This is a continuation-in-part of Serial No.

08/823,794, filed March 24, 1997.

Background of the Invention: Although various bleaches have long been used in numerous applications, hypochlorite based bleaches generally provide superior bleaching performance over competing varieties of bleach such as perborates, percarbonates, and peracids. The most widely used hypochlorite bleach is common household liquid bleach which is approximately a 5.25 weight percent aqueous solution of sodium hypochlorite. As a salt of a weak acid (hypochlorous acid) and a strong base (i.e. sodium hydroxide), hypochlorite (-OCl) exists in equilibrium (pKa m 7.5) with hypochlorous acid (HOCl). Although both are relatively unstable, hypochlorite is more stable of the two and may be further stabilized by basic solutions. As a result, the pH of common household bleach is usually maintained at alkaline levels between

about 10.5 and 12 where virtually all of the species are in the hypochlorite form.

In addition to a cleaning agent, hypochlorite bleach is also used as a germicide. Interestingly, hypochlorous acid has the potential to be a much more potent germicide than hypochlorite. At least one study has shown that hypochlorite has only 1/80th of the germicidal potency of hypochlorous acid. Block, Disinfectants and Antiseptics, Lea & Febiger (publ.), (1991), 131-138.

Although many applications could use hypochlo- rite's combination of germicidal efficacy and superior bleaching ability, there may be certain limitations of use. For example, sodium hypochlorite is primarily available in liquid form. While liquids are convenient to use, unartful application can result in unintended splashing and spillage. As a result, a need exists for an alternative delivery of a hypochlorite based bleach- ing system.

Summary of the Invention: It is an object of the present invention to provide a non-alkaline enzymatic bleaching composition wherein hypochlorous acid and hypochlorite are generated either immediately prior to delivery or in situ.

It is a further object of the invention to provide methods for cleaning a fabric or surface using the enzymatic composition for applications that require a bleaching agent or a germicidal agent or both.

In one aspect of the present invention, a composition comprises a chloroperoxidase enzyme, a hydrogen peroxide source, a chloride salt and an adhering agent which tends to localize the enzyme to the fabric or surface being treated. The composition may be formed either before delivery to the fabric or surface, or in the alternative, formed at the site being treated.

When the enzymatic components (chloro- peroxidase and its substrates hydrogen peroxide and chloride ion) are brought together in aqueous solution to clean a fabric or surface, the enzyme catalyzes a reaction which results in the formation of hypochlorous acid. Depending on the pH of the environment, some fraction of the hypochlorous acid (pKa = 7.5) will then ionize to form hypochlorite. However, because the present invention is in the non-alkaline pH range (preferably between about 4 and about 8), at least about 10%, and more preferably at least about 20% of the hypochlorous acid will remain in the non-ionized form.

Because the enzyme will continue to catalyze the reaction until all the substrates are depleted, the components must be stored in such a way to prevent the enzymatic reaction from occurring prematurely. One method for achieving this purpose is by separately storing the enzyme from at least one of its substrates.

In the alternative, chloroperoxidase, hydrogen peroxide source, chloride salt, and adhering agent all may be in a non-liquid form, thus preventing the enzymatic reaction from appreciably occurring until the composi- tion is dissolved in aqueous solution.

Brief Description of the Drawing: Figure 1: The amount of hypochlorous acid generated by myeloperoxidase as a function of pH is graphically illustrated.

Detailed Description of the Invention: The present invention relates to compositions and methods for using an enzymatic bleaching system for generating hypochlorous acid and hypochlorite either immediately prior to delivery or in situ. An embodiment of the invention comprises a chloroperoxidase enzyme, a hydrogen peroxide source, a chloride salt, and an

adhering agent which tends to localize the enzyme to the fabric or surface being treated. However, for the enzymatic reaction to occur, the composition must be present in aqueous solution.

Because the use of the present invention "cleans" (referring to the removal of soils without the use of an oxidizing agent), "bleaches" (referring to the removal of stains using an oxidizing agent), and "disinfects" (referring to the destruction or prevention of the growth of microorganisms), these terms and their various conjugations are used interchangeably for convenience, unless implicitly or explicitly rendered otherwise. Similarly, "disinfectant" and "germicide" (referring to an agent that kills microorganisms) and their various conjugations are also used interchangeably unless implicitly or explicitly rendered otherwise.

