SCHNEIDER PALLE (DK)
CONRAD LARS SPARRE (DK)
EBDRUP SOEREN (DK)
SPILE NICKIE INGER (DK)
DAMHUS TURE (DK)
SCHNEIDER PALLE (DK)
CONRAD LARS SPARRE (DK)
EBDRUP SOEREN (DK)
SPILE NICKIE INGER (DK)
| WO1994012621A1 | 1994-06-09 | |||
| WO1996012845A1 | 1996-05-02 | |||
| WO1996012846A1 | 1996-05-02 | |||
| WO1992018683A1 | 1992-10-29 |
| 1. | A process for bleaching stains present on fabric, comprising contacting, in an aqueous medium, the fabric with a phenol oxidizing enzyme system and a mediator for a sufficient period of time, wherein said mediator can be described by one of the following formulas : I: B0 / \ HO O A \ /' CO in which formula A is a group such as COX, CH=CHX, CH=CH CH=CHX, CH=CHCOX, S02X, POXZ, in which X and Z may be identical or different and selected from the group consisting of H, OH, CnH2n+1, OCnH2n+ι and NEF, in which E and F may be identical or different and selected from the group consisting of H and CnH2n+ι; 1 < n < 5; and B and C may be the same or different and selected from CmH2m.ι; 1 ≤ m < 5; or in which formula X represents (0) or (S) , and the substituent groups R^R9, which may be identical or different, independently represents any of the following radicals: hydrogen, halogen, hydroxy, formyl, carboxy, and esters and salts hereof, carbamoyl, sulfo, and esters and salts hereof, sulfamoyl, nitro, amino, phenyl, CιCi4alkyl, CιC5alkoxy, carbonylCiCsalkyl, arylCιC5alkyl; which carbamoyl, sulfa moyl, and amino groups may furthermore be unsubstituted or substituted once or twice with a substituent group R10; and which phenyl may furthermore be unsubstituted or substituted with one or more substituent groups R10; and which CιCι4alkyl, C1C5 alkoxy, carbonylCiCsalkyl, and arylCιC5alkyl groups may be saturated or unsaturated, branched or unbranched, and may furthermore be unsubstituted or substituted with one or more substituent groups R10; which substituent group R10 represents any of the following radicals: halogen, hydroxy, formyl, carboxy and esters and salts hereof, carbamoyl, sulfo and esters and salts hereof, sulfamoyl, nitro, amino, phenyl, aminoalkyl, piperidino, piperazinyl, pyrrolidino, CiCsalkyl, CiCsalkoxy; which carbamoyl, sulfamoyl, and amino groups may furthermore be unsubstituted or substituted once or twice with hydroxy, C1C5 alkyl, CιC5alkoxy; and which phenyl may furthermore be substituted with one or more of the following radicals: halogen, hydroxy, amino, formyl, carboxy and esters and salts hereof, carbamoyl, sulfo and esters and salts hereof, and sulfamoyl; and which CιC5alkyl, and CιC5alkoxy groups may furthermore be saturated or unsaturated, branched or unbranched, and may furthermore be substituted once or twice with any of the following radicals: halogen, hydroxy, amino, formyl, carboxy and esters and salts hereof, carbamoyl, sulfo and esters and salts hereof, and sulfamoyl; or in which general formula two of the substituent groups R1R9 may together form a group B, in which B represents any of the following the groups: (CHR10N=N) , (CH=CH)n, ( CH=N)n or (N=CR10NR11) , in which groups n represents an integer of from 1 to 3, R10 is a substituent group as defined above and R11 is defined as R10. |
| 2. | A process according to claim 1, in which the phenol oxidizing enzyme system is a peroxidase and a hydrogen peroxide source. |
| 3. | A process according to claim 2, wherein the peroxidase is horseradish peroxidase, soybean peroxidase or a peroxidase enzyme derived from Coprinus, Bacillus or Myxococcus . |
| 4. | A process according to claim 2 or 3, wherein the hydrogen peroxide source is hydrogen peroxide or a hydrogen peroxide precursor. |
| 5. | A process according to claim 1, wherein the aqueous medium contains H202 or a precursor for H202 in a concentration corresponding to 0.00125 mM H202. |
| 6. | A process according to claim 1, in which the phenol oxidizing enzyme system is a laccase or a laccase related enzyme together with oxygen. |
| 7. | A process according to claim 6, wherein the laccase is derived from Trametes, Coprinus, or Myceliophthora . |
| 8. | A process according to claim 1, wherein the concentration of the phenol oxidizing enzyme corresponds to 0.001100 mg of enzyme protein per liter of aqueous medium. |
| 9. | A process according to claim 1, wherein the mediator belongs to the group consisting of acetosyringone, syringaldehyde, methylsyringate and syringic acid. |
| 10. | A process according to claim 1, wherein the mediator belongs to the group consisting of 10methylphenothiazine, phenothiazine10propionic acid, phenoxazine10propionic acid, phenoxazine10hydroxyethyl, phenothiazine10ethyl4carboxy, promazine hydrochloride and phenothiazine10ethylalcohol. |
| 11. | A process according to claim 1, wherein the mediator in the aqueous medium is present in concentrations of from 0.01 to 5000 μM. |
| 12. | A process according to claim 1, wherein the phenol oxidizing enzyme system in the aqueous medium is present in concentrations of from 0.001100 mg of enzyme protein per liter. |
FIELD OF INVENTION
The present invention relates to a process for removing coloured stains present on fabric at low washing temperatures .
