NCTC 900 strains were used in this example. The bacteria were cultured for 15-18 hours at 30 ° C in a Brain Heart Infusion (BHI) broth environment. After

For 24 cultures, the strains were washed twice with citrate buffer pH 5.5 (20 mmol / l, Na-Citran-NaOH + 10 mmol / l NaCl). The bacterial suspensions were centrifuged in an Eppendorf centrifuge (14000 rpm for 5 minutes). Then, the supernatants were discarded and then the bacterial pellet was resuspended in citrate buffer. This washing procedure was then repeated with each bacterial suspension again. The twice washed bacterial suspension was then diluted with citrate solution at pH

Q

5.5 to give a suspension of approximately 10 bacteria per _Λ ml. During the washing steps, cells were kept on ice. Buffers and BSA-solution (1% w / v in citrate solution pH 5.5) were sterilized by filtration and stored at 4 ° C. The hypochlorite solutions were made from the stock solution (107000 ppm) by dilution with sterilized demineralized water. H 2 O 2 solutions were prepared from a 30% stock solution by dilution with sterile demineralized water. Casitone was purchased from Difco. The chloroperoxidase from Curvularia inaequalis was purified according to van Schijndel et al. (1993). Glucose oxidase from Aspergillus niger was purchased from Sigma.

methodology:

Using a sterile technique, a suspension was prepared

Q approximately 10 bacteria per ml in citrate buffer pH 5.5. From this suspension, 1.3 ml aliquots were added to sterile reaction vials, which were stirred continuously with a magnetic stirrer during the experiment. Then 0.5 ml of a 0.5 mg / ml casitone solution (in citrate buffer pH »5.5) was added. 0.2 ml was then added to the reaction vials

V-CPO solutions to give a final concentration of 6.5 ppm ppm of hypochlorite. Comparative samples were also prepared which contained 0.2 ml of sterile buffer instead of 0.2 ml of V-CPO. The reaction vials were incubated at 30 C. C. Then 0.5 ml of one of the following three hydrogen peroxide generating systems was added:

1. H2O2 from a 3 mmol / l stock solution;

2. Sodium percarbonate from a 3 mmol / l stock solution;

3. Mixture of glucose oxidase (0.39 units / ml) and 75 mg / ml glucose.

The samples were incubated for exactly 5 minutes at 30 ° C. After this incubation period, 1 ml of the reaction mixture was withdrawn and added to 9 ml of cold BSA (1% w / v) solution and immediately placed on ice. This was done to stop the hypochlorite from reacting with the microorganisms. Survival as the number of colony forming units (CFU) per ml was determined by diluting the sample from 10 µM to 10 -6 and loading 100 µL samples of various dilutions on labeled BHI-agar plates. Plates were incubated for 15-18 hours at 30 ° C. If no CFU was detectable after this incubation period, the plates were incubated again for another 24 hours at 30 C.

The MIC value obtained was compared to the MIC value obtained when equal amounts of hypochlorite were added by a micropump. It was done as follows:

Using a sterile technique, a suspension was prepared

About 10 bacteria per ml in citrate buffer pH 5.5. From this suspension, 1.3 ml aliquots were added to sterile tubes, which were stirred continuously with a magnetic stirrer during the experiment. Then 0.5 ml of casitone solution (0.5 mg / ml in citrate buffer pH 5.5) was added. Subsequently, 0.2 ml of sterile citrate buffer pH 5.5 was added to the reaction vials. The reaction vials were incubated at 30 ° C. Then a flow of 0.5 ml of a 32.5 ppm hypochlorite solution was applied for 5 minutes (flow rate: 0.1 ml per minute) at 30 ° C. After an incubation period of 5 minutes, 1 ml of the reaction mixture was collected and added to 9 ml of cold BSA (1% w / v) solution and immediately placed on ice. This was done to stop the hypochlorite from reacting with the microorganisms. Survival as the number of colony forming units (CFU) per ml was determined by diluting the sample from 10 “10 to 10 6 6 and loading 100 μΐ samples of various dilutions onto labeled BHI-agar plates. Plates were incubated for 15-18 hours at 30 ° C. If no CFU was detectable after this incubation period, the plates were incubated again for another 24 hours at 30 C. The results are shown in Table V.