Chloroperoxidase Enzvme Any chloroperoxidase which results in the formation of hypochlorous acid in the presence of hydrogen peroxide and a chloride salt may be used. The chloroperoxidase of the present invention may be purified from known sources or may be produced using recombinant means by methods known in the art. See, for example, B.W. Griffin, Peroxidases in Chemistry and Biology, Volume 2, chapter 4, pages 86-131 (1991), incorporated herein by reference. Preferred chloro- peroxidases are those whose optimal pH for activity is in the range of about 4 to about 8. Especially pre- ferred chloroperoxidases are those that exhibit good thermostability as well as good stability towards commonly used components in detergents and other cleaning formulations. Particularly preferred chloro- peroxidases are myeloperoxidases. Cloroperoxidases from Curvularia inaequalis or Caldariomyces fumago are also preferred.

Hydrogen Peroxide Hydrogen peroxide or a generator capable of generating hydrogen peroxide may be used. Hereinafter, the term "hydrogen peroxide" is used to describe both hydrogen peroxide itself and a source for hydrogen peroxide unless implicitly or explicitly rendered otherwise. For example, sodium peroxide, sodium perborate or other salts of hydrogen peroxide are sources of hydrogen peroxide since each forms hydrogen peroxide upon dissolution. Another example of a source of hydrogen peroxide is an oxidase with a suitable substrate. Examples include but are not limited to: D-glucose oxidase and glucose; hexose oxidase and hexose; cholesterol oxidase and cholesterol; galactose oxidase and D-galactose; pyranose oxidase and pyranose; choline oxidase and choline; pyruvate oxidase and pyruvate; oxalate oxidase and oxalate; glycolate oxidase and glycolate; and D-amino acid oxidase and D-amino acid. If the desired hydrogen peroxide source is an enzyme, it may either be purified from known sources or may be produced using recombinant means by methods known in the art.

Chloride Salt Any water soluble chloride salt may be used.

The chloride salt may be inorganic such as MgCl2, KCl, NaCl, ZnCl2, Caul2, and NE Cl. Generally, metal inor- ganic salts are preferred. Alternatively, the chloride salts may be organic. Illustrative examples include but are not limited to triethylammonium chloride, pyridinium chloride, and tolonium chloride. However, for reasons of ready availability and economy, sodium chloride is particularly preferred.

quantity of the Enzymatic Components The quantities of the enzymatic components (chloroperoxidase, hydrogen peroxide, and chloride salt) will depend on a number of inter-related factors.

However, because the present invention generally relies on generating the bleaching agents at the site being treated, substantially lesser amounts of bleaching agents are required to achieve the same bleaching activity as prior art hypochlorite solutions. In other words, since the effects of the bleaching agents are localized to the surface of the stain being treated, the effective concentration of the generated bleaching agents is substantially higher than the actual concen- trations of the bleaching agents in solution.

Because the cost of the chloroperoxidase enzyme is more than that of any of the other components, it is preferred to minimize the amount of enzyme necessary. Not surprisingly, the amount of enzyme will vary depending on the particular application. For example, if the intended use of the inventive composi- tion is as a kitchen cleaner, then the bleaching agents need to be generated relatively quickly. However, if the intended use of the inventive composition is treating a laundry stain prior to washing, then the bleaching agents may be generated over a longer period of time. With these parameters in mind, typically, the amount of enzyme in the inventive compositions is between about 1 pg/ml and about 10 mg/ml. Depending on the particular applications, ranges yielding between about 5 pg/ml and about 10 pg/ml, about 100 pg/ml and about 500 pg/ml, and about 1 mg/ml to about 5 mg/ml of chloroperoxidase in solution are preferred.

Once the amount of enzyme is known, the optimal amounts of hydrogen peroxide and chloride salt depend on the desired pH of the aqueous solution where the enzymatic reaction is to take place. As stated

earlier, the pH range for the present invention is preferably between about 4 and about 8 depending upon the ratio of hypochlorous acid to hypochlorite that is desired. For example, since hypochlorous acid is a much more potent germicide, if the intended application is primarily as a disinfectant, a more acidic pH between about 4 and about 5 will be desired. However, if intended application is primarily as a bleaching agent and a dual bleaching system is desired, a pH near the pKa or approximately 7.5 will be desired. In any event, in the pH range of the present invention, the amount of hypochlorous acid that remains non-ionized will be at least about 10% and more preferably at least about 20%.