BACKGROUND ART
When washing clothes and other types of fabric there are some coloured stains which are problematic to remove: These stains typically originate from red wine, fruit such as black currant, cherry, strawberry and tomato (in particular ketchup and spaghetti sauce) , vegetables such as carrots and beetroot, tea, coffee, spices such as curry and paprika, grass, or ball pens/ink.
Commercial detergents normally include bleaching agents to solve the above mentioned problem. Traditionally, bleaching agents incorporated in detergent compositions are compounds which are precursors of hydrogen peroxide; hydrogen peroxide is then formed in the course of the washing procedure. Perborates and percarbonates are the most important examples of such hydrogen peroxide precursors . Perborate and percarbonate may also be combined with a peracid-forming bleach activator such as TAED (tetraacetylethylenediamine) or NOBS (nonanoyloxybenzenesulfonate) .
These traditional bleaching systems function quite satisfactorily at high temperatures, the optimum operation temperature of these systems being typically above 60°C.
An important challenge during the last years has been to provide efficient bleaching systems which function at low temperatures (10°C - 50°C) .
In WO 89/09813 it is suggested to remove stains by using peroxidase. The process has an effect but a more efficient
stain bleaching system useful at low temperatures is still needed.
SUMMARY OF THE INVENTION
It has now surprisingly been found possible to create a stain bleaching process which effectively removes stains at low temperatures, said process comprising contacting, in an aqueous medium, the fabric with a phenol oxidizing enzyme system and a mediator for a sufficient period of time, wherein said mediator can be described by one of the following formulas:
I:
B-0
/ \ HO- O -A
\ /
C-O
in which formula A is a group such as -CO-X, -CH=CH-X, -CH=CH-
CH=CH-X, -CH=CH-CO-X, -S0 2 -X, -PO-XZ, in which X and Z may be identical or different and selected from the group consisting of
-H, -OH, -C n H 2n +ι , -OC n H n+ ι and -N-EF, in which E and F may be identical or different and selected from the group consisting of
-H and C n H 2n+ ι; 1 < n < 5; and B and C may be the same or different and selected from C m H 2m+ ι; 1 < m < 5; or
in which formula X represents (-0-) or (-S-) , and the
substituent groups R 1 -.. 9 , which may be identical or different, independently represents any of the following radicals: hydrogen, halogen, hydroxy, formyl, carboxy, and esters and salts hereof, carbamoyl, sulfo, and esters and salts hereof, sulfamoyl, nitro, amino, phenyl, Cι-Cι 4 -alkyl, Cι-C 5 -alkoxy, carbonyl-Cι-C 5 -alkyl, aryl-Cι-C 5 -alkyl; which carbamoyl, sulfa¬ moyl, and amino groups may furthermore be unsubstituted or substituted once or twice with a substituent group R 10 ; and which phenyl may furthermore be unsubstituted or substituted with one or more substituent groups R 10 ; and which Ci-Cn-alkyl, C1-C5- alkoxy, carbonyl-Ci-Cs-alkyl, and aryl-Ci-Cs-alkyl groups may be saturated or unsaturated, branched or unbranched, and may furthermore be unsubstituted or substituted with one or more substituent groups R 10 ; which substituent group R 10 represents any of the following radicals: halogen, hydroxy, formyl, carboxy and esters and salts hereof, carbamoyl, sulfo and esters and salts hereof, sulfamoyl, nitro, amino, phenyl, aminoalkyl, piperidino, piperazinyl, pyrrolidino, Cι-C 5 -alkyl, Cι-C 5 -alkoxy; which carbamoyl, sulfamoyl, and amino groups may furthermore be unsubstituted or substituted once or twice with hydroxy, CJ-C5- alkyl, Ci-Cs-alkoxy; and which phenyl may furthermore be substituted with one or more of