JPH09511396A - Enzymatic antibacterial composition - Google Patents

Abstract

  (57) [Summary] An antibacterial composition comprising vanadium haloperoxidase, a halide source and hydrogen peroxide or a hydrogen peroxide source is provided. The vanadium haloperoxidase is preferably chloroperoxidase obtained from Curvularia inaequalis.

Description

Detailed Description of the Invention                         Enzymatic antibacterial composition Technical field   The present invention provides an enzymatic antimicrobial composition (enzymatic antimicrobial). l composition) and its use. More specifically, Ming contains vanadium haloperoxidase, hydrogen peroxide source and halide source. The present invention relates to an enzymatic antibacterial composition. The present invention further provides for the fermentation using recombinant DNA technology. It relates to the production of vanadium haloperoxidase which can be used in elementary antibacterial compositions.Background and prior art   Various enzymatic antimicrobial compositions are known in the art. For example, WO-A-9 4/04217 is an antibacterial effective concentration of hypothiocyanate (OSCN).^- ) Is disclosed which discloses a stabilized toothpaste composition. The composition is hydrogen peroxide -Producing oxidoreductase and thiocyanate ion normally present in saliva Antibacterial hypothiocyanite (OSCN^-) Peroxidase Contains enzymes. Suitable peroxidases include lactoperoxidase, Eloperoxidase, saliva peroxy Includes dases and chloroperoxidase.   An enzymatic antibacterial composition containing haloperoxidase is disclosed in EP-A-5003877. (Exoxemis). Haloperoxidase is a peroxide And selectively binds to the target microorganism in the presence of a halide to prevent its growth It is stated that. EP-A-500387 is a suitable haloperoxider. As myeloperoxidase (MPO), eosinophil oxidase (EPO) , Lactoperoxidase (LPO) and chloroperoxidase (CPO) It has been described. Stabilization of haloperoxidase by the ratio of halide to hydrogen peroxide It was reported to be an important factor regarding sex and functionality. Hydrogen peroxide has a very low ratio Rate can inhibit haloperoxidase function, whereas halides It can block enzymatic reactions at extremely high rates. The ratio can vary within wide limits, It is preferable to keep it at 50 or more.   The practice of hydrogen peroxide in antimicrobial compositions due to the undesirable side reactions of hydrogen peroxide The concentration of is expected to be lower than expected. Accordingly, Applicants have found that antibacterial compositions are customary. High starting concentrations of hydrogen peroxide can be used in combination with a dose of halide I found it more desirable to do so.   Furthermore, an enzyme having a spectrum of antibacterial activity different from known enzymatic antibacterial compositions There is a need for a specific antibacterial composition. The composition comprises microorganisms that are difficult to eradicate, such as S It may have antibacterial activity against Treptococcus faecalis preferable. Under other circumstances, it can also spoil foods and, as a result, also cause non-pathogenic microorganisms. It is desirable to eradicate it.   Accordingly, it is an object of the present invention to provide an enzymatic antimicrobial composition that eliminates one or more of the above drawbacks. Is to provide.   Surprisingly, the Applicant has found that with vanadium haloperoxidase Have found that a particularly effective enzymatic antimicrobial composition can be formulated.   The above-mentioned prior art includes various haloperoxidases for use in antibacterial compositions. However, to date, vanadium haloperoxidase species have received particular attention. Did not.   In vanadium haloperoxidase, the prosthetic group in the enzyme is vanadic acid (vanadium). It has similar structural properties to Nadium V), while other haloperoxidas Is different from other haloperoxidases in that it is a heme peroxidase. Has become.   Another object of the present invention is to identify the gene encoding vanadium haloperoxidase. The gene is cloned in another microorganism that is cloned and sequenced to favor growth. And increase the amount of enzyme that can be produced using recombinant DNA technology That is.   Certain vanadium bromoperoxidases are naturally found on seaweed surfaces. (Wever et al., 1991). In healthy plants in seawater, vanadium bromope Luoxidase is accessible to the added substrate and upon addition of hydrogen peroxide HOBr can be released. Although the role of the enzyme has not been elucidated, it has a high level in seawater. Formation of highly oxidative HOBr inhibits the growth of microorganisms or fungi on the surface of plants It is considered to be part of the plant's defense system.   Non-heme vanadium bromope from the seaweed Ascophyllum nodosum Many other seagrass species have also contained these enzymes since the discovery of luoxidase. Rukoto has been shown. In particular, A. Bromoperoxida obtained from nodosum Extensive research has been carried out on these cases to determine their characteristics. See pages 2-3). The prosthetic group in these enzymes is vanadic acid (vanadium). Dium It has structural properties similar to V). The catalytic mechanism induced for the enzyme Hydrogen peroxide reacts with the enzyme to form a hydrogen peroxide / enzyme complex, after which , Bromide and protons react with the complex to form an enzyme / HOBr complex . It was shown that when the complex collapses, it produces an enzyme and HOBr (De Bo er et al., 1988). These vanadium bromoperoxidases are aqueous and It has also been shown to have high operational stability in organic media (De Boer et al., 198). 8). For example, these enzymes were stable for 3 weeks under turnover conditions and Solvents such as acetone, methanol, ethanol (maximum 60v / v% present It can be stored for 1 month or more without losing its activity in 1-propanol and 1-propanol. there were. However, the enzyme has potential applications where bromide is present or added, The genes encoding these bromoperoxidases obtained from seaweed are cloned. And the attempt to determine its amino acid sequence will not work. It has drawbacks.   Existing known heme-containing from Caldariomyces fumago Chloroperoxidase has an inherent instability and a low optimal p of 2.75. For H, Its use is very limited and not well suited for the production of enzymatic antibacterial compositions. For the same reason, human leukocyte-derived enzyme myeloperoxida capable of generating HOCl The use of nuclease (MPO) is prevented (ARJ Bakkenist et al., 19). 80).   Hypomyctes, a family of Dematium, are extremely stable. The paper that secretes luoxidase (see references 7 and 8) has already been published. I have. In particular, the terrestrial fungus Curvularia inaequalis It has a high affinity for substances and has an optimum pH of about 5.5 for chlorination reactions. It has been shown to secrete vanadium chloroperoxidase (J.W.P. M. Van Schijndel et al., 1993). Bromoper obtained from seaweed As with oxidases, the prosthetic group in chloroperoxidase is vanadium. It has structural properties similar to diacid (vanadium V). More detailed research after that (JWPM Van Sijjndel et al., 1994). The enzymatic reaction rate of chloride oxidation by hydrogen oxide was determined by seaweed A. obtained from nodosum Is shown to be similar to that of vanadium bromoperoxidase. Was. Furthermore, by three different methods, the enzyme reacts with chlorine oxide species as a reaction product. Produced (HOCl) and was shown to be resistant to the product itself Was. The enzyme has high thermostability (Tm 90 ° C.), 40% methanol, ethanol It exhibits high stability in organic solvents such as nor and propanol.Definition of invention   The present invention provides, in a first aspect, a vanadium haloperoxidase, a halogen And an enzymatic antibacterial composition comprising hydrogen peroxide or a hydrogen peroxide source.   