CZ288041B6 - Enzymatic antimicrobial preparation - Google Patents

Abstract

In the present invention there is disclosed an enzymatic antimicrobial preparation containing vanadium halogen peroxidase, a halide source and hydrogen peroxide or a hydrogen peroxide source. A preferred enzyme is chloroperoxidase of Curvularia inaequalis.

Description

Enzymatic antimicrobial agent

Technical field

The invention relates to an enzymatic antimicrobial composition comprising a vanadium haloperoxidase, a hydrogen peroxide source and a halide source. The enzyme can be obtained by genetic engineering.

BACKGROUND OF THE INVENTION

A variety of enzymatic antimicrobial agents are known. For example, WO-A-94/04217 discloses a stabilized dentifrice composition which provides an antimicrobially effective concentration of hypothiocyanite ions, OSCN. The composition comprises an oxidoreductase to obtain hydrogen peroxide and a peroxidase capable of oxidizing the thiocyanate ions commonly present in the saliva of the above-mentioned antimicrobial ions. Suitable peroxidases for this use are lactoperoxidase, myeloperoxidase, saliva peroxidase and chloroperoxidase.

Enzymatic antimicrobial agents containing haloperoxidase are also described in EP-A-500 387 (Exoxemis). Halogen peroxidases have been reported to be capable of selectively binding and to inhibit growth of target microorganisms in the presence of peroxide and halide. In EP-A-500 387, myeloperoxidase MPO, eosinophil oxidase, EPO, lactoperoxidase, LPO, and chloroperoxidase, CPH are mentioned as suitable haloperoxidase. The ratio of halide to hydrogen peroxide is said to be a critical factor for the stability and functionality of the haloperoxidase. At very low ratios, hydrogen peroxide may inhibit the function of the enzyme, while at very high ratios the halide may block the enzymatic reaction. Said ratio may vary within wide limits, but is preferably greater than 50.

Since hydrogen peroxide can cause undesirable side reactions, its concentration in the antimicrobial should be kept as low as possible. However, it has now been found desirable to use higher starting concentrations of hydrogen peroxide in the composition when combined with an appropriate amount of halide.

Further, it would be desirable to have enzymatic antimicrobial compositions with a different spectrum of activity than the known compositions. In particular, compositions effective against microorganisms whose growth is difficult to inhibit, such as Streptococcus faecalis, should be provided. In some cases, it may be desirable to suppress non-pathogenic organisms as well, as they may degrade food.

SUMMARY OF THE INVENTION It is an object of the present invention to provide an enzymatic antimicrobial composition devoid of the above disadvantages.

A number of haloperoxidases suitable for use in antimicrobial agents have been described in the literature, but so far no attention has been paid to the group of enzymes referred to as vanadium haloperoxidases.

Said enzymes differ from other haloperoxidases in that their prosthetic group has properties similar to vanadate (pentavalent vanadium), while the other haloperoxidases are haem peroxidase.

It is also an object of the present invention to provide a method of cloning a gene for said peroxidases and to determine the sequence of the gene so that it can be expressed in microorganisms that are easy to grow and thus obtain large amounts of enzymes using recombinant DNA.

-1 CZ 288041 B6

Some enzymes of this type can be found naturally on the surface of some seaweed (Wever et al., 1991). In intact seaweed in seawater, said enzyme may release HOBr upon addition of hydrogen peroxide. The role of this enzyme has not been elucidated yet, but it is likely that the formation of highly oxidizing HOBr in seawater is part of the defense system of these plants against attack by microorganisms or fungi.

After the discovery of vanadium bromoperoxidase from the seaweed Ascophyllum nodosum, different from conventional peroxidases, it has been shown that a large number of other algae also contain these enzymes. In particular, A. nodosum bromoperoxidase has been studied and its properties have been determined in detail, which have been described in more detail in particular in references 2 and 3 below. The prosthetic group of these enzymes has a vanadate-like structure. In the catalytic mechanism derived from this enzyme, hydrogen peroxide reacts with the enzyme to form a hydrogen peroxide-enzyme complex and then reacts the bromide and proton with the complex to form an enzyme-HOBr complex. It has been shown (DeBoer et al., 1988) that this complex decomposes to form the enzyme and HOBr. The same publication demonstrates that said enzymes are very stable in both aqueous and organic media. For example, the enzymes were stable for 3 weeks and could be stored for more than a month in organic solvents such as acetone, methanol, ethanol (up to 60% by volume) and 1-propanol without loss of potency. However, these enzymes have the disadvantage of requiring the presence or addition of bromide and, moreover, attempts to clone these marine algae genes and determine their amino acid sequence have not been successful.

Known existing chloroperoxidases containing heme from Caldariomyces fumago are less suitable for the preparation of enzymatic antimicrobial agents due to their instability and low optimal pH of 2.75 (J. R. Kanofsky, 1984), which severely limits their use. Similar reasons prevent the use of the enzyme myeloperoxidase MPO from human white blood cells, this peroxidase may also form HOC1 (A.R.J. Bakkenist et al., 1980).

References 8 and 9 below disclose reports of halper peroxidases with high stability from fungal hyphomyctes. In particular, it is a fungus Curvularia inaequalis which secretes vanadium chloroperoxidase (J.W.P.M. Van Schjindel et al., 1993), which has a high affinity for chlorides and its optimum pH for the chlorination reaction is about 5.5. As with seaweed bromoperoxidase, the prosthetic group of these enzymes has structural properties similar to vanadate, i.e., pentavalent vanadium. In further detailed studies by the same authors, 1994, it was shown that the kinetics of these enzymes in the oxidation of chlorides by hydrogen peroxide is similar to the kinetics of vanadium bromoperoxidases from the A. nodosum marine series. In addition, it has been shown by three different procedures that the enzyme produces an oxidized HOC1 group as a reaction product and is itself resistant to the action of this product. The enzyme is stable up to 90 ° C and is also very stable in organic solvents, for example 40% methanol, ethanol and propanol.

SUMMARY OF THE INVENTION

The present invention provides an enzymatic antimicrobial composition comprising a vanadium haloperoxidase, a halide source and hydrogen peroxide or a source of hydrogen peroxide.

The invention also relates to a process for the production of vanadium haloperoxidase by transforming a suitable host with an expression vector comprising an origin of replication, transcriptional and transcriptional termination control chains, and at least a portion of the vanadium haloperoxidase DNA coding strand, the transformed host being grown under conditions where to express the structural gene and the resulting enzyme is isolated.

The individual components of the antimicrobial composition of the present invention will be described in more detail below.

-2GB 288041 B6

(a) Vanadium haloperoxidase

The enzyme antimicrobial composition contains as its essential component the enzyme vanadium haloperoxidase, which may be selected from various known enzymes of this type. For example, a heme-free haloperoxidase produced by Curvularia inaequalis according to US-A-4,707,466 can be used. It is also possible to isolate and purify chloroperoxidase from Curvularia inaequalis, CBS 102.42 according to the method of J.W.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 subpapendorfn, 656.74, Embelisia hyacinthi, 416.71, Embarisia, 459, Embarisia, 45, Embarisia, .

Vanadium haloperoxidase can also be obtained by genetic engineering in the manner described above, which will be described in more detail in the Examples section of the application.

b) Halide ion source

A second component of the composition of the invention is a source of halide ions. These may be any halide ions, but the source of iodide or chloride ions is particularly preferred, since these ions are most effective. Sodium chloride is the most preferred source of halide ions. The halide may be added to the composition of the invention or a halide conventionally present in tap water in an amount of usually 2 to 5 mM may be used.

(c) Source of hydrogen peroxide

The antimicrobial compositions of the invention further comprise a hydrogen peroxide source or hydrogen peroxide generating system. Examples of such a system are perborates or percarbonates, for example sodium percarbonate or sodium perborate.