the following radicals: halogen, hydroxy, amino, formyl, carboxy and esters and salts hereof, carbamoyl, sulfo and esters and salts hereof, and sulfamoyl; and which Cι-C 5 -alkyl, and Ci-Cs-alkoxy groups may furthermore be saturated or unsaturated, branched or unbranched, and may furthermore be substituted once or twice with any of the following radicals: halogen, hydroxy, amino, formyl, carboxy and esters and salts hereof, carbamoyl, sulfo and esters and salts hereof, and sulfamoyl; or in which general formula two of the substituent groups R -R 9 may together form a group -B-, in which B represents any of the following the groups: (-CHR 10 -N=N-) , (-CH=CH-) n , (- CH=N-) n or (-N=CR 10 -NR n -) , in which groups n represents an integer of from 1 to 3, R 10 is a substituent group as defined
above and R 11 is defined as R 10 .
DETAILED DESCRIPTION OF THE INVENTION
According to the present invention it may be possible to remove coloured stains present on fabric at low washing temperatures, in particular at washing temperatures of from 5°C to 50°C, preferably at washing temperatures of from 10°C to _0°C, more preferably at temperatures of from 10°C to 30°C, even more preferably at temperatures of from 10°C to 20°C, by using a bleaching process comprising contacting, in an aqueous medium, the fabric with a phenol oxidizing enzyme and a mediator for a sufficient period of time.
Stains which may be removed according to the present invention typically originate from for example spices such as curry and paprika or vegetables/fruits, e.g., carrots.
The bleaching time for removing the stain (s) may vary; the fabric may be soaked for one or two days or the bleaching may be performed within a shorter period, typically for a period of 1 to 90 minutes, preferably for a period of 1 to 30 minutes.
Phenol Oxidizing Enzymes
By the term "a phenol oxidizing enzyme" is meant an enzyme, which by using hydrogen peroxide or molecular oxygen, is capable of oxidizing organic compounds containing phenolic groups. Examples of such enzymes are peroxidases and oxidases.
If the phenol oxidizing enzyme requires a source of hydrogen peroxide, the source may be hydrogen peroxide or a hydrogen peroxide precursor for in situ production of hydrogen peroxide, e.g., percarbonate or perborate, or a hydrogen peroxide generating enzyme system, e.g., an oxidase and a substrate for the oxidase, or an amino acid oxidase and a suitable amino acid, or a peroxycarboxylic acid or a salt thereof. Hydrogen peroxide may be added at the beginning of or during the process, e.g., in a concentration corresponding to
0.001-25 mM H 2 0 2 .
If the phenol oxidizing enzyme requires molecular oxygen, molecular oxygen from the atmosphere will usually be present in sufficient quantity. In the context of the present invention the enzyme of the phenol oxidizing enzyme may be an enzyme possessing peroxidase activity or a laccase or a laccase related enzyme as described below.
According to the invention the concentration of the phenol oxidizing enzyme in the aqueous medium where the stain bleaching of the fabric is taking place, may be of from 0.001- 100 mg of enzyme protein per liter, in particular of from 0.01- 50 mg of enzyme protein per liter, even more preferably of from 0.1-10 mg of enzyme protein per liter.
Peroxidases and Compounds possessing Peroxidase Activity
An enzyme exhibiting peroxidase activity may be any peroxidase enzyme comprised by the enzyme classification (EC 1.11.1.7), or any fragment derived therefrom, exhibiting peroxidase activity, or synthetic or semisynthetic derivatives thereof (e.g. porphyrin ring systems or microperoxidases, cf. e.g. US 4,077,768, EP 537,381, WO 91/05858 and WO 92/16634) .