The present invention provides, according to a second aspect, an origin of replication, a transcription / termination regulatory sequence, and vanadium An expression vector containing at least a portion of the DNA sequence encoding muhaloperoxidase. A suitable host can be transformed with a vector to express a structural gene By culturing under conditions and isolating vanadium haloperoxidase, It relates to the production of haloperoxidase.Detailed description of the invention (A)Vanadium haloperoxidase   The enzymatic antibacterial composition of the present invention comprises vanadiu as the first component. Contains muhaloperoxidase. As a rule, vanadium haloperoxidase From various vanadium haloperoxidases disclosed in the art. You can choose. For example, Curvul as described in US-A-4707466. Vanadium (non-heme) haloper produced from aria inaequalis An oxidase may be used. Alternatively, J. W. P. M. Van Schijn Curvularia inaequa according to the method of del et al. (1993). Isolation and purification of chloroperoxidase from lis (CBS 102.42) Good.   Other sources of vanadium haloperoxidase include Drechslera bis eptata (CBS 371.72), Drechslera fugas (CBS)  509.77), Drechslera nicotiae (CBS 655.74). , Drechslera subpapendorfii (656.74), E mbelisia hyacinthi (416.71), Embelisia   didymospora (CBS 766), Ulocladium char tarum (200.67) and Ulocladium botrytis (4 52.72) are included.   Alternatively, the origin of replication, transcriptional and termination regulatory sequences, and vanadium haloperoxynucleotides. An expression vector containing a DNA sequence encoding a sidase is used to transform a suitable host. After transformation, the host is cultured under conditions that allow expression of the structural gene, and vanadium haloperoxide is added. Recombinant DNA technology by isolating oxidase Loperoxidase can be produced. This is described in detail in the examples below. (B)Halide ion source   The second component of the hygiene composition of the present invention is a source of halide ions. These are These halide ions may be used, but iodide is used because it is the most effective. Or chloride ions are preferred. Most preferred halide is sodium chloride On source. Halides may be added to the enzymatic antimicrobial composition of the present invention and water may be added. Using halides that naturally occur in water and are usually on the order of 2-5 mM Good. (C)Hydrogen peroxide source   The hygiene composition of the present invention further comprises a source of hydrogen peroxide or a hydrogen peroxide generating system. Suitable Examples of such hydrogen peroxide generating systems are perborates or percarbonates, preferably sodium percarbonate. Rium or excess It is sodium borate.   Hydrogen peroxide can also be supplied by an enzymatic hydrogen peroxide generating system. Enzymatic peracid As a general rule, the hydride generation system is based on various enzymatic peracids disclosed in the art. It may be selected from hydrogen fluoride generating systems. For example, amine oxidase and amine, amino Acid oxidase and amino acid, lactate oxidase and lactate, cholesterol oxy Dose and cholesterol, urate oxidase and uric acid, or xanthine oxidase Zeze and xanthine can be used. However, preferred is glucose oxidase It is a combination of glucose.   The amount of glucose oxidase depends on its specific activity and possible residual catalase. Depending on the activity, for example, generally the detergent composition of the present invention comprises 1 g or 1 g of the composition. 10 to 1000, preferably 20 to 500 units of glucose per ml It can be said that it contains sidase. One unit of enzyme activity is 1 μm per minute under standard conditions It is defined as the amount required to convert ol substrate. (D)Other ingredients   The enzymatic antimicrobial composition of the present invention is generally 0.01 to 50% by weight, preferably 0. . 1 to 5.0% by weight of one or more fields It contains a surface-active agent or a wetting agent. Suitable surfactants or cleaning active compounds are Soken or non-soap anionic surfactant, nonionic surfactant, cationic interface Activators, zwitterionic or zwitterionic compounds. Surfactant system is usually one or more Of anionic surfactants and one or more nonionic surfactants. Interactivity The activator system may further comprise an amphoteric or zwitterionic surfactant compound, It is usually undesirable because the compounds are relatively expensive.   Generally, surfactant-based nonionic and anionic surfactants are commercially available from Schwart. Volume 1 of "Surface Active Agents" by z and Perry , Schwartz, Perry and Berch Interscience e 1949, Volume 2, Interscience 1958, Manufact. "McCutche" published by Uring Configurations. The latest version of on's Emulsifiers and Detergents ”, Or "Tenside-Taschenbuch", H.M. Stage, Volume 2, Selected from the surfactants described by Carl Hauser Verlag, 1981. obtain.   Suitable nonionic detergent compounds that can be used include, among others, hydrophobic groups and reactive hydrogen sources. Compounds having offspring, such as aliphatic alcohols, acids, amides or alkylphenos And alkylene oxide, especially ethylene oxide alone or propylene oxide. Included are reaction products with ethylene oxide, including xides. Specific non-ionic detergent The compounds are generally 5 to 25 EO, ie 5 to 25 units of ethylene oxide per molecule. Cide of Cide₆-C_{twenty two}Alkylphenol / ethylene oxide condensate, and aliphatic C₈-C₁₈Primary or secondary straight or branched chain alcohols and generally 5-4 It is a condensation product of 0EO with ethylene oxide.   Suitable anionic detergent compounds that can be used typically contain from about 8 to about 22 carbon atoms. Water-soluble organic sulfuric acid and sulfonic acid alkali metal salt having an alkyl group containing Good. The term alkyl is used to include the alkyl portion of higher acyl groups. It has been. An example of a suitable synthetic anionic detergent compound is sodium alkylsulfate. And potassium, especially higher C produced, for example, from beef tallow or coconut oil₈-C₁₈A Alkyl C, obtained by sulfolating rucor₉-C₂₀Benzenesulfonic acid Thorium and potassium, especially linear second Grade alkyl C_Ten-C_FifteenSodium benzene sulfonate; and beef tallow or coconut oil Higher alcohol ethers derived from petroleum and synthetic alcohols derived from petroleum It is. Preferred anionic detergent compounds are C₁₁-C_FifteenAlkyl benzene sulfone Sodium acid and C₁₂-C₁₈It is sodium alkylsulfate.   Further usable one is EP-A-328177 (Univer). Surfactants exhibiting salting-out resistance such as those described, EP-A-070074. The listed alkyl polyglycoside surfactants and alkyl monoglycosides.   A preferred surfactant system is a combination of anionic and nonionic detersive actives. Mixtures, in particular the anios described in EP-A-346995 (Unilever). And nonionic surfactants and examples thereof. Particularly preferred is C₁₆ -C₁₈Primary alcohol Alkali metal sulfate and C₁₂-C_FifteenPrimary alcohol 3 -7 is a surfactant system that is a mixture with EO ethoxylates.   The nonionic detergent is in an amount greater than 10% by weight of the surfactant system, eg 25-90. It is preferably present in an amount of% by weight. The anionic surfactant is, for example, in an amount ranging from about 5 to about 40% by weight of the surfactant system. May exist.   The enzymatic detergent compositions of the present invention are commonly used in antimicrobial compositions such as thickeners. Other components may be included. Particularly useful in this regard is EP-A-3142. No. 32 (Unilever), which is a combination of surfactants, The combination becomes a thickened gel when dissolved in water.   Use of the antimicrobial composition of the present invention for cleaning hard surfaces and cleaning textiles. Not only does it provide hygiene benefits, but also hygiene and The benefits of cleaning as well as cleaning and disinfection of medical devices are also obtained. Other uses are dairy For disinfection of milking equipment in. The antibacterial composition is capable of combating bacteria that cause an offensive odor. Due to its ability to destroy, it can also be used successfully in deodorants.   