WO2001011969A1

WO2001011969A1 - ENZYMATIC METHOD FOR KILLING OR INHIBITING MICROBIAL CELLS AT HIGH pH

Info

Publication number: WO2001011969A1
Application number: PCT/DK2000/000451
Authority: WO
Inventors: Charlotte Johansen
Original assignee: Novozymes A/S
Priority date: 1999-08-13
Filing date: 2000-08-11
Publication date: 2001-02-22
Also published as: AU6558100A

Abstract

An enzymatic method for killing or inhibiting microbial cells at high pH comprising a haloperoxidase, a hydrogen peroxide source, and a bromide and/or iodide source.

Description

ENZYMATIC METHOD FOR KILLING OR INHIBITING MICROBIAL CELLS AT HIGH pH

The present invention relates to a very effective enzymatic method for killing or inhibiting microbial cells or microorganisms at high pH, more specifically microbial cells or microorganisms present in laundry, on hard surfaces, in water systems, m the pulp and paper industry, m the oil industry, on skin, teeth or mucous membranes; and for preserving food products, cosmetics, paints, coatings, etc.

BACKGROUND OF THE INVENTION

Various enzymatic antimicrobial compositions are known in the art. For instance, WO 94/04127 discloses stabilized dentifrice compositions which are capable of producing antimicrobially effective concentrations of hypothiocyanite ions. The compositions comprise an oxidoreductase capable of producing hydrogen peroxide and a peroxidase enzyme capable of oxidizing thiocyanate ions, which are normally present in saliva, to antimicrobial hypothiocyanite ions. Suitable peroxidases include lactoperoxidase, myeloperoxidase, salivary peroxidase and chloroperoxidase .

EP-A-0 500 387 discloses enzymatic antimicrooial compositions comprising a haloperoxidase, e.g., myelo- peroxidase, eosinophil oxidase, lactoperoxidase and chloroperoxidase, which selectively binds to and inhibits the growth of target microorganisms in the presence of peroxide and halide.

WO 95/27046 discloses an antimicrobial composition comprising a Vanadium chloroperoxidase, halide ions, and hydrogen peroxide or a hydrogen peroxide-generating agent.

WO 96/38548 discloses an antimicrobial composition comprising a haloperoxidase, a halide ion, a peroxide generating agent and an amino acid type. WO 97/09447 discloses an analytical composition for providing a chemiluminescent signal having a pH of from 6.5 to 10, comprising a chemiluminescent signal-generating reagent, a halogen, and a peroxide. WO 97/31090 discloses a cleaning composition comprising a peroxidase, an enhancer and a source of hydrogen peroxide. The peroxidase is incorporated into a special release agent.

The ob_ject of the present invention is to provide a method for killing or inhibiting microbial cells at high pH, i.e., for disinfection or preservation, which is easy to use and an effective alternative to the known disinfecting and preserving compositions and methods.

SUMMARY OF THE INVENTION

It has been found that the combined action of a haloperoxidase, a hydrogen peroxide source and a halide source selected from bromide and iodide results in an improved antimicrobial effect at high pH. Thus, based on these findings, the present invention provides, in a first aspect, a method for killing or inhibiting microbial cells comprising treating said microbial cells in an aqueous solution at a pH in the range of 8 to 11 with a haloperoxidase, a halide source selected from bromide and iodide, and a hydrogen peroxide source.

The method of the invention is useful as an antimicrobial treatment wherever such a treatment is needed, for example, for the preservation of food, beverages, cosmetics, deodorants, contact lens products, food ingredients or enzyme compositions; as a disinfection for use, e.g., on human or animal skin, hair, oral cavity, mucous membranes, wounds, bruises or in the eye; for killing microbial cells in laundry; and for disinfection of hard surfaces, in the pulp and paper industry, in the oil industry, or for water treatment. In a second aspect, the present invention provides a detergent composition comprising a haloperoxidase, a halide source selected from bromide and iodide, a hydrogen peroxide source, and a surfactant, wherein said detergent composition in an aqueous solution has a pH in the range of 8 to 11. The composition of the invention is useful as an antimicrobial agent wherever such an agent is needed, for example, for the preservation of food, beverages, cosmetics, deodorants, contact lens products, food ingredients or enzyme compositions; as a disinfectant for use, e.g., on human or animal skin, hair, oral cavity, mucous membranes, wounds, bruises or m the eye; for killing microbial cells in laundry; and for incorporation in cleaning compositions or disinfectants for hard surface cleaning, in the pulp and paper industry, in the oil industry, for water treatment, or for disinfection.