Preferably, the peroxidase employed in the method of the invention is producible by plants (e.g. horseradish or soybean peroxidase) or microorganisms such as fungi or bacteria. Some preferred fungi include strains belonging to the sub¬ division Deuteromycotina, class Hyphomycetes, e.g. Fusarium, Humicola , Tricoderma , Myrothecium, Verticillum, Arthromyces, Caldariomyces, Ulocladium, Embellisia , Cladosporium or Dre- schlera, in particular Fusarium oxysporum (DSM 2672), Humicola insolens, Trichoderma resii, Myrothecium verrucaria (IFO 6113) , Verticillum alboa trum, Verticillum dahlie, Arthromyces ramosus (FERM P-7754), Caldariomyces fumago, Ulocladium chartarum, Embellisia alii or Dreschlera halodes . Other preferred fungi include strains belonging to the subdivision Basidiomycotina, class Basidiomycetes, e.g.
O 97/11217 PC17DK96/00390
Coprinus, Phanerochaete, Coriolus or Trametes, in particular Coprinus cinereus f . microsporus (IFO 8371), Coprinus macror- hizus, Phanerochaete chrysosporium (e.g. NA-12) or Trametes (previously called Polyporus) , e.g. T. versicolor (e.g. PR4 28-A) .
Further preferred fungi include strains belonging to the subdivision Zygomycotina, class Mycoraceae, e.g. Rhizopus or Mucor, in particular Mucor hiemalis .
Some preferred bacteria include strains of the order Actinomycetales, e.g. Streptomyces spheroides (ATTC 23965), Streptomyces thermoviolaceus (IFO 12382) or Streptoverticill um verticillium ssp. verticillium .
Other preferred bacteria include Bacill us pumil us (ATCC 12905), Bacillus stearothermophil us, Rhodobacter sphaeroides, Rhodomonas pal ustri, Streptococcus lactis, Pseudomonas purrocinia (ATCC 15958) or Pseudomonas fl uorescens (NRRL B-ll) .
Further preferred bacteria include strains belonging to Myxococcus, e . g . M. virescens . The peroxidase may furthermore be one which is producible by a method comprising cultivating a host cell trans¬ formed with a recombinant DNA vector which carries a DNA sequence encoding said peroxidase as well as DNA sequences encoding functions permitting the expression of the DNA sequence encoding the peroxidase, in a culture medium under conditions permitting the expression of the peroxidase and recovering the peroxidase from the culture.
Particularly, a recombinantly produced peroxidase is a peroxidase derived from a Coprinus sp., in particular C. macrorhizus or C. cinereus according to WO 92/16634, or a variant thereof, e.g., a variant as described in WO 93/24618 and WO 95/10602.
In the context of this invention, compounds possessing peroxidase activity comprise peroxidase enzymes and peroxidase active fragments derived from cytochromes, haemoglobin or peroxidase enzymes, and synthetic or semisynthetic derivatives
thereof, e.g., iron porphyrins, and iron phthalocyanine and derivatives thereof.
Determination of Peroxidase Activity (PODU) 1 peroxidase unit (PODU) is the amount of enzyme that catalyzes the conversion of 1 μmole hydrogen peroxide per minute at the following analytical conditions: 0.88 mM hydrogen peroxide, 1.67 mM 2, 2 ' -azinobis (3-ethylbenzothiazoline-6- sulfonate) , 0.1 M phosphate buffer, pH 7.0, incubated at 30°C, photometrically followed at 418 nm.