Hydrogen peroxide may also be formed by the systems producing this substance. This system can be selected from the various systems already described. For example, amino oxidase and amine, amino acid oxidase and amino acid, lactate oxidase and lactate, cholesterol oxidase and cholesterol, uric acid oxidase and uric acid or xanthine oxidase and xanthine may be used. Particularly preferred is a combination of glucose oxidase and glucose.

The amount of enzyme, for example glucose oxidase, will depend on the specific activity of the enzyme and the activity of any remaining catalase that may be present; generally, the composition of the invention will contain 10 to 1000, preferably 20 to 500 glucose oxidase units per g or ml of a composition, wherein the enzyme activity unit is defined as that amount of enzyme required to convert 1 micromole of substrate per minute under standard conditions.

d) Other components

The enzymatic antimicrobial composition of the invention may generally contain 0.01 to 50, preferably 0.1 to 5.0% by weight of at least one wetting agent. A suitable wetting agent is, for example, a soap or other anionic, nonionic, cationic, amphoteric or zwitterionic ionic compounds. The surfactant system typically comprises at least one anionic surfactant and at least one nonionic surfactant. In addition, the system may contain amphoteric or zwitterionic substances, but this is not common due to the relatively high cost of these substances.

Typically, nonionic and anionic surfactants may be selected from Surface Active Agents, Vol. 1, Schwartz and Foams, Interscience 1949, Vol. 2, Schwartz, Perry and Berch, Interscience 1958, in the current edition of McCutcheon's Emulsifiers and Detergents, Manufac

-3EN 288041 B6 turing Confectioners Company or Tenside-Taschenbuch, H. Stache, 2nd edition, Carl Hauser

Verlag, 1981.

Suitable nonionic compounds which can be used for this purpose are, for example, the reaction products of compounds having a hydrophobic group and a reactive hydrogen atom, such as aliphatic alcohols, acids, amides or alkylphenols with alkylene oxides, especially ethylene oxide per se or mixed with propylene oxide. Specific nonionic surfactants are condensates of alkylphenols having an alkyl moiety of 6 to 22 carbon atoms with ethylene oxide, usually containing from 2 to 25 units of ethylene oxide per molecule, and condensation products of straight or branched aliphatic alcohols of 8 to 18 carbon atoms with ethylene oxide, usually 5 to 40 units of ethylene oxide (EO) per molecule.

Suitable anionic surfactants which can be used for this purpose are the water-soluble alkali metal salts of organic sulfates and sulfonates having alkyl radicals of 8 to 22 carbon atoms, the alkyl moiety being the alkyl portion of the higher acyl radicals. Examples of suitable synthetic anionic surfactants are sodium or potassium alkyl sulphate, in particular the products of sulphation of higher alcohols having from 8 to 18 carbon atoms, obtained, for example, from tallow or coconut oil, sodium or potassium alkylbenzene sulphonates having an alkyl moiety of 9 to 20 atoms. The fatty alcohols, especially the linear secondary alkylbenzene sulphonates of 10 to 15 carbon atoms and the sodium alkyl glyceryl ether sulphates, in particular the higher alcohol ethers, derived from tallow or coconut oil, are also suitable synthetic alcohols derived from naphtha. Preferred anionic surfactants are sodium alkylbenzene sulfonates of 11 to 15 carbon atoms in the alkyl moiety and sodium alkyl sulfates of 12 to 18 carbon atoms in the alkyl moiety.

Wetting agents described in EP-A-328 177 (Unilever) which are resistant to salts and the alkylpolyglycoside surfactants described in EP-A-070 074 as well as alkyl monoglycosides can also be used.

Preferred surfactant systems are mixtures of anionic and nonionic surfactants, especially the groups described in EP-A-346 995 (Unilever). Particularly preferred is a wetting system consisting of a mixture of alkali metal salts of primary alcohol sulfates of 16 to 18 carbon atoms and ethoxylates of primary alcohols of 12 to 15 carbon atoms of 3 to 7 EO.

The nonionic surfactant is preferably present in an amount of greater than 10, for example 25 to 90% by weight based on the weight of the surfactant system. For example, anionic surfactants may be present in an amount of 5 to 40% by weight based on the weight of the surfactant system.

The enzymatic composition of the invention may contain still other components usually present in antimicrobial agents, for example thickeners. Particularly suitable in this regard are the surfactant combinations disclosed in EP-A-314 232 (Unilever) which form thickening gel-like substances upon dissolution in water.

The antimicrobial compositions of the present invention can be used to clean hard surfaces and wash fabrics, but also for industrial use, in hospitals, and to clean and disinfect medical instruments. Another possible field of application is dairy. The antimicrobial agents can also be successfully used as deodorants in order to suppress the growth of odorous bacteria.

The antimicrobial compositions of the invention may take the form of powders intended to be dissolved in water prior to use, but may also be directly formulated into liquid products or gels. In these products, it is important that hypohalite formation does not occur earlier than before use of the composition. This can be achieved by physically separating the enzyme from its substrate, for example by encapsulating the enzyme in a known manner.

-4GB 288041 B6

In use, the composition of the invention is diluted 5-10x with water to form an antimicrobial agent. The diluted composition is then contacted with the surface to be disinfected.

List of literary data

1. R. Wever, M.G.M. Hiker. B.E. Xrenn A.Jarjani and She. van Tol. Brominating activity of Ascochvllum nodosum: Impact on the biosphere. Envir. Sc. Techn. 25 (1991) 446-449.

2. R. Wever and K. Kustin. Vanadium: a biologically relevant element. Adv. Zncrg. Chem. 35 (1990) 81-115.

D. Render. The bioinorganic chemiscry of vanadium.

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

4. Ξ. de Baer and R. Wever - The reacticr. meohanism of the novel vanadium bromoperoxidase, and staady-scata kinetic analysis. J. Biol. Chem. 263 (1988), 12325-12332.

5. S. de Boer, H. Plac, M.G.M. Tromp, M.C.R. Franssen, H.C. var. der Plas, Ξ.Μ. Meijer and Κ.Ξ. Scčoemaker. Vanadium-containing bromoperoxidase: an example of an oxidoreducase with high operational stability in aaueous and organic media. Biotechn 3ioeng. 30 (1987) 607-610.

6. J.R. Kanofsky. Singlet oxygen produced by chloroperoxidase-hydrogen peroxide-syscams. J. Biol. Chem. 259 (1984) 5596-5600.

7. A.R.J. Bakkenisc, J.2.G. de Boer, H. Plat, and R. Wever. The halide complexas cf myeloperoxidase and the meohanism of the halcgenation raaczicns. Biochim. Biophvs. Aera, 613 (1980), 337-348.

and J.C. Huntar-Cavera and L. Satos. Scraening for a new enzyme in nature: hailopercxidase prcducticn by Deach Valley demathiaceous hyphomycetes. Microb. Scol. 12 (1986) 121-127.

9. Τ-Ν.Ξ. Liu, T. M'Timkulu, J. Geigert, B.Wolf, S.L. Neidleman, □. Silva and J.C. Huntar-Cavera. Isolation and characterization of novel non-heme chloroperoxidase. Biochem. Biopnys. Res. Commun. 142 (1987) 329-333.

10. J.W.P.M. van Schijndel, E.G.M. VcLLenbroek and R. Wever. Curvularia inaecrualis; and a novel vanadium enzyme. Biochim. Biopnys. Acza 1151 (1993) 249-256.

-5GB 288041 B6

11. Van Schijndel, Barnen - 1, Rcelse, J.,

VoLlanfaraek, E.G.M. and Wever, R. < tb > Vanadium chlorcceroxidase front of the fungus " Curvularia inaecrualis " stability and staady-state kinetics (1994) Eur. J. Biachem. 225, 151-157.

12. H. Schagger and G. Von Jagow (1937) Anal.Bicchem. 156: 363-379.

12.?. Matsudaira (1937) J.Bial.Cham. 262, 10035-10033.

14. Sambrook, J., Fritch, E.F. and Maniatis, T. (1989)

Molacular Cloning: a Laboratory Manual, 2nd edh., Cold

Spring Harbor Laboratory, Cold Sering Barier, MY.