Table V

V-CPO reaction with various sources of H ₂ 0 ₂

Source of hydrogen peroxide concentric tration casitone (Mg / ml) log reduction with 6.5 ppm of hypochlorite log reduction with 6.5 ppm of hypochlorite gen. V-CPO stock solution h ₂ o ₂ 0.0 8.2 (= Total slaughter) 8.0 (= Total slaughter) peroxouhli- carbonate 0.0 8.0 (= Total slaughter) 8.2 (= Total slaughter) oxidase glucose 0.0 6.4 (= Total slaughter) 6.4 (= Total slaughter) stock solution h ₂ o ₂ 0.1 1.0 8.0 (= Total slaughter) peroxouhli- carbonate 0.1 0.8 8.2 (= Total slaughter) oxidase glucose 0.1 1.0 6.4 (= Total slaughter)

Example 6

Method for determining the coding sequence of the chloroperoxidase gene (cDNA) and the gene from Curvularia inaequalis (Central Bureau voor

Schimmelcultures, Netherlands, strain no. 102.42) and possible expression systems.

Chloroperoxidase was isolated and purified from liquid cultures of C. inaequalis as described by Van Schijndel et al. (1993) except that two additional purification steps were performed after DEAE chromatography using the FPLC system (Pharmacia LKB). First, a phenyl-sepharose C1-4B hydrophobic interaction column was used to bind the enzyme in the presence of 2 mol / l

NaCl in 50 mmol / l Tris-HCl (pH 8.3), followed by a decreasing gradient from 2 M NaCl in 50 mmol / l Tris-HCl (pH 8.3). For final purification an MonoQ HR 5/5 anion exchanger column (ex Pharmacia LKB) was used to bind the enzyme, followed by elution with a gradient from 0 mol / l to 0.5 mol / l NaCl in 20 mmol / l piperazine-HCl (pH 5.4). Subsequent concentration of the enzyme was carried out by rotary evaporation, followed by dialysis into 50 mM Tris-SO ₄ buffer (pH 8). Purified chloroperoxidase was enzymatically digested with proteases Staphylococcus V8 and trypsin according to standard procedures known in the art, or chemically digested with CNBr (Gross, E. (1967), Methods Enzymology 11, 238-255). The resulting peptides were separated by SDS-PAGE according to Laemmli (Laemmli, UK (1970) Nature 277. 680-685) or on a Tricine gel according to Schagger and von Jagow (1987) and subsequently transferred to PVDF membranes (Immobilon-P ex Millipore). using CAPS transfer buffer (10 mmol / l 3- [cyclohexylamino] -1-propanesulfonic acid, 10% methanol, pH 11) as described by Matsudair (1987). After electrophoretic elution, the membrane was rinsed for 5 minutes with deionized water, stained with 0.1% Coomassie Blue R-250 in 50% methanol for 5 minutes and bleached with 50% methanol, 10% acetic acid for 10 minutes at room temperature and air dried. The peptide bonds were subjected to automatic Edmann sequencing on the Porton LF 3000 protein sequencer (Beckmann Instruments, Inc., USA). The results of the amino acid sequence determination are summarized in Figure 1.

Fully degenerate oligonucleotides were designed based on the amino acid sequences of the peptides (see Table VII). These degenerate primers were used in Polymerase Chain Reaction (PCR) reactions, using single-stranded cDNA as a template. A single stranded cDNA was prepared as follows:

To isolate RNA, C. inaequalis spores were inoculated into a ferraentation medium containing 4 g of yeast extract and 2 ml of microelement solution (Van Schijndel et al., 1993) per liter. After several days of growth, the mycelium was collected by filtration and lyophilized. The lyophilized C. inaequalis mycelium was ground in liquid nitrogen. RNA was extracted by adding RNA extraction buffer (42 mmol sodium citrate pH 7, 0.83% N-lauryl sarcosine, 50 mmol / l betamercaptoethanol, 1% Triton X-100 and 4 mol / l guanidine isothiocyanate), and incubating for 1 hour. hours at room temperature. 0.1 volume of 2M sodium acetate was added. (pH 4) and 1 volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) were placed on ice for 15 minutes. After centrifugation for 10 minutes at 10,000 xg (4 ° C), the aqueous phase was separated, 1 volume of absolute alcohol was added and the mixture was incubated for 1 hour at -20 ° C, followed by brief centrifugation at 10,000 x g. The pellet was resuspended in a suitable volume of RNA extraction buffer and fractionated by cesium chloride gradient ultracentrifugation (Sambrook et al., 1989). The pellet was carefully washed and stored in a 75% ethanol solution at 70 ° C. To isolate mRNA, RNA was precipitated and resuspended in RNAse-free water, after which the mRNA was extracted using the PolyAtract mRNA isolation kit (Promega Corporation, USA). Synthesis of single-stranded cDNA was performed on isolated mRNA from C. inaequalis using a Pharmacia single-stranded cDNA synthesis kit (Pharmacia Biotech). Based on the amino acid sequences of the chloroperoxidase peptides, four 20-mer degenerate oligonucleotides (see also Table VII) were designed and used as primers in polymerase chain reactions with single-stranded C. inaequalis cDNA as a template. PCR was performed using a thermocycler (Eppendorf Mastercycler 5330) and Taq polymerase (Promega ^r Cor poration). For optimal amplification of the chloroperoxidase encoding cDNA using degenerate primers, the polymerase chain reaction was performed at 46 ° C (amplification step) for 30 cycles. The two resulting specific fragments were ligated to the pUC18 vector, cloned and sequenced from both strands. The following two specific primers were designed based on DNA sequencing results:

CATAGCGATAGCGACGCGGA the 5 '-3'

CTAACCCCGGCGCCAACATC the 5 '-3'

These two primers were used as primers in polymerase chain reactions with single-stranded C. inaequalis cDNA as a template. This gave a gene-specific DNA fragment linking the two known DNA sequences. This fragment was cloned in the pUC18 vector and subsequently sequenced. The 5'-Amplifinder Raca kit (Clonetech Corporation) was used to obtain the 5 'region of the chloroperoxidase coding mRNA. The genomic chloroperoxidase gene from C. inaequalis was isolated as follows:

C. inaequalis genomic DNA was isolated from lyophilized mycelium, which was ground in liquid nitrogen and extracted with an appropriate amount of extraction buffer (200 mM Tris-HCl, pH 8.5, 25 mM EDTA, 250 mM NaCl, 1% SDS and 0.2 mg per ml proteinase K). After incubation overnight at room temperature, 0.7 volumes of phenol and 0.3 volumes of chloroform were added and mixed vigorously. The tubes were centrifuged at 10,000 x g and the aqueous layer was transferred to a clean tube. Genomic DNA was precipitated with 2 volumes of absolute ethanol. After centrifugation for 5 minutes at 5000 x g, the pellet was resuspended in 2 ml of 10 mM Tris-HCl, pH 8.0, 1 mM EDTA and treated with RNAse (Boehringer Mannheim) as recommended by the manufacturer. The solution containing genomic DNA was extracted with phenol: chloroform: isoamyl alcohol (25: 24: 1) and, after ethanol precipitation, was finally dissolved in a suitable volume of 10 mM Tris-HCl pH 8, 1 mM EDTA buffer. For Southern analysis of genomic DNA, the DNA was digested with several combinations of restriction enzymes and, after agarose gel electrophoresis, applied to a nitrocellulose membrane (Sambrook et al., 1989). Hybridization was performed using a radiolabeled gene specific fragment (amplified with two specific primers as described above), which was performed by random initiation with alpha-32P labeled dATP (Sambrook et al., 1989). Based on the results obtained, a mini library was made using genomic DNA cut with Pst I, which was inserted into the pUC18 vector. The library was screened with the same probe as described for the Southern test. Positive clones were isolated and also partially sequenced from both strands to confirm the cDNA sequencing results. Gén kó. The present C. inaequalis chloroperoxidase and its putative gene products are described in Figure 2.