The antibacterial composition of the present invention may be used in the form of a powder that is dissolved in water before use It may be formulated as a product or gel. In the product form, when using the composition It is important that the production of hypohalite does not start until It is important. This is done, for example, by encapsulating the enzyme in a known manner, Physically separate from the substrate Can be achieved by   When using the enzymatic antibacterial composition of the present invention, water is added to adjust the composition to 5-100. Diluting twice gives a medium with effective antimicrobial activity. Then erase the medium Touch the surface to be poisoned.   The invention is illustrated in detail by the following non-limiting examples.Example 1 Minimum inhibitory concentration (MIC) of hypochlorite material :   The bacterial strain used in this example was Escherichia coli NCTC. 900, Pseudomonas aeruginosa ATCC 15442 , Staphylococcus aureus ATCC 13565, Str eptococcus faecalis NCTC 1092 and Lister ia innocua ATCC 33090 serotype 6B. Bacteria Rain Heart Infusion (BHI) Broth Medium at 30 ° C for 15-18 hours Propagated. After culturing, citrate buffer pH 5.5 (20 mM Na_ThreeCitric acid / N The bacterial strain was washed twice in aOH buffer + 10 mM NaCl). Bacterial suspension Centrifuge (14,000 rpm for 5 minutes) in a Schoendorf centrifuge and then the supernatant After removal of the bacteria, the bacterial pellet was resuspended in citrate buffer. Then each bacterial suspension This washing procedure was repeated once for the suspension. Then, the bacteria suspension washed twice Dilute the suspension with citrate buffer pH 5.5 to about 10⁷ A suspension of bacteria / ml is obtained. Cells were kept on ice during the wash steps. Buffer solution And BSA solution (1% w / v in citrate buffer pH 5.5) by filter sterilization, Stored at 4 ° C. Dilute the stock solution (107,000 ppm) with sterile deionized water To prepare a hypochlorite solution.Method :   Using sterile technique, citrate buffer pH About 10 out of 5.5⁷A suspension of bacteria / ml was prepared. 1.9 ml aliquot from the suspension The recoat was added to a sterile tube. Then, to obtain the dilution range of hypochlorite, To the tube was added 0.1 ml of cold hypochlorite solution of various concentrations. The tube with a magnetic stirrer It was continuously stirred. In addition, add only sterile buffer instead of the hypochlorite solution. A blank measurement was also performed. The sample was incubated at 30 ° C. for exactly 5 minutes. After the incubation, 1 ml was taken from the reaction mixture and 9 ml of cold BSA (1 % W / v) solution and immediately placed on ice. Now with hypochlorite and microbes Stopped the reaction. 10 samples^-1-10^-FiveDiluted to 100 from various dilutions The μl sample was placed on a labeled BHI-agar plate and colony-forming single cells per ml were formed. Rank (CF The survival rate as U) was measured. The plate is then incubated at 30 ° C for 15-18 hours. Incubated. There is no detectable CFU after this incubation period If not, the plates are incubated at 30 ° C for an additional 24 hours.Definition : The minimum inhibitory concentration (MIC value) is as used herein under the experimental conditions used. Reduce at least log 6 colony forming units of the particular microorganism tested It is defined as the concentration of chlorite.   The results obtained are shown in Table I. Detected values are in the same range as described in the literature . Example 2 Hypochlorite and Curvularia inaequa enzymatically produced by vanadium chloroperoxidase (V-CPO) from lis Minimum inhibitory concentration of grown hypochlorite   In this example, the bactericidal action of hypochlorite was determined by Curvularia ina. vanadium chloroperoxidase (V-CPO) obtained from Equalis To compare with the bactericidal action of hypochlorite produced enzymatically. Enable comparison For this, a micropump was used. This determines the concentration of hypochlorite during the experiment. , To achieve the same profile as the situation in which hypochlorite is produced enzymatically. Was. In addition, in order to know the amount of hypochlorite present at each point in the experiment, the experimental conditions The V-CPO activity was measured very carefully below.material :   The bacterial strain used in this example was Escherichia coli NCTC. 900, Pseudomonas aeruginosa ATCC 15442 , Staphylococcus aureus ATCC 13565, Str eptococcus faecalis NCTC 1092 and Lister ia innocua ATCC It was 33090 serotype 6B. Bacteria, Brain Heart Infusion ( BHI) Broth medium was grown at 30 ° C. for 15-18 hours. After culturing, relax citric acid Buffer pH 5.5 (20 mM Na_ThreeCitric acid / NaOH buffer + 10 mM NaCl ), The bacterial strain was washed twice. Centrifuge the bacterial suspension in an Eppendorf centrifuge ( (14,000 rpm for 5 minutes), then remove the supernatant and then cleave the bacterial pellet. Resuspended in enoic acid buffer. Then repeat this washing procedure for each bacterial suspension. Repeated once. The bacterial suspension washed twice was then citrate buffer pH 5.5. Diluted with about 10⁷A suspension of bacteria / ml was obtained. Keep cells on ice during the wash step Maintained. Buffer and BSA solution (1% w / v in citrate buffer pH 5.5) Filter sterilized and stored at 4 ° C. Aseptic stock solution (107,000 ppm) A hypochlorite solution was prepared by diluting with deionized water. Aseptically remove 30% stock solution Dilute with on-water H₂O₂A solution was prepared. From Difco to Cassitone got one). According to Van Schijndel et al. (1993), Cu Purification of chloroperoxidase from Rvularia inaequalis Was. Using a spectrophotometer, Sodium phosphate buffer pH 5.5, 10 mM NaCl, 100 μM H₂O₂, 5 Monochlorodimedone (e 290 nm = 0 μM monochlorodimedone at 30 ° C.) 20.2 mM^-1. cm^-1) To dichlorodimedone (e 290 nm = 0.2 mM^{- 1} . cm^-1) To Cl,^-To HOCl conversion Loperoxidase activity was measured. 1 unit of chloroperoxidase for 1 minute The amount of enzyme that converts 1 μmol of monochlorodimedone (Sigma) per Define.Method :   About 10 in citrate buffer pH 5.5 using aseptic method⁷Prepare a suspension of bacteria / ml Made. A 1.8 ml aliquot from the suspension was added to a sterile reaction vessel, which was The magnetic stirrer was continuously stirred for the duration of the experiment. Then obtain the dilution range of V-CPO To the reaction vessel at various concentrations (see below for calculation) of V-CPO. 0.2 ml of solution was added. Also, instead of 0.2 ml of V-CPO solution, 0.2 Blank measurements were also performed with the addition of ml of sterile buffer. Incubate the reaction vessel at 30 ° C. Bate, then 0.5 ml H₂O₂Initiate V-CPO reaction by adding stock solution did. Used H₂ O₂Stock solution concentration depends on the concentration of hypochlorite produced, H₂O₂Final concentration of There is a 5-fold molar excess (at the end of the flux) with respect to the final concentration of hypochlorite. Selected to. The sample was incubated at 30 ° C. for exactly 5 minutes. ink After incubation, 1 ml of reaction mixture was removed and 9 ml of cold BSA (1% w / v) Added to the solution and immediately placed on ice. Now, the reaction between hypochlorite and microorganisms Stopped. 10 samples^-1-10^-FiveTo 100 μl from various dilutions. Material on labeled BHI-agar plates and colony forming units (C The survival rate as FU) was measured. Then plate at 30 ° C. for 15-18 hours Incubated. There is detectable CFU after this incubation period If not, the plates are incubated at 30 ° C for an additional 24 hours.   When using a micropump and adding the same amount of hypochlorite to the obtained MIC value It was compared with the MIC value obtained in. This was done as follows.   