Laccase and Laccase Related Enzymes
In the context of this invention, laccases and laccase related enzymes comprise any laccase enzyme comprised by the enzyme classification (EC 1.10.3.2), any catechol oxidase enzyme comprised by the enzyme classification (EC 1.10.3.1), any bilirubin oxidase enzyme comprised by the enzyme classification (EC 1.3.3.5) or any monophenol monooxygenase enzyme comprised by the enzyme classification (EC 1.14.18.1) . The above mentioned enzymes may be derived from plants, bacteria or fungi (including filamentous fungi and yeasts) and suitable examples include a laccase derivable from a strain of Aspergillus, Neurospora, e.g., N. crassa , Podospora, Botrytis, Collybia , Fomes, Lentinus, Pleurotus, Trametes , e.g., T. villosa and T. versicolor, Rhizoctonia , e.g., R . solani , Coprinus, e.g., C. plicatilis and C. cinereus, Psa tyrella , Myceliophthora , e.g., M. thermophila , Schytalidium, Polyporus, e.g., P. pinsitus, Phlebia, e.g., P. radita (WO 92/01046), or Coriolus, e.g., C. hirsutus (JP 2-238885) . The laccase or the laccase related enzyme may furthermore be one which is producible by a method comprising cultivating a host cell transformed with a recombinant DNA vector which carries a DNA sequence encoding said laccase as well as DNA sequences encoding functions permitting the expression of the DNA sequence encoding the laccase, in a cul-
ture medium under conditions permitting the expression of the laccase enzyme, and recovering the laccase from the culture.
Determination of Laccase Activity (LACU) Laccase activity is determined from the oxidation of syringaldazin under aerobic conditions. The violet colour produced is photometered at 530 nm. The analytical conditions are 19 μM syringaldazin, 23.2 mM acetate buffer, pH 5.5, 30°C, 1 min. reaction time. 1 laccase unit (LACU) is the amount of enzyme that catalyses the conversion of 1.0 μmole syringaldazin per minute at these conditions.
Mediators According to the present invention a mediator is any compound that enhances the bleaching process. The enhancing agent will typically be an organic compound, e.g., an organic compound described by one of the following formulas:
B-0
/ \ HO- O -A
/ C-O
in which formula A is a group such as -CO-X, -CH=CH-X, -CH=CH- CH=CH-X, -CH=CH-CO-X, -S0 2 -X, -PO-XZ, in which X and Z may be identical or different and selected from the group consisting of -H, -OH, -C n H 2n+ ι , -OC n H 2n+ ι and -N-EF, in which E and F may be identical or different and selected from the group consisting of -H and C n H 2n+ ι; 1 < n < 5; and B and C may be the same or different and selected from C m H 2 m+ι; 1 ≤ m < 5.
In the above mentioned formula A may be placed meta to the hydroxy group instead of being placed in the para-position as shown.
In particular embodiments, the mediator is acetosyringone, syringaldehyde, methylsyringate, syringic acid, ethylsyringate, propylsyringate, butylsyringate, hexylsyringate, octylsyringate or ethyl 3- (4-hydroxy-3, 5- dimethoxyphenyl) acrylate.
The mediator used in the present invention may also be described by the following formula:
in which formula X represents (-0-) or (-S-) , and the substituent groups R^R 9 , which may be identical or different, independently represents any of the following radicals: hydrogen, halogen, hydroxy, formyl, carboxy, and esters and salts hereof, carbamoyl, sulfo, and esters and salts hereof, sulfamoyl, nitro, amino, phenyl, Cι-Cι -alkyl, Ci-Cs-alkoxy, carbonyl-Ci-Cs-alkyl, aryl-Cι-C 5 -alkyl; which carbamoyl, sulfamoyl, and amino groups may furthermore be unsubstituted or substituted once or twice with a substituent group R 10 ; and which phenyl may furthermore be unsubstituted or substituted with one or more substituent groups R 10 ; and which Cι-Cι 4 -alkyl, C1-C5- alkoxy, carbonyl-Ci-Cs-alkyl, and aryl-Cι-C 5 -alkyl groups may be saturated or unsaturated, branched or unbranched, and may furthermore be unsubstituted or substituted with one or more substituent groups R 10;, which substituent group R 10 represents any of the following radicals: halogen, hydroxy, formyl, carboxy and esters and salts hereof, carbamoyl, sulfo and esters and salts hereof, sulfamoyl, nitro, amino, phenyl, aminoalkyl, piperidino, piperazinyl, pyrrolidino, d-C 5 -alkyl, Cι-C 5 -alkoxy; which
carbamoyl, sulfamoyl, and amino groups may furthermore be unsubstituted or substituted once or twice with hydroxy, C 1 -C 5 - alkyl, Cι-C 5 -alkoxy; and which phenyl may furthermore be substituted with one or more of the following radicals: halogen, hydroxy, amino, formyl, carboxy and esters and salts hereof, carbamoyl, sulfo and esters and salts hereof, and sulfamoyl; and which Cι-C 5 -alkyl, and Cι-C 5 -alkoxy groups may furthermore be saturated or unsaturated, branched or unbranched, and may furthermore be substituted once or twice with any of the following radicals: halogen, hydroxy, amino, formyl, carboxy and esters and salts hereof, carbamoyl, sulfo and esters and salts hereof, and sulfamoyl; or in which general formula two of the substituent groups R x -R 9 may together form a group -B-, in which B represents any of the following the groups: (-CHR 10 -N=N-) , (-CH=CH-) n , (- CH=N-) n or (-N=CR 10 -NR n -) , in which groups n represents an integer of from 1 to 3, R 10 is a substituent group as defined above and R 11 is defined as R 10 .