* The chloroperoxidase production system in Saccharomyces cerevisiae was done as follows:

The well known GALI inducible yeast promoter was obtained as an EcoR1 BamH1 fragment from the 5th cerevisiae wild type GALI gene (Molecular and Cellular Biology 10: 4757-4769, 1990) and cloned at EcoRl BamH1 sites corresponding to plasmids YCplac33 and YEplac95 (Gietz and Sugino ( 1988, Gene 74, 527-534). The plasmids obtained were named TNT1 and TNT1 respectively. TNT2. The BamH1 restriction site was created opposite the 5 'origin of the C. inaequalis chloroperoxidase gene by performing a PCR experiment using the Pst EcoRl 5' fragment of the C. inaequalis chloroperoxidase gene subcloned on pUC18 as a template and as primers: primer M13 / pUC 22-mer reverse sequence and primer

5 ’GAG AGA GGA TCC ACT CAC TAC CAA TCA CAC 3’

The amplified fragment was digested with BamHI and EcoR1. The EcoR1 PvuII fragment from the C. inaequalis chloroperoxidase gene containing 3 'of the gene was subcloned into EcoRl SmaI digested with pUC18.

* A fragment containing part 3 'of the C. inaequalis chloroperoxidase gene was purified from this clone after digestion with EcoRl and XbaI.

Three-point binding was performed, which included either TNT1 or TNT2 digested with BamH1 and XbaI, and a 5 'BamH1 EcoR1 fragment and a 3' EcoR1 XbaI fragment. After binding and cloning, the plasmids obtained were checked for identity. The plasmids thus obtained were termed TNT3 (obtained from TNT1), respectively. TNT4 (obtained from TNT2).

Yeast strain BJ1991 was transformed with plasmids TNT3, respectively. TNT 4, according to procedures known in the art, and selected for ura + transformants. Ura + transformants were replicated on YP-plates containing either glucose (2% by weight) or galactose (2% by weight). After cultivation, part of the cells were removed from the plates and resuspended in 200 μΐ 20 mmol / L Tris-HCl pH 8.1. After incubation for 5 minutes, 10 μΐ was removed from the liquid and applied as a spot and nitrocellulose filter. The nitrocellulose filter was incubated in 100 mmol / l sodium acetate buffer (pH 5.5), 1 mmol / l ortodianisidine, 100 mmol / l KBr and 2 ml H ₂ O ₂ . Clear staining was observed on all spots obtained from yeast strains cultured on galactose, whereas no color formation was observed for yeast cultured on glucose. This indicates that a galactose-inducible production system for the Curvularia inaequalis chloroperoxidase gene on the yeast Saccharomyces cerevisiae was created. A similar assay employing a buffer of 100 mmol / 1 potassium phosphate (pH 6.5), 100 mmol / 1 potassium bromide, 1 mmol / 1 H ₂ 0 ₂ and 40 pmol / l phenol red (BDH), clearly showed a blue purple color formation with fluids from yeast cultured on galactose, while color changes were observed with yeast liquids cultured on glucose. To further confirm that a heterogeneous chloroperoxidase gene expression system, which constitutes a C. inaequalis chloroperoxidase gene with the same functionality as the C. inaequalis chloroperoxidase gene, was prepared in yeast, the recombinant enzyme was purified from TNT3 or TNT4 transformed yeast strains. After cultivation in medium containing galactose, the yeast cells were harvested and resuspended in 20 mM Tris-HCl buffer (pH 8.1). Sterile glass beads were then added and the suspension was shaken vigorously. After centrifugation for 15 minutes at 10,000 xg, the supernatant was collected and loaded onto a DEAE column and the recombinant enzyme purified using essentially the same purification procedure as for the wild-type C. inaequalis enzyme (see above). After purification, recombinant chloroperoxidase was obtained with a specific activity of 22 U per mg protein (determined in 100 mmol / l sodium acetate buffer pH 5.5, 1 mmol / l H ₂ O ₂ , 5 mmol / l potassium chloride and 50 gmol / l). MCD, see also van Schijndel et al., 1993), which corresponded well to a specific activity of approximately 20 U per g of protein obtained with purified (see above) chloroperoxidase from C. inaequalis alone. The pH profile of the activity of wild-type chloroperoxidase and yeast-derived recombinant chloroperoxidase is shown in Figure 3. Figure 3 further provides evidence that the recombinant yeast-produced enzyme has the same functionality as the wild-type enzyme.