About 10 in citrate buffer pH 5.5 using aseptic method⁷Prepare a suspension of bacteria / ml Made. Add a 1.8 ml aliquot from this suspension to a sterile reaction vessel and add During the experiment Stir continuously with a magnetic stirrer. Incubate the reaction vessel at 30 ° C, then , 0.2 ml H₂O₂Stock solution was added. Used H₂O₂Stock solution concentration is H depends on the hypochlorite concentration₂O₂The final concentration of (at the end of the flux) is , A 5-fold molar excess with respect to the final concentration of hypochlorite. Then , 0.5 ml of a flux of hypochlorite solution of known concentration at 30 ° C over 5 minutes (Flow rate: 0.1 ml / min). Various hypochlorite solutions with different concentrations A series of experiments were performed using the solution to obtain the final concentration range of hypochlorite (for calculation See below). After incubating for 5 minutes, remove 1 ml of reaction mixture It was then added to 9 ml of cold BSA (1% w / v) solution and immediately placed on ice. with this , The reaction between the hypochlorite and the microorganism was stopped. 10 samples^-1-10^-FiveDiluted to 100 μl samples from various dilutions were loaded onto labeled BHI-agar plates for 1 m Viability as colony forming units (CFU) per liter was measured. Then, The rates were incubated at 30 ° C for 15-18 hours. This incubation If no detectable CFU was present after the period, plate at 30 ° C. Incubate for 24 hours.   Identical experimental setup incorporated into the spectrophotometer (in the case of blank experiments, (Not added), the hypochlorite concentration within a certain period of time  290nm = 20.2mM^-1・ Cm^-1) To dichlorodimedone (e 290 nm = 0.2 mM^-1・ Cm^-1) To observe at 290 nm From V-CPO and hypochlorite from stock solutions, respectively, in the above experiment. The similarity of the resulting hypochlorite profiles was confirmed.Calculation :   The final concentration of hypochlorite produced by V-CPO was calculated as follows: :   When 0.01 U / ml V-CPO was used, 0 added to the initial volume of 2.0 ml. . 0.05 μmol hyposulfite per 5 minutes due to the dilution effect of 5 ml of flux 0.01 μmol hypochlorite / ml / min is produced corresponding to chlorate / ml . The final concentration after 5 minutes is (2.0 / 2.5) * 0.05 μmol / ml = after 5 minutes 0.04 μmol of hypochlorite / ml. This concentration is then adjusted to 0.0 4 μmol hypochlorite / ml = 0.04 * 52.5 ppm hypochlorite = 2. It can be expressed as 10 ppm hypochlorite. V-C The PO concentration was increased or decreased to obtain final concentrations of other hypochlorites. Added hypochlorite The final concentration of was calculated as follows:   Since 0.5 ml of flux is added to 2.0 ml of reaction volume, the final volume is 2 . 5 ml, which corresponds to a 5-fold dilution. 2.10 ppm hypochlorite A stock solution of 10.50 ppm was used to obtain the final concentration. Hypochlorite storage Other hypochlorite concentrations were obtained by increasing or decreasing the stock solution.Definition : The minimum inhibitory concentration (MIC value) is as used herein under the experimental conditions used. Reduce at least log 6 colony forming units of the particular microorganism tested It is defined as the concentration of chlorite.   The results are shown in Table II. Produced by V-CPO or hypochlorite flakes To compare the bactericidal activity of different final concentrations of hypochlorite produced at the end of You. As we can conclude from Table II, hypothalias already enzymatically produced by V-CPO. The chlorate has a low hypochlorite concentration of about 0.4 ppm, and thus Staphyloco Completely inhibits the growth of all tested microorganisms except ccus aureus In contrast, in Staphylococcus aureus, similar growth inhibition was observed. 2ppm hypochlorite is required to obtain I need it. These results indicate that vanadium-containing haloperoxidase Demonstrated to be a more effective hygiene ingredient than predicted from chlorate production alone ing. Furthermore, all tested V-CPO, whether pathogenic or non-pathogenic Heme-containing haloperoxidase, which kills microorganisms in It is clear that it only sterilizes only the rice efficiently (EP-A-500387). No.). Example 3 Enzymatically produced by V-CPO in the presence of hypochlorite and protein hydrolysates Of sterilized hypochlorite   To obtain hygiene, under actual circumstances, under-dosing hypochlorite It is known that giving is necessary. This is because the reactive molecule not only reacts with the microorganism, but also with other compounds present. Because it reacts with. Thus, for example, the bar in a liquid containing protein hydrolyzate It was important to test the behavior of nadium-containing haloperoxidase.material :   The bacterial strain used in this example was Escherichia coli NCTC. It was 900. Brain Heart Infusion (BHI) Broth Culture It was grown at 30 ° C. in the ground for 15 to 18 hours. After culturing, citrate buffer pH 5.5 ( 20 mM Na_Three2 bacterial strains in citric acid / NaOH buffer + 10 mM NaCl) Washed twice. Centrifuge the bacterial suspension in an Eppendorf centrifuge (14,000 rp m) for 5 minutes, and then, after removing the supernatant, the bacterial pellet was resuspended in citrate buffer. Suspended. This washing procedure was then repeated once for each bacterial suspension. The twice-washed bacterial suspension was then diluted with citrate buffer pH 5.5 to about 1 0⁸A suspension of bacteria / ml was obtained. Cells were kept on ice during the wash steps. Buffer And BSA solution (1% w / v in citrate buffer pH 5.5) by filter sterilization Store at 4 ° C Was. Stock solution (107,000ppm) was diluted with sterile deionized water to prepare hypochlorous acid. A salt solution was prepared. Dilute a 30% stock solution with sterile deionized water to remove H₂O₂Prepare the solution Made. Cassitone was obtained from Difco. Van Sijjndel et al., 19 According to 93 from Chlorvularia inaequalis The sidase was isolated and purified.Method :   About 10 in citrate buffer pH 5.5 using aseptic method⁸Prepare a suspension of bacteria / ml Made. A 1.3 ml aliquot from the suspension was added to a sterile reaction vessel, which was The magnetic stirrer was continuously stirred for the duration of the experiment. Then 0.5 ml of 0.5 mg / ml Cassitone (in citrate buffer pH 5.5) solution and 0.5 ml citric acid Buffer pH 5.5 was added. Then, 3.2 ppm or 6.5 ppm of hypochlorous acid Different concentrations (see Example 2 for calculations) to obtain the final concentration of acid salt. 0.2 ml of the V-CPO solution of was added to the reaction vessel. Also, 0.2 ml of V-CP A blank measurement was also performed in which 0.2 ml of sterile buffer was added instead of the O solution. reaction Incubate the container at 30 ° C., then add 0.5 ml H 2₂O₂Stock solution (5 times Flux is a molar excess of hydrogen peroxide The concentration was chosen so that it was obtained at the end of the) and the V-CPO reaction was initiated. . The sample was incubated at 30 ° C. for exactly 5 minutes. After incubation, 1 ml of reaction mixture was removed and added to 9 ml of cold BSA (1% w / v) solution, Immediately placed on ice. This stopped the reaction between the hypochlorite and the microorganism. sample 10^-1-10^-6100 μl samples from various dilutions are labeled BHI- Plate as a colony forming unit (CFU) per ml on agar plates The survival rate was measured. The plates were then incubated at 30 ° C for 15-18 hours Was. If there was no detectable CFU after this incubation period Incubate the plate at 30 ° C. for an additional 24 hours.   When the same amount of hypochlorite was added to the obtained sterilization efficiency using a micropump It was compared with the sterilization efficiency obtained in. This was done as follows.   About 10 in citrate buffer pH 5.5 using aseptic method⁸Prepare a suspension of bacteria / ml Made. Add a 1.3 ml aliquot from this suspension to a sterile reaction vessel and add this The magnetic stirrer was continuously stirred for the duration of the experiment. Then 0.5 ml of 0. 