In particular embodiments, the mediator is 10-methyl- phenothiazine, 10-phenothiazine-propionic acid, N-hydroxy¬ succinimide-10-phenothiazine-propionate, 10-ethyl-4-pheno- thiazine-carboxylic acid, 10-ethylphenothiazine, 10-propyl- phenothiazine, 10-isopropylphenothiazine, methyl-10-pheno- thiazinepropionate, 10-phenylphenothiazine, 10-allylpheno- thiazine, 10- (3- (4-methyl-l-piperazinyl)propyl)phenothiazine, 10- (2-pyrrolidinoethyl)phenothiazine, chlorpromazine, 2-chloro- 10-methylphenothiazine, 2-acetyl-10-methylphenothiazine, 4- carboxy-10-phenothiazine, 10-methylphenoxazine, 10-ethyl- phenoxazine, 10-phenoxazine-propionic acid or 4-carboxy-10- phenoxazine-propionic acid.
The mediator of the invention may be present in concentrations of from 0.01 to 5000 μM, preferably in concentrations of from 0.1 to 1000 μM, even more preferably in concentrations of from 1 to 500 μM.
Preparation of Mediators
The mediators described in the present application may be prepared using methods well known to those skilled in the art; some of the mediators are also commercially available. Some of the mediators may be obtained from Sigma-
Aldrich, Janssen Chimica, Kodak, Tokyo Kasai Organic Chemicals, Daiichi Pure Chemicals Co. or Boehringer Mannheim.
N-methylated derivatives of phenothiazine and phenoxazine may be prepared by methylation with methyliodide as described by Cornel Bodea and loan Silberg in "Recent Advances in the Chemistry of Phenothiazines" (Advances in heterocyclic chemistry, 1968, Vol. 9, pp. 321-460); B. Cardillo & G. Casnati in Tetrahedron, 1967, Vol. 23, p. 3771. Phenothiazine and phenoxazine propionic acids may be prepared as described in J_. Org. Chem. 15, 1950, pp. 1125-1130. Hydroxyethyl and hydroxy¬ propyl derivatives of phenothiazine and phenoxazine may be prepared as described by G. Cauquil in Bulletin de la Society Chemique de France, 1960, p. 1049.
Methylsyringate, ethylsyringate, propylsyringate, butylsyringate, hexylsyringate and octylsyringate may be produced by using the method disclosed in Chem. Ber. 67, 1934, p. 67.
Ethyl 3- (4-hydroxy-3, 5-dimethoxyphenyl) acrylate was synthesised from syringaldehyde and triethyl phosphonoacetate in ethanol/sodium ethanolate. The product was after purification characterised by 1 H-NMR and 13 C-NMR (showing spectra as expected) and the melting point was 68-70°C.
The invention is further illustrated in the following examples which are not intended to be in any way limiting to the scope of the invention as claimed.
EXAMPLE 1
Stain Bleaching with mediated laccase systems
To investigate the stain bleaching effect of mediated laccase systems, small-scale washing trials were carried out. Test pieces of fabric soiled with beta-carotene, curry, and paprika, respectively, were treated in beakers with magnetical stirring in borate solution with several combinations of laccases and mediators.
The reference treatment in each case was a wash under the same conditions with no laccase-mediator system present.
After the wash, the test pieces of fabric were thoroughly rinsed in cold tap water and air-dried in the dark overnight. The bleaching effect of any given laccase-mediator system was then evaluated by using a Datacolor Elrepho 2000 (remission measurements at 460 nm) or by using a Minolta Chroma Meter (colourspace XYZ) using D65 as the light source.