Example 7

Search for suitable haloperoxidases in other microorganisms.

In this example, the microorganisms Curvularia inaequalis (CBS 102.42), Drechslera biseptata (CBS 371.72), Drechslera fugax (CBS 509.77), Drechslera nicotiae (CBS 655.74), Drechslera subpapendorfii (CBS 654.76iaisia, Embiisisisisisisisia, Embelisisisisisia, Embelisisisisia), CBS 766.79), and Ulocladium chartarum (200.67) and Ulocladium botrytis (452.72). Individual fungi were grown on agar plates. When growth was complete, extracellular proteins were transferred to a nitrocellulose filter (replication spots) that had been prewetted with 50 mM Tris-HCl buffer (pH 8.3). After 15 minutes incubation on agar plates, the filter was tested for haloperoxidase activity by soaking the filter in 100 mM sodium acetate buffer (pH 5.5) or potassium phosphate buffer (pH 6.5 and 7.5), 1 mM orthodianisidine, 2. mmol / l hydrogen peroxide in the presence and absence of 0.1 mol / l potassium bromide. Thus, production of bromoperoxidase and / or chloroperoxidase was detected. To test whether the haloperoxidase formed is vanadium containing peroxidase, the test described above was repeated in the presence and absence of 10 and 100 gmol / l sodium vanadate. In the case of vanadium-containing haloperoxidases, an increase in signal should be observed in vanadate delivery situations. To test whether the identified chloroperoxidases are indeed similar to vanadium-containing haloperoxidases from C. inaequalis, small amounts of chloroperoxidases were purified (essentially as described in van Schijndel et al., 1993) from Ulocladium. chartarum, Embelisia didymospora and Drechslera subpapendorfii. The optimum pH for these enzymes varied from 4.5 to 5.5. The chlorination activity of these enzymes increased after the addition of vanadate, which clearly shows that these enzymes are indeed vanadium haloperoxidases. To further test the similarity • to the enzymes identified as C. inaequalis vanadium chloroperoxidase, one of the identified halogen peroxoxidases was further characterized. Chloroperoxidase produced by the fungus Drechsler biseptata (CBS 371.72) was selected for this. It has properties similar to chloroperoxidase from Curvularia inaequalis, ie high thermal stability and high affinity to the respective substrates. The EPR spectrum of the purified enzyme was also recorded. As with other vanadium haloperoxidases (de Boer et al., 1988; Vever et al., 1988), the oxidized enzyme EPR is inactive; but after reduction with sodium bisulfite, a typical EPR spectrum of vanadyl is observed (data not shown). The isotropic parameters g _Q 1.969 and Aq 9.0 mT are almost the same as those for the enzyme from C. inaequalis (Vever et al., 1985). Further, the purified enzyme was separated into peptides by proteases and cyanobromide. The peptide map shows the same image, confirming that the two enzymes have large sequence homology. This is the case where two separate enzyme peptides, which were obtained by protease treatment and which were purified, were sequenced. The sequences show very large homology and therefore can be closed, these two enzymes are very similar.

&

•

Amino acid sequence of C.inaequalis peptide:

(asp) -leu-arg-gln-pro-tyr-asp-pro-thr-ala-pro-ile-glu-asp-gln-pro-gly-ile-val-arg-thrThe amino acid sequence of a similar peptide from D. biseptata : (asp) -leu-arg-gln-pro-tyr-asp-pro-thr-ala-pro-ile-glu-glu-gln-pro-gly-ile-val-arg-thr-34 Suitable vanadium containing haloperoxidases thus, they can be identified using a replication technique in which an increase in activity upon addition of vanadate is tested and / or (partially) by purification of the enzyme and looking for an increase in activity after addition of vanadate.