15. R. Wever, H. Plat and De-Boer (1985), 3.cem.

Biconys. Acta 830, 131-136.

The following examples are intended to illustrate the invention.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Minimum inhibitory concentration of MIC hypochlorite

Materials:

The bacterial strains used in this example were Escherichia coli NCTC 900, Pseudomonas aeruginosa ATCC 15442, Staphylococcus aureus ATCC 13565, Streptococcus faecalis NCTC 1092, and Listeria innocua ATCC 33090, serotype 6B. Bacteria were grown for 15-18 hours at 30 ° C in a broth with brain and heart broth, BHI. After this time, the bacteria were washed twice with citrate buffer pH 5.5 (20 mM sodium citrate and NaOH + 10 mM NaCl). The bacterial suspension was then centrifuged in an Eppendorf centrifuge for 5 minutes at 14,000 rpm, the supernatants were removed, and the bacterial pellet was resuspended in citrate buffer. This wash is repeated once again for each bacterial suspension. The twice washed bacterial suspension is then diluted with citrate buffer pH 5.5 to a suspension containing approximately 10 ⁷ bacteria / ml. During washing, the cell material is stored on ice. Buffers and BSA solution (1% citrate buffer pH 5.5) are filter sterilized and stored at 4 ° C. Hypochlorite solutions are prepared from a stock solution of 107,000 ppm by dilution with sterile demineralized water.

Method:

Under sterile conditions, a suspension is formed containing approximately 10 ⁷ bacteria / ml citrate buffer pH 5.5. From this suspension 1.9 ml aliquots were added to sterile tubes. 0.1 ml of cold hypochlorite solution of varying concentration is then added to the tubes to form a dilution series of hypochlorite. The contents of the tubes are constantly stirred with a magnetic stirrer. Controls to which only sterile buffer is added instead of hypochlorite solution are also used. The samples are incubated for exactly 5 minutes at 30 ° C, then 1 ml of the reaction mixture is removed, added to 9 ml of cold 1% BSA solution and immediately placed on ice to stop the hypochlorite reaction with the micro-organism.

-6GB 288041 B6 nisms. Then, survival of bacteria is determined so as to determine the remaining number of colony forming units, CFU / ml by diluting the sample in the range 10 ^-1 to 10 ^-5 and plating 100 l samples of the various dilutions on labeled BHI-agar plates. The plates are incubated at 30 ° C for 15-18 hours.

If no CFU is detected after this time, the plates are incubated for 24 hours at 0 ° C.

The minimum inhibitory concentration, the MIC value, is defined as that concentration of hypochlorite which, under given conditions, results in at least a log 6-reduction of colony-forming units in the case of the microorganism under investigation.

The results obtained are summarized in the following Table I. The values obtained corresponded to those reported in the literature.

Table I

MIC values for various microorganisms

Microorganism _____________________________MIC in ppm

Escherichia coli2-3

Staphylococcus aureus2-3

Listeria innocua2-3

Pseudomonas aeruginosa2-3

Streptococcus faecalis 2-3

Example 2

Minimum inhibitory concentration of hypochlorite as compared to hypochlorite produced enzymatically by vanadium chloroperoxidase V-CPO from Curvularia inaequalis.

This example compares the effect of hypochlorite with the effect of hypochlorite produced enzymatically by vanadium chloroperoxidase V-CPO from Curvularia inaequalis. A micro pump was used to make the comparison. Indeed, it was necessary to ensure the same concentration profile during the experiment for the hypochlorite produced by the enzyme. The efficacy of V-CPO was determined very carefully during the experiment to know the amount of hypochlorite at each time of the experiment.

Materials:

In this example, bacterial strains of Escherichia coli NCTC 900, Pseudomonas aeruginosa ATGG 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 a broth containing BHI broth and heart. After this time, the bacterial strains were washed twice with citrate buffer pH 5.5 (20 mM sodium citrate and NaOH + 10 mM NaCl). The bacterial suspension was then centrifuged for 5 minutes in an Eppendorf centrifuge at 14,000 rpm, the supernatant was removed, and the bacterial pellet was resuspended in citrate buffer. This wash was repeated once more. The washed bacterial suspension was then diluted with citrate buffer pH 5.5 to a suspension containing approximately 10 ⁷ bacteria / ml. In these steps, the cell material was stored on ice. Buffers and 1% BSA in citrate buffer pH 5.5 were filter sterilized and stored at 4 ° C. Hypochlorite solutions were prepared from a stock solution containing 107,000 ppm by dilution with sterile demineralized water.

Hydrogen peroxide solutions were prepared from a 30% stock solution by dilution with sterile demineralized water. Casitone was obtained from Difco. Curvularia inaequalis chloroperoxidase was purified according to Schijndel et al., 1993. The efficacy of chloroperoxidase in converting chlorine to HOC1 was determined spectrophotometrically in 20 mM sodium citrate buffer pH5.5 containing 10 mM NaCl, 100 microM hydrogen peroxide and 50 microM monochlordimedone at 30 ° C, follows the conversion of monochlorodimedone (e 290nm = 20.2 mM ^-1. cm ^-1) to dichlorodimedone (e 290 nm = 0.2 mM ^-1. cm ^-1). One unit of chloroperoxidase is the amount of enzyme that converts one micromole of monochlorodisone (Sigma) per minute.

Method:

Using sterile conditions, a suspension of approximately 10 ⁷ bacteria / ml citrate buffer pH 5.5 is formed. From this suspension, 1.8 ml aliquots are added to a sterile reaction vessel in which the contents are stirred under sterile conditions with a magnetic stirrer throughout the experiment. 0.2 ml of a V-CPO solution of varying concentration (calculated below) is then added to form a dilution series. In addition, a control assay was included to which 0.2 ml of sterile buffer was added instead of 0.2 ml of V-CPO solution. The samples were incubated at 30 ° C and then 0.5 ml of hydrogen peroxide stock solution was added to initiate the reaction. The hydrogen peroxide concentration used depended on the hypochlorite concentration and was chosen such that the final hydrogen peroxide concentration was five times the molar excess, based on the final hypochlorite concentration. The samples were incubated for exactly 5 minutes at 30 ° C. After this incubation period, 1 ml of the reaction mixture was collected and added to 9 ml of cold 1% BSA solution, the sample was immediately placed on ice to stop the hypochlorite reaction with the microorganism. The survival of the microorganisms, expressed as the number of colony forming units, CFU / ml was determined by diluting the samples between 10 * ¹ and 10 * ⁵ and sample 100 microliters of various dilutions on the labeled BHI-agar plates. The plates were then incubated at 30 ° C for 15-18 hours. If CFU was not detectable after this time, the plates were incubated for 24 hours at 30 ° C.

MIC values were compared to those obtained by adding the same amounts of hypochlorite using a micro pump. The procedure was performed as follows:

Under sterile conditions, a suspension of approximately 10 ⁷ bacteria / ml opH5.5 citrate buffer was prepared. From this suspension, 1.8 ml aliquots were placed in a sterile reaction vessel which was stirred under sterile conditions with a magnetic stirrer during the experiment. The mixture is incubated at 30 ° C and 0.2 ml of hydrogen peroxide stock solution is added, the concentration of which is selected depending on the concentration of hypochlorite, so that the final concentration of hydrogen peroxide is five times the molar amount of hypochlorite. Then, 0.5 ml of a hypochlorite solution of known concentration is added over a period of 5 minutes at a rate of 0.1 ml / min at 30 ° C. A number of experiments are performed, each using a hypochlorite solution of different concentration to produce a series of increasing concentrations of hypochlorite (calculation below). The mixture is incubated for 5 minutes and then 1 ml of the reaction mixture is withdrawn and added to 9 ml of cold 1% BSA solution, then the samples are immediately placed on ice to stop the hypochlorite reaction with the microorganisms. Survival of microorganisms in the form of colony forming units, CFU / ml is determined by diluting the sample in the range of 10 ^-1 to 10 ^-5 and each 100 microliters of various dilutions are applied to labeled BHI agar plates, which are then incubated for 13-18 hours at 30 ° C. If CFU is not detectable after this time, the plates are incubated for an additional 24 hours at 30 ° C.

