Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~ WO 96/10079 PCT/DK95100384
220 13 34
ENHANCERS SUCH AS ACETOSYRINGONE
= FIELD OF INVENTION
The invention relates to a method of oxidizing a
compound with a phenol oxidizing enzyme and an enhancing
agent. The invention also relates to a detergent additive and
to a detergent composition.
BACKGROUND ART
By a phenol oxidizing enzyme is meant an enzyme
which by using hydrogen peroxide or molecular oxygen, is
lo capable of oxidizing organic compounds containing phenolic
groups. Examples of such enzymes are peroxidases and
oxidases.
It has earlier been found that coloured substances
leached from dyed fabrics could be bleached by means of a
phenol oxidizing enzyme. The use of peroxidases or oxidases
for inhibiting dye transfer in this way is described in wo
91/05839.
Certain oxidizable substances, e.g., metal ions and
phenolic compounds such as 7-hydroxycoumarin, vanillin, and
p-hydroxybenzenesulfonate, have been described as
accelerators or enhancing agents able to enhance enzymatic
bleaching reactions (cf. e.g. WO 92/18683, WO 92/18687, and
Kato M and Shimizu S, Plant Cell Physiol