5 mg / ml Cassitone solution (in citrate buffer pH 5.5) and 0.5 ml Citrate buffer pH 5.5 was added. Then 0.2 ml of sterile citrate buffer pH 5.5 was added. The reaction vessels were incubated at 30 ° C and then each Final concentration of hypochlorite of 3.2ppm and 6.5ppm (concentration calculation To obtain a mixture of 0.5 ml of hypochlorite solution, Was added at 30 ° C. over 5 minutes (flow rate: 0.1 ml / min). Incubate for 5 minutes 1 ml of reaction mixture was taken out and dissolved in 9 ml of cold BSA (1% w / v). It was added to the liquid and immediately placed on ice. This stops the reaction between hypochlorite and microorganisms did. 10 samples^-1-10^-6100 μl samples of various dilutions are labeled B Place on HI-agar plate to make colony forming units (CFU) per ml The survival rate was measured. The plate is then incubated at 30 ° C for 15-18 hours. I started. There was no detectable CFU after this incubation period In some cases, plates are incubated at 30 ° C. for an additional 24 hours. The results are shown in Table II. Shown in I. Example 4 Bactericidal efficacy of vanadium-containing haloperoxidase and heme-containing haloperoxidase Rate comparison material :   The bacterial strain used in this example was Escherichia coli NCTC. 900, Streptococcus faecalis NCTC 1092 and And Listeria innocua ATCC 33090 serotype 6B. Was. Bacteria in Brain Heart Infusion (BHI) Broth at 30 ° C Allowed to grow for 15-18 hours. After culturing, citrate buffer pH 5.5 (20 mM Na_Three Bacterial strains were washed twice in citrate / NaOH buffer + 10 mM NaCl). Centrifuge the bacterial suspension using an Eppendorf centrifuge. Bacterial pellet after hearting (5 min at 14,000 rpm) and then removing the supernatant Was resuspended in citrate buffer. This washing procedure is then carried out for each bacterial suspension. Repeated once more. The bacterial suspension washed twice was then citrate buffer pH 5 . Dilute with 5 to about 10⁷A suspension of bacteria / ml was obtained. Ice cells during the wash step Kept on. Buffer and BSA solution (1% w / v in citrate buffer pH 5.5) ) Was filter sterilized and stored at 4 ° C.   Curvularia according to Van Schijndel et al. (1993). Chloroperoxidase was purified from inaequalis. Heme-containing chloro Peroxidase was added to Sigma (Caldariomyces fumago) I came from). Spectrophotometer, 20 mM sodium citrate buffer pH 5.5 10 mM NaCl, 100 μM H₂O₂, 50 μM 3 in monochlorodimedone Monochlorodimedone (e 290 nm = 20.2 mM at 0 ° C)^-1・ Cm^-1) From Dichlorodimedone (e 290 nm = 0.2 mM^-1/ Cm^-1) To the conversion Guess Cl^-Of chloroperoxidase activity in the conversion of HO to HOCl Specified. 1 unit of chloroperoxidase hits for 1 minute Defined as the amount of enzyme that converts 1 μmol of monochlorodimedone (Sigma) You. The same assay for both enzymes (and so that the two enzymes with equal activity can be administered) And the definition of activity) were used.Method :   Using aseptic method, 20 mM sodium citrate buffer pH 5.5, 10 mM N About 10 in aCl⁷A suspension of bacteria / ml was prepared. 1.9 ml aliquot from the suspension The recoat was added to a sterile tube. Then, each vanadium-containing chloroperoxy Add 0.1 ml from stock solution of chloroperoxidase containing dase and heme to the tube In addition, a dilution range was obtained for both enzymes. Also, instead of the enzyme stock solution, A blank measurement in which only the bacterial buffer was added was also performed. Then 0.5 ml of 25 mM H₂O₂Stock solution was added. The sample was incubated at 30 ° C. for exactly 5 minutes. After incubation, 1 ml of reaction mixture was removed and 9 ml of cold BSA (1 % W / v) solution and immediately placed on ice. 10 samples^-1-10^-FiveDiluted to 100 μl samples from various dilutions were loaded onto labeled BHI-agar plates for 1 m Viability was measured as colony forming units (CFU) per liter Was. The plates were then incubated at 30 ° C for 15-18 hours. This inn If no detectable CFU was present after the cubation period, plate Are incubated at 30 ° C. for a further 24 hours.   The results are shown in Table IV. The data show that vanadium-containing chloroperoxidase It is far more effective than heme-containing chloroperoxidase even when administered isocratically. It clearly shows that it provides a living system. Example 5 Effect of various hydrogen peroxide sources on the blocking action of V-CPO   In the examples so far, hydrogen peroxide, which is one of the substrates in the V-CPO reaction, is used. Was added from a stock solution, but in this example the effect of using other hydrogen peroxide sources was Describe the fruit. Citrate buffer pH 5.5 (20 mM Na as a buffer)_ThreeQuen Acid / NaOH buffer + 10 mM NaCl) to monitor oxygen consumption. Physical Oxygen Monitor (Biological Oxygen Monitor) Y Oxidase in SI model 5300 (oxygen chamber model 5301) at 30 ° C Was assayed. The buffer was saturated with air at 30 ° C. For glucose oxidase As a substrate of 15 g. 1^-1(Final concentration) glucose was used. In this example The bacterial strain used was Escherichia coli NCTC 900. . Bacteria were incubated in Brain Heart Infusion (BHI) Broth medium at 30 ° C for 1 hour. Allowed to grow for 5-18 hours. After culturing, citrate buffer pH 5.5 (20 mM Na_Three Bacterial strains were washed twice in citrate / NaOH buffer + 10 mM NaCl). Eppendorf centrifugation of bacterial suspension Centrifuge in a separator (14,000 rpm for 5 minutes), then remove the supernatant and The bacterial pellet was resuspended in citrate buffer. Then for each bacterial suspension The washing procedure was repeated once. The bacterial suspension, which had been washed twice, was then washed with citric acid. Approximately 10 after diluting with a pH 5.5⁸A suspension of bacteria / ml was obtained. During the cleaning phase , Cells were kept on ice. Buffer and BSA solution (in citrate buffer pH 5.5) 1% w / v) was filter sterilized and stored at 4 ° C. Stock solution (107,000) ppm) was diluted with sterile deionized water to prepare a hypochlorite solution. 30% storage Dilute the solution with sterile deionized water and add H₂O₂A solution was prepared. Oak from Difco Got the tone. Curv according to Van Schijndel et al. (1993). Isolation and purification of chloroperoxidase from ularia inaequalis did. Glucose oxidase derived from Aspergillus niger Obtained from igma.Method :   Aseptic method, about 10 in citrate buffer pH 5.5⁸Bacteria / ml suspension Prepared. Add a 1.3 ml aliquot from the suspension to a sterile reaction vessel and add During the experiment Stir continuously with a magnetic stirrer. Then 0.5 ml of 0.5 mg / ml casito Solution (in citrate buffer pH 5.5) was added. Then add 0.2 to the reaction vessel. ml of V-CPO solution was added to give a final concentration of 6.5 ppm hypochlorite. Also, 0.2 ml of sterile buffer solution was added instead of 0.2 ml of V-CPO solution. A blank measurement was also performed. The reaction vessel was incubated at 30 ° C and then 0.5 ml of one of the three hydrogen peroxide generating systems was added: H from 1.3 mM stock solution₂O₂; Sodium percarbonate from a 2.3 mM stock solution; 3. Glucose oxidase (0.39 units / ml) and 75 mg / ml gluco A mixture of sugars.   The sample was incubated at 30 ° C. for exactly 5 minutes. After incubation, 1 ml of reaction mixture was removed and added to 9 ml of cold BSA (1% w / v) solution, Immediately placed on ice. This stopped the reaction between the hypochlorite and the microorganism. Sample 10^-1-10^-6100 μl samples from various dilutions are labeled BHI-cold. Survival as colony forming units (CFU) per ml on top plates The rate was measured. The plate is then incubated at 30 ° C for 15-18 hours. Incubated. There is no detectable CFU after this incubation period If not, the plates are incubated at 30 ° C for an additional 24 hours. Ma The value obtained was measured using an icro pump when exactly the same amount of hypochlorite was added. It was compared with the MIC value obtained. This was done as follows.   