The similarity of the V-CPO-released hypochlorite effect profile and the hypochlorite from the stock solution was confirmed by the same experiment using a spectrophotometer in which the hypochlorite concentration was monitored over time at 290 nm based on the monochlordimedone conversion (e 290 nm = 20.2 mM). ¹ cm @ ^-1 ) to dichlorodisone (e 290 nm = 0.2 mM @ ¹ cm @ ^-1 ).

-8EN 288041 B6

Calculation:

The final concentration of hypochlorite formed by V-CPO is calculated as follows:

Using 0.01 units of V-CPO / ml, 0.01 micromoles of hypochlorite is produced per minute in 1 ml of solution, equivalent to 0.05 micromoles / ml over 5 minutes due to the dilution effect of 0.5 ml of solution added to 2.0 ml of initial volume so that the final concentration after 5 minutes is (2.0 / 2.5). 0.05 micromol / ml = 0.04 micromol of hypochlorite / ml after 5 minutes. This concentration can be expressed as 0.04.52.5 ppm hypochlorite, i.e. 20.10 ppm hypochlorite. Additional final hypochlorite concentrations were prepared by increasing or decreasing the V-CPO concentration. The final hypochlorite concentration was calculated as follows: since 0.5 ml of the solution was added to the 2.0 ml reaction volume, the final volume was 2.5 ml corresponding to a 5-fold dilution. In order to prepare a hypochlorite solution with a final concentration of 2.10 ppm, it is necessary to use 10.50 ppm of hypochlorite in the stock solution. Additional concentrations may be prepared by increasing or decreasing the hypochlorite content of the stock solution.

The minimum inhibitory concentration of MIC is defined as that concentration of hypochlorite that results in at least a log 6-colony count reduction of the microorganism of interest under the test conditions.

The results of these experiments are summarized in Table II, which compares the results of exposure to final concentrations of hypochlorite produced by V-CPO or added from the stock solution. As shown in Table II, enzymatically released hypochlorite after V-CPO treatment completely inhibits growth at a low concentration of 0.4 ppm in all organisms except Staphylococcus aureus, which requires 2 ppm of hypochlorite for complete inhibition. Thus, vanadium haloperoxidase is a far more potent component of the composition than would be expected only by its ability to form hypochlorite. It is also evident that all microorganisms, both pathogenic and non-pathogenic, can be killed using the enzyme, whereas in the case of heme-containing haloperoxidases, only the ability to suppress the growth of pathogenic bacteria is described in EP-A-500 387.

Table II

MIC values for various microorganisms

MIC for hypochlorite ppm MIC for 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

Efficacy of added hypochlorite and hypochlorite produced by V-CPO in the presence of protein hydrolyzate

It is known that higher doses of hypochlorite are generally required to achieve the desired effect, since this reactive agent reacts not only with microorganisms but also with various compounds. For this reason, the behavior of vanadium haloperoxidase in the presence of protein hydrolyzate was investigated.

Materials:

The bacterial strain used was Escherichia coli NCTC 900. The bacteria were grown for 15-18 hours at 30 ° C in a BHI broth with broth from the heart and brain. After cultivation, the bacteria were washed twice with citrate buffer pH 5.5 (20 mM sodium citrate with NaOH + 10 mM NaCl). The bacterial suspension was then centrifuged for 5 minutes in an Eppendorf centrifuge at 14,000 rpm, the supernatants were removed, and the bacterial pellet was resuspended in citrate buffer. This wash was repeated once for each bacterial suspension. The twice washed bacterial suspensions were then diluted with citrate buffer pH 5.5 to a bacterial suspension of approximately 10 ⁸ / ml. During washing, the cell material was stored on ice. Buffers and 1% BSA in citrate buffer pH 5.5 were filter sterilized and stored at 4 ° C. Hypochlorite solutions were prepared from a stock solution containing 107,000 ppm by dilution with sterile solutions. Hydrogen peroxide solutions were prepared from a 30% stock solution by dilution with sterile demineralized water. Casitone was obtained from Difco. Chloroperoxidase was isolated from Curvularia inaequalis and purified according to Van Schiindel et al., 1993.

Method:

Under sterile conditions, a suspension of bacteria of about 10 ⁸ / ml in opH 5.5 citrate buffer was prepared. Aliquots of 1.3 ml of this suspension were placed in a sterile reaction vessel whose contents were continuously stirred with a magnetic stirrer during the experiments. Then 0.5 ml of a casitone solution containing 0.5 mg / ml in citrate buffer pH 5.5 was added, the control solution contained only 0.5 ml citrate buffer pH 5.5. Then 0.2 ml of a different concentration of V-CPO solution (calculations are given in Example 2) was added to achieve a final concentration of 3.2 ppm or 6.5 ppm hypochlorite. 0.2 ml of sterile buffer was added to the control solutions instead of 0.2 ml of V-CPO solution. The samples were incubated at 30 ° C and then 0.5 ml of hydrogen peroxide solution was added to give a 5-fold molar excess of hydrogen peroxide at the end of the addition, resulting in a V-CPO reaction. The samples are incubated for exactly 5 minutes at 30 ° C. After this time, 1 ml of the reaction mixture was withdrawn and added to 9 ml of cold 1% BSA solution, whereupon the samples were immediately placed on ice to stop the hypochlorite reaction with the microorganisms. Bacterial survival is determined based on the number of colony forming units, CFU, which are determined by diluting samples between 10 ^L and 10 ^-6 and loading 100 microliters of sample with various dilutions on labeled BHI-agar plates. The plates are then incubated at 30 ° C for 15-18 hours. If CFU is not detectable after this time, the plates are incubated for an additional 24 hours at 30 ° C.

The efficacy achieved in this way was compared with the efficacy of the added hypochlorite in varying amounts when using a micro pump as follows:

Under sterile conditions, a suspension of bacteria of about 10 ⁸ / ml in citrate buffer pH 5.5 is prepared. 1.3 ml aliquots of this suspension were added to a sterile reaction vessel whose contents were continuously stirred with a magnetic stirrer during the experiment. Then a solution of 0.5 ml of casitone containing 0.5 mg of this substance in 1 ml of citrate buffer pH 5.5 is added, as a control solution 0.5 ml of citrate buffer pH 5.5 is added. 0.2 ml of sterile citrate buffer pH 5.5 is then added. The contents of the reaction vessels are incubated at 30 ° C and then 0.5 ml of hypochlorite solution is added to achieve a final concentration of 3.2 ppm or 6.5 ppm of hypochlorite (for the calculation of the concentration the conditions are given in Example 2) for 5 minutes at a rate 0.1 ml / min at 30 ° C. After an incubation period of 5 minutes, 1 ml of the reaction mixture was removed and added to 9 ml of a 1% cold BSA solution, and the samples were immediately placed on ice to stop the hypochlorite reaction with the microorganisms. Bacterial survival was determined in colony forming units, CFU, by diluting the sample in the range of 10 ^-1 to 10 · ⁶ with 100 µl sample loading.

B6 microliters at various dilutions on labeled BHI-agar plates were incubated for 15-18 hours at 30 ° C. If CFU was not detectable after this time, the plates were incubated for an additional 24 hours at 30 ° C. The results are summarized in Table III.

