CN1146782A

CN1146782A - Enzymatic antimicrobial compositions

Info

Publication number: CN1146782A
Application number: CN95192407A
Authority: CN
Inventors: P·巴内特; D·H·洪曼; L·H·西蒙斯; P·F·特施蒂格; R·韦弗
Original assignee: Unilever NV
Current assignee: Unilever NV
Priority date: 1994-03-31
Filing date: 1995-03-31
Publication date: 1997-04-02
Also published as: BR9507226A; PL316571A1; CA2182966A1; PL181397B1; WO1995027046A2; SK123096A3; AU2215495A; EP0753055A1; JPH09511396A; CZ288041B6; HU9602673D0; NL9401048A; WO1995027046A3; CZ285096A3; PL181389B1; HUT74967A

Abstract

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

Description

The enzymatic antimicrobial composition that contains the halo peroxidase

Technical field

The present invention relates to enzymatic antimicrobial composition and application thereof.More particularly, relate to and contain vanadium-halogenated peroxidase (Vanadium haloperoxidase), the enzymatic antimicrobial composition of hydrogen peroxide cource and halide source.The invention still further relates to and adopt recombinant DNA technology to produce the vanadium-halogenated peroxidase, this salt can be used in the enzymatic antimicrobial composition.

The prior art background

Various enzymatic antimicrobial composition are known in the art.For example, WO-A-94/04217 discloses stable dentifrice composition, and said composition can produce the low thiocyanic acid ion (OSCN of antimicrobial effective concentration ^-).Said composition contains the oxydo-reductase that produces hydrogen peroxide and thiocyanate ion can be oxidized to antimicrobial low thiocyanic acid ion (OSCN ^-) peroxidase, said thiocyanate ion is present in the saliva usually.Suitable peroxidase comprises lactoperoxidase, myeloperoxidase, salivary peroxidase and chloroperoxidase.

The enzymatic antimicrobial composition that contains the halo peroxidase is also disclosed at EP-A-500387 (Exoxemis).The document has been described at halo peroxidase in the presence of superoxide and the halogenide and optionally has been attached on the target microorganism and suppresses its growth.The suitable halo peroxidase that EP-A-500387 introduces is myeloperoxidase (MEP), eosinophilic granulocyte (EPO), lactoperoxidase (LPO), and chloroperoxidase (CPO).Reported that the ratio of halogenide and hydrogen peroxide is a key factor with regard to the stability of halo peroxidase and function.When low-down ratio, hydrogen peroxide can suppress the function of halo peroxidase, and when very high ratio, halogenide can hinder enzymatic reaction.This ratio can change in a wide region, but preferably is maintained at about more than 50.

Owing to there is the undesirable side reaction of hydrogen peroxide, concentration of hydrogen peroxide actual in this antimicrobial composition is lower than what expect.Therefore, the halogenide of the hydrogen peroxide of inventor's discovery high initial concentration of use in this antimicrobial composition and convention amount may be more satisfactory.

Moreover, need the anti-microbial activity of a kind of and known enzymatic antimicrobial composition to compose different enzymatic antimicrobial composition.Preferably, said composition should have and resists for example anti-microbial activity of streptococcus faecium of the microorganism be difficult to resist.Under other environment, it also is desired can also resisting non-pathogenic microorganism, because these microorganisms can cause the corruption of food.

Therefore the purpose of this invention is to provide a kind of enzymatic antimicrobial composition, said composition has overcome top said one or more shortcomings.

We are surprised to find and use the vanadium-halogenated peroxidase can prepare especially effectively enzymatic antimicrobial composition now.

The prior art of introducing above comprises the various halo peroxidase that is used for antimicrobial composition, but does not pay particular attention to the vanadium-halogenated peroxide enzyme up to now.

The vanadium-halogenated peroxide enzyme is different from other halo peroxidase, and difference is that the prothetic group of these enzymes has the constitutional features that is similar to vanadate (vanadium V), and other halo peroxidase is the protoheme peroxidase.

Further purpose of the present invention is that its sequence is cloned and measured to the gene of a kind of vanadium-halogenated peroxidase of coding so that it is expressed in the microorganism that other is convenient to grow, and utilizes recombinant DNA technology to improve the output of this enzyme.

Some vanadium bromine peroxide enzymes (Wever et al., 1991) have been found on the surface of natural seaweed.The vanadium bromine peroxide enzyme is easy to be subjected to the influence of substance and can discharges HOBr by adding hydrogen peroxide in the whole plant in seawater.Still do not establish the effect of this enzyme, but the HOBr that forms the hyperoxia voltinism in seawater may be a part that hinders the system of defense that microorganism or fungi grow in its surface as plant.

After from tubercle capsule leaf algae, finding non-heme vanadium bromine peroxide enzyme, proved that a large amount of marine alga kinds also contains these enzymes.Particularly, the bromine peroxide enzyme of finding extensive studies and qualitative (as comment, document sees reference) have been carried out from tubercle capsule leaf algae.The prothetic group of these enzymes has the constitutional features that is similar to vanadate (vanadium V).The catalytic mechanism of these enzymes is: peroxidase and this enzyme react to form hydrogen peroxide-enzyme complex, and a bromide and a proton and this mixture react to form an enzyme-HOBr mixture afterwards.Verified (De Boer et al., 1988) this mixture decay forms enzyme and HOBr.Prove that also these vanadium bromine peroxide enzymes (De Boer et al., 1988) have operational high stability in water and organic medium.For example, these enzymes are stable and at organic solvent acetone for example, methyl alcohol can store the time more than one month in ethanol (content is up to 60%) and the 1-propyl alcohol in three weeks under the turnover condition, and loss of activity not.But these enzymes have disadvantageous one side, and the existence or the adding of bromide promptly must be arranged when practical application, and the gene clone of the bromine peroxide enzyme of coding marine alga and the trial of measuring its aminoacid sequence are not achieved success.

Because its genetic instability and its low optimal pH are 2.75 (J.R.Kanofsky, 1984) (these characteristics have seriously limited it and used) existing known chloroperoxidase that contains protoheme from dark brown Ka Erhei enzyme is suitable for preparing the enzymatic antimicrobial composition hardly, similar arguement has hindered the application from the myeloperoxidase of people's white cell (MPO), this enzyme also can produce HOCl (A.R.J.Bakkenist et al., 1980).

Also the someone reports the halo peroxidase that (

document

8,9 sees reference) dark-coloured spore thread fungus secretion has obvious stability.Proved that particularly the Lu Sheng fungi do not wait a kind of vanadium chloroperoxidase of the mould secretion of curved spore people such as (, 1993) J.W.P.M.Van Schijndel, this enzyme and muriate have high-affinity and have optimal pH 5.5 when participating in chlorination reaction.When the bromine peroxide enzyme that is used to from marine alga, the prothetic group of chloroperoxidase has the structural performance that is similar to vanadate (vanadium V).More studying in great detail in (people such as J.W.P.M.Van Schijndel, 1993) subsequently, be similar to vanadium bromine peroxide enzyme from tubercle capsule leaf algae by the muriatic enzyme kinetics of hydrogen peroxide oxidation.In addition, three kinds of diverse ways show that this enzyme produces a kind of reaction product oxidized chlorine class (HOCl) and this product itself is had resistance.This enzyme shows high heat stability (Tm90 ℃) and at organic solvent 40% methyl alcohol for example, has the stability of height in ethanol and the propyl alcohol.

Summary of the invention

First aspect the present invention relates to a kind of enzymatic antimicrobial composition, and it contains the vanadium-halogenated peroxidase, halogenide and hydrogen peroxide cource or hydrogen peroxide cource.

According to second aspect, the present invention relates to the production of vanadium-halogenated peroxidase, comprise that transforming appropriate host by means of a kind of expression vector (contains a replication origin in the carrier, transcribe and stop control sequence and to the dna sequence dna of small part coding vanadium-halogenated peroxidase), under the condition that allows expression of structural gene, cultivate this host and separate the vanadium-halogenated peroxidase.

Detailed Description Of The Invention

(a) vanadium-halogenated peroxidase

Enzymatic antimicrobial composition of the present invention contains the vanadium-halogenated peroxidase as first composition.This vanadium peroxidase is selected from disclosed in the prior art various vanadium-halogenated peroxidase in principle.For example can use the vanadium-halogenated peroxidase (non-heme) that never waits the mould generation of curved spore, describe as US-A-4707466.Another kind of available method is that the description according to people (1993) such as J.W.P.M.Van Schijndel never waits isolated or purified chloroperoxidase in the curved spore mould (CBS102.42).

The source of other vanadium-halogenated peroxidase comprises Drechslera biseptata (CBS 371.72), Drechslera fuqax (CBS 509.77), Drechslera nicotiae (CBS 655.74), Drechslera subpapendorfii (656.74), Embelisia hyacinthi (416.71), Embelisia didymospora (CBS 766), Ulocladium chartarum (200.67) and Ulocladium botrytis (452.72).