Aseptic method, about 10 in citrate buffer pH 5.5⁸Bacteria / ml suspension Prepared. Add a 1.3 ml aliquot from the suspension to a sterile reaction vessel and add The magnetic stirrer was continuously stirred during the experiment. Then 0.5 ml of Kasitone A solution (0.5 mg / ml in citrate buffer pH 5.5) was added. Then, 0. 2 ml of sterile citrate buffer pH 5.5 was added. Incubate the reaction vessel at 30 ° C. I got it. Then 32.5 ppm hypochlorite solution 0.5 ml flack Was added at 30 ° C. over 5 minutes (flow rate: 0.1 ml / min). Incubate for 5 minutes 1 ml of reaction mixture was taken out and dissolved in 9 ml of cold BSA (1% w / v). It was added to the liquid and immediately placed on ice. This will stop the reaction between the hypochlorite and the microorganisms. Was. 10 samples^-1-10^-6Label 100 μl samples from various dilutions BHI- Plate as a colony forming unit (CFU) per ml on agar plates The survival rate was measured. The plates were then incubated at 30 ° C for 15-18 hours Was. If there was no detectable CFU after this incubation period Incubate the plate at 30 ° C. for an additional 24 hours. The results are shown in Table V. . Example 6 Chloroperoxidase gene (cDNA) and Curvularia ina equalis (Central Bureau voor Simmel cultures, the Netherlands, strain no. 102. 42) Method for determining the coding sequence of the gene obtained as well as possible expression systems   C. V. Schijndel et al. (1993). inaequa Chloroperoxidase was isolated and purified from a liquid culture of Lis, except that D After EAE chromatography, FPLC system (Pharmacia LKB) Was used to perform a two-step additional purification. First, phenyl sepharose Cl-4 Using a B hydrophobic interaction column, 50 mM Tris- in the presence of 2M NaCl. Enzyme was bound in HCl (pH 8.3), then 50 mM Tris-HCl. Elution with a decreasing gradient from 2M NaCl in (pH 8.3). As final purification, MonoQ HR 5/5 anion exchange column (Pharmacia LKB) To bind the enzyme, then 20 mM piperazine-HCl (pH 5.4) Eluted with a 0 M to 0.5 M NaCl gradient in. Then, the rotary evaporation method (rotat Ion evaporation) to concentrate the enzyme, then 50 mM T ris-SO_FourIt was dialyzed against a buffer solution (pH 8). Standard hands known in the art According to the order, purified chloroperoxidase was added to protease Stap hylococcu enzymatically digested with sV8 and trypsin, or CNBr [Gross, E . (1967), Methods Enzymology 11, 238-255. ], It chemically cut. Laemmli [Laemmli, U .; K. (1970 ) Nature 227, 680-685], using SDS-PAGE. Or according to Schagger and Von Jagow (1987). After isolation of the resulting peptide on a gel, Matsudoira (1987) ), The CAPS transfer buffer (10). mM 3- [cyclohexylamino] -1-propanesulfonic acid, 10% methano PVDF membrane (Millipore Immobilo) n-P). After electrophoretic elution, the membrane is rinsed with deionized water for 5 minutes, 50 Stained with 0.1% Coomassie Blue R-250 in 5% methanol for 5 minutes, 50% It was decolorized in methanol, 10% acetic acid at room temperature for 10 minutes and air dried. Peptide band And a Porton LF 3000 protein sequencer (Beckman I nstruments, Inc. , USA) Fully automatic Edmann sequence I went to Thing. The results of the amino acid sequencing are summarized in Figure 1.   Design all degenerate oligonucleotides based on the amino acid sequence of the peptide (See Table VII). Use these degenerate primers as the template for the first strand cDNA It was used for polymerase chain reaction (PCR). The first strand cDNA is as follows Created:   To isolate RNA, C.I. 1 liter of spores of inaequalis 4 g of yeast extract and 2 ml of a trace ingredient solution (Van Schijndel Et al., 1993). After a few days of growth, the mycelium was filtered Harvested and freeze dried. Lyophilized C.I. inaequalis mycelium in liquid nitrogen Ground down. RNA extraction buffer (42 mM sodium citrate pH 7, 0. 83% N-lauryl sarcosine, 50 mM β-mercaptoethanol, 1% Riton X-100 and 4M guanidine isothiocyanate) was added to Extracted and incubated at room temperature for 1 hour. 0.1 volume of 2M sodium acetate ( pH 4) and 1 volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) was added and the mixture was placed on ice for 15 minutes. 10,000 × g (4 ℃) After centrifuging at 10 minutes, collect the aqueous phase, add 1 volume of anhydrous alcohol and mix. Incubate for 1 hour at -20 ° C, then centrifuge briefly at 10,000 xg did. The pellet was resuspended in an appropriate amount of RNA extraction buffer and a cesium chloride gradient (Sa Fractionation by ultracentrifugation in mbrook et al., 1989). Wash pellets carefully It was purified and stored in 75% ethanol solution at -70 ° C. to isolate mRNA RNA was then precipitated, resuspended in RNAse-free water, and then PolyAt ract mRNA isolation kit (Promega Corporation, US MRNA was extracted using A). Pharamacia First Strand cDNA Synthesis Key (Pharmacia Biotech) and C.I. inaequali First-strand cDNA synthesis was performed on mRNA isolated from s. Chloroperoxida 4 20-mer degeneracy based on the amino acid sequence of the enzyme peptide (see also Table VII). An oligonucleotide was designed and used as a template for C.I. No. obtained from inaequalis It was used as a primer in the polymerase chain reaction using single-stranded cDNA. Thermo Cycler (Eppendorf Master Cycler 5330) and Taq Poly Polymerase sequence using merase (Promega Corporation) The chain reaction was carried out. Use degenerate primer To obtain optimal amplification of the cDNA encoding chloroperoxidase. Limerase chain reaction was carried out at 46 ° C. (annealing step) for 30 cycles. Profit The two specific fragments obtained were ligated into the pUC18 vector, cloned and And sequenced from both strands. Based on the results of the DNA sequence, the following two features are Designed constant primers: as well as   Polymerase using these two primers and first strand cDNA as template Used for chain reaction. In this way, gene features that bind to two known DNA sequences A foreign DNA fragment was obtained. This fragment was cloned into pUC18 vector. After sequencing, it was sequenced. Of the mRNA encoding chloroperoxidase To obtain the 5'region, the 5'-Amplifier RACE kit (Cl Onetech Corporation) was used. C. inaequali The genomic chloroperoxidase gene was isolated from s as follows:   C. inaequalis genomic DNA was isolated from freeze-dried mycelium and Grinding under a vacuum, a proper amount of extraction buffer (200 mM Tris-HCl, pH 8.5, 25 mM EDTA, 250 mM NaCl, 1% SDS and 0.2 mg / ml Extracted with proteinase K). After overnight incubation at room temperature, Phenol and 0.3 volume of chloroform were added and vigorously stirred. Tube for 10,000 Centrifuge at 0xg and transfer the aqueous phase to a clean tube. Genomic DN with 2 volumes of absolute ethanol A was allowed to settle. After centrifugation at 5,000 xg for 5 minutes, the pellet is resuspended in 2 ml of 1 ml. Resuspended in 0 mM Tris-HCl, pH 8.0 and 1 mM EDTA for manufacturing With RNAse (Boehringer Mannheim) according to the recommendation did. A solution containing genomic DNA is phenol: chloroform: isoamyl alcohol. (25: 24: 1) and precipitated with ethanol. It was dissolved in 0 mM Tris-HCl pH 8, 1 mM EDTA buffer. genome To carry out a Southern analysis of DNA, the DNA was combined with several restriction enzyme combinations. Digested and blotted on nitrocellulose membrane after agarose gel electrophoresis (Sa mbrook et al., 1989).   α-³²Random Plot Using P-Labeled dATP (Sambrook et al., 1989) Created by liming (amplified using the above two specific primers) Hybridization was performed using a radiolabeled gene-specific fragment. Was. Based on the results obtained, it was digested with Pst I and inserted into the vector pUC18 A mini library was prepared using the genomic DNA. Suther the library Screened with the same probes as described for immunoblotting . Positive clones were isolated and partially sequenced from both strands to obtain cDNA The sequence results were confirmed. C. inaequalis chloroperoxidase The gene to be cloned and its putative gene product are shown in FIG.   