Table III

Effect of casitone on HC10 efficacy generated by V-CPO

casitone mg / ml log reduction with 3.2 ppm HClOzV-CPO log reduction with 3.2 ppm HCl log reduction with 6.5 ppm HClOzV-CPO log reduction with 6.5 ppm HCl 0.0 8.0 (complete extermination) 8.2 (complete extermination) 8.0 (complete extermination) 8.2 (complete extermination) 0.1 5.0 0.5 8.0 (complete extermination) 1.0

Example 4

Comparison of the efficacy of vanadium haloperoxidase and heme-containing haloperoxidase

Materials:

The bacterial strains Escherichia coli NCTC 900, Streptococcus faecalis NCTC 1092 and Listeria innocua ATCC 33090, serotype 6B were used for the experiment. The bacteria were grown for 15-18 hours at 30 ° C in BHI broth. After this time, the strains were washed twice with citrate buffer pH 5.5 (20 mM sodium citrate with NaOH + 10 mM NaCl). Then, the bacterial suspensions were centrifuged for 5 minutes in an Eppendorf centrifuge at 14,000 rpm, the supernatants were removed, and the bacterial sediments were resuspended in citrate buffer. This wash was in each case repeated once more. The twice washed bacterial suspensions were then diluted with citrate buffer pH 5.5 to suspensions containing approximately 10 ⁷ / ml bacteria. During washing, the cell material was stored on ice. Buffers and 1% BSA in citrate buffer pH 5.5 were filter sterilized and stored at 4 ° C. Chloroperoxidase from Curvularia inaequalis was purified according to Van Schijndel et al., 1993. Heme-containing chloroperoxidase was obtained from Sigma (from Caldotiomyces fumago). The efficacy of chloroperoxidase in the conversion of chlorine to HOC1 was determined spectrophotometrically in 20 mM citrate buffer pH 5.5 with 10 mM NaCl, 100 microM hydrogen peroxide and 50 microM monochlordimedone at 30 ° C by monitoring the conversion of monochlordimedone (e 290 nm - 20.2 mM). ¹ cm @ ^-1 ) to chlordimedone (e 290 nm = 0.2 mM @ ¹ cm @ ^-1 ). One unit of chloroperoxidase is defined as that amount of enzyme that converts 1 micromole of monochlorodisone per minute. The same assay was used for both enzymes to ensure the same effective dose for both.

Method:

Under sterile conditions, a suspension of bacteria containing about 10 ⁷ / ml in 20 mM sodium citrate buffer pH 5.5 with 10 mM NaCl is prepared. From this suspension, aliquots of 1.9 ml are placed in sterile tubes. Thereafter 0.1 ml of a stock solution of vanadium chloroperoxidase or chloroperoxidase containing heme is added to the tubes to obtain a dilution series for both enzymes. Control tubes contained only sterile buffer without the addition of enzyme stock. 0.5 ml of a 25 mM hydrogen peroxide stock solution is then added. The samples are incubated for exactly 5 minutes at 30 ° C. After this incubation period, 1 ml of the reaction mixture is withdrawn and added to 9 ml of cold 1% BSA solution, and the samples are immediately placed on ice. Bacterial survival is determined by the number of colony forming units, CFU / ml, determined by

-11 CZ 288041 B6, the sample is diluted in the range of Io ^{'1-10' 5} and 100 microliters of sample at various dilutions is applied to a labeled

BHI-agar plates. These plates are then incubated at 30 ° C for 15-18 hours. If GFU cannot be detected after this time, the plates are incubated for an additional 24 hours at 0 ° C.

The results are summarized in Table IV below. Obviously, vanadium chloroperoxidase is a far more potent system than chloroperoxidase containing haem when both enzymes are used at the same dose.

Table IV

10mMNaCl vanadium chloroperoxidase units / ml heme chloroperoxidase units / ml log reduction E. coli 0,000 0.0 0.00156 4.0 0,0030 5.0 0.0060 7.0 * 0.012 7.0 * 0,025 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 0,025 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 * 0.012 7.0 * 0,025 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 0,025 0.0 0.050 0.0 0.10 0.0

- 12GB 288041 B6

10 mM NaCl vanadium chloroperoxidase units / ml heme chloroperoxidase units / ml log reduction S. faecalis 0,000 0.0 0.00156 5.0 0,0030 5.0 0.0060 7.0 * 0.012 7.0 * 0,025 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 0,025 0.1 0.050 0.1 0.10 0.9

* = complete eradication

Example 5

Effect of various hydrogen peroxide sources on V-CPO inhibitory activity

In the previous examples, hydrogen peroxide, which is one of the substrates in the V-CPO reaction, was added as a stock solution. In this example, the effect of other hydrogen peroxide sources was observed. The oxidases were tested at 30 ° C in an Oxygen Consumption Monitor, Biological Oxygen Monitor YSI Model 5300 (Oxygen chamber model 5301) using a pH 5.5 citrate buffer (20 mM sodium citrate with NaOH + 10 mM NaCl). The buffer was air saturated at 30 ° C. Glucose at a final concentration of 15 g / liter was used as a substrate for glucose oxidase. The bacterial strain Escherichia coli NCTC 900 was used. The bacteria were grown for 15-18 hours at 30 ° C in BHI broth. After this time, the strains were washed twice with citrate buffer pH 5.5 (20 mM sodium citrate with NaOH + 10 mM NaCl). The bacterial suspensions were centrifuged for 5 minutes in an Eppendorf centrifuge at 14,000 rpm, the supernatant removed and the bacterial pellet resuspended in citrate buffer. This wash was repeated once for each bacterial suspension. The twice washed bacterial suspensions were then diluted with citrate buffer pH 5.5 to give a bacterial suspension of approximately 10 ⁸ / ml. During washing, the cell material was placed on ice. Buffers and 1% BSA in citrate buffer pH 5.5 were filter sterilized and then stored at 4 ° C. Hypochlorite solutions were prepared from a stock solution of this substance containing 107,000 ppm by dilution with sterile, demineralized water. Hydrogen peroxide solutions were also prepared from a 30% stock solution by dilution with sterile demineralized water. Casein hydrolyzate Casitone was obtained from Difco. Chloroperoxidase from Curvularia inaequalis was isolated and purified according to the method of Van Schijndel et al., 1993. Glucose oxidase from Aspergillus niger was obtained from Sigma.

Method:

Under sterile conditions, a suspension of bacteria of approximately 10 ⁸ / ml citrate buffer pH 5.5 is prepared. 1.3 ml aliquots of this suspension were added to a sterile reaction vessel whose contents were continuously stirred with a magnetic stirrer during the experiment. 0.5 ml of a solution containing 0.5 mg / ml casitone in citrate buffer pH 5.5 is then added. Then 0.2 ml of V-CPO solution is added

-13GB 288041 B6 to a final concentration of 6.5 ppm hypochlorite. Blank controls are also included in which 0.2 ml of sterile buffer is added instead of 0.2 ml of V-CPO solution. The reaction vessels were incubated at 30 ° C. Then 0.5 ml of one of the following three hydrogen peroxide containing systems is added:

1. hydrogen peroxide from a 3 mM stock solution;

2. Sodium percarbonate from a 3 mM stock solution;

3. a mixture of 0.39 units / ml glucose oxidase mixed with 75 mg / ml glucose.

The samples are incubated for exactly 5 minutes at 30 ° C. After this incubation period, 1 ml of the reaction mixture is withdrawn and added to 9 ml of cold 1% BSA solution and then the samples are immediately placed on ice to stop the hypochlorite reaction with the microorganisms. Survival of the microorganisms is monitored on the basis of colony forming units, CFU / ml, determined by diluting the samples in the range of 10 ' to 10 < ⁶ > and then loading 100 microliters of these solutions at various dilutions on labeled BHI-agar plates. . These plates are then incubated at 30 ° C for 15-18 hours. If CFU is not detectable after this incubation period, the plates are incubated for an additional 24 hours at 30 ° C. The values obtained are compared with the MIC values when the same amount of hypochlorite was directly added using a micro pump as follows:

Under sterile conditions, a suspension of bacteria containing approximately 10 ⁸ / ml in citrate buffer pH 5.5 is prepared. 1.3 ml aliquots of this suspension were added to a sterile reaction vessel whose contents were continuously stirred with a magnetic stirrer during the experiment. Then 0.5 ml of a casitone solution containing 0.5 mg / ml of this substance in citrate buffer pH 5.5 is added. 0.2 ml of sterile citrate buffer pH 5.5 is then added. The reaction vessels were incubated at 30 ° C. Then, 0.5 ml of a 32.5 ppm hypochlorite solution was added over 5 minutes at a rate of 0.1 ml / min at 30 ° C. After an incubation period of 5 minutes, 1 ml of the reaction mixture is removed and added to 9 ml of cold 1% BSA solution and the samples are immediately placed on ice to stop the hypochlorite reaction with the microorganisms. The surviving microorganisms was determined based on colony forming units, CFU / ml and determined such that the sample is diluted in the range 10 ^1-1 θ ^'6 and 100 l samples of the various dilutions on labeled BHI-agar plates. These plates are then incubated at 30 ° C for 15-18 hours. If CFU is not detectable after this time, the plates are incubated for an additional 24 hours at 30 ° C. The results obtained are summarized in Table V below.