In further aspects, the present invention provides a method of inhibiting microorganisms present m laundry, wherein the laundry is treated with the composition of the invention e.g. in a soaking, washing or rinsing liquor; a method of inhibiting microbial growth on a hard surface, wherein the surface is contacted with the composition of the invention; a method of inhibiting microbial cells present in industrial water lines; and a method of killing microbial cells present on human or animal skin, mucous membranes, teeth, wounds, bruises or in the eye or inhibiting the growth thereof, wherein the cells to be killed or inhibited on the skin, mucous membrane, teeth, wound or bruise are/is contacted with the composition of the invention.

DETAILED DESCRIPTION OF THE INVENTION

In the context of the present invention the term "antimicrobial" is intended to mean that there is a bactericidal and/or a bacteriostatic and/or fungicidal and/or fungistatic effect and/or a virucidal effect, wherein

The term "bactericidal" is to be understood as capable of killing bacterial cells.

The term "bacteriostatic" is to be understood as capable of inhibiting bacterial growth, i.e. inhibiting growing bacterial cells.

The term "fungicidal" is to be understood as capable of killing fungal cells.

The term "fungistatic" is to be understood as capable of inhibiting fungal growth, i.e. inhibiting growing fungal cells. The term "virucidal" is to be understood as capable of inactivating virus.

The term "microbial cells" denotes bacterial or fungal cells, and the term "microorganism" denotes a fungus (including yeasts) or a bacterium.

In the context of the present invention the term "inhibiting growth of microbial cells" is intended to mean that the cells are in the non-growing state, i.e., that they are not able to propagate.

In the context of the present invention the term "log reduction" is defined as a logarithmic reduction of the number of living cells, e.g. 1 log reduction corresponds to a reduction in living cell number of Escherichia coli DSM1576 or Entβrococcus faecalis DSM2570 from Y x 10^x CFU/M (CFU: Colony Forming Units, M: ml or g) to Y x lO^"1 CFU/M, where X can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, and Y can be any number from 0 to 10. The number of living bacteria is to be determined as the number of E. coli or E. faecalis, respectively, which can grow on Tryptone Soya Agar (#CM129, Oxoid, England) plates at 30°C.

The term "hard surface" as used herein relates to any surface which is essentially non-permeable for microorganisms. Examples of hard surfaces are surfaces made from metal, e.g., stainless steel, plastics, rubber, board, glass, wood, paper, textile, concrete, rock, marble, gypsum and ceramic materials which optionally may be coated, e.g., with paint, enamel and the like. The hard surface can also be a process equipment, e.g., a cooling tower, an osmotic membrane, a water treatment plant, a dairy, a food processing plant, a chemical plant, a pharmaceutical process plant, a pulp and paper plant or an oil processing plant. Accordingly, the composition according to the present invention is useful in a conventional cleaning-in-place (C-I-P) system.

Haloperoxidases

In the context of the present invention the term "haloperoxidase" is intended to mean an enzyme selected from the group consisting of chloride peroxidase (EC 1.11.1.10), bromide peroxidase, and iodide peroxidase (EC 1.11.1.8), preferably "haloperoxidase" is a bromide peroxidase.

A chloride peroxidase is an enzyme capable of oxidizing chloride, bromide and iodide ions with the consumption of H₂0₂ . A bromide peroxidase is an enzyme capable of oxidizing bromide and iodide ions with the consumption of H₂0₂.

A iodide peroxidase is an enzyme capable of oxidizing iodide ions with the consumption of H₂0₂. According to the invention Vanadium haloperoxidases are preferred. Vanadium haloperoxidases are different from other haloperoxidases m that the prosthetic group in theses enzymes has structural features similar to vanadate (vanadium V) and contain vanadium, whereas the other haloperoxidases are hemeperoxidases . The Vanadium haloperoxidases disclosed in WO 95/27046 are preferred.

Haloperoxidases form a class of enzymes which are able to oxidize halides (X = C1-, Br-, or I-) m the presence of hydrogen peroxide to the corresponding hypohalous acid (HOX) according to:

H₂0₂ + X- + H+ -> H₂0 + HOX

If an appropriate nucleophilic compound is present, a reaction will occur with HOX, which has an antimicrobial effect.

Haloperoxidases have been isolated from various organisms: mammals, marine animals, plants, algae, a lichen, fungi and bacteria (for reference see Biochimica et Biophysica Acta 1161,

1993, pp. 249-256). It is preferred that the haloperoxidase is obtained from fungi, bacteria or algae. It is generally accepted that haloperoxidases are the enzymes responsible for the formation of halogenated compounds in nature, although other enzymes may be involved.