Example 8 »Further search for suitable chloroperoxidases in other microorganisms using antibodies.

r

In this example, strains were used: Curvularia inaequalis (CBS 102.42), Drechslera biseptata (CBS 371.72), Drechslera subpapendorfii (CBS 654.74), Embelisia didymospora (766.79), and Uococladium chartarum (200.67).

The microorganisms were cultured in two phases. First, 50 ml of sterile germination medium (as described in Van Schijndel et al., 1993) was inoculated with a spore mass of microorganisms. The culture was shaken for 3 days at 23 ° C, after which it was transferred to a 3 liter Erlenmeyer flask containing 1 liter fermentation broth (5 g casein hydrolyzate (Gibco BRL), 3 g yeast extract and 1 g fructose per liter deionized water). The medium that was shaken at 23 ° C was collected after 14 to 17 days, filtered and the chloroperoxidases were purified essentially according to van Schijndel et al. (1994). Polyclonal antibodies (using Freund's complete adjuvant in the first injection and Freund's incomplete adjuvant in the booster injection) against purified (according to van Schijndel et al., ® 1994) chloroperoxidase from Curvularia inaequalis in a rabbit (2 month old female) were raised. Rabbits were bled 6 days after the last booster injection. The serum was heated for 30 minutes at 56 ° C to inactivate the additional ingredients and then centrifuged. The supernatant was collected. A series of dilutions of each purified chloroperoxidase as well as bromoperoxidase from Ascophylum nodosum (purified according to Vever et al., 1985), starting with 50 μΐ proteins of approximately 0.1 mg per ml, are made, and the following sample is diluted twice. Diluted samples were spotted onto a nitrocellulose filter, washed with 2% BSA and incubated sequentially with 1: 800 diluted rabbit anti-chloroperoxidase antiserum, biocin goat anti-rabbit serum (1: 3000 dilution), alkaline phosphatase conjugate (streptavidin conjugate-conjugate) (1: 2000 dilution) and staining reagent (5-bromo-4-chloro-3-indolylphosphate, 4-nitroblue-tetrazolium chloride (Boehringer Mannheim)) All steps were carried out according to standard procedures. listed in Table VI.

Table VI

haloperoxidase from cross-reactivity with polyclonal antibodies raised against C. inaequalis chloroperoxidase C. inaequalis Yes D. biseptata Yes D. subpapendorfii Yes E. didymospora Yes U. chartarum Yes bromoperoxidase from A. nodosum not

Based on the results presented in Table VI, it can be concluded that an immunoassay with antibodies raised against C. inaequalis chloroperoxidase is suitable for identifying other suitable vanadium-containing haloperoxidases. These haloperoxidases can be either crude, partially or totally purified. Purification techniques can be any known technique such as gel filtration, ion exchange chromatography, hydrophobic interaction chromatography, precipitation techniques, (ultra) filtration techniques, affinity chromatography, gel electrophoresis, and others.

Example 9

Further search for suitable chloroperoxidases in other microorganisms.

In this example, it is described how a radioactive probe derived from the chloroperoxidase gene from Curvularia inaequalis can be used to detect homologous genes in other microorganisms. It was done as follows:

r Chromosomal DNA from Curvularia inaequalis (CBS 102.42), »Embelisia didymospora (200.67), Drechslera biseptata (CBS)

371.72), were purified essentially as described for chromosomal DNA from Curvularia inaequalis (as described in Example 6). For Southern analysis of genomic DNA, the DNA was digested with several combinations of restriction enzymes and after electrophoresis on an agarose gel it was loaded onto a nitrocellulose membrane (Sambrook et al., 1989). Hybridization was performed using a radiolabeled gene-specific fragment, which was performed by random initiation with alpha-32p labeled dATP (Sambrook et al., 1989). The gene-specific fragment used was amplified (before radiolabeling) in the polymerase chain reaction, using a single-stranded cDNA (see Example 6) as a template, and using the following primers:

5’-CACGATGGGTCCCGTTACAC a

β 5’-GTACCGCTATCGCTGCGCCTG.