Table V

Reaction of V-CPO with various sources of hydrogen peroxide

source of hydrogen peroxide casitone concentration mg / ml log reduction at 6.5 ppm hypochlorite log reduction at 6.5 ppm hypochlorite from V-CPO H ₂ O ₂ stock solution 0.0 8.2 (complete extermination) 8.0 (complete extermination) percarbonate 0.0 8.0 (complete extermination) 8.2 (complete extermination) glucose oxidase 0.0 6.4 (complete extinction) 6.4 (complete extinction) H ₂ O ₂ stock solution 0.1 1.0 8.0 (complete extermination) percarbonate 0.1 0.8 8.2 (complete extermination) glucose oxidase 0.1 1.0 6.4 (complete extinction)

-14GB 288041 B6

Example 6

Determination of Chloroperoxidase and Curvularia inaequalis (cDNA) Coding Sequence (Centraal Bureau voor Schimmelcuktures, The Netherlands, Strain No. 102.42) and Applicable Expression Systems

Chloroperoxidase was isolated and purified from C. inaequalis cultures in a liquid culture medium according to Van Schijndel et al., 1993, except that two more purification steps were performed after DEAE chromatography using the FPLC system (Pharmacia LKB). First, a C1-4B hydrophobic phenylsepharose column was used to bind the enzyme in the presence of 2M NaCl in 50 mM tris-HCl pH 8.3, then eluting with a descending gradient of 2M NaCl in 50 mM tris-HCl pH 8.3. Final purification was performed by using an MonoQ HR 5.5 anion exchange resin column (Pharmacia LKB) to bind the enzyme, eluting with a gradient of 0 to 0.5 M NaCl in 20 mM piperazine-HCl pH 5.4 . Subsequent enzyme concentration was performed on a rotary evaporator followed by dialysis against 50 mM tris-SO ₄ buffer at pH 8. Purified chloroperoxidase was enzymatically digested with proteases from Staphylococcus V8 and trypsin by conventional procedures or chemically digested with CNBr according to Gross E., 1967 Methods Enzymology, 11, 238-255. The resulting peptides were resolved using SDS-PAGE according to Laemmli, UK, 1970, Nature 227, 680-685 or on a Tricine gel according to Schagger and Von Jagow, 1987 and transferred to PVDF membranes (Immobilon-P, Millipore) at using CAPS transfer buffer containing 10 mM 3- (cyclohexylamino) -1-propanesulfonic acid and 10% methanol opH 11 according to Matsudair, 1987. After electrophoretic elution, the membrane was flushed with deionized water for 5 minutes, then stained for 0.1 minutes with 0.1. % Coomassie R-250 blue in 50% methanol, the dye was removed with 50% methanol with 10% acetic acid for 10 minutes at room temperature, then the membrane was air dried. Peptide bands were subjected to automatic Edmann sequence analysis on a Porton LF 3000 (Beckman Instruments, Inc., USA). The results of the amino acid sequence determination are shown in Figure 1.

Depending on the amino acid sequence of the peptides, fully degenerate oligonucleotides were constructed (Table VII). These degenerate primers were used for the PCR reaction using the first strand cDNA as a template. The first strand cDNA was prepared as follows. For RNA isolation, C. inaequalis spores were inoculated into a fermentation broth containing 4 g yeast extract and 2 ml microelement solution per liter according to Van Schijndel et al., 1993. After several days of cultivation, the mycelium was separated by filtration and lyophilized. The lyophilized C. inaequalis mycelium was ground in liquid nitrogen. RNA was extracted with RNA extraction buffer containing 42 mM sodium citrate pH 7, 0.83% N-laurysarcosine, 50 mM betamercaptoethanol, 1% triton X-100 and 4 M guanidinium isothiocyanate, and incubated for 1 hour at room temperature. Then, 0.1 volume of 2M sodium acetate pH4 and one volume of 25: 24: 1 phenol / chloroform / isoamyl alcohol were added to the mixture and stored on ice for 15 minutes. The mixture is centrifuged for 10 minutes at 4 ° C at 10,000g, the aqueous phase is separated, 1 volume of absolute alcohol is added and the mixture is incubated at -20 ° C for 1 hour, then centrifuged briefly again at 10,000g. The pellet is resuspended in an appropriate volume of RNA extraction buffer and fractionated in an ultra centrifuge using a cesium chloride gradient (Sambrook et al., 1989). The pellet was washed thoroughly and stored in 75% ethanol at -70 ° C. For mRNA isolation, RNA is precipitated and resuspended in ribonuclease-free water, whereupon the mRNA is extracted using a mRNA isolation package, polyAtract (Promega corporation, USA). First strand cDNA synthesis is performed using mRNA isolated from C. inaequalis using a first strand cDNA synthesis kit (Pharmacia Biotech). Depending on the amino acid sequence of the chloroperoxidase peptides, four degenerate 20 nucleotide oligonucleotides were constructed (Table VII) and were used as primers in PCR together with the first C. inaequalis cDNA strand as a matrix. The PCR reaction was performed using a thermal cycling apparatus (Eppendorf mastercycler 5330) and Taq polymerase (Promega corporation). For optimal code amplification

The PCR reaction was carried out at 46 ° C (in the binding step) for 30 cycles using chloroperoxidase cDNA using degenerate primers. The two resulting specific fragments were ligated to the pUC18 vector, cloned, and the sequences in both strands analyzed. Depending on the DNA sequence, the following two specific primers were constructed:

5’-CATAGCGATAGCGACGCGGA-3 ’a

5’-CTAACCCCGGCGCCAACATC-3 ’.

These two primers were used in the PCR reactions together with the first strand cDNA as a template. In this way, a gene-specific DNA fragment was obtained by joining two known DNA strands. This fragment was cloned in the pUC18 vector and its sequence analyzed. A 5‘-AmpIifinder RACE package (Clonetech corporation) was used to obtain the 5 'region of the chloroperoxidase-encoded mRNA. The chloroperoxidase gene from the C. inaequalis genome was isolated as follows:

C. inaequalis genome DNA is isolated from lyophilized mycelium, ground in liquid nitrogen and extracted with an appropriate amount of extraction buffer containing 200 mM tris-HCl pH 8.5, 25 mM EDTA, 250 mM NaCl, 1% SDS and 0.2 mg. / ml proteinase K. Incubate overnight at room temperature, then add 0.7 volume of phenol and 0.3 volume of chloroform and mix vigorously. Then centrifuge the contents of the tubes at 10,000 g and transfer the aqueous layer to a clean tube. The genome DNA is then precipitated by adding 2 volumes of absolute ethanol. After centrifugation for 5 minutes at 5000 g, the pellet is resuspended in 2 ml of 10 mM trisHCl pH 8.0 and 1 mM EDTA and ribonuclease (Boehringer Mannheim) is added according to the manufacturer's instructions. The DNA containing genome solution was extracted with 25: 24: 1 phenol / chloroform / isoamyl alcohol and, after ethanol precipitation, the material was dissolved in an appropriate volume of 10 mM tris-HCl pH 8 with 1 mM EDTA. To perform Southem analysis, DNA is digested with several combinations of restriction enzymes and, after agarose gel electrophoresis, the material is transferred to a nitrocellulose membrane (Sambrook et al., 1989).

Hybridization was performed using a radiolabeled specific fragment of the gene (amplified using the two specific primers described above), obtained using alpha- ³² P-labeled dATP (Sambrook et al., 1989). Based on the obtained results, a minibank was prepared using the Pst I digested DNA genome, and the material was inserted into the pUC18 vector. The flask was serially screened with the same probe as described for the South blot. A positive clone was isolated and the sequence of both strands was also partially analyzed in order to confirm the results of the cDNA analysis. The C. inaequalis chloroperoxidase gene and its gene product are shown in Figure 2 below.

A system for producing chloroperoxidase in Saccharomyces cerevisiae was prepared as follows:

The well-known inducible yeast GAL1 promoter was obtained as a fragment after digestion with EcoRI and BamHI from the wild-type GAL1 gene of S. cerevisiae (Molecular and Cellular Biology, 10, 4757-4769, 1990) and was cloned at the same enzymatic site in YCplac33 and YEplac95 (Gietz and Sugino, 1988, Gene, 74, 527-534). The plasmids obtained were designated TNT1 and TNT2. A BamHI restriction enzyme site was created at the 5'-end of the C. inaequalis chloroperoxidase gene by PCR using a fragment of the C. inaequalis chlorperoxidase gene from the 5 'region after digestion with PstI and EcoRI as a matrix, which was subcloned in plasmid pUC18 using primer M13 / pUC (22-mer with reverse sequence) and the following primer

5‘-GAG AGA GGA TCC ACT CAC TAC CAA TCA CAC 3 ‘

The amplified fragment was digested with BamHI and EcoRI. The fragment of the C. inaequalis gene for chloroperoxidase after digestion with EcoRI and PvuII, containing the 3‘-part of this gene, was

-28GB 288041 B6 was subcloned in the digested plasmid pUC18 after digestion with EcoRI and SmaI. This clone was isolated and purified after digestion with EcoRI and an XbaI fragment containing the 3 'portion of the C.peraequalis chloroperoxidase gene.

Three components were then coupled, namely TNT1 or TNT2, each after digestion with BamHI and XbaI, a 5‘ fragment after digestion with BamHI and EcoRI and a 3‘ fragment after digestion with EcoRI and XbaI. After binding and cloning of the plasmids obtained, their identity was verified. The resulting plasmids were designated TNT3 (derived from TNT1) and TNT4 (derived from TNT2).

The yeast strain BJ1991 is transformed with plasmid TNT3 or TNT4 in a known manner and selected for ura + transformants. These transformants were replicated on YP-plates containing 2% glucose or 2% galactose. Once the yeast has grown, part of the cells are removed from the plates and suspended in 200 µl of 20 mM Tris-HCl, pH 8.1. After incubation for 8 minutes, 10 microliters of material was removed and stained on a nitrocellulose filter. The nitrocellulose filters are incubated in a buffer of 100 mM sodium acetate pH 5.5 containing 1 mM ortodianisidine, 100 mM KBr and 2 mM hydrogen peroxide. A color spot appears at all sites taken from the galactose plates, whereas no color develops from the yeast from the glucose plates. This demonstrates that a production system for the galactose-inducible chloroperoxidase gene was constructed in the yeast Saccharomyces cerevisiae. In a similar assay using 100 mM potassium phosphate buffer pH 6.5, 100 mM KBr, 1 mM hydrogen peroxide and 40 microM BDH phenol red, blue-purple color formation was observed using yeast from galactose media, yeast from glucose media. there is no discoloration. In order to further confirm the production of a system for the expression of a heterologous C. inaequalis chloroperoxidase gene with the same functional properties, the recombinant enzyme was purified from yeast strains transformed with plasmid TNT3 or TNT4 after galactose induction. After culturing in a galactose-containing medium, the yeast was separated and resuspended in 20 mM Tris-HCl, pH 8.1. Sterile glass beads were then added and the suspension vigorously shaken. After centrifugation for 15 minutes at 10,000 g, the supernatant was collected and loaded onto a DEAE column, and the recombinant enzyme was purified essentially as described above for the wild-type C. inaequalis enzyme. After purification, recombinant chloroperoxidase was obtained with a specific efficiency of 22 units / mg protein as determined in 100 mM sodium acetate buffer pH 5.0 containing 1 mM hydrogen peroxide, 5 mM potassium chloride and 50 microM MCD, according to Van Schijndel et al. , 1993. This finding is in very good agreement with the specific efficacy of approximately 20 units / mg protein as demonstrated above for purified C. inaequalis chloroperoxidase. The pH-dependent activity is shown in Figure 3 for the wild-type chloroperoxidase and for the recombinant yeast enzyme. This efficiency is further evidence that the recombinant yeast-derived enzyme has the same functional properties as the wild-type enzyme.

Example 7

Monitoring of haloperoxidases in other microorganisms

Other investigated microorganisms were Curvularia inaequalis CBS 102.42, Drechslera biseptat CBS 371.72, Drechslera fugax CBS 509.77, Drechslera nicotiae CBS 655.74, Drechslera subpapendorfii 656.74, Embelisia hyacintlocladium 416.71.79, Embelisiumium Cis.77.79.99, Embelisiumium6.67.99, Embelisiumisisumisisumisisumisis6.77. Mushrooms were grown on agar plates. After growth, extracellular proteins were transferred in duplicate to nitrocellulose filters pre-wetted with 50 mM Tris-HCl buffer pH 8.3. After 15 minutes incubation on agar plates, the filters were tested for haloperoxidase activity by immersing the filter in 100 mM sodium acetate at pH 5.5 or in potassium phosphate at pH 6.5 and 7.5 and 1 mM orthodianisidine and 2 mM. hydrogen peroxide in the presence or absence of 0.1 M potassium bromide. In this way, the formation of bromoperoxidase and / or chloroperoxidase can be detected. To determine whether the haloperoxidase produced was vanadium halide peroxidase, the experiment was repeated in the presence or absence of 10 and 100 microM sodium vanadate. In the case of vanadium haloperoxidase, signal amplification was observed after the addition of vanadate. To determine whether the identified chloroperoxidase was truly similar to vanadium haloperoxidase from C. inaequalis, small amounts of chloroperoxidase were purified according to Van Schijndel et al., 1993, enzymes from Ulocladium chartarum, Embelisia didymospora and Drechslera subpapendorfii. The optimum pH for these enzymes was in the range of 4.5 to 5.5. The chlorination activity of these enzymes increased after the addition of vanadate, clearly demonstrating that they are vanadium haloperoxidases. In order to further demonstrate the similarity of the enzymes to the C. inaequalis enzyme, one of the identified halogen peroxidases was further analyzed. It was an enzyme from Drechslera biseptata CBS 371.72. The properties of this enzyme are similar to those of Curvularia inaequalis chloroperoxidase, which has high thermal stability and high affinity for its substrates. The EPR spectrum of the purified enzyme was also recorded. As with other vanadium haloperoxidases according to de Boer et al., 1988 and Wever et al., 1988, the oxidized enzyme does not show up in this spectrum, but after reduction with sodium dithionite, a typical spectrum for the vanadyl group appears (data not shown). Isotropic ERR- parameters, go - _about 1,969 and A - of 9.0 mT are almost the same parameters for the enzyme from C. inaequalis according to Wever et al, 1985. Furthermore, the purified enzyme has been split into peptides by treatment with proteases and cyanogen bromide. The peptide maps are identical for both enzymes, so these enzymes are likely to have great homology to their sequences. Therefore, the sequence of two peptides, one peptide from each enzyme after protease digestion and purification, was analyzed. The obtained sequences showed great homology and it can be concluded that both enzymes are very similar.

The C. inaequalis peptide has the following amino acid sequence: (Asp) -leu-arg-gln-pro-tyr-asp-pro-market-ala-pro-ile-glu-asp-gln-pro-gly-ile-val- arg-thrThe similar peptide of D. biseptata has the following amino acid sequence:

Thus, asp-leu-arg-gln-pro-tyr-asp-pro-thr-ala-pro-ile-glu-glu-glu-gln-pro-gly-ile-val-arg-thrVanadium haloperoxidase can be identified using the technique of which shows an increase in potency after the addition of vanadate, or it is also possible to partially purify the enzyme before monitoring the increase in potency after the addition of vanadate.

Example 8

Tests with chloroperoxidases of other microorganisms using antibodies

The strains used were Curvularia inaequalis CBS 102.42, Drechslera biseptata CBS 371.72, Drechslera subpapendorfii CBS 656.74, Embellissia didymospora CBS 766.79 and Ulocladium chartarum CBS 200.67.

Microorganisms were grown in two phases. First, 50 ml of sterile medium according to Van Schijndel et al., 1993 were inoculated with spores of the microorganism. The culture was grown for three days with constant shaking at 23 ° C and then transferred to a 3 liter Erlenmeyer flask containing 1 liter fermentation broth containing 5 g casein hydrolyzate (Gibco BRL), 3 g yeast extract and 1 g fructose per liter of deionized water. The medium was maintained at 23 ° C for 14-17 days with shaking, then filtered, and the chloroperoxidases purified according to Van Schijndel et al., 1994. The rabbits were induced to produce polyclonal antibodies against this purified chloroperoxidase from Curvularia inaequalis. Two month old female rabbits were used, the first injection received complete Freund's adjuvant and repeated administration with incomplete Freund's adjuvant. After the last injection, rabbits were bled 6 days later, the serum was heated at 56 ° C for 30 minutes to inactivate the complement, and then

- 18GB 288041 B6 serum is centrifuged. The supernatant was collected and processed into a dilution series. The dilution series was also prepared for bromoperoxidase from Ascophylum nodosum, purified according to Wever et al., 1985. The starting material was 50 microliters of each protein containing approximately 0.1 mg of this protein per ml of solution, and each sample was diluted twice in succession. These dilutions were then applied to a nitrocellulose filter using a dot-blot (bio-Rad), and after washing with 2% BSA filters, the filters were incubated sequentially with a 1: 800 dilution of anti-chloroperoxidase rabbit antisera with a biotinylated goat anti-rabbit tissue antibody at dilution. 1: 3000, with an alkaline phosphatase conjugated to 1: 2000 streptavidin and a color reaction reagent consisting of 5-bromo-4-chloro-3-indolylphosphate and tetrazolium chloride 4-nitromodor (Boehringer Mannheim). All steps were performed in a standard manner. The results of these tests are summarized in Table VI below.

Table VI

Halogen peroxidase from cross-reactivity with polyclonal antibodies against chloroperoxidase from C. inaequalis C. inaequalis D. biseptata D. subpapendorfii E. didymospora U. chartarum yes yes yes yes yes bromoperoxidase from A. nodosum No

From the results shown in Table VI, it can be concluded that immunoassays with antibodies against C. inaequalis chloroperoxidase are useful for identifying other vanadium-containing haloperoxidases. These haloperoxidases may be in crude form or may be partially or fully purified. Purification can be accomplished by any known method, such as gel filtration, ion exchange chromatography, hydrophobic interaction chromatography, precipitation, filtration, ultrafiltration, affinity chromatography, gel electrophoresis, and the like.

Example 9

Search for chloroperoxidases from other microorganisms

This example describes the possibility of using a radiolabeled probe derived from the chloroperoxidase gene from Curvularia inaequalis to detect homologous genes in other microorganisms. The procedure was performed as follows: Chromosomal DNA from C. inaequalis CBS 102.42, Embellissia didymospora CBS 766.79 and Drechslera biseptata CBS 371.72 were purified as described in Example 6 for C. inaequalis chromosomal DNA. For Southem's DNA genome analysis, the DNA was digested with several combinations of restriction enzymes, and after agarose gel electrophoresis, the fractions were stained on a nitrocellulose membrane (Sambrook et al., 1989). Hybridization was performed using a radiolabeled specific gene fragment using alpha- ³² P-labeled dATP (Sambrook et al., 1989). The specific gene fragment used was PCR amplified prior to radiolabeling using the first strand cDNA as template as in Example 6 using the following primers:

5‘-CACGATGGGGTCCGTTACAC a

5‘-GTACCGCTATCGCTGCGCCTG

- 19GB 288041 B6

Hybridization was performed as follows: For pre-hybridization and hybridization, a mixture of 6 x SSPE, 5 x Denhards, 0.5% SDS with 10 mg salmon sperm DNA was used as medium. Pre-hybridization was carried out for 1 hour at 55 ° C, then the radioactive probe was boiled for 1 minute and then used directly. Hybridization was performed overnight. The autoradiograph obtained using DNA from Curvularia inaequalis and Drechslera biseptata is shown in Figure 4, where the individual features are as follows: lane 1: lambda DNA, lane 2: uncleaved DNA of the C. inaequalis genome, lane 3: same material, digested with EcoRI, lane 4: same after digestion with BamHI, lane 5: same after digestion with EcoRI and BamHI, lane 6: same after digestion with XbaI, lane 7: same after digestion with PstI, lane 8: same after digestion with XbaI and PstI, lanes 9-14: digestion material from D. biseptata by the same enzymes in the same order. As can be seen from the drawing, the positive signal was obtained from chromosomal DNA from Drechsler biseptata, demonstrating the high similarity of the two genes. Similar results were also obtained using DNA. Similar results were obtained using DNA from Embellissia didymospora. Thus, it can be concluded that a chloroperoxidase gene, a portion of that gene, or a probe prepared based on the C. inaequalis chloroperoxidase gene sequence can be used to detect other similar vanadium haloperoxidases from other microorganisms.

Table VII

Oligonucleotide primers (20-mers), prepared based on the amino acid sequence of vanadium chloroperoxidase from Curvularia inaequalis

Oligonucleotide 1:

5'-T A C / T A T G A A / G C CIG TIG A A / G C A-3 ‘

Oligonucleotide 2:

5'-A G / A T / C T G IG C / A / T AIG C / A / T T / A C-3 ‘

Oligonucleotide 3:

5‘-G A / G G A / G A CIG CIG A / G T A C / T G A-3 ‘

Oligonucleotide 4:

5‘-A G / A IG C T / C T GIG C I C G / A / T / C C A T-3 ’

Explanatory notes to Table VII:

I = inosine

A / G: A mixture of A and G with the same amount of both components was used in this position.

C / T: at this position, a mixture of C and T with the same amount of both components was used.

G / A / T / C: a mixture of G, A, C and T with the same amount of all components was used in this position.

Claims (6)

PATENT CLAIMS An enzymatic antimicrobial composition comprising vanadium haloperoxidase, a halide source and hydrogen peroxide or a source of hydrogen peroxide. Enzymatic antimicrobial composition according to claim 1, characterized in that it contains chloroperoxidase as vanadium haloperoxidase. Enzymatic antimicrobial composition according to claim 1 or 2, characterized in that it contains chloroperoxidase from Curvularia inaequalis as vanadium haloperoxidase. Enzymatic antimicrobial composition according to one of claims 1 to 3, characterized in that it contains chloroperoxidase with cross-immunological reactivity with chloroperoxidase from Curvularia inaequalis CBS 102.42 as vanadium haloperoxidase. The enzymatic antimicrobial composition according to any one of claims 1 to 4, characterized in that it comprises a hydrogen peroxide generating enzyme system as the hydrogen peroxide source. The enzymatic antimicrobial composition according to claim 5, characterized in that it contains glucose and glucose oxidase or lactate and lactate oxidase as the hydrogen peroxide generating enzyme system.