In addition to controlling the hypochlorous acid to hypochlorite ratio, the pH also affects the enzyme's affinity for its substrates. For example, the enzyme's affinity for clcride ion varies with pH and the concentration of hydrogen peroxide. Similarly, the enzyme's affinity for hydrogen peroxide also varies with pH and the concentration of chloride ion. As a result, for optimal enzyme efficiency, it is preferred to have the chloride ion and hydrogen peroxide concentrations necessary for optimal enzyme activity at the particular pH. In the case of myeloperoxidase, the ratio of chloride and hydrogen peroxide concentration may be derived from the following formula: pH = log[Cl ] + log (560/[H202]) where 560 represents the experimentally derived propor- tionality coefficient. Zoliczynski et al., Procedures of the Society for Experimental Biology and Medicine, 154 (1977), 418-422. Keeping this relationship in mind, typically, the chloride salt should be present such that the chloride concentration is between about 0.1 and about 15 weight percent, and preferably is between about

2 and about 10 weight percent. Similarly, typical values for hydrogen peroxide concentration are between about 0.001 and about 1 weight percent in the aqueous solution.

Adhering Anent Any compound or material that tends to localize the enzyme to the fabric or surface being treated may be used as an adhering agent. By keeping the enzymes at or relatively near the site being treated, adhering agents significantly increase the effective concentration of hypochlorous acid and hypochlorite at the desired site. The adhering agent may be present in an amount between about 0.01 and about 10 weight percent, and preferably between about 0.1 and about 5 weight percent.

Many suitable adhering agents are surfactants.

Surfactants useful in the present invention are gener- ally either anionic or non-ionic and may be used alone or as mixtures of various surfactants. Representative examples of anionic surfactants include but are not limited to: ammonium, substituted ammonium (for example, mono-, di-, and triethanolammonium), alkali metal and alkaline earth metal salts of C6-C18 fatty acids and resin acids; linear and branched alkyl benzene sulfo- nates; alkyl sulfates; alkyl ether sulfates, alkane sulfonates, olefin sulfonates, hydroxyalkane sulfonates, acyl sarcosinates, and acyl N-methyltaurides.

Representative examples of non-ionic surfact- ants include linear ethoxylated alcohols such as those sold by Shell Chemical Company under the brand name NEODOLTM. Other non-ionic surfactants include various linear ethoxylated alcohols with an average length of from about 6 to 16 carbon atoms and averaging about 2 to 20 moles of ethylene oxide per mole of alcohol; linear and branched, primary and secondary ethoxylated,

propoxylated alcohols with an average length of about 6 to 16 carbons and averaging 0 to 10 moles of ethylene oxide and about 1 to 10 moles of propylene oxide per mole of alcohol; linear and branched alkyl- phenoxy(polyethoxy) alcohols, otherwise known as ethoxylated alkylphenols with an average chain length of 8 to 16 carbon atoms and averaging 1.5 to 30 moles of ethylene oxide per mole of alcohol.

Other ingredients The inventive composition may include other ingredients known in the art to either maintain or enhance the performance of the enzyme or the generated bleaching agents. For example, buffering agents may be used to maintain the aqueous solution at the desired pH.

Illustrative examples of buffering agents include but are not limited to carbonates, phosphates, silicates, borates, and mixtures thereof. Buffering agents may be present in an amount between about 0.1 and about 30 weight percent. Preferably, the buffering agent is present in an amount between about 0.5 and about 10 weight percent in the aqueous solution where the enzymatic reaction is to occur.

Builders or chelating agents may also be added to the composition. If a builder or chelating agent is desired, typical amounts range from about 0.1 to about 30 weight percent, and preferably from about 1 to about 20 weight percent. Examples of builders include but are not limited to ethylenediaminetetraacetic acid, tartaric acid, citric acid, nitrilotriacetic acid, sodium carboxymethylsuccinic acid, sodium N-(2-hydroxy- ethyl)ethylenediaminetriacetic acid, N-dithyleneglycol- N,N-diacetic acid, diethylenetriaminepentaacetic acid, and mixtures thereof.

Potentiators, agents that enhance the efficacy of enzyme activity and/or the generated bleaching

agents, may also be included in an amount between about 0.1 to about 30 weight percent. Potentiators are well known in the art and include builders, chelating agents, and enzyme stabilizers such as diethylaminoethanol.

Other optional ingredients include those that enhance the aesthetic appeal of the inventive composi- tion such as fragrances and coloring agents. Fra- grances, such as those commercially available from International Flavors and Fragrance Inc., may be included in an amount ranging from about 0.001 to about 2.0 weight percent. Preferably, a fragrance or mixture of fragrances is present in an amount from about 0.1 to about 1 weight percent. Similarly, coloring agents may be included in small amounts. Illustrative examples of widely used coloring agents include but are not limited to ultramarine blue and copper pthalocyanines and may be included in an amount from about 0.001 to about 0.1 weight percent.