Another kind of available method, can adopt recombinant DNA technology to prepare the vanadium-halogenated peroxidase, comprise by means of a kind of expression vector and transform appropriate host, said carrier contains a kind of replication origin, transcribe or stop the dna sequence dna of control sequence and coding vanadium-halogenated peroxidase, under the condition that allows expression of structural gene, cultivate this host and separate the vanadium-halogenated peroxidase.To be described in detail among the embodiment hereinafter.

(b) source of halide ions

Second moiety of health composition of the present invention is a source of halide ions.They can be any source of halide ions, but iodide or chloride ion are preferred, because they are the most effective.Sodium-chlor is the most effective source of halide ions.This halogenide can be added in the enzymatic antimicrobial composition of the present invention, the perhaps available natural halogenide that is present in the tap water replaces, and its consumption is generally 2-5mM.

(c) hydrogen peroxide cource

Health composition of the present invention further comprises hydrogen peroxide cource or produces the system of hydrogen peroxide.The system of suitable generation hydrogen peroxide is peroxyboric acid or percarbonate, preferably SPC-D or Sodium peroxoborate.

Also can provide hydrogen peroxide by enzymatic peroxidation hydrogen generation system.Enzymatic peroxidation hydrogen generation system can be selected from the antibiotic superoxide of disclosed in the prior art various enzymatics and produce system in principle.For example, can use amine oxidase and amine, amino-acid oxidase and amino acid, Lactate Oxidase and lactic acid, rCO and cholesterol, urico-oxidase and uric acid or XOD and xanthine, preferably, the combination of glucose oxidase and glucose.

The amount of glucose oxidase will depend on that it is than any residual Peroxidase activity of living and may exist, in general every milliliter of detergent compositions of the present invention or every gram contain 10-1000, the glucose oxidase of 20-500 unit preferably, the definition of per unit enzymic activity is the required amount of substrate that per minute transforms 1 μ mol under standard state.

(d) other composition

It is 0.01-50% that enzymatic antimicrobial composition of the present invention contains weight percent usually, and preferably one or more tensio-active agents of 0.1-5.0% are as moistening agent.Suitable tensio-active agent or stain remover active compound are soap or non-soap anionic, nonionic, positively charged ion, both sexes or zwitterionic compound.Surfactant system contains a kind of and multiple anion surfactant and one or more nonionogenic tensides usually.This surfactant system also contains both sexes or zwitterionic detergent compound in addition, but because its expense is higher relatively, usually can not be satisfactory.

In general, the nonionic of this surfactant system and anion surfactant can be selected from by Schwartz﹠amp; Perry, Interscience 1949, Vol.1 " tensio-active agent " and by Schwartz, Perry ﹠amp; Berch, Interscience 1958, Vol.2, " McCutcheon ' sEmulsifiers and Detergents " current version or " Tenside-Taschenbuch " that publishes by Manufacturing Confectioners Company, H.Stache, second edition., Carl Hauser Verlag, 1981 tensio-active agents of describing.

Operable suitable nonionic detergent compound is particularly including the fatty alcohol for example of the compound with a hydrophobic grouping and a hydrogen atoms, acid, acid amides or alkylphenol and alkene oxide, the reaction product of the oxyethane of especially independent oxyethane or coupling collar Ethylene Oxide.Specific nonionic detergent compound is C ₆-C ₂₂Alkylphenol-ethylene oxide condensate (per molecule contains 5-25EO usually, i.e. the oxyethane of 5-25 unit) and aliphatic C ₈-C ₁₈The reaction product of one-level or secondary linearity or branched-chain alcoho and oxyethane, 5-40 EO usually.

Operable suitable anionic detersive immunomodulator compounds is the water-soluble alkali metal salts of organo-sulfate and sulphonate normally, and it contains the alkyl of 8-22 the carbon atom of having an appointment, and employed term alkyl comprises the moieties of senior acyl group.The example of suitable synthetic anionic stain remover compound is that sodium alkyl sulfate and potassium are (especially by making high-grade C ₈-C ₁₈The alcohol sulfation produces from for example fatty oil or theobroma oil), alkyl C ₉-C ₂₀Benzene sulfonic acid sodium salt and potassium, particularly linear secondary alkyl C ₁₀-C ₁₅Benzene sulfonic acid sodium salt and alkyl glycerol ether sodium sulfate are especially from the ether of fatty oil or theobroma oil deutero-higher alcohols with from the ether of the synthol of petroleum derivation.Preferred anionic surfactants stain remover compound is C ₁₁-C ₁₅Sodium alkyl benzene sulfonate and C ₁₂-C ₁₈Sodium alkyl sulfate.

Also can use those tensio-active agents (these tensio-active agents show the resistance of having saltoutd) of in EP-A-328177 (Unilever), describing, alkyl poly glucoside tensio-active agent of in EP-A-070074, describing and alkyl list glucoside.

The preferred surfactants system is the mixture of negatively charged ion and nonionic detergent active substance, particularly negatively charged ion and nonionogenic tenside group and the example of pointing out at EP-A-346995 (Unilever).Especially preferred is C ₁₆-C ₁₈Monohydroxy-alcohol sulfuric acid an alkali metal salt and 3-7 EO ethoxylation C ₁₂-C ₁₅The surfactant system of monohydroxy-alcohol mixture.

Preferably this nonionic detergent in surfactant system shared weight greater than 10%, 25-90% for example.For example anion surfactant shared weight in surfactant system is in the scope of about 5%-40%.

Enzymatic detergent compositions of the present invention can also comprise other composition that is generally used in the antimicrobial composition, for example thickening material.Relevant this point particularly suitable be combination at the disclosed tensio-active agent of EP-A-314232 (Unilever), the document provides the thickening gel in the water soluble.

Antimicrobial composition of the present invention can be used for cleaning rigid surface and laundering of textile fabrics, but also can be used for industry or research institution for example hospital health and cleaning and be used for the cleaning and disinfection medical facilities.Other application is at the industrial sterilization system milk equipment that is used for of system milk.Cause the ability of malodorous bacterium because it has to resist, this antimicrobial composition also can be successfully used to deodorizing.

Antimicrobial composition of the present invention can use to need powder type soluble in water before using, but also can be mixed with liquid product or gel.The generation that can not start hypohalite in those product forms before said composition is used is important.By enzyme and its substrate are separated, for example by adopting known technology that the enzyme bag is got up to accomplish this point.

When using antibiotic enzymatic composition, by thin up 5-100 doubly so that preparation has the medium of imitating anti-microbial activity.Then it is contacted with the surface that is allowed to use with to be sterilized.Reference

1.R.Wever, the bromination activity of M.G.M.Tromp.B.E.Krenn A.Marjani and M.van Tol. tubercle capsule leaf algae: to the influence in biosphere.Envir.Sc.Techn.25(1991)，446-449.

2.R.Wever and K.Kustin. vanadium: a kind of biological coherent element.Adv.Inorg.Chem.35(1990)，81-115.

3.D.Rehder. the bio-inorganic chemistry of vanadium.Ang.Chem.Ed.Engl.30(1991)148-167.

4.E.de the reaction mechanism of the vanadium bromine peroxide enzyme that Boer and R.Wever. are new, a kind of stability kinetics analysis.J.Biol.Chem.263(1988)，12326-12332.

5.E.de Boer, H.Plat, M.G.M.Tromp, M.C.R.Franssen, H.C.van derPlas, E.M.Meijer and H.E.Schoemaker. contain the bromine peroxide enzyme of vanadium: the example at the stable oxydo-reductase of water and organic medium camber.BiotechnBioeng.30(1987)，607-610.

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

7.A.R.J.Bakkenist, J.E.G.de Boer, the halogenide mixture of H.Plat and R.Wever. chloroperoxidase and halogenating reaction mechanism.Biochim.Biophys.Acta613(1980)，337-348.

8.J.C.Hunter-Cevera and L.Sotos. screens " newly " enzyme at occurring in nature: prepare the halo peroxidase with Death Valley dematiaceous hyphomycetes.Microb.Ecol.12(1986)121-127.

9.T-N.E.Liu, T.MTimkulu, J.Geigert, B.Wolf, S.L.Neidleman, the separating and sign of the non-heme chloroperoxidase that D.Silva and J.C.Hunter-Cevera. are new.Biochem.Biophys.Res.Commun.142(1987)，329-333.

10.J.W.P.M.Van Schijndel, E.G.M.Vollenbroek and R.Wever. are from not waiting mould chloroperoxidase of curved spore; A kind of new vanadium enzyme.Biochim.Biophys.Acta.1161(1993)，249-256.

11.Van Schijndel, J.W.P.M., Barnett, P., Roelse, J., Vollenbroek, E.G.M. and Wever, R. is from not waiting mould vanadium chloroperoxidase of curved spore; Stability and stability kinetics (1994) Eur.J.Biochim.225,151-157.

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

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

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

15.R.Wever，H.Plat?and?E.de?Boer(1985)，Biochem.Biophys.Acta830，181-186.

Can further set forth the present invention by means of following non-limiting example.