Haloperoxidases have been isolated from many different fungi, in particular from the fungus group dematiaceous hypho- mycetes, such as Caldariomyces, e.g., C. fumago, Al ternaπa , Curvulaπa , e.g., C. verruculosa and C. maequalis, Drechslera , e.g., D. hartlebn, Ulocladium, Phaeotπchoconis, e.g., P. crotalaπae, Gemculosporium, Dendryphiella, e.g., D. salma , and Botrytis (see US Patent No. 4,937,192).

According to the present invention a haloperoxidase obtainable from Curvulaπa , in particular C. verruculosa is preferred such as C. verruculosa CBS 147.63 or C. verruculosa CBS 444.70, or a haloperoxidase which is lmnaunologically cross- reactive with C. verruculosa CBS 147.63. Curvulaπa haloperoxidase and recombinant production hereof is described in WO 97/04102. SEQ ID NO:l and SEQ ID NO:2 of the present invention are also described m WO 97/04102.

Haloperoxidase has also been isolated from bacteria such as Pseudomonas, e.g., P. pyrrocima (for reference see The Journal of Biological Chemis try 263, 1988, pp. 13725-13732) and Streptomyces , e.g., S . a ureofaciens (for reference see Struc- tural Biology 1 , 1994, pp. 532-537).

Bromide peroxidase has been isolated from algae (see US Patent No. 4,937,192) .

In a preferred embodiment the haloperoxidase of the invention is an isolated polypeptide having an ammo acid sequence which has a degree of identity to the ammo acid sequence of SEQ ID NO:2 of at least about 60%, preferably at least about 70%, more preferably at least about 80%, most preferably at least about 90%, and in particular at least about 95%, and have haloperoxidase activity (hereinafter "homologous haloperoxidases"). In a preferred embodiment, the homologous haloperoxidases have an amino acid sequence which differs by five am o acids, preferably by four ammo acids, more preferably by three ammo acids, even more preferably by two ammo acids, and most preferably by one ammo acid from the amino acid sequence of SEQ ID NO: 2. For purposes of the present invention, the degree of identity between two ammo acid sequences is determined by using GAP version 8 from the GCG package (Genetics Computer Group, 575 Science Drive, Madison, WI 53711, USA) with standard penalties for proteins: GAP weight 3.00, length weight 0.100, Matrix described m Gribskov and Burgess, Nucl . Acids Res. 14(16); 6745-6763 (1986).

Preferably, the haloperoxidases of the present invention comprise the amino acid sequence of SEQ ID NO: 2 or a fragment thereof that has haloperoxidase activity. In a more preferred embodiment, the haloperoxidases of the present invention comprise the ammo acid sequence of SEQ ID NO: 2. In another preferred embodiment, the haloperoxidases of the present invention consist of the amino acid sequence of SEQ ID NO: 2 or a fragment thereof that has haloperoxidase activity. In another preferred embodiment, the haloperoxidases of the present invention consists of the ammo acid sequence of SEQ ID NO: 2.

In use, the concentration of the haloperoxidase may be varied m order to achieve the desired antimicrobial effect in the desired time frame. However, according to the invention the haloperoxidase will normally be present in a concentration of 0.01-100 mg enzyme protein per liter, preferably m a concentration of 0.05-10 mg enzyme protein per liter, more preferably in a concentration of 0.1-5 mg enzyme protein per liter, and most preferably m a concentration of 0.1-1 mg enzyme protein per liter.

Hydrogen Peroxide Sources According to the invention the hydrogen peroxide needed for the reaction with the haloperoxidase may be achieved in many different ways: It may be hydrogen peroxide or a hydrogen peroxide precursor, such as, e.g., percarbonate or perborate, or a peroxycarboxylic acid or a salt thereof, or it may be a hydrogen peroxide generating enzyme system, such as, e.g., an oxidase and its substrate. Useful oxidases may be, e.g., a glucose oxidase, a glycerol oxidase or an ammo acid oxidase. An example of an amino acid oxidase is given in WO 94/25574.

It may be advantageous to use enzymatically generated hydrogen peroxide, since this source results in a relatively low concentration of hydrogen peroxide under the biologically relevant conditions. Low concentrations of hydrogen peroxide result in an increase in the rate of haloperoxidase-catalysed reaction. According to the invention the hydrogen peroxide source needed for the reaction with the haloperoxidase may be present in a concentration corresponding to a hydrogen peroxide concentration in the range of from 0.01-1000 mM, preferably in the range of from 0.05-500 mM, more preferably in the range of from 0.1-100 mM, even more preferably in the range of from 0.5- 50 mM, and most preferably m the range of from 1-10 mM.

Halide Sources According to the invention the halide source needed for the reaction with the haloperoxidase is selected from bromide and iodide, preferably the halide source is bromide. It may be achieved in many different ways, e.g., by adding a bromide salt and/or a iodide salt: e.g., sodium bromide, potassium bromide, sodium iodide, or potassium iodide.