Storage Aids Because the present composition is an enzy- matic one, proper care must be taken to prevent the enzymatic substrates from prematurely reacting. If the substrates are permitted to interact with the enzyme substantially before the inventive composition's intended use, the composition's effectiveness as a bleaching agent will degrade over time as the concentra- tions of the substrates are depleted.

To prevent this from occurring, either the chloroperoxidase or one of the substrates may be separately stored from the remainder of the composition.

When the components are separately stored, the enzymatic composition may be formed in situ by delivering the components to the site in rapid succession. Alterna- tively, the components may be admixed immediately before delivering the composition to the affected area. If the

separate-storage strategy is used, a convenient storage means is a multiple chambered dispenser such as that disclosed in Beacham et al., U.S. Patent 4,585,150, issued April 29, 1986, and commonly assigned to The Clorox Company.

Another strategy is to store the enzyme, hydrogen peroxide source, and the chloride salt in a non-liquid form since the enzymatic reaction does not appreciably occur in the absence of a suitable aqueous medium. Illustrative uses for the non-liquid form of the inventive composition include uses as an additive to dry laundry detergent or to a type of scouring powder for cleaning kitchens and bathrooms. In this context, the enzymatic reaction generating hypochlorous acid and hypochlorite will not occur until the composition comes into contact with an aqueous medium.

In the laundry pretreatment context, addi- tional flexibility exists since lag time may be permis- sible between the delivery of the separately stored components of the inventive composition. For example, the inventive composition may be in the form of a solid, pre-treatment stick and either a liquid or dry laundry detergent. In an illustrative embodiment, the chloro- peroxidase and one or more adhering agents may be part of the pre-treatment stick which is rubbed on the stain or area being treated, with the remaining ingredients being a part of the laundry detergent. When the soiled fabric is subsequently washed either minutes or days later, the enzymatic reaction will occur as part of the washing process. Suitable stick formulations are known in the art such as those described in Sabol, U.S. Patent 4,842,762; Barrett, Jr. et al., U.S. Patent 3,953,353; and Borrello, U.S. Patent 4,396,521. For example, one preferred embodiment for a pre-treatment stick includes propylene glycol, nonylphenol ethoxylate, linear alcohol

ethoxylate, dodecylbenzenesulfonic acid, and stearic acid.

Methods of Use The inventive composition may be used in any application where a bleaching agent is desired.

Illustrative examples include but are not limited to use as a laundry additive, as a laundry pre-treatment agent, a kitchen or bathroom all-purpose scouring powder, a liquid kitchen or bathroom cleaner and disinfectant, a mildew remover, a toilet bowl cleaner, and a general purpose germicide.

Generally, the inventive composition is used to clean a fabric or a surface by contacting the fabric or surface being treated with a composition that comprises an aqueous solution, a chloroperoxidase, a hydrogen peroxide source, and a chloride salt. The addition of an adhering agent such as a surfactant promotes the tendency of the chloroperoxidase to adhere to the fabric or surface being treated. When the composition is delivered, enzymatic reaction will occur, generating hypochlorous acid in situ. As previously discussed, a subset of the hypochlorous acid will ionize to form hypochlorite depending on the pH of the sur- rounding environment.

Many of the benefits result from the in situ generation of the bleaching agents. As an initial matter, only a fraction of the amount of hypochlorous acid and hypochlorite needs to be generated to achieve equivalent bleaching outcomes of standard liquid bleach.

Because the localized concentration of the bleaching agents is substantially greater than the actual concen- tration of the bleaching agents, the characteristic bleach odor is mitigated. In addition, because the bleaching agents are formed at or near the desired site of action, the possibilities for unproductive oxidation

by the bleaching agents are also minimized. In other words, there is a greater likelihood of the bleaching agents oxidizing the source of the stain and not some other oxidizable material in the aqueous solution.

Moreover, because the inventive composition can be delivered as a solid or granular product, the unintended spilling or splashing through unartful use of a liquid product can be avoided.

Aspects of the invention will now be illus- trated by the following examples. It will be understood that these examples are intended to illustrate, and not to limit, the present invention.

EXAMPLE 1 Taurine Chloramine Assay The concentration of hypochlorous acid generated by myeloperoxidase, hydrogen peroxide and sodium chloride was measured using a taurine chloramine assay. Briefly, taurine (also known as 2-aminoethane- sulfonic acid, "TauNH2") reacts with the hypochlorous acid to form taurine chloramine (TauNHCl) which in turn reacts to form dithionitrobenzoic acid (DTNB), the formation of which is measured by absorption spectros- copy.