Embodiment 1

The minimum inhibitory concentration of hypochlorite (MIC)

Material:

The bacterial isolates of Shi Yonging is intestinal bacteria NCTC900 in the present embodiment, Pseudomonas aeruginosa ATCC15442, streptococcus aureus ATCC13565, streptococcus faecium NCTC1092 and harmless Li Site bacterium ATCC33090 serotype 6B.Soak in juice (BHI) broth culture at the brain heart, 30 ℃ with after microbial culture 15-18 hour, with bacterial strain washed twice in pH5.5 citrate buffer solution (20mM trisodium citrate-NaOH damping fluid+10mM NaCl).With bacterial suspension centrifugal on the Eppendorf whizzer (14000rpm, 5 minutes), remove supernatant liquor then and bacterial precipitation is suspended in the citrate buffer solution.Various bacterial suspensions are repeated this washing step once.Then with the pH5.5 citrate buffer solution dilute this through the bacterial suspension of twice washing to obtain every milliliter about 10 ⁷The suspension of bacterium.Carry out in the process at washing step, cell remains in the ice bath.With damping fluid and BSA solution (1%w/v is dissolved in the pH5.5 citrate buffer solution) filtration sterilization and be stored in 4 ℃.By with the dilution of aseptic deionized water from storage liquid (107,000ppm) preparation hypochlorite solutions.

Method:

Utilize aseptic technique, preparation is dissolved in every milliliter about 10 in the pH5.5 citrate buffer solution ⁷The suspension of bacterium.Getting this suspension 1.9ml joins in the sterile test tube.0.1ml the cold hypochlorite solutions of various concentration is added in these test tubes, to obtain various dilution hypochlorites.Use a bar magnet agitator respectively with these test tube mixings then.Blank determination is to use sterile buffer to substitute hypochlorite solutions.At 30 ℃ sample accurately is incubated 5 minutes.Get the 1ml reaction mixture then, join in the cold BSA of 9ml (1%w/v) solution and place ice bath immediately, to stop the reaction of hypochlorite and microorganism.With sample from 10 ^-1Be diluted to 10 ^-5And 100 μ l samples of each diluent are placed on the BHI agar plate of mark and cultivate, be expressed as motility rate with the clonogenic unit (CFU) in every milliliter.30 ℃ flat board cultivated 15-18 hour after, if do not detect CFU, then flat board was cultivated 24 hours again at 30 ℃.

Definition: minimum inhibitory concentration (MIC value) is defined as the concentration that the clonogenic unit that causes the specified microorganisms that detected under employed experiment condition reduces the required hypochlorite of six orders of magnitude at least herein.

The results are shown in table 1.The scope of numerical value wherein is identical with bibliographical information.

The MIC value of the various microorganisms of table 1.

Microorganism	The MIC value is represented with ppm
Microorganism	The MIC value is represented with ppm	Intestinal bacteria	????????2-3
Streptococcus aureus	????????2-3	Intestinal bacteria	????????2-3
Streptococcus aureus	????????2-3	Harmless Li Site bacterium	????????2-3
Pseudomonas fluorescens	????????2-3	Harmless Li Site bacterium	????????2-3
Pseudomonas fluorescens	????????2-3	Streptococcus faecium	????????2-3

Embodiment 2

The minimum inhibitory concentration of hypochlorite and by from the hypochlorite that does not wait mould vanadium chloroperoxidase (V-CPO) enzymatic of curved spore to produce

In the present embodiment, compare with the lethal effect of hypochlorite and by lethal effect from the hypochlorite that does not wait mould vanadium chloroperoxidase (V-CPO) enzymatic of curved spore to produce.In order to compare, used a Micropump.This step can be according to being similar to the Experiment Preparation hypochlorite concentration that enzymatic produces hypochlorite.Under this experiment condition, also very carefully determine the V-CPO activity, thereby can know the amount of the hypochlorite that exists at each time point of testing.

Material:

The bacterial isolates of Shi Yonging is intestinal bacteria NCTC900 in the present embodiment, Pseudomonas aeruginosa ATCC15442, streptococcus aureus ATCC13565, streptococcus faecium NCTC1092 and harmless Li Site bacterium ATCC33090 serotype 6B.Soak in juice (BHI) broth culture at the brain heart, 30 ℃ with microbial culture 15-18 hour.After cultivating, with bacterial strain washed twice in pH5.5 citrate buffer solution (20mM trisodium citrate-NaOH damping fluid+10mM NaCl).With bacterial suspension centrifugal on the Eppendorf whizzer (14000rpm, 5 minutes), remove supernatant liquor then and subsequently bacterial precipitation is suspended in the citrate buffer solution.Repeat this washing step once for various bacterial suspensions then.Then with the pH5.5 citrate buffer solution dilute this through the bacterial suspension of twice washing to obtain every milliliter about 10 ⁷The suspension of bacterium.Carry out in the process at washing step, cell remains in the ice bath.With damping fluid and BSA solution (1%w/v is dissolved in the pH5.5 citrate buffer solution) filtration sterilization and be stored in 4 ℃.By with the dilution of aseptic deionized water from storage liquid (107,000ppm) preparation hypochlorite solutions.By preparing superoxol from 30% storage liquid with the aseptic deionized water dilution.Casein hydrolysate obtains from Difco.According to people such as Van Schijndel, the method for (1993) never waits the mould purifying chloroperoxidase of curved spore.30 ℃ at 20mM pH5.5 sodium citrate buffer solution, 10mM sodium-chlor, 100 μ M hydrogen peroxide, in 50 μ M, the one chlorine methone by with a chlorine methone (e290nm=20.2mM ^-1.cm ^-1) be converted into dichloro methone (e 290nm=0.2mM ^-1.cm ^-1) determine that with spectrophotometer chloroperoxidase is with Cl afterwards ^-Be converted into the activity among the HOCl.The definition of the chloroperoxidase of a unit is the required enzyme amount of a chlorine methone (Sigma) that transforms 1 μ mol at per minute.

Method:

Utilize aseptic technique, preparation is dissolved in every milliliter about 10 in the pH5.5 citrate buffer solution ⁷The suspension of bacterium.Get the 1.8ml sample from this suspension and join the aseptic reaction vessel, stir continuously with the bar magnet agitator at whole experimental session.The cold V-CPO solution of the various concentration of 0.2ml is added in these containers subsequently, to obtain different dilution V-CPO solution (its method of calculation see below).Blank determination is to substitute 0.2mlV-CPO solution with adding the 0.2ml sterile buffer.At 30 ℃ reaction vessel is incubated.The hydrogen peroxide storage liquid that adds 0.5ml then is so that start the V-CPO reaction.The concentration of employed hydrogen peroxide storage liquid decide according to the hypochlorite concentration that is produced, and is that the mode of five times of molar excess is selected according to the final concentration of comparing hydrogen peroxide with the final concentration of hypochlorite (when flowing end).At 30 ℃ sample accurately is incubated 5 minutes.Get the 1ml reaction mixture then, join in the cold BSA of 9ml (1%w/v) solution and place ice bath immediately, to stop the reaction of hypochlorite and microorganism.With sample from 10 ^-1Be diluted to 10 ^-5And 100 μ l samples of each diluent are placed on the BHI agar plate of mark and cultivate, (CFU) is expressed as motility rate with every milliliter clonogenic unit.At 30 ℃ flat board was cultivated 15-18 hour then.If do not detect CFU, then flat board was cultivated 24 hours again at 30 ℃.

MIC value that is obtained and the MIC value that the hypochlorite that uses a Micropump to add same amount obtains are compared.This step is finished as follows:

Utilize aseptic technique, preparation is dissolved in every milliliter about 10 in the pH5.5 citrate buffer solution ⁷The suspension of bacterium.Get the 1.8ml sample from this suspension and join the aseptic reaction vessel, stir continuously with the bar magnet agitator at whole experimental session.At 30 ℃ reaction vessel is incubated.The hydrogen peroxide storage liquid that adds 0.2ml then, the concentration of employed hydrogen peroxide storage liquid decide according to the hypochlorite concentration that is produced, and is that the mode of five times of molar excess is selected according to the final concentration of comparing hydrogen peroxide with the final concentration of hypochlorite (when flowing end).In 30 ℃ of times of 5 minutes, the hypochlorite solutions 0.5ml effluent (flow velocity 0.1ml/ minute) of concentration known is gone up sample then.Utilize the various hypochlorite solutions of different concns to finish a series of experiment respectively, so that obtain a series of hypochlorite final concentration (its calculating sees below).After being incubated 5 minutes, get the 1ml reaction mixture, join the cold BSA of 9ml (1%w/v) solution and place ice bath immediately, to stop the reaction of hypochlorite and microorganism.With sample from 10 ^-1Be diluted to 10 ^-5And 100 μ l samples of each diluent are placed on the BHI agar plate of mark and cultivate, be expressed as motility rate with the clonogenic unit (CFU) in every milliliter.At 30 ℃ flat board was cultivated 15-18 hour then.If do not detect CFU, then flat board was cultivated 24 hours again at 30 ℃.