The halide source will typically be present m a concentration corresponding to 0.01-1000 mM halide, preferably corresponding to 0.05-500 mM halide, more preferably corresponding to 0.1-100 mM halide, even more preferably corresponding to 0.5-50 mM halide, and most preferably corresponding to 1-10 mM halide.

The method The present invention provides a method for killing or inhibiting microbial cells comprising treating said microbial cells m an aqueous solution at a pH m the range of 8 to 11 with:

- a haloperoxidase, - a halide source selected from bromide and iodide, and

- a hydrogen peroxide source

In another aspect is provided a method of killing or inhibiting microorganisms present laundry, wherein the laundry is treated with a soaking, washing or rinsing liquor comprising an effective amount of the detergent composition of the invention. Preferably the laundry is treated in a washing machine .

In still other aspects are provided a method of preserving a cosmetic product, wherein an effective amount of the detergent composition of the invention is incorporated into the cosmetic product; and a method of cleaning, disinfecting or inhibiting microbial growth on a hard surface, wherein the surface is contacted with the detergent composition of the invention. As an "effective amount" is meant an amount suitable for obtaining the required antimicrobial activity in the chosen application; e.g. to reduce the number of living cells to 10%, 1% or less than 1%; or to prevent the number of living cells from doubling during 12 hours, 1 day, 5 days, 30 days or more than 30 days

When the methods of the invention are applied, they result in an antimicrobial activity of at least 1 log reduction, preferably an antimicrobial activity of at least 2 log reductions, and more preferably an antimicrobial activity of at least 3 log reductions .

When the methods of the invention are applied, treatment with the hydrogen peroxide component can be done separately from treatment with the other components.

The methods of the invention may further comprise treatment with auxiliary agents such as wetting agents, thickening agents, buffer, stabilisers, perfume, colourants, fillers and the like. Useful wetting agents are surfactants, i.e., non-ionic, anionic, amphoteric or zwitteπonic surfactants.

In the context of the present invention the term "treating" comprise contacting, reacting, acting on, or in other ways having an effect on. The pH of the methods of the invention is in the range of from pH 8 to 11, preferably in the range of from pH 8.6 to 10.5, more preferably in the range of from pH 9 to 10.5, and most preferably m the range of from pH 9 to 10.

The composition

The detergent composition comprising the haloperoxidase, the hydrogen peroxide source, the halide source, and the surfactant preferably has an antimicrobial activity of at least 1 log reduction, more preferably an antimicrobial activity of at least 2 log reductions, and most preferably an antimicroDial activity of at least 3 log reductions.

The detergent composition may be formulated as a solid or a liquid.

When formulated as a solid all components may be mixed together, e.g., as a powder, a granulate, a gelled, or a dry product .

When other than dry form compositions are used and even in that case, it is preferred to use a two-part formulation system having the hydrogen peroxide separate from the other components.

The composition of the invention may further comprise auxiliary agents such as wetting agents, thickening agents, buffer, stabilisers, perfume, colourants, fillers and the like. Useful wetting agents are surfactants, i.e., non-ionic, anionic, amphoteric or zwitterionic surfactants.

The composition of the invention may be a concentrated product or a ready-to-use product. In use, the concentrated product is typically diluted with water to provide a medium having an effective antimicrobial activity, applied to the object to be disinfected or preserved, and allowed to react with the micro-organisms present.

The pH of a 1% aqueous solution of the composition is in the range of from pH 8 to 11, preferably m the range of from pH 8.6 to 10.5, more preferably m the range of from pH 9 to 10.5, and most preferably in the range of from pH 9 to 10.

The composition of the invention may also be formulated as a two part system wherein one part is the haloperoxidase and the surfactant; the other part is the hydrogen peroxide source; and the halide source may then come from tap water or otherwise be naturally present.

Uses

The haloperoxidase, halide source and hydrogen peroxide source (hereinafter denoted "the antimicrobial system") of the invention may be incorporated into a detergent or cleaning composition comprising more enzyme types useful in detergent or cleaning compositions, preferably at least one further enzyme selected from the group consisting of proteases, carbohydrases, amylases, cutmases, peroxidases, oxidases, laccases, cellulases, pectmase, mannanases, arabmases, galactanases, xylanases, and lipases.

When used for preservation of food, beverages, cosmetics such as lotions, creams, gels, ointments, soaps, shampoos, conditioners, antiperspirants, deodorants, mouth wash, contact lens products, enzyme formulations, or food ingredients, the composition used in the method of the present invention may be incorporated into the e.g. unpreserved food, beverages, cosme- tics, contact lens products, food ingredients or anti flammatory product in an amount effective for killing or inhibiting growth of microbial cells.

Thus, the composition used in the method of the invention may by useful as a disinfectant, e.g., m the treatment of acne, infections in the eye or the mouth, skin infections; in antiperspirants or deodorants; in foot bath salts; for cleaning and disinfection of contact lenses, hard surfaces, teeth (oral care), wounds, bruises and the like. The composition may also be used for the manufacture of a medicament for therapeutic treatment, such as the treatments mentioned above.

In general it is contemplated that the composition of the present invention is useful for cleaning, disinfecting or inhibiting microbial growth on any hard surface. Examples of surfaces, which may advantageously be contacted with the composition of the invention are surfaces of process equipment used n e.g. dairies, chemical or pharmaceutical process plants, water sanitation systems, oil processing plants, paper pulp processing plants, water treatment plants, and cooling towers. The composition of the invention should be used in an amount, which is effective for cleaning, disinfecting or inhibiting microbial growth on the surface question.

Further, it is contemplated that the composition of the invention can advantageously be used in a cleaning-in-place (C.I. P.) system for cleaning of process equipment of any kind.

The method of the invention may additionally be used for cleaning surfaces and cooking utensils in food processing plants and any area in which food is prepared or served such as hospitals, nursing homes, restaurants, especially fast food restaurants, delicatessens and the like. It may also be used as an antimicrobial in food products and would be especially useful as a surface antimicrobial cheeses, fruits and vegetables and food on salad bars.

It may also be used as a preservation agent or a disinfection agent in water based paints.

The composition of the present invention is also useful for microbial control of water lines, and for disinfection of water, m particular for disinfection of industrial water. Detergent Compositions

The antimicrobial system of the invention may be added to and thus become a component of a detergent composition. The detergent composition of the invention may for example be formulated as a hand or machine laundry detergent composition including a laundry additive composition suitable for pre- treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use m general household hard surface cleaning operations, or be formulated for hand or machine dishwashing operations.

In a specific aspect, the invention provides a detergent additive comprising the antimicrobial system of the invention and a surfactant. The detergent additive as well as the detergent composition may comprise one or more other enzymes such as a protease, a lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabmase, a galactanase, a xylanase, an oxidase, e.g., a laccase, and/or a peroxidase. In general the properties of the chosen enzyme (s) should be compatible with the selected detergent, (i.e. pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme (s) should be present in effective amounts. Proteases : Suitable proteases include those of animal, vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included. The protease may be a seπne protease or a metallo protease, preferably an alkaline microbial protease or a trypsm-like protease. Examples of alkaline proteases are subtilisins, especially those derived from Bacill us, e.g., subtilism Novo, subtilism Carlsberg, subtilism 309, subtilism 147 and subtilism 168 (described in WO 89/06279). Examples of trypsm- like proteases are trypsm (e.g. of porcine or bovine origin) and the Fusarium protease described in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants with substitutions in one or more of the following positions: 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and 274.

Preferred commercially available protease enzymes include Alcalase™, Sav ase™, Pπmase™, Duralase™, Esperase™, and Kannase™ (Novo Nordisk A/S) , Maxatase™, Maxacal™, Maxapem™, Properase™, Purafect™, Purafect OxP™, FN2™, and FN3™ (Genencor International Inc.). Lipases : Suitable l pases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful lipases include lipases from Humi cola (synonym Thermomyces ) , e.g. from H. lanugmosa [ T . lanugmosus ) as described in EP 258 068 and EP 305 216 or from H. insolens as described in WO 96/13580, a Pseudomonas lipase, e.g. from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. stutzeπ (GB 1,372,034), P. fluorescens, Pseudomonas sp . strain SD 705 (WO 95/06720 and WO 96/27002), P. wisconsmensis (WO 96/12012), a Bacillus lipase, e.g. from B . subtilis (Dartois et al . (1993), Biochemica et Biophysica Acta, 1131, 253-360), B . stearothermophilus (JP 64/744992) or B . pumilus (WO 91/16422) .

Other examples are lipase variants such as those described m WO 92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202.

Preferred commercially available lipase enzymes include Lipolase™ and Lipolase Ultra™ (Novo Nordisk A/S) . Amylases : Suitable amylases ( and/or β) include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, α-amylases obtained from Bacill us, e.g. a special strain of B . licheniformis, described m more detail in GB 1,296,839.

Examples of useful amylases are the variants described in WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants with substitutions m one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391, 408, and 444.

TM TM

Commercially available amylases are Duramyl , Termamyl , Fungamyl™ and BAN™ (Novo Nordisk A/S) , Rapidase™ and Purastar™ (from Genencor International Inc.) . Cellulases : Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacill us , Pseudomonas , Humicola , Fusarium, Thielavia , Acremonium, e.g. the fungal cellulases produced from Humicola msolens , Myceliophthora thermophila and Fusarium oxysporum disclosed m US 4,435,307, US 5,648,263, US 5,691,178, US 5,776,757 and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral cellulases having colour care benefits. Examples of such cellu- lases are cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299. Commercially available cellulases include Celluzyme™, and Carezyme™ (Novo Nordisk A/S), Clazmase™, and Puradax HA™ (Genencor International Inc.), and KAC-500(B)™ (Kao Corporation) . Peroxidases/Oxidases : Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Copπnus, e.g. from C. cmereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257. Commercially available peroxidases include Guardzyme™ (Novo Nordisk A/S) .

The detergent enzyme (s) may be included m a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes. A detergent additive of the invention, i.e. a separate additive or a combined additive, can be formulated e.g. as a granulate, a liquid, a slurry, etc. Preferred detergent additive formulations are granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids, or slur^¬

Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991 and 4,661,452 and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly (ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and m which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglyceπdes of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given m GB 1483591. Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Protected enzymes may be prepared according to the method disclosed in EP 238,216. The detergent composition of the invention may be m any convenient form, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid. A liquid detergent may be aqueous, typically containing up to 70 % water and 0-30 % organic solvent, or non- aqueous . The detergent composition comprises one or more surfactants, which may be non-ionic including semi-polar and/or aniomc and/or catiomc and/or zwitterionic. The surfactants are typically present at a level of from 0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1% to about 40% of an aniomc surfactant such as linear alkylbenzenesulfonate, alpha-olef sulfonate, alkyl sulfate (fatty alcohol sulfate) , alcohol ethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsucc ic acid or soap. When included therein the detergent will usually contain from about 0.2% to about 40% of a non-ionic surfactant such as alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylam eoxide, ethoxylated fatty acid monoethanol- amide, fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamme ("glucamides") .

The detergent may contain 0-65 % of a detergent builder or complexmg agent such as zeolite, diphosphate, tπphosphate, phosphonate, carbonate, citrate, nitπlotriacetic acid, ethylenediammetetraacetic acid, diethylenetπammepentaacetic acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst) . The detergent may comprise one or more polymers. Examples are carboxymethylcellulose, poly (vinylpyrrolidone) , poly

(ethylene glycol) , poly(vmyl alcohol), poly (vmylpyridme-N- oxide) , poly (vmylimidazole) , polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system which may comprise a H₂0₂ source such as perborate or percarbonate which may be combined with a peracid-formmg bleach activator such as tetraacetylethylenediamme or nonanoyloxybenzenesulfonate . Alternatively, the bleaching system may comprise peroxyacids of e.g. the amide, lmide, or sulfone type.

The enzyme (s) of the detergent composition of the invention may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in e.g. WO 92/19709 and WO 92/19708. The detergent may also contain other conventional detergent ingredients such as e.g. fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil- suspendmg agents, anti-soil redeposition agents, dyes, bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.

It is at present contemplated that in the detergent compositions any enzyme, in particular the haloperoxidase of the invention, may be added in an amount corresponding to 0.01-100 mg cf enzyme protein per liter of wash liqour, preferably 0.05- 10 mg of enzyme protein per liter of wash liqour, more preferably 0.1-5 mg of enzyme protein per liter of wash liqour, and most preferably 0.1-1 mg of enzyme protein per liter of wash liqour.

The antimicrobial system of the invention may additionally be incorporated m the detergent formulations disclosed m WO 97/07202 which is hereby incorporated as reference.

The present invention is further illustrated m the following examples which are not any way intended to limit the scope of the invention as claimed.

EXAMPLES Materials and methods

The Malthus Flexi M2060 instrument is available from Malthus

Instruments Limited, England.

The Curvulaπa verruculosa recombmant peroxidase is available from Novo Nordisk A/S, Denmark. NOPA V0054 powder detergent is available from Nordisk Detergent

A/S, Denmark.

Brain Heart Infusion Broth (#CM225) and Tryptone Soya Agar

(#CM129) is available from Oxoid, England.

The buffers (0.0005 M) used were: pH 5 Homopipes (#6047H, Research Organics, U.S.) pH 6 MES (#M2250, Sigma) pH 7 HEPES (#H3375, Sigma) pH 8 HEPES (#H3375, Sigma) pH 9 HEPES (#H3375, Sigma) + CAPS (#C2632, Sigma] pH 10: CAPS (#C2632, Sigma)

CFU/ml: Colony Forming Units per ml

The term "log reduction" is defined as a logarithmic reduction of the number of living cells, e.g. 1 log reduction corresponds to a reduction in living cell number of Escherichia coli DSM1576 or Enterococcus faecalis DSM2570 from Y x 10^x CFU/M (CFU: Colony Forming Units, M: ml or g) to Y x 10^x"1 CFU/M, where X can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, and Y can be any number from 0 to 10. The number of living toacteπa are to be determined as the number of E . coli or E . faecal is , respectively, which can grow on Tryptone Soya Agar plates at 30°C.

EXAMPLE 1

Antibacterial activity of Curvulaπa verruculosa recoπibinant haloperoxidase with chloride, iodide and bromide against S . epidermidis at high pH

The antibacterial activity of Curvulaπa verruculosa reco bmant peroxidase (rCvP) was evaluated at pH 5-10 with chloride, iodide and bromide as halide sources. The antibacterial activity of haloperoxidase (1 mg/1) was tested against Staphylococcus epidermidis DSM20042 with sodium chloride (80 mM) as electron donor, and hydrogen peroxide (0.5 mM) was added as electron acceptor in the presence of NH₄ ⁺ (8 mM) . When iodide (0.5 mM) or bromide (4 mM) was used as halide source, no NH₄ ⁺ was present. S . epidermidis was grown Brain

Heart Infusion Broth (BHI) at 30°C and diluted in the buffers, respectively to a concentration of approximately 10⁶ CFU/ml. The cell suspensions were incubated with enzyme for 15 mm at 40°C.

The bactericidal activity was determined by incubation in a Malthus instrument. The detection times measured by the Malthus instrument were converted to CFU/ml by a calibration curve. Either direct or indirect Malthus measurements were used when enumerating total survival cells. By the direct measurements, the cell metabolism was determined by conductance measurements in the growth substrate. By the indirect measurements, 3 ml of growth medium was transferred to the outer chamber of the indirect Malthus cells, and 0.5 ml of sterile KOH (0.1 M) was transferred to the inner chamber. The cell suspensions were after enzyme treatment transferred to the outer chamber of the Malthus cell. As cells are growing m the outer chamber they produce C0₂ which will dissolve in the KOH in the inner chamber and tnereby change the conductance of the KOH. The amount of C0₂ formed by the respiring cells surviving the enzyme treatment was used for estimating the number of viable cells. When the conductance change is measurable by the Malthus instrument, a detection time (dt) will be recorded. The dt s were converted to colony counts by use of a calibration curve relating CFU/ml to dt.

At pH values above 7, no significant antimicrobial activity was obtained with chloride as halide source (table 1) . At high pH values, bromide is the most effective halide source.

Table 1

EXAMPLE 2

Antibacterial activity m detergent of haloperoxidase using bromide as electron donor The antibacterial activity of haloperoxidase (rCvP) was tested in NOPA V0054 powder detergent. pH of the detergent was measured as approximately 9.9, antimicrobial activity was evaluated in the detergent at pH 9.9, 9, and 8 where pH was adjusted. Antimicrobial activity was determined by using a factorial experimental design (2*3⁴ factorial experiment) where the factors are rCvP (0, 0.5, 1 mg/1), KBr (0, 4, 8 mM) , (NH₄)₂S0₄ (0, 1, 2 mM) and H₂0₂ (0, 0.5, 1 mM) .

Microbial cells ( Escheπ chia col i DSM1576 and En terococcus faecalis DSM2570) were grown over night in Tryptone Soy Broth, these two strains was not found to be sensitive to the detergent when no enzyme was present. Cells were suspended NOPA detergent (6 g/L) to the cell concentration of approximately 10^{^}-10⁸ CFU/ml, followed by addition of the enzyme system. After incubation at 35°C for 12 mm, the number of living microorganisms was determined by use of a Malthus instrument. Results obtained without NH₄ ⁺ are shown in table 2. A total kill of both E. coli and E . faecalis was obtained in the detergent when the bromide system (KBr, rCvP, H₂0₂) was present, and no significant activity was obtained in the controls where one or two of the compounds were missing. When NH₄ ⁺ was present the antimicrobial activity was increased.

Table 2.

* corresponds to a total kill

EXAMPLE 3

Antibacterial activity in detergent of haloperoxidase using bromide, chloride and iodide as electron donor The antibacterial activity of haloperoxidase (rCvP) was tested in NOPA 2 powder detergent. pH of the detergent was adjusted to pH 8 or 9 and antimicrobial activity was evaluated in the detergent as described above. Antimicrobial activity was determined by using a factorial experimental design (2*2³ factorial experiment) where the factors are rCvP (0 and 5 mg/L) ; halide: KBr, KC1, KI (0 and 2 mM) ; H₂0₂ (0 and 0.5 mM) .

Microbial cells ( Escheπ chia col i DSM1576 and En terococcus faecalis DSM2570) were grown over night in Tryptone Soy Broth, these two strains were found not to be sensitive to the detergent when no enzyme was present. Cells were suspended in NOPA 2 (5 g/L) to the cell concentration of approximately 10⁷- 10⁸ CFU/ml, followed by addition of the enzyme system. After incubation at 40°C for 20 mm, the number of living microorganisms was determined by use of Malthus. Results are shown in table 3. A total kill of both E . coli and E . faecalis was obtained in the detergent at both pH 8 and 9 when the bromide system (KBr, rCvP, H₂0₂) was present, and no significant activity was obtained in the controls where one or two of the compounds were missing. Chloride resulted in a very low bactericidal activity at high pH, whereas Iodide resulted in a significant kill, however, iodide was also slightly bactericidal by oxidation by hydrogen peroxide without enzyme. In general, bromide as enhancer was significantly more bactericidal than iodide and chloride in detergent at high pH .

Table 3.

* corresponds to a total kill,

EXAMPLE 4

Antibacterial activity in detergent of different haloperoxidases using bromide as electron donor

The antibacterial activity of four haloperoxidases was tested in NOPA 2 powder detergent. pH of the detergent was measured and antimicrobial activity of the haloperoxidases was determined in the detergent solution as well as in a borate buffer (0.05 M) with the same pH as the detergent solution (pH 9.8). Antimicrobial activity was determined by using a factorial experimental design (2*2³ factorial experiment with center points) where the factors are haloperoxidase (0 and 5 mg/L), KBr (0 and 8 mM) , and H₂0₂ (0 and 1 mM) . The following haloperoxidases were compared: Phaeotrichoconis crotalariae (DSM13441), Geniculosporium sp . (DSM13442), Dendryphiella salina (DSM13443) and Curvularia verruculosa (rCvP) . Microbial cells { Escherichia coli DSM1576) were grown over night in Tryptone Soy Broth. Cells were washed in saline and suspended in NOPA 2 (5 g/L) to the cell concentration of approximately 10⁷-10⁸ CFU/ml, followed by addition of the enzyme system, or cells were suspended in the borate buffer. After incubation at 40°C for 20 min, the number of living microorganisms was determined by use of Malthus. Results are shown in table 4. A significantly higher bactericidal activity was determined in detergent compared to buffer, thus a synergistic effect was observed when combining haloperoxidases with detergent.

Table 4.

* corresponds to a total kill.

** corresponds to a total kill in one of the doublets

Claims

1. A method for killing or inhibiting microbial cells comprising treating said microbial cells m an aqueous solution at a pH in the range of 8 to 11 with:

- a haloperoxidase,

- a halide source selected from bromide and iodide, and

- a hydrogen peroxide source.

2. The method according to claim 1, wherein the haloperoxidase is obtainable from fungi.

3. The method according to claim 2, wherein the haloperoxidase is obtainable from a fungus selected from the group consisting of Curvularia , Phaeotrichoconis, Geniculosporium and

Dendryphiella .

4. The method according to claim 3, wherein the haloperoxidase is obtainable from Curvularia verruculosa .

5. The method according to claim 1, wherein the haloperoxidase is a Vanadium peroxidase.

6. The method according to claim 1, wherein the haloperoxidase is a bromide peroxidase.

7. The method according to claim 1, wherein the haloperoxidase is a polypeptide having an ammo acid sequence which has a degree of identity to the ammo acid sequence of SEQ ID NO: 2 of at least about 60%, and has haloperoxidase activity.

8. The method according to claim 1, wherein the halide source is a bromide salt.

9. A detergent composition comprising:

- a haloperoxidase,

- a halide source selected from bromide and iodide,

- a hydrogen peroxide source, and - a surfactant, wherein said detergent composition in an 1% aqueous solution has a pH in the range of 8 to 11.

10. The detergent composition according to claim 9, wherein the haloperoxidase is the haloperoxidase of any of claims 2-7.

11. The detergent composition according to claim 9, wherein the halide source is a bromide salt.

12. The detergent composition according to any of claims 9-11, which further comprises one or more other enzymes, in particular selected from proteases, lipases, amylases, cellulases, cutinases, carbohydrases, pectinases, mannanases, arabinases, galactanases, xylanases, oxidases, peroxidases, and laccases .

13. A method of killing or inhibiting microorganisms present in laundry, wherein the laundry is treated with a soaking, washing or rinsing liquor comprising an effective amount of the detergent composition according to claim 9.

14. A method of preserving a cosmetic product, wherein an effective amount of the detergent composition according to claim 9 is incorporated into the cosmetic product.

15. A method of cleaning, disinfecting or inhibiting microbial growth on a hard surface, wherein the surface is contacted with the detergent composition according to claim 9.

16. Use of the detergent composition according to claim 9 for cleaning or disinfection of contact lenses.