HOC1 + TauNH2 < TauNHCl + H2O TauNHCl + 2TNB o DTNB + Cl + TauNH2 Standard absorbance curves were generated by measuring the absorbance at varying pHs of known concentrations of taurine chloramine. There was a generally linear relationship between absorbance and concentration of taurine chloramine. By comparing the absorbance of a myeloperoxidase reaction mixture with the appropriate standard curve, the amount of hypochlo-

rous acid generated by the myeloperoxidase may be inferred.

Assays were performed by pipetting 1 ml samples into 12 x 75 mm test tubes and then adding 0.125 ml of 1 mM thionitrobenzoic acid ("TNB") to each sample.

The resulting solutions were thoroughly mixed and allowed to stand in the dark for 10 to 15 minutes. The samples were mixed again before 200 pl of each sample were pipetted into triplicate microplate wells. The microplates were read at 412 nm. Table 1 shows results from representative myeloperoxidase reaction conditions.

TABLE 1 NaCl & H2o2 pH 5 [chlo- pH 6 [chlo- pH 7 [chlo- pH 8 [chlo- Concentratn. ramine] ramine] ramine] ramine] 1 mM NaCl 17.9 µM 26.2 µM 4.0 µM 7.9 µM 100 µM H2O2 1 mM NaCl 20.9 pM 24.4 pM 3.8 pM 5.7 µM 200 µM H2O2 1 mM NaCl 14.4 uM 23.6 pM 4.2 pM 10.1 pM 400 pM H202 10 mM NaCl | 13.9 µM | 22.2 µM | 5.2 µM | 8.8 µM 100 pM H2°2 10 mM NaCl 13.6 µM 20.9 µM 5.3 µM 7.6 µM 200 µM H2O 10 mM NaCl 19.1 µm 22.5 µM 4.9 µM 7.4 µM 400 µM H2O2 25 mM NaCl 14.1 µM 26.1 µM 6.4 µM 10.9 µM 100 µM H2O2 25 M NaCl 17.3 µM 31.6 µM 4.6 µM 6.3 µM 200 µM H2O2 25 mM NaCl 9.4 µM 24.4 µM 4.6 µM 7.4 µM 400 µM H2O2 50 mM NaCl 14.6 µM 25.0 µM 7.2 µM 8.0 µM 100 µM H2O2 50 mM NaCl 11.5 µM 23.6 µM 4.6 µM 7.5 µM 200 µM H2O2 50 mM NaC1 15.7 µM 23.1 pM 5.3 µM 8.3 pM 400 µM H2O2 Table 2 illustrates results using larger concentrations of substrates. Either 5 µg/ml myelo- peroxidase or 10 µg/ml myeloperoxidase ("MPO") was tested at 400 mM NaCl and 830 µM H202.

TABLE 2 pH 5 pg/ml MPO 10 pg/ml MPO [chloramine] [chloramine] 5 811 pM 841 pM 6 791 pM 842 µM 7 200 µM 319 µM 8 124 µM 145 µM Fig. 1 graphically illustrates the data in Table 2, displaying the taurine chloramine generation by myeloperoxidase as a function of pH.

EXAMPLE 2 Prespotter Test for Stain Removal Fabric swatches spotted with ball point ink and blood were treated with an embodiment of the inventive composition which comprised of a 0.1 M phosphate buffer solution at pH 6.0 containing 10 pg/ml of myeloperoxidase, 0.83 mM H202, and 0.4 M NaCl.

Because normal washings are generally insufficient, pretreatment is usually necessary for effective stain removal. Substantially improved results for both ink and blood stains were obtained using the inventive composition over the buffer control.

In general, 2.5 ml solution of 0.1 M phosphate buffer (pH 6.0) containing 0.8 M NaCl, and 1.66 mM H202 was delivered to a swatch, followed by 2.5 ml solution of 0.1 M phosphate buffer (pH 6.0) containing 20 pg/ml myeloperoxidase. Mixing at the fabric interface results in the following concentrations: 0.1 M phosphate buffer (pH 6.0); 10 Ug/ml myeloperoxidase; 0.83 mM H202; and 0.4 M NaCl.

Table 3 describes representative results with ball point ink stains. The solution containing 0.1 M phosphate buffer (pH 6.0), 0.4 M NaCl, and 0.83 mM H202 is referred to as the buffer control.