Use same experiment setting (with regard to blank assay, not adding microorganism), and spectrophotometer, by with a chlorine methone (e 290nm=20.2mM ^-1.cm ^-1) be converted into dichloro methone (e 290nm=0.2mM ^-1.cm ^-1), measure hypochlorite concentration at the 290nm place and the similar hypochlorite of storing liquid that comes from of hypochlorite that obtains with V-CPO respectively that confirms to describe hereinbefore on time.

Calculate:

The final concentration of the hypochlorite that produces with V-CPO calculates as follows:

With the V-CPO of every milliliter of 0.01U, every milliliter of hypochlorite that produces 0.01 μ mol in per minute is equivalent to the hypochlorite that every ml in per 5 minutes produces 0.05 μ mol.Because the dilution effect (it joins in the initial volume of 2.0ml) of 0.5ml effluent, the final concentration after 5 minutes produces 0.04 μ mol hypochlorite for every ml after (2.0/2.5) * 0.05 μ mol/ml=5 minute.Subsequently this concentration is expressed as 0.04 μ mol hypochlorite/ml=0.04 * 52.5ppm hypochlorite=2.10ppm hypochlorite.Obtain other hypochlorite final concentration by improving or reduce V-CPO concentration.The hypochlorite final concentration that is added calculates as follows: owing to added the 0.5ml effluent in the reaction volume of 2.0ml, final volume is 2.5ml, is equivalent to dilute 5 times.In order to obtain the hypochlorite final concentration of 2.10ppm, use 10.50ppm storage liquid.By improving or reduce the hypochlorite concentration that hypochlorite storage liquid obtains other.

The results are shown in the Table II.Various final concentrations that will be produced by V-CPO or the hypochlorite when hypochlorite flows out end compare.From Table II as can be seen, the hypochlorite that is produced by the V-CPO enzymatic has the growth-inhibiting effect to all organisms except that streptococcus aureus when hypochlorite concentration is low to moderate 0.4ppm, need the 2ppm hypochlorite and obtain same growth inhibitory effect.The halo peroxidase that this demonstration contains vanadium is than the more effective health composition of its hypochlorite.Obviously V-CPO also has and kills that all that detected are caused a disease and abilities of non-pathogenic microorganism, and it is said that the halo peroxidase that contains protoheme only has the effectiveness (referring to EP-A-500387) of killing malignant bacteria.

The MIC value of the various microorganisms of Table II

Microorganism	MIC value (ppm) with hypochlorite	MIC value (ppm) with V-CPO
Microorganism	MIC value (ppm) with hypochlorite	MIC value (ppm) with V-CPO	Intestinal bacteria	????????2	??????0.4
Streptococcus aureus	???????2-3	??????1.6	Intestinal bacteria	????????2	??????0.4
Streptococcus aureus	???????2-3	??????1.6	Harmless Li Site bacterium	????????2	??????0.4
Pseudomonas fluorescens	????????2	??????0.4	Harmless Li Site bacterium	????????2	??????0.4
Pseudomonas fluorescens	????????2	??????0.4	Streptococcus faecium	????????2	??????0.4

Embodiment 3

The deadly effectiveness of hypochlorite and the hypochlorite that in the presence of protein hydrolystate, produces by the V-CPO enzymatic

In order to reach the purpose of health, known to actual situation of running into, it is necessary using excessive hypochlorite, because this reactive behavior molecule not only reacts with microorganism, and reacts with other compound that exists.Therefore the behavior that detects the halo peroxidase that contains vanadium in containing the fluid of protein hydrolystate for example is important.

Material:

The bacterial isolates of Shi Yonging is intestinal bacteria NCTC900 in the present embodiment.Soak in juice (BHI) broth culture at the brain heart, 30 ℃ with microbial culture 15-18 hour.After cultivating, with bacterial strain washed twice in pH5.5 citrate buffer solution (20mM trisodium citrate-NaOH damping fluid+10mM NaCl).With bacterial suspension centrifugal on the Eppendorf whizzer (14000rpm, 5 minutes), remove supernatant liquor then and subsequently bacterial precipitation is suspended in the citrate buffer solution.Repeat this washing step once for various bacterial suspensions then.Then with the pH5.5 citrate buffer solution dilute this through the bacterial suspension of twice washing to obtain the suspension of every milliliter of about 108 bacteriums.Carry out in the process at washing step, cell remains on ice.With damping fluid and BSA solution (1%w/v is dissolved in the pH5.5 citrate buffer solution) filtration sterilization and be stored in 4 ℃.By with the dilution of aseptic deionized water from storage liquid (107,000ppm) preparation hypochlorite solutions.By preparing superoxol from 30% storage liquid with the aseptic deionized water dilution.Casein hydrolysate obtains from Difco.Method according to people (1993) such as Van Schijndel never waits the mould purifying chloroperoxidase of curved spore.

Method:

Utilize aseptic technique, preparation is dissolved in every milliliter about 10 in the pH5.5 citrate buffer solution ⁸The suspension of bacterium.Getting the 1.3ml sample from this suspension joins the aseptic reaction vessel, stir continuously with the bar magnet agitator at whole experimental session, 0.5mg/ml casein hydrolysate (the being dissolved in the pH5.5 citrate buffer solution) solution that adds 0.5ml subsequently respectively, 0.5ml pH5.5 citrate buffer solution solution, cold each 0.2ml of V-CPO solution (its method of calculation see below) that in these containers, adds various concentration in the mode of the concentration that obtains 3.2ppm or 6.5ppm hypochlorite.Blank determination is only to add the sterile buffer of 0.2ml to substitute the V-CPO solution of 0.2ml.At 30 ℃ reaction vessel is incubated.The superoxol that adds 0.5ml then is so that start the V-CPO reaction.The selection of the concentration of superoxol is according to the hydrogen peroxide that obtains five times of molar excess when flowing end.At 30 ℃ sample accurately is incubated 5 minutes.Get the 1ml reaction mixture after cultivating, join the cold BSA of 9ml (1%w/v) solution and place ice bath immediately, to stop the reaction of hypochlorite and microorganism.With sample from 10 ^-1Be diluted to 10 ^-5And 100 μ l samples of each diluent are placed on the BHI agar plate of mark and cultivate, be expressed as motility rate with the clonogenic unit (CFU) in every milliliter.At 30 ℃ flat board was cultivated 15-18 hour then.If do not detect CFU, then flat board was cultivated 24 hours again at 30 ℃.

Lethal effect that is obtained and the lethal effect that the hypochlorite that uses a Micropump to add same amount obtains are compared.This step is finished as follows:

Utilize aseptic technique, preparation is dissolved in every milliliter about 10 in the pH5.5 citrate buffer solution ⁸The suspension of bacterium.Getting the 1.3ml sample from this suspension joins the aseptic reaction vessel, stir continuously with the bar magnet agitator at whole experimental session, 0.5mg/ml casein hydrolysate (the being dissolved in the pH5.5 citrate buffer solution) solution that adds 0.5ml subsequently respectively, the pH5.5 citrate buffer solution solution of 0.5ml.The aseptic pH5.5 citrate buffer solution that adds 0.2ml then.Reaction vessel is 30 ℃ of insulations.Then in 30 ℃ of times of 5 minutes with sample on the effluent of the hypochlorite solutions of 0.5ml, produce the hypochlorite final concentration (calculating of concentration is referring to embodiment 2) of 3.2ppm or 6.5ppm.Get the 1ml reaction mixture after the insulation, join the cold BSA of 9ml (1%w/v) solution and place ice bath immediately, to stop the reaction of hypochlorite and microorganism.With sample from 10 ^-1Be diluted to 10 ^-6And 100 μ l samples of each diluent are placed on the BHI agar plate of mark and cultivate, (CFU) is expressed as motility rate with every milliliter of clonogenic unit.At 30 ℃ flat board was cultivated 15-18 hour then.If do not detect CFU, then flat board was cultivated 24 hours again at 30 ℃.The results are shown in Table III.The Table III casein hydrolysate is to the influence of the deadly effectiveness of the HOCl that produced by V-CPO

The concentration of casein hydrolysate (mg/ml)	Use the log value of the 3.2ppm hypochlorite reduction that produces by V-CPO	Use the log value of the hypochlorite reduction that produces by 3.2ppm	Use the log value of the 6.Sppm hypochlorite reduction that produces by V-CPO	Use the log value of the hypochlorite reduction that produces by 6.5ppm
The concentration of casein hydrolysate (mg/ml)					?0.0	(8.0=complete lethal)	(8.2=complete lethal)	(8.0=complete lethal)	(8.2=complete lethal)
?0.1	??????5.0	?????0.5	(8.0=complete lethal)	?????1.0	?0.0	(8.0=complete lethal)	(8.2=complete lethal)	(8.0=complete lethal)	(8.2=complete lethal)

Embodiment 4

The halo peroxidase and the deadly comparison of rendeing a service of the halo peroxidase that contains protoheme that contain vanadium

Material:

The bacterial isolates of Shi Yonging is intestinal bacteria NCTC900 in the present embodiment, streptococcus faecium NCTC1092 and harmless Li Site bacterium ATCC33090 serotype 6B.Soak in juice (BHI) broth culture at the brain heart, 30 ℃ with microbial culture 15-18 hour.After cultivating, with bacterial strain washed twice in pH5.5 citrate buffer solution (20mM trisodium citrate-NaOH damping fluid+10mM NaCl).With bacterial suspension centrifugal on the Eppendorf whizzer (14000rpm, 5 minutes), remove supernatant liquor then and subsequently bacterial precipitation is suspended in the citrate buffer solution.Repeat this washing step once for various bacterial suspensions then.Then with the pH5.5 citrate buffer solution dilute this through the bacterial suspension of twice washing to obtain every milliliter about 10 ⁷The suspension of bacterium.Carry out in the process at washing step, cell remains in the ice bath.With damping fluid and BSA solution (1%w/v is dissolved in the pH5.5 citrate buffer solution) filtration sterilization and be stored in 4 ℃.Method according to people (1993) such as Van Schijndel never waits the mould purifying chloroperoxidase of curved spore.The chloroperoxidase that contains protoheme is to obtain (from dark brown Ka Er black mould) from Sigma.30 ℃ at 20mM pH5.5 sodium citrate buffer solution, 10mM sodium-chlor, 100 μ M hydrogen peroxide, in 50 μ M, the one chlorine methone with a chlorine methone (e 290nm=20.2mM ^-1.cm ^-1) be converted into dichloro methone (e 290nm=0.2mM ^-1.cm ^-1), use the spectrophotometric determination chloroperoxidase at Cl ^-Be converted into the activity among the HOCl.The definition of the chloroperoxidase of a unit is the required enzyme amount of a chlorine methone (Sigma) that transforms 1 μ mol at per minute.Two kinds of enzymes are used identical analysis (with the activity definition), so that can when identical activity, determine the dosage of two kinds of enzymes.

Method:

Utilize aseptic technique, preparation is dissolved in 20mM pH5.5 sodium citrate buffer solution, in the 10mM sodium-chlor every milliliter about 10 ⁷The suspension of bacterium.Getting the 1.9ml sample from this suspension joins the sterile test tube.Subsequently to obtain each 0.1ml of storage liquid that two kinds of dilution modes of enzyme add the chloroperoxidase that contains vanadium and contain the peroxidase of protoheme.The superoxol that adds 0.5ml 25mM then accurately is incubated 5 minutes at 30 ℃ with sample.Get the 1ml reaction mixture, join the cold BSA of 9ml (1%w/v) solution and place ice bath immediately, to stop the reaction of hypochlorite and microorganism.With sample from 10 ^-1Be diluted to 10 ^-5And 100 μ l samples of each diluent are placed on the BHI agar plate of mark and cultivate, be expressed as motility rate with the clonogenic unit (CFU) in every milliliter.At 30 ℃ flat board was cultivated 15-18 hour then.If do not detect CFU, then flat board was cultivated 24 hours again at 30 ℃.

The results are shown in the Table IV.Add fashionablely with identical activity even these results show significantly, the chloroperoxidase that contains vanadium also can provide than the more effective sanitation system of the chloroperoxidase that contains protoheme.

Table IV

??10mM?NaCl	Vanadium chloroperoxidase (U/ml)	Protoheme chloroperoxidase (U/ml)	Log reduction value
??10mM?NaCl	Vanadium chloroperoxidase (U/ml)	Protoheme chloroperoxidase (U/ml)	Log reduction value	Intestinal bacteria	?????0.000 ?????0.00156 ?????0.0030 ?????0.0060 ?????0.012 ?????0.025 ?????0.050 ?????0.10	?????0.000 ?????0.00156 ?????0.0030 ?????0.0060 ?????0.012 ?????0.025 ?????0.050 ?????0.10	0.0 4.0 5.0 7.0 (==complete lethal) 7.0 (==complete lethal) 7.0 (==complete lethal) 7.0 (==complete lethal) 7.0 (==complete lethal) 0.0 0.1 0.1 0.1 0.2 1.0 1.2 1.2
Harmless Li Site bacterium	?????0.000 ?????0.00156 ?????0.0030 ?????0.0060 ?????0.012 ?????0.025 ?????0.050 ?????0.10	?????0.000 ?????0.00156	0.0 7.0 (==complete lethal) 7.0 (==complete lethal) 7.0 (==complete lethal) 7.0 (==complete lethal) 7.0 (==complete lethal) 7.0 (==complete lethal) 7.0 (==complete lethal) 0.0 0.0	Intestinal bacteria

		???0.0030 ???0.0060 ???0.012 ???0.025 ???0.050 ???0.10	????0.0 ????0.0 ????0.0 ????0.0 ????0.0 ????0.0
		???0.0030 ???0.0060 ???0.012 ???0.025 ???0.050 ???0.10	????0.0 ????0.0 ????0.0 ????0.0 ????0.0 ????0.0	Streptococcus faecium	????0.000 ????0.00156 ????0.0030 ????0.0060 ????0.012 ????0.025 ????0.050 ????0.10	0.000 0.00156 0.0030 0.0060 0.012 0.025 0.050 0.10	0.0 5.0 5.0 7.0 (==complete lethal) 7.0 (==complete lethal) 7.0 (==complete lethal) 7.0 (==complete lethal) 7.0 (==complete lethal) 0.0 0.0 0.1 0.1 0.1 0.1 0.1 0.9

Embodiment 5

The hydrogen peroxide in various sources is to the inhibiting influence of V-CPO

Among the embodiment in front, from storage liquid, add as the hydrogen peroxide of one of V-CPO reaction substrate.The effect of the hydrogen peroxide in other source has been described in this embodiment.In a biological oxygen monitor YSI5300 type (oxygen storehouse 5301), analyze oxydase at 30 ℃, wherein utilize the consumption of pH5.5 citrate buffer solution (20mM Trisodium Citrate-sodium hydrate buffer solution+10mM sodium-chlor) as damping fluid monitoring oxygen.At 30 ℃ with this damping fluid of air saturation.Utilize the substrate of 15g/l (final concentration) glucose as glucose oxidase.The bacterial isolates of Shi Yonging is intestinal bacteria NCTC900 in this embodiment.Soak in juice (BHI) broth culture at the brain heart, 30 ℃ with microbial culture 15-18 hour.After cultivating, with bacterial strain washed twice in pH5.5 citrate buffer solution (20mM trisodium citrate-NaOH damping fluid+10mM NaCl).With bacterial suspension centrifugal on the Eppendorf whizzer (14000rpm, 5 minutes), remove supernatant liquor then and subsequently bacterial precipitation is suspended in the citrate buffer solution.Repeat this washing step once for various bacterial suspensions then.Then with the pH5.5 citrate buffer solution dilute this through the bacterial suspension of twice washing to obtain every milliliter about 10 ⁸The suspension of bacterium.Carry out in the process at washing step, cell remains in the ice bath.With damping fluid and BSA solution (1%w/v is dissolved in the pH5.5 citrate buffer solution) filtration sterilization and be stored in 4 ℃.By with the dilution of aseptic deionized water from storage liquid (107,000ppm) preparation hypochlorite solutions.By preparing superoxol from 30% storage liquid with the aseptic deionized water dilution.Casein hydrolysate obtains from Difco.Method according to people (1993) such as Van Schijndel never waits mould separation of curved spore and purifying chloroperoxidase.From the glucose oxidase of Sigma acquisition from aspergillus niger.

Method:

Utilize aseptic technique, preparation is dissolved in every milliliter about 10 in the pH5.5 citrate buffer solution ⁸The suspension of bacterium.Getting the 1.3ml sample from this suspension joins the aseptic reaction vessel, stir continuously with the bar magnet agitator at whole experimental session, 0.5mg/ml casein hydrolysate (the being dissolved in the pH5.5 citrate buffer solution) solution that adds 0.5ml subsequently adds V-CPO solution 0.2ml to obtain the hypochlorite that final concentration is 6.5ppm in this reaction vessel.Blank determination is only to add the sterile buffer of 0.2ml to substitute the V-CPO solution of 0.2ml.At 30 ℃ reaction vessel is incubated.Add following three kinds of each 0.5ml of peroxidation hydrogen generation system subsequently:

1. store the hydrogen peroxide of liquid from 3mM;

2. store the Sodium Hydrogen Carbonate of liquid from 3mM;

3. the mixture of glucose oxidase (0.39U/ml) and 75mg/ml glucose.

At 30 ℃ sample accurately is incubated 5 minutes.Get the 1ml reaction mixture, join the cold BSA of 9ml (1%w/v) solution and place ice bath immediately, to stop the reaction of hypochlorite and microorganism.With sample from 10 ^-1Be diluted to 10 ^-5And 100 μ l samples of each diluent are placed on the BHI agar plate of mark and cultivate, (CFU) is expressed as motility rate with every milliliter of clonogenic unit.At 30 ℃ flat board was cultivated 15-18 hour then.If do not detect CFU, then flat board was cultivated 24 hours again at 30 ℃.Value that is obtained and the MIC value that the hypochlorite that uses a Micropump to add same amount obtains are compared.This step is finished as follows:

Utilize aseptic technique, preparation is dissolved in every milliliter about 10 in the pH5.5 citrate buffer solution ⁸The suspension of bacterium.Getting the 1.3ml sample from this suspension joins the aseptic reaction vessel, stir continuously with the bar magnet agitator at whole experimental session, casein hydrolysate (being dissolved in the 0.5mg/ml pH5.5 citrate buffer solution) solution that adds 0.5ml subsequently adds the aseptic pH5.5 citrate buffer solution solution of 0.2ml then.The hypochlorite solutions effluent (flow velocity 0.1ml/ minute) of sample 0.5ml32.5ppm on then in 30 ℃ of times of 5 minutes.Get the 1ml reaction mixture after being incubated 5 minutes, join the cold BSA of 9ml (1%w/v) solution and place ice bath immediately, to stop the reaction of hypochlorite and microorganism.With sample from 10 ^-1Be diluted to 10 ^-6And 100 μ l samples of each diluent are placed on the BHI agar plate of mark and cultivate, be expressed as motility rate with the clonogenic unit (CFU) in every milliliter.At 30 ℃ flat board was cultivated 15-18 hour then.If do not detect CFU, then flat board was cultivated 24 hours again at 30 ℃.The results are shown in the Table V.The hydrogen peroxide of Table V different sources and the reaction of V-CPO

The hydrogen peroxide source	The concentration of casein hydrolysate (mg/ml)	The reduction of log value when the 6.5ppm hypochlorite	The reduction of log value during the 6.5ppm hypochlorite that produces by enzymatic
The hydrogen peroxide source	The concentration of casein hydrolysate (mg/ml)	The reduction of log value when the 6.5ppm hypochlorite		Hydrogen peroxide storage liquid	?????0.0	(8.2=complete lethal)	(8.0=complete lethal)
Hydrocarbonate	?????0.0	(8.0=complete lethal)	(8.2=complete lethal)	Hydrogen peroxide storage liquid	?????0.0	(8.2=complete lethal)	(8.0=complete lethal)
Hydrocarbonate	?????0.0	(8.0=complete lethal)	(8.2=complete lethal)	Glucose oxidase	?????0.0	(6.4=complete lethal)	(6.4=complete lethal)
Hydrogen peroxide storage liquid	?????0.1	???????1.0	(8.0=complete lethal)	Glucose oxidase	?????0.0	(6.4=complete lethal)	(6.4=complete lethal)
Hydrogen peroxide storage liquid	?????0.1	???????1.0	(8.0=complete lethal)	Hydrocarbonate	?????0.1	???????0.8	(8.2=complete lethal)
Glucose oxidase	?????0.1	???????1.0	(6.4=complete lethal)	Hydrocarbonate	?????0.1	???????0.8	(8.2=complete lethal)

Embodiment 6

Determine chloroperoxidase gene (cDNA) encoding sequence method and from not waiting mould gene of curved spore (Centraal Bureau voor Schimmelcultules, rheNetherlands, strain No 102.42) and possible expression system

Method according to people (1993) such as Van Schijndel never waits the mould liquid culture of curved spore to separate and the purifying chloroperoxidase, and different is to use FPLC system (Pharmacia LKB) to carry out two other purification steps after the DEAE chromatography.At first use phenyl-Sepharose Cl-4B hydrophobic interaction post desmoenzyme in the presence of the 2M NaCl in being dissolved in 50mM Tris-HCl (pH8.3), the gradient of using the 2MNaCl from be dissolved in 50mM Tris-HCl (pH8.3) to descend is subsequently carried out wash-out.For last purification step,, carry out wash-out with the 0M-0.5M NaCl that is dissolved among 20mM piperazine-HCl (pH5.4) subsequently with MonoQ HR 5/5 anion-exchange column (ex Pharmacia) desmoenzyme.Utilize rotary evaporation that enzyme is concentrated subsequently, subsequently at 50mM Tris-SO ₄Dialysis in the damping fluid (pH8).Use SP V8 and trypsinase enzymatic digestion respectively according to standard method known in the art, perhaps use CNBr chemical cracking (Gross, E. (1976), Methods Enzymology 11,238-255) chloroperoxidase of purifying.According to Laemmli (Laemmli, U.K. (1970) Nature227,680-685) utilize SDS-PAGE that the peptide that obtains is separated or separates on the Wheat flavone gel with the method for Von Jagow (1987) according to Schagger, CAPS transfering buffering liquid (the 10mM 3-(hexahydroaniline)-1-propane yellow acid that utilizes Matsudaira (1987) to describe then, 10% methyl alcohol PH11) is transferred to (Immobilon-P ex Millipore on the pvdf membrane.Behind electrophoresis elution, this film was embathed 5 minutes, at room temperature dyeed 5 minutes, and at 50% methyl alcohol, decolouring is 10 minutes and air-dry in 10% acetate with the 0.1% Coomassie brilliant blue R-250 that is dissolved in 50% methyl alcohol with distilled water.(Backman Instruments, Inc. USA) carries out the Edmann automatic sequencing with the peptide bands of a spectrum with Porton LF 3000 protein sequencers.The determined amino acid sequence result is summarized in Fig. 1.

Design the oligonucleotide (seeing Table VII) of complete degeneracy according to the aminoacid sequence of this peptide, utilize the first chain cDNA in polymerase chain reaction (PCR), to use the primer of these degeneracys as template.The first chain cDNA is by being prepared as follows: for isolation of RNA, will not wait the mould spore inoculating of curved spore in a kind of fermention medium, every liter of this fermention medium contains trace element solution people such as (, (1993)) Van Schijndel of 4 gram yeast extracts and 2ml.After growth several days by filtering collection mycelium and freeze-drying.In liquid nitrogen, the freeze dried mould mycelium of curved spore that do not wait is ground.Extract damping fluid (42mM Trisodium Citrate PH7,8.3%M-Sarkosyl L, 50mM beta-mercaptoethanol, 1%Triton x-100 and 4M guanidinium isothiocyanate) by adding RNA, and at room temperature be incubated 1 hour extraction RNA.Add the 2M sodium acetate (PH4) of 0.1 part of volume and the phenol of a volume: chloroform: primary isoamyl alcohol (25: 24: 1) and this mixture placed 15 minutes on ice.After centrifugal 10 minutes of 10000 * g (4 ℃), collect water, add the dehydrated alcohol of 1 times of volume and subsequently with 10000 * g carried out centrifugal in 1 hour this mixture insulation at-20 ℃.The RNA that this precipitation is suspended in suitable volumes again extracts in the damping fluid and surpass centrifugal classification people such as (, 1989) Sambrook in the cesium chloride gradient.Should precipitate and wash carefully and be stored in 75% ethanolic soln at-70 ℃.For separating mRNA, with RNA precipitation and be suspended in again in the water that does not have the RNA enzyme, (Promega company USA) extracts mRNA to use polyAtract mRNA separating kit afterwards.Utilize the Pharmacia first chain cDNA synthetic agent box (Pharmacia BioTech) never waiting the synthetic first chain cDNA on the mould isolating mRNA of curved spore.Aminoacid sequence according to the chloroperoxidase peptide designs 4 20 aggressiveness degenerate oligonucleotides (referring to Table VII) and is being used as primer not wait in the mould polymerase chain reaction of the first chain cDNA as template of curved spore.Utilize thermal cycler (Eppemdorfmastercycler5330) and Taq polysaccharase (Promega company) to finish polymerase chain reaction.Obtain maximum amplification for the primer that utilizes these degeneracys makes the cDNA of coding chloroperoxidase, make polymerase chain reaction circulation 30 times at 46 ℃ (annealing steps).Two specific fragments that obtain are connected on the pUC18 carrier, and the clone also checks order to two chains, according to two kinds of special primers below the dna sequencing consequence devised:

5’-CATAGCGATAGCGACGCGGA-3’

With

5’-CTAACCCCGGCGCCAACATC-3’

These two kinds of primers are used for the polymerase chain reaction as template with the first chain cDNA.Therefore obtain the gene specific dna fragmentation by connecting two known dna sequence dnas.This fragment cloning to the pUC18 carrier, is checked order subsequently.In order the 5 ' district of the mRNA of the chloroperoxidase that obtains to encode to use 5 ' amplification RACE test kit (Clometech company).Saidly never wait curved spore to separate the chloroperoxidase genomic gene in mould by following: freeze driedly not wait the mould mycelium of curved spore to separate not wait the mould genomic dna of curved spore from what liquid nitrogen, grind, and extraction damping fluid (200mM Tris-HCl with appropriate amount, PH8.5,25mMEDTA, 250mM NaCl, 1%SDS and 0.2mg/ml Proteinase K) extract.At room temperature after the incubated overnight, add the phenol of 0.7 volume and the chloroform of 0.3 volume and thoroughly mix.With 10000 * g centrifuge tube and water layer is transferred in the test tube of a cleaning.Dehydrated alcohol precipitation genomic dna with 2 times of volumes.After centrifugal 5 minutes with 5000 * g.Again the 10mM Tris-HCl that is suspended in 2ml, pH8.0,1mM EDTA will be precipitated.And recommendation RNA enzyme (Boehringer, Mannheim) processing by manufacturers.Use phenol: chloroform: primary isoamyl alcohol (25: 24: 1) extracts the solution that contains genomic dna, after ethanol sedimentation, is dissolved in the 10mM Tris-HCl of suitable volumes at last, and pH8.0 is in the 1mM edta buffer liquid.For the Southern that carries out genomic dna analyzes, unite dna digestions with several limit endoenzymes, after agarose gel electrophoresis, inhale and print on the nitrocellulose filter (Sambrook et al., 1989).Utilize radiolabeled specific fragment (two special primers with above group description increase) to finish hybridization, said fragment causes at random with the dATP (Sambrook et al., 1989) of α-32P mark.Prepare little library based on the result who obtains with the genomic dna that is inserted into the PstI digestion on the pUC18 carrier.Screen this library with analyzing identical probe with Southern.Separate positive colony and also two chains are checked order confirmation cDNA sequencing result.Having provided coding in Fig. 2 does not wait the gene of the mould chloroperoxidase of curved spore and infers gene product.

The chloroperoxidase that is prepared as follows cereuisiae fermentum produces system:

From wild-type cereuisiae fermentum GAL1 gene (Molecular and Cellular Biology 10,4757-4769,1990) obtain the known GAL1 induction type of EcoR1-BamH1 fragment Yeast promoter, and be cloned into plasmid YCplac33 and YEplac95 (Gietz and Sugino (1988) Gene 74, EcoR1 527-534), BamH1 site respectively.The plasmid that is obtained is called TNT1 and TNT2.By utilizing the PstI-EcoRI5 ' fragment that do not wait curved spore mould chloroperoxidase gene of subclone in the pUC18 to carry out the PCR experiment as primer and at BamH1 restriction site of the front of the 5 ' starting point that does not wait the mould chloroperoxidase gene of curved spore preparation as template with M13/pUC22-aggressiveness reverse sequence primer, and primer is in the front of the 5 ' starting point that does not wait the mould chloroperoxidase gene of curved spore:

5’GAG?AGA?GGA?TCC?ACT?CAC?TAC?TTA?CAA?TCA?CAC3’

Fragment with EcoR1 and BamH1 digest amplification.The pUC18 that digests to EcoR1-SmaI from the EcoRI-PvuII fragment subclone that does not wait the mould chloroperoxidase gene of curved spore that will contain 3 ' part of this gene.After EcoRI and XbaI digestion, go out to contain not wait the mould chloroperoxidase gene 3 ' part of curved spore from this clone purification.

Finish 3 ligations, wherein contain the TNT1 or the TNT2 that digest with BamH1 and XbaI respectively, 5 ' BamH1-EcoRI fragment and 3 ' EcoRI-XbaI fragment.After connecting and cloning, check the identity property of the plasmid that obtains.Thus obtained plasmid is called TNT3 (being derived by TNT1) and TNT4 (being derived by TNT2).

According to the method known in the art plasmid TNT3 and TNT4 transformed yeast bacterial strain BJ1991 and selection ura+ transformant.The ura+ transformant is copied on the YP flat board that contains glucose (2%) and semi-lactosi (2%).After growth, obtain cell and be suspended in 200 μ l 20mM Tris-HCl again, among the pH8.1 from flat board.After cultivating 5 minutes, get 10 μ l liquid and point sample to nitrocellulose filter.At 100mM sodium acetate buffer (pH5.5), the positive dianisidine of 1mM is incubated nitrocellulose filter in 100mM KBr and the 2mM hydrogen peroxide.The yeast strain that grows in semi-lactosi have a few and observe clearly color and generate, and grow in glucose yeast strain have a few and do not observe color.This shows made up the semi-lactosi induction type production system that does not wait the mould chloroperoxidase gene of curved spore in cereuisiae fermentum.Use 100mM potassium phosphate buffer (pH6.5), the similar analysis that 100mM KBr and 1mM hydrogen peroxide and 40 μ M phenol red (BDH) carry out clearly illustrates that the liquid from the yeast strain that grows in semi-lactosi forms indigo plant/purple, and is used for not having change in color from the liquid that grows in the yeast strain of glucose.Has the allos chloroperoxidase expression of gene system that does not wait the mould chloroperoxidase function of curved spore that is similar in order further to confirm in yeast, to have produced to express, from the yeast strain purification of Recombinant enzyme of semi-lactosi inductive TNT3 or TNT4 conversion.On containing the substratum of semi-lactosi, after the growth, collect yeast cell and be suspended in 20mM Tris-HCl again, (pH8.1) in.Add aseptic granulated glass sphere then, suspension thoroughly vibrates.After centrifugal 15 minutes with 10,000 * g, obtain supernatant liquor and be added on the DEAE post, utilize to be substantially similar to this recombinase of purification process purifying that wild-type does not wait the mould enzyme of curved spore (above).After purifying, obtaining specific activity is that the proteinic reorganization chloroperoxidase of 22U/mg is (at 100mM sodium acetate buffer pH5.0, the 1mM hydrogen peroxide, measure among 5mM Repone K and the 50 μ M MCD, also can be referring to van Schijndel et al., 1993), it is equivalent to never to wait the proteinic specific activity of 20U/mg of the chloroperoxidase of the mould purifying of curved spore own.The active situation of pH that in accompanying drawing 3, has shown wild-type chloroperoxidase and reorganization chloroperoxidase.The enzyme that accompanying drawing 3 further provides recombination yeast to produce has the function that is similar to wild-type enzyme.

Embodiment 7

The suitable halo peroxidase of screening in other microorganism

The microorganism of Shi Yonging is not wait curved spore mould (CBS 371.72) in the present embodiment, Drechslera biseptata (CBS 371.72), Drechslera fuqax (CBS 509.77), Drechslera nicotiae (CBS 655.74), Drechslera subpapendorfii (656.74), Embelisia hyacinthi (416.71), Embelisia didymospora (CBS 766), Ulocladium chartarum (200.67) and Ulocladium botrytis (452.72).Various fungies are grown on agar plate.To nitrocellulose filter, this filter membrane soaks in 50mM Tris-HCl damping fluid (8.3) in advance after growth is finished, extracellular protein to be shifted (duplicate and inhale seal).After on agar plate, cultivating 15 minutes, by being with or without in the presence of the 0.1M Potassium Bromide filter membrane at 100mM sodium acetate (5.5) or potassium phosphate buffer (pH6.5 and 7.5), imbibition in positive dianisidine of 1mM and the 2mM hydrogen peroxide and detect the halo peroxidase activity of filter membrane.Therefore can detect the bromine peroxide enzyme and/or the chloroperoxidase of generation.For whether the halo peroxidase that detects generation is the halo peroxidase that contains vanadium, repeat above-described detection in the presence of the 10 and 100 μ M vanadic acid sodiums having and do not have.For the halo peroxidase that contains vanadium, under the situation of replenishing vanadate, observe the growth signal.Whether be similar to the vanadium-halogenated peroxidase that does not wait curved spore mould really in order to detect the chloroperoxidase that identifies, according to van Schijndel et al., 1993 method is from Ulocladiumchartarum, Embelisia didymospora, the chloroperoxidase of Drechslera subpapendorfii purify small quantities.The optimal pH of these enzymes is 4.5-5.5.Increase owing to add the chlorization activity of these enzymes of vanadic salts, obviously the peroxidase of vanadium-halogenated really of these enzymes.Whether be similar to and do not wait the mould chloroperoxidase of curved spore in order further to detect these enzymes of being discerned, further carry out qualitative a kind of halo peroxidase of being discerned.Select chloroperoxidase by fungi Drechslera biseptata (CBS 371.72) generation for this reason.It has and is similar to the characteristic that does not wait the mould chloroperoxidase of curved spore, has high thermal stability and to the high-affinity of its substrate.For other vanadium-halogenated peroxidase (de Boer et al., 1988; Wever etal., 1988), oxidized enzyme does not have epr signal; But after the SODIUM HYDROSULPHITE sodium reduction, observe typical vanadium base EPR spectrum (result does not show).Isotropy EPR parameter g ₀=1.969 and A ₀=9.0 almost with identical (Wever et al, 1985) of not waiting the mould enzyme of curved spore.In addition, utilize proteolytic enzyme and cyanogen bromide the enzymatic lysis of purifying to be peptide.Peptide structure collection of illustrative plates shows identical form, shows that these two kinds of enzymes have very big sequence homology.Really for this situation, two peptide chains of the enzyme that obtains with protease treatment and purifying are checked order.This two sequence shows very large homology and can sum up two kinds of enzymes is very similar.

Derive from the aminoacid sequence that does not wait the mould peptide of curved spore:

(Asp)-leu-arg-gln-pro-tyr-asp-pro-thr-ala-pro-ile-glu-asp-gln-pro-gly-ile-val-arg-thr-

Aminoacid sequence from the similar peptide of Drechslera biseptata:

Asp-leu-arg-gln-pro-tyr-asp-pro-thr-ala-pro-ile-glu-glu-gln-pro-gly-ile-val-arg-thr-

Therefore by using replica technique (after adding vanadic salts, detecting active the raising) and/or can differentiating the suitable vanadium-halogenated peroxidase that contains by (part) purifying enzyme and in the raising of detection of active afterwards of adding vanadic salts.

Embodiment 8

Utilize the further suitable chloro peroxidase of screening in other microorganism of antibody

The bacterial strain of Shi Yonging is not wait curved spore mould (CBS102.42) in the present embodiment, Drechslera biseptata (CBS 371.72), Drechslerasubpapendorfii (CBS 656.74), Embelisia didymospora (CBS 766.79), UloCladium chartarum (200.67).Microorganism is pressed two growth periods.At first, use the spore inoculating sterile propagation substratum (Van Schijndel et al., 1993) of microorganism.23 ℃ of shaking culture 3 days, culture is transferred to contained in 1 liter of fermention medium, 3 liters of triangular flasks of (containing 5 gram caseins (Gibco BRL) in every liter of distilled water, 3 gram yeast extracts and 1 gram fructose) afterwards.After 23 ℃ of shaking culture 14-17 days, collect substratum, filter and substantially according to Van Schijndel et al. 1994 method purifying chloroperoxidase.The preparation antivenom purification is (according to Van Schijndel et al. in a rabbit (does at two monthly ages), 1994 method) the polyclonal antibody that does not wait the mould chloroperoxidase of curved spore (the complete auxiliary agent of Fu Shi is used in injection for the first time, and the incomplete auxiliary agent of Fu Shi is used in booster shots).Booster shots were got blood from rabbit in 6 days afterwards the last time.At 56 ℃ serum is heated 30 minutes so that complement inactivation and centrifugal then.Obtain supernatant liquor.The bromine peroxide enzyme for preparing various purifying chloroperoxidases and Ascophylum nodosrm is (according to Wever et al, 1985 purifying) serial dilutions, each protein (about 0.1mg/ml) since 50 μ l, and each sample dilutes twice successively.Utilize a dot blotting instrument (Bio-Rad) on nitrocellulose filter, to put diluent, with the 2%BSA washing and successively with the anti-chloroperoxidase serum of rabbit, biotinylated goat antirabbit (diluting 1: 3000), 1: 800 diluent and the developer (5-bromo-4-chloro-3-indoles phosphoric acid, 4-nitroblue tetrazolium drone muriate) (Boehinger Mannheim) of alkaline phosphate bonded streptavidin (diluting 1: 2000) are incubated.All steps are carried out according to standard method.The results are shown in the Table VI.

Table VI

The halo peroxidase comes from	Do not wait the polyclonal antibody of the mould chloroperoxidase of curved spore that cross reaction is arranged with anti-
The halo peroxidase comes from		Do not wait curved spore mould	Be
????Drechslera?biseptata	Be	Do not wait curved spore mould	Be
????Drechslera?biseptata	Be	??Drechslera?subpapendorfii	Be
????Embelisia?didymospora	Be	??Drechslera?subpapendorfii	Be
????Embelisia?didymospora	Be	?????Ulocladium?chartarum	Be
Bromine peroxide enzyme from Ascophylum nodosrm	Not	?????Ulocladium?chartarum	Be

Based on disclosed result in the Table VI, can sum up the chloroperoxidase that carries out with the anti-antibody that does not wait the mould chloroperoxidase of curved spore and be suitable for discerning other suitable halo peroxidase that contains vanadium.These halo peroxidase can be rough forms, part or all of purified form.Purification technique is a technology known in the art, as gel-filtration, and ion exchange chromatography, hydrophobic interaction chromatography, precipitation technology, (surpassing) filtering technique, affinity chromatography, gel electrophoresis and other technology.

Embodiment 9

The suitable chloroperoxidase of screening in other microorganism

Described what use is made of in the present embodiment and in other microorganism, detected homologous gene from the radioactive probe that does not wait the mould chloroperoxidase gene of curved spore.Can finish as described below:

Substantially do not wait curved spore mould (CBS102.42) according to the purification process that does not wait the mould chromosomal DNA of curved spore (being described in embodiment 6) purifying, Drechslera biseptata (CBS 371.72), Embelisia didymospora (CBS 766.79), chromosomal DNA.Southern for genomic dna analyzes, and prints on the Nitrocellulose filter membrane (Sambrook et al., 1989) in conjunction with digesting this DNA, inhaling after agarose gel electrophoresis with several Restriction Enzymes.Utilize radiolabeled gene specific fragment to hybridize, described fragment utilize α- ³²The dATP of P mark (Sambrook et al., 1989) causes at random.Utilize the first chain cDNA (referring to embodiment 6) as template and use following primer (before radio-labeling) the employed gene specific fragment that in polymerase chain reaction, increases:

5’-CACGATGGGGTCCGTTACAC

With

5’-GTACCGCTATCGCTGCGCCTG

Hybridization conditions is as follows:

In prehybridization and hybridization, utilize 6 * SSPE, 5 * Denharts, 0.5%SDS and 10mg salmon sperm dna are as damping fluid.55 ℃ of prehybridizations 1 hour.Then radioactive probe is boiled and directly added again in 1 minute.Continuing hybridization subsequently spends the night.Provided in the accompanying drawing 4 with not waiting radioautogram that curved spore is mould and Drechslera biseptata obtains.In the figure, swimming lane 1: λ DNA; Swimming lane 2: non-digestion do not wait the mould genomic dna of curved spore; Swimming lane 3:idem digests with EcoRI; Swimming lane 4: digest with BamH1; Swimming lane 5: with EcoRI and BamH1 digestion; Swimming lane 6: digest with XbaI; Swimming lane 7: digest with PstI; Swimming lane 8: with XbaI and PstI digestion; Swimming lane 9-14: use Drechslera biseptata, idem.As seeing in the figure, use chromosomal DNA to obtain positive colony from Drechslera biseptata, show that two kinds of gene height are similar.Use DNA to obtain analog result from Embelisiadidymospora.Therefore we sum up to utilize and do not wait the mould chloroperoxidase gene of curved spore, or this gene deutero-part or can detect the vanadium-halogenated peroxidase of two kinds of other microorganisms based on the probe of the sequence preparation that does not wait the mould chloroperoxidase gene of curved spore.

Table VII is based on two kinds of Oligonucleolide primers (20 aggressiveness) that do not wait the mould chloroperoxidase aminoacid sequence of curved spore.

I: inosine

A/G: use at this position A and impartial mixing of G.

C/T: use at this position C and impartial mixing of T.

G/A/T/C: at this position G, A, the impartial mixing of C and T uses.

Oligonucleotide 1:

5’??-TAC/TATGAAA/GCCIGTIGAA/GCA-3’

Oligonucleotide 2:5 '-AG/AT/CTGGCG/ATAIGCG/ATTG/ATC-3 '

Oligonucleotide 3:5 '-GAC/TGAA/GACIGCIGAA/GTAC/TGA-3 '

Oligonucleotide 4:5 '-AG/AIGCT/CTGIGCICCG/A/T/CCCCAT-3 '

Claims

1. the enzymatic antimicrobial composition contains the vanadium-halogenated peroxidase, halogenide and hydrogen peroxide cource or hydrogen peroxide cource.

2. the enzymatic antimicrobial composition of claim 1, wherein the vanadium-halogenated peroxidase is a chloroperoxidase.

3. the enzymatic antimicrobial composition in the arbitrary claim in front, wherein the vanadium-halogenated peroxidase is the chloroperoxidase that never waits the mould acquisition of curved spore.

4. the enzymatic antimicrobial composition in the arbitrary claim in front, wherein the vanadium-halogenated peroxidase is a chloroperoxidase, it has immunological cross-reaction with the chloroperoxidase that never waits the mould CBS102.42 of curved spore to obtain.

5. the enzymatic antimicrobial composition in the arbitrary claim in front, wherein hydrogen peroxide cource is an enzymatic hydrogen peroxide generation system.

6. the enzymatic antimicrobial composition of claim 5, wherein enzymatic hydrogen peroxide generation system is means of glucose/glucose oxidase or lactic acid/Lactate Oxidase system.

7. the enzymatic antimicrobial composition in the arbitrary claim in front, described composition does not have the katalaze enzyme activity basically.

8. suppress the method for microorganism growth, comprise the front described composition of arbitrary claim is applied to surface to be sterilized.

9. the vanadium-halogenated peroxidase is as the application of sanitizer.

10.DNA sequence contains the structure gene of coding from the vanadium chloroperoxidase that does not wait the mould CBS102.42 of curved spore.

11.DNA sequence as shown in Figure 2, contains the structure gene of coding from the vanadium chloroperoxidase that does not wait the mould CBS102.42 of curved spore.

12. expression vector contains a replication origin, transcribes and stop the control sequence and the part of claim 10 or 11 dna sequence dna at least.

13. prepare the method for vanadium-halogenated peroxidase, comprise that the expression vector with claim 12 transforms appropriate host, under the condition that allows expression of structural gene, cultivate the host and separate the vanadium-halogenated peroxidase.