The hybridization conditions were as follows:

In the prehybridization and hybridization, 6 * buffer was used

SSPE, 5 * Denhardts, 0.5% SDS and 10 mg salmon sperm DNA. Prehybridization was performed for 1 hour at 55 ° C. The radioactive sample was boiled for 1 minute and then added directly. Subsequently, overnight hybridization continued. The autoradiogram obtained with DNA from Curvularia ina-37 equalis and Drechsler biseptata is shown in Figure 4. In this Figure, lane 1 is lambda DNA; lane 2: uncleaved C. inaequalis genomic DNA; lane 3: as above, digested with EcoRI; lane 4: digested with BamHI; lane 5: digested with EcoRI and BamHI; lane 6: cleaved with XbaI; lane 7: PstI; lane 8: XbaI and PstI; lanes 9-14: as before using D. biseptata. As shown in the figure, a positive signal was obtained with chromosomal DNA from Drechsler biseptata, indicating a high degree of similarity between the two genes. Similar results were obtained with DNA from Embelisia didymospora. Therefore, it can be concluded that the chloroperoxidase gene, or a portion derived therefrom, or a probe based on the C. inaequalis chloroperoxidase gene sequence can be used to detect suitable vanadium haloperoxidases from other microorganisms.

Table VII

Oligonucleotide primers (20-mer) based on the amino acid sequence of vanadium chloroperoxidase ex Curvularia inaequalis

I: inosine

A / G: A and G mixture 1: 1 C / T was used at this position: C and T 1: 1 mixture G / A / T / C was used at this position: G, A, C and T 1: 1: 1: 1.

Oligo 1:

5 '-T A C / T A T G A A / G C I G T I G A / G C A - 3'

Oligo 2:

5’- A G / A T / C T G I G C G / A T A I G C G / A T T G / A T C - 3 ’ oligo 3: 5’- G C / T G A A / G A C I G C I G A A / G T A C / T G A - 3 ’ oligo 4: 5’- A G / A I G C T / C T G I G C I C C G / A / T / C C C C A T - 3 ’

Z> -9c

Claims (13)

PATENT CLAIMS An enzymatic antimicrobial composition comprising vanadium haloperoxidase, a halide source and hydrogen peroxide or a source of hydrogen peroxide. e Enzymatic antimicrobial composition according to claim 1, characterized in that the vanadium haloperoxidase is chloroperoxidase. Enzymatic antimicrobial composition according to any one of the preceding claims, characterized in that the vanadium haloperoxidase is a chloroperoxidase obtainable from Curvularia inaequalis. Enzymatic antimicrobial composition according to any one of the preceding claims, characterized in that the vanadium haloperoxidase is chloroperoxidase, which is immunologically cross-reactive with chloroperoxidase from Curvularia inaequalis CBS 102.42. Enzymatic antimicrobial composition according to any one of the preceding claims, characterized in that the source of hydrogen peroxide is an enzyme system generating hydrogen peroxide. The enzymatic antimicrobial composition of claim 5, wherein the hydrogen peroxide generating enzyme system is a glucose / glucose oxidase system or a lactate / lactate oxidase system. Enzymatic antimicrobial composition according to any one of the preceding claims, characterized in that the composition is substantially free of catalase activity. 8. A method of inhibiting microbial growth, comprising applying the composition of any of the preceding claims to a surface to be disinfected. 9. Use of vanadium haloperoxidases as sanitizing reagents. 10. DNA sequence comprising a structural gene encoding a vanadium chloroperoxidase from Curvularia Inequalis CBS 102.42. 11. A DNA sequence comprising the vanadium chloroperoxidase structural gene from Curvularia Inequalis CBS 102.42 as shown in FIG. Second An expression vector comprising the origin of replication, transcription and termination sequences and at least a portion of the DNA sequence of claims 10 or 11. A method for preparing a vanadium haloperoxidase by converting a suitable host using the expression vector of claim 12, wherein the host is cultured under conditions that permit expression of a structural gene and isolation of the vanadium haloperoxidase.