Positive values of more than 2-3 units indicate significant bleaching effects in both systems.
The mediators used are abbreviated as follows:
PPT: phenothiazine-10-propionic acid EPC: 4-carboxyphenothiazine-10-propionic acid POP: phenoxazine-10-propionic acid AS: acetosyreingone,
(4-hydroxy-3, 5-dimethoxyphenyl) ethan-1-one MS: methyl syringate,
4-hydroxy-3, 5-dimethoxybenzoic acid methyl ester
Wash trial 1. Laccase from Trametes villosa .
Medium: 25 mM borate (from boric acid with NaOH), pH 7. Temperature: 40 C C, 15°C, 10°C. Duration of wash: 30 min.
Laccase preparation obtained in the following way: 800 ml culture broth of Trametes villosa , CBS 678.70, was filtered with filter aid to give a clear filtrate, which was concentrated and washed by ultrafiltration on a membrane with a cut-off of 6-8 kDa. One ml samples of concentrated preparation was applied onto a Q-Sepharose HP column (Pharmacia, Sweden) equilibrated with 0.1 M fosfate pH 7, and the laccase was eluted with a flat NaCl gradient around 0.25 M. Fractions with laccase activity from 10 runs were pooled and concentrated by ultrafiltration.
Laccase dossage: 1 mg/l washing liquor.
Mediator dosage: 120 μM. Mediators predissolved in ethanol.
Bleaching Results (40"C) :
Delta (remission (%) at 460 nm)
Betacarotene Curry Paprika
P PPPTT 1 144 6 22
EPC 7 5 20
POP 3 2 16
AS 5 6 15
MS 8 7 19
Bleaching results (15 * C) :
Calculated Delta-E (colourspace XYZ)
Paprika*
PPT 18
Bleaching results (lO'C) :
Calculated Delta-E (colourspace XYZ)
Paprika*
PPT 21
*) : The paprika test pieces of fabric were obtained from Center for Test materials, WFK P (P043) .
It can be seen from the results presented above that paprika is bleached very effectively even at 10-15°C.
Wash trial 2. Laccase from Coprinus cinereus .
Medium: 25 mM borate (from boric acid with NaOH), pH 8.5.
Temperature: 40° C.
Duration of wash: 30 min.
Laccase preparation obtained in the following way: Coprinus cinereus (IFO 30116 - freely available to the public from Institute of Fermentation, Osaka (IFO) under the indicated deposit number) was inoculated from a PDA agar slant (PDA: 39 g/1 potato dextrose agar) into a 100 ml shake flask containing medium A (Medium A is described below) . The culture was cultivated for 6 days at 26°C and 100 rpm. A 10-liter fermentor containing medium A was inoculated with the 100 ml culture broth. The fermentation ran for 6 days at 26°C and 100 rpm. The culture broth was filtrated and concentrated by ultrafiltration. Further purification was carried out using hydrophobic interaction chromatography followed by anionic exchange chromatography. This process resultated in at preparation with a laccase activity of 3.6 LACU/ml. The estimated purity was >80 on a protein basis.
Medium A: Soja meal 30 g/1
Maltodextrin 15 g/1 bacto peptone 5 g/1 pluronic 0.2 g/1
Laccase dosage: 1 mg/l washing liquor.
Mediator dosage: 120 μM. Mediators predissolved in ethanol.
Bleaching Results:
Delta (remission (%) at 460 nm)
Betacarotene Paprika
PPT 16 5
EPC 12 8
AS 5 1
MS 15 6
Wash trial 3. Laccase from Myceliophthora thermophila .
Medium: 25 mM borate (from boric acid with NaOH) pH 8.5 at 40°C;
25 mM borate (from boric acid with NaOH) pH 7 at 15°C and at
10°C. Temperature: 40°C, 15°C, 10°C.
Duration of wash: 30 min.
Laccase preparation obtained as described in PCT/US95/06815.
Laccase dosage: 1 mg/l washing liquor.
Mediator dosage: 120 μM. Mediators predissolved in ethanol.