TABLE 3 Percent Strain Treatment Removal 15 Minute Pre-treatment Buffer Control 3.2 Inventive Embodiment 23.1 Overnight Pre-treatment Buffer Control 7.9 Inventive Embodiment 46.2 Because hydrogen peroxide is also a bleaching agent, some of the ink stain is removed with the buffer control. However, in contrast to the slight bleaching action of the buffer control, the ink stain is signifi- cantly improved with the inventive embodiment.

EXAMPLE 3 Hard Surface Testing for Stain Removal The stain removal ability of the inventive compositions was tested on hard surfaces stained with grape, tea, and mildew spores. In order to simulate dual bottle dispensing, the enzymatic compositions were mixed immediately prior to delivery. The estimated amount of bleaching agent based upon the taurine chloramine assay was approximately 3.5 ppm.

All five solutions were based upon the solution (hereinafter referred to as "buffer control") comprising the following: 0.4 M phosphate buffer (pH 5.5); 1.6 M Nail; and 33.2 mM H2O2. Using the buffer

control as a base, the following solutions were tested against the grape, tea, and mildew spores: 1. bleach control; 2. buffer control; 3. inventive embodiment A: buffer control with 1.3 mg/ml myeloperoxidase; 4. inventive embodiment B: buffer control with 1.3 mg/ml myeloperoxidase and 0.4% SDS; and, 5. inventive embodiment C: buffer control with 1.3 mg/ml myeloperoxidase and 0.4% NEODOLTM.

After 15 minutes, the ability of the compositions to remove the grape, tea, and mildew spore stains was assessed. In all cases, the inventive embodiments outperformed a comparable amount of liquid hypochlorite bleach, providing superior cleaning performance. In addition, SDS and NEODOLTM appear to enhance the overall cleaning performance of the inventive compositions.

EXAMPLE 4 Surface Antimicrobial Action The surface antimicrobial action of the inventive embodiments were tested. The basic solution (referred to as "basic solution") comprised: 0.1 M phosphate buffer (pH 5.5); 0.4 M NaCl; and 0.83 mM HzO2.

Inventive embodiment D includes basic solution with 10 pg/ml of myeloperoxidase. Inventive embodiment E is 10 zg/ml of myeloperoxidase and 1% surfactant (NEODOLTM) in basic solution.

The results of the antimicrobial actions of the inventive compositions are summarized in Table 4.

Good results were seen against gram-positive and gram- negative bacteria. Moreover, the antimicrobial effi- cacies were not diminished by the presence of soil which was simulated by the addition of 5% serum.

TABLE 4 Surface/ Con- % Reduction Composition tact Time Bacteria vs. Control Inventive Em- hard surface Staphylococcus aureus 80.6% bodiment D 5 minutes (gram positive) Inventive Em- fabric Enterbacteraerogenes 100% bodiment D 10 minutes (gram negative) Inventive Em- hard surface Enterbacteraerogenes 99.8% bodiment E 5 minutes (gram negative) Inventive Em- fabric Staphylococcus aureus 99.8% bodiment E 10 minutes (gram positive) Inventive Em- fabric Klebsiella pneumoniae 100% bodiment E ~ 10 minutes (gram negative) EXAMPLE 5 As explained previously, because at least 10% of the generated hypochlorous acid remains non-ionized, the inventive compositions are powerful germicidal agents. This increased germicidal efficacy may be exploited in many potential applications especially since microbes attached to surfaces are harder to kill than the same microbes in solution. An inventive embodiment, which was used to demonstrate this phenome- non, comprised: 0.3 pg/ml of myeloperoxidase, 10 RM H202 and 0.01 M NaCl in 0.02 M phosphate buffer (pH 7.0).

When the above embodiment was tested against Streptococcus faecalis bacteria in solution and on surfaces, a five-minute period killed approximately 99.9% of the bacteria in solution, but only killed approximately 87.9% of the bacteria seeded on porous tile. Even when the enzyme is pre-adsorbed, bacteria attached to surfaces are harder to kill than bacteria in solution. When the myeloperoxidase was allowed to pre- adsorb to the bacteria, 100% of the bacteria in solution were killed in five minutes, while 96.29% of the bacteria on porous tile, 96.61% on fabric, and 99.43% on glass were killed in five minutes.

It is to be understood that while the inven- tion has been described above in conjunction with preferred specific embodiments, the description and examples are intended to illustrate and not to limit the scope of the invention, which is defined by the scope of the appended claims.