In Saccharomyces cerevisiae as follows A chloroperoxidase production system was created.   S. cerevisiae wild type GAL1 gene (Molecular an d Cellular Biology 10, 4757-4769, 1990). As an EcoRI-BamHI fragment from the well-known GAL1-inducible yeast plasmid. Obtained the lomotor and designated it as plasmid YC plac33 and YEplac95 [Gietz and Sugino (1988) Gene 74,527-534] at the EcoRI-BamH1 site. did. The obtained plasmids were named TNT1 and TNT2, respectively. pUC1 Subcloned into C. inaequalis chloroperoxidase inheritance Using the PstI EcoRI 5'fragment of the offspring as a template and as a primer M13 / pUC22mer reverse sequence primer and the following primers: PCR experiments were performed using C. inaequalis chloroperoxidase A BamHI restriction site was formed before the 5'start site of the gene.   The amplified fragment was digested with BamHI and EcoRI. C. inae EcoRI containing the 3'portion from the qualis chloroperoxidase gene -The PvuII fragment was subcloned into EcoRI SmaI digested pUC18. It has become After digesting this clone with EcoRI and XbaI, the clone To C. inaequalis contains the 3'portion of the chloroperoxidase gene flag The menthol was purified.   TNT1 or TNT2 digested with BamHI and XbaI, respectively, and 5 'BamHI-EcoRI fragment and 3'EcoRI-XbaI fragment A three-part ligation reaction was performed. Obtained after ligation and cloning The plasmid was identified. Each of the thus obtained plasmids was TN They were named T3 (derived from TNT1) and TNT4 (derived from TNT2).   Yeast strain BJ1991 was transformed into plasmid TNT3 according to procedures known in the art. And TNT4 and selected for ura + transformants. ura + YP-prey containing transformants containing glucose (2%) or galactose (2%) Duplicated. After growth, a few cells were removed from the plate and 200 μl of 2 Resuspended in 0 mM Tris-HCl pH 8.1. Incubated for 5 minutes After that, 10 μl was taken from the solution and spotted on a nitrocellulose filter. Was. Use a nitrocellulose filter with 100 mM sodium acetate buffer (pH 5). . 5) 1 mM orthodianisidine, 100 mM KBr and 2 mM H₂O₂During ~ Incubated at. Galactose forms a transparent color It was found in all spots derived from growing yeast, whereas it was found in glucose-producing yeast. I couldn't. This is the yeast Saccharomyces cerevisia Curvularia inaequalis chloroperoxidase It shows that a galactose-inducible production system of genes has been constructed. 100 mM Potassium phosphate buffer (pH 6.5), 100 mM KBr, 1 mM H₂O₂as well as In a similar assay with 40 μM Phenol Red (BDH) A blue-purple color was formed in the solution obtained from the yeast that grew with the glucose, whereas glucose It was clearly shown that the solution obtained from the cultured yeast did not undergo a color change. yeast Heterologous chloroperoxidase gene expression system in C. inaequalis C. which has the same functionality as loroperoxidase. inaequalis chlorope In order to further confirm that it produced ruoxidase, galactose-induced T Recombinant enzymes were purified from yeast strains transformed with NT3 or TNT4. Galacto Yeast cells were harvested after growth in medium containing glucose and 20 mM Tris-HC 1 (pH 8.1). Then add sterile glass beads and intensify the suspension. It was shaken. 15 minutes at 10,000 xg After the preparation, the supernatant was taken and added to the DEAE column, and wild type C. inaequali recombinant enzyme using substantially the same purification protocol as for the s enzyme (see above). Purified. After purification, the specific activity of 22 U / mg protein (100 mM sodium acetate Um buffer pH 5.0, 1 mM H₂O₂5 mM potassium chloride and 50 μM M Recombinant with measurements in CD, see also Van Schijndel et al., 1993) Obtained chloroperoxidase. The activity is C. inaequalis itself Specific activity obtained with chloroperoxidase purified from Very similar to 20 U / mg protein. Wild type chloroperoxider PH activity profile of recombinant chloroperoxidase derived from zebrafish and yeast Is shown in FIG. FIG. 3 shows that the recombinant yeast-produced enzyme has the same functionality as the wild-type enzyme. Further demonstrates thatExample 7 Screening for suitable haloperoxidases in other microorganisms   The microorganism used in this example was Curvularia inaequalis. (CBS 102.42), Drech slera biseptata (CBS 371.72), Drechsler a fugax (CBS 509.77), Drechslera nicoti ae (CBS 655.74), Drechslera subpapendor fii (656.74), Embelisia hyacinthi (416. 71), Embelisia didymospora (CBS 766), Ul. ocladium chartarum (200.67) and Ulocladi um botrytis (452.72). Various fungi on agar plate Propagate with. When the growth is completed, the extracellular protein is added to 50 mM Tri in advance. Nitrocellulose fill moistened in s-HCl buffer (pH 8.3) Transfer to the target (replica blotting). Incubate for 15 minutes on agar plate And 100 mM sodium acetate in the presence or absence of 0.1 M potassium bromide. Thorium (pH 5.5) or potassium phosphate (pH 6.5 and 7.5), 1 mM  Dip the filter in orthodianisidine, 2 mM hydrogen peroxide, and The filters were tested for luoxidase activity. In this way, bromo Production of peroxidase and / or chloroperoxidase Raw can be detected. The haloperoxidase produced was vanadium-containing haloperoxidase. To test for oxidase, 10 μM and 100 μM vana The above test was repeated in the presence and absence of sodium dinate. Contains vanadium In the case of haloperoxidase, the addition of vanadate increases the signal. Admitted. The identified chloroperoxidase is actually C.I. inaequal Test for similarity to vanadium haloperoxidase obtained from is. To strike, Ulocladium chartarum, Embelis ia didymospora and Drechslera Subpapendo A small amount of chloroperoxidase was purified from rfii (Van Schijn (substantially the same as described by del et al., 1993). The optimum pH for these enzymes is , PH fluctuated in the range of 4.5 to 5.5. The chlorinating activity of the enzyme It increased with the addition of the enzyme because the enzyme was actually a vanadium haloperoxide. It is clearly shown that it is ZE. The identified enzyme and C.I. inaequalis Identification to further test its similarity to vanadium chloroperoxidase Type of haloperoxidase Was further characterized. For this purpose, the fungus Drechs lera bisep chloroperoxidase produced by Tata (CBS 371.72) Selected. The chloroperoxidase is highly thermostable and highly stable to its substrate. Chloroperioa from Curvularia inaequalis with affinity It has similar properties to xydase. In addition, the EPR spectrum of the purified enzyme Toll was also recorded. Other vanadium haloperoxidases (de Boer et al., 1 988; Wever et al., 1988), oxidases are observed in EPR strains. It does not show any vector, but when reduced with sodium dithionite, it shows typical A Nazil EPR spectrum was observed (data not shown). g₀Is 1.969 , A₀Is 9.0 mT isotropic EPR parameter is C.I. inaequalis (Wever et al., 1985). In addition, the purified enzyme was separated into peptides using protease and cyanogen bromide. Released. The peptide maps show the same pattern, which indicates that these two enzymes It suggests that the two have high sequence homology. This is actually a protease Processed and purified That is, two separate peptides of the isolated enzyme were sequenced. these The sequences show very high homology, so these two enzymes are very similar. Can be concluded.   C. Amino acid sequence of the peptide derived from inaequalis: (Asp) -leu-arg-gln-pro-tyr-asp-pro-th r-ala-pro-ile-glu-asp-gln-pro-gly-il e-val-arg-thr-   D. Amino acid sequence of a similar peptide obtained from biseptata: asp-leu-arg-gln-pro-tyr-asp-pro-thr- ala-pro-ile-glu-glu-gln-pro-gly-ile- val-arg-thr-   Therefore, a suitable vanadium-containing haloperoxidase is suitable after addition of vanadate. And / or (partially) purifying the enzyme using a replica method to test for increased activity of Can be identified by making and examining the increase in activity after addition of vanadate.Example 8 Further screening of suitable chloroperoxidases in other microorganisms using antibodies. Earning   The strain used in this example was Curvularia inaequalis (C BS 102.42), Drechsler biseptata (CBS 3 71.72), Drechslera subpapendorfii (CBS 656.74), Embellissia didymospora (CBS 7). 66.79) and Ulocladium chartarum (CBS200. 67).   The microorganism was grown in two stages. In the first stage, 50 ml of sterile germination medium (Va n Schijndel et al., 1993) was inoculated with a spore population of the microorganism. 2 After shaking at 3 ° C. for 3 days, the culture was treated with 1 liter of fermentation medium [1 liter of deionized water. In tor, 5 g casein hydrolyzate (Gibco BRL), 3 g yeast extract and 1 g of fructose] was transferred to a 3 liter Erlenmeyer flask. Shake at 23 ° C The collected medium was collected after 14 to 17 days, filtered, and substantially Van Schijnd. Chloroperoxidase was purified according to el et al. (1994). (2 months old Female) in a rabbit, C purified from Urvularia inaequalis (Van Schijn del et al., 1994) produced polyclonal antibodies against chloroperoxidase. Born (Freund's complete adjuvant for the first injection, booster injection Used Freund's incomplete adjuvant). 6 days after the last booster injection I bled the rabbit. Heat serum at 56 ° C for 30 minutes to inactivate complement, then I centrifuge. The supernatant was removed. Purified chloroperoxidase and A, respectively Bromoperoxidase from Scophylum nodosum (Weber Et al., 1985), 50 μl of each protein (about 0.1 mg / Ml) was prepared and each sample was serially diluted 2-fold. Dot Diluted solution on a nitrocellulose filter using a lot machine (Bio-Rad) Spots, wash with 2% BSA, and then sequentially extract rabbit anti-chloroperoxidase. Antiserum (dilution ratio 1: 800), biotinylated goat anti-rabbit (dilution ratio 1: 3000) ), Alkaline phosphatase-conjugated streptavidin (dilution ratio 1: 2000) and And color formers [5-bromo-4-chloro-3-indolyl phosphate, 4-Nit roBlue Tetrazolium Chloride (Boehringer Mannheim)] did. All steps were performed according to standard protocols. The results obtained are shown in Table VI. You.   Based on the results shown in Table VI, C.I. inaequalis Chloroperoki Immunoassays using antibodies raised against sidase have been performed with other suitable vanadies. It can be concluded that it is suitable for the identification of halo-containing haloperoxidases. These halos Peroxidase may be in crude or partially or fully purified form. You may. Purification methods include gel filtration, ion exchange chromatography, hydrophobic Action chromatography, sedimentation method, (ultra) filtration method, Affini No known in the art such as T-chromatography, gel electrophoresis etc. The offset method may be used.Example 9 Further screening for suitable chloroperoxidases in other microorganisms   In this example, the chlorope from Curvularia inaequalis Of other microorganisms using radioactive probes derived from the A method for detecting a seed gene is described. This was done as follows.   C. inaequalis (CBS 102.42), Embelissia didymospora (CBS 766.79) and Drechslera b The chromosomal DNA from iseptata (CBS 371.72) is substantially . inaequalis as described for chromosomal DNA (see Example 6). Purified). Several types of DNA for Southern analysis of genomic DNA Digested with a combination of restriction enzymes and subjected to agarose gel electrophoresis, followed by nitrocellulose Membranes (Sambrook et al., 1989) were blotted. α-³²P-labeled dATP Produced by random priming used (Sambrook et al., 1989) Hybridization was performed using a radiolabeled gene-specific fragment. Was. The gene-specific fragment used was used as a template for the first-strand cDNA (Example 6). See below) and use the following primers: Was amplified by polymerase chain reaction (before radiolabeling).   Hybridization conditions were as follows.   6 * SS in prehybridization and hybridization PE, 5 * Denhardts, 0.5% SDS and 10 mg salmon sperm DNA Was used as a buffer. Pre-hybridize at 55 ° C for 1 hour and then Then, the radioactive probe was boiled for 1 minute and added as it was. After that, the hybrid The iza- tion continued overnight. Curvularia inaequalis and And the autora obtained using the DNA of Drechler biseptata The geograph is shown in FIG. In the figure, lane 1: λDNA; lane 2: non-quenched C. inaequalis genomic DNA; lane 3: the digested with EcoRI DNA; lane 4: said DNA digested with BanHi; lane 5: EcoRI and BamHI digested DNA; Lane 6: XbaI digested DNA; 7: the DNA digested with PstI; lane 8: digested with XbaI and PstI Lanes 9 to 14: D. Same as above using biseptata It is something. As can be seen in the figure, Drechslera bisepta A chromosomal DNA from ta gives a positive signal, which is high in both genes. The degree of similarity is shown. From Embellisia didymospora Similar results were obtained with the obtained DNA. Therefore, the chloro derived from the gene Peroxidase gene or part thereof or C.I. inaequalis chloro From other microorganisms using a probe based on the peroxidase gene sequence It was concluded that it was possible to detect the appropriate vanadium haloperoxidase in obtain.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI // (C12N 15/09 ZNA C12R 1: 645) (81) Designated country EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, MW, SD, SZ, UG), AM, AT, AU, BB, BG, BR, BY, CA, CH, CN, CZ, DE, DK, EE, ES , FI, GB, GE, HU, IS, JP, KE, KG, KP, KR, KZ, LK, LR, LT, LU, LV, MD, MG, MN, MW, MX, NL, NO NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TT, UA, UG, US, UZ, VN (72) Inventor Simmons, Lambertus Henricus Netherlands, 1183. Kerr Ha Amstelveen, Camera Opps Claran 368 (72) Inventor Tel Steff, Peter Huerdinand The Netherlands, 2803 El Her Gouda, Kerf El Harde 5 (72) Inventor Weber, Ronald The Netherlands, 1622 Har Hae Horn, Hout Tour Himlen 62

Claims (1)

[Claims] 1. Vanadium haloperoxidase, halide source and hydrogen peroxide or peracid An enzymatic antibacterial composition comprising a hydrogen fluoride source. 2. The vanadium haloperoxidase is chloroperoxidase. The enzymatic antibacterial composition according to 1. 3. Vanadium haloperoxidase is Curvularia inaequa A chloroperoxidase obtainable from lis, according to claim 1 or 2. The enzymatic antibacterial composition described. 4. Vanadium haloperoxidase is Curvularia inaequ Chloroperoxy immunologically cross-reactive with alis CBS 102.42 The enzymatic antibacterial composition according to any one of claims 1 to 3, which is a dase. 5. The hydrogen peroxide source is an enzymatic hydrogen peroxide generating system, according to any one of claims 1 to 4. The enzymatic antibacterial composition according to the item 1. 6. The enzymatic hydrogen peroxide generating system is glucose / glucose oxidase or lactic acid. The enzymatic antibacterial composition according to claim 5, which is a salt / lactate oxidase. 7. The method according to claim 1, which has substantially no catalase activity. 7. The enzymatic antibacterial composition according to any one of 6 to 6. 8. Applying a composition according to any one of claims 1 to 7 to the surface to be sterilized And a method of inhibiting the growth of microorganisms. 9. Use of vanadium haloperoxidase as a sanitizer. 10. Curvularia inaequalis CBS 102.42 derived bar A DNA sequence containing a structural gene encoding nadium chloroperoxidase. 11. Curvularia inaequalis CBS shown in FIG.  Structural gene encoding vanadium chloroperoxidase derived from 102.42 A DNA sequence containing 12. Origin of replication, transcriptional and termination regulatory sequences, and D according to claim 10 or 11. An expression vector comprising at least part of an NA sequence. 13. A suitable host is transformed with the expression vector according to claim 12 to obtain a structure. The host is cultivated under conditions that allow the gene to be expressed, and vanadium haloperoxide A method for producing vanadium haloperoxidase by isolating zease.