Bleaching Results (40°C) :
Delta (remission (%) at 460 nm)
Betacarotene Paprika
PPT 4 0
EPC 3 3
AS 7 4 MS 7 8
Bleaching results (15'C) :
Calculated Delta-E (colourspace XYZ)
Paprika*
MS 21
Bleaching results (lO'C) :
Calculated Delta-E (colourspace XYZ)
Paprika*
PPT 17
*) : The paprika test pieces of fabric were obtained from Center for Test aterials, WFK P (P043) .
It can be seen from the results presented above that paprika is bleached very effectively even at 10-15°C.
EXAMPLE 2
Stain bleaching with mediated peroxidase systems To investigate the stain bleaching effect of mediated peroxidase systems, a small-scale washing trial was carried out analogously to the ones described in Example 1.
Wash trial. Peroxidase from Coprinus cinereus .
Medium: 25 mM borate (from boric acid with NaOH), pH 7. Temperature: 40°C, 15°C, 10°C. Duration of wash: 30 min.
Peroxidase preparation obtained as described in WO 94/12621 Peroxidase dosage: 0.2 mg/l washing liquor.
Mediator dosage: 120 μM. Mediators predissolved in ethanol. Hydrogen peroxide: 0.5 mM.
Bleaching Results (40"C)
Delta (remission (%) at 460 nm)
Betacarotene Curry Paprika
PPT 12 9 25
EPC 13 8 21
POP 4 4 18
AS 0 8 13
MS 9 9 20
Bleaching results (15'C)
Calculated Delta-E (colourspace XYZ)
Paprika*
PPT 28
Bleaching Results (10'Q :
Calculated Delta-E (colourspace XYZ)
Paprika*
MS 23
*) : The paprika test pieces of fabric were obtained from Center for Testmaterials, WFK P (P043) .
It can be seen from the results presented above that paprika is bleached very effectively even at 10-15°C.
EXAM LE 3
Stain bleaching with mediated peroxidase systems
Peroxidase-mediator systems identical to or similar to the ones studied in Example 2 were subjected to a Launder-ometer wash trial to investigate the effects of having a closed system and a different mechanical treatment. The Launder-ometer is a laboratory-scale set-up for simulating conditions in a traditional front-loaded European washing machine.
Wash trial. Peroxidase from Coprinus cinereus ,
Medium: 25 mM borate (from boric acid with NaOH), pH 7. Temperature: 40°C. Duration of wash: 5 min preheating from 35°C to 40°C, then 30
min . wash at 40°C .
Peroxidase preparation obtained as described Example 2. Peroxidase dosage: 120 μM. Mediators predissolved in ethanol. Hydrogen peroxide: 0.5 mM.
Mediators tested: the above ones (see Example 1) , except AS, and additionally MPT, 10-methylpenothiazine.
Bleaching results:
Delta (remission (%) at 460 nm)
Betacarotene Curry Paprika
P PPPTT 1 111 6 11
EPC 10 5 10
MPT 8 4 6
POP 6 3 10
MS 7 7 10
It is seen that trends are much the same as in the small-scale washing test in Example 2. (The absolute numbers obtained depend on, among other things, wash load and mechanical action on the fabrics in the wash and are not directly com- parable between the two examples here.)
EXAMPLE 4
Stain bleaching with a laccase and a series of homologous alkyl syringates as mediators
To investigate the effect on stain bleaching properties of homologous alkyl syringates mediators, a series of alkyl syringates was tested in an experiment much like the one in Example 3, except that the enzyme was a laccase instead of a peroxidase.
Wash trial. Laccase from Trametes villosa .
Medium: 25 mM borate (from boric acid with NaOH), pH 7. Temperature: 40°C. Duration of wash: 5 min preheating from 35°C to 40°C, then 30 min wash at 40°C.
Laccase preparation obtained as described in Example 1. Laccase dosage 1 mg/l washing liquor. Mediator dosage: 120 μM. Mediators predissolved in ethanol.
Mediators tested: methyl, ethyl, propyl, butyl, hexyl, and octyl syringate.
Bleaching results:
Delta (remission (%) at 460 nm)
Betacarotene methyl 13 ethyl 13 propyl 32 butyl 26 hexyl 8 octyl 2
It is seen that a marked optimum is reached at chain lengths 3 and 4 of the alkyl group. All these syringates except the octyl homologue achieve significant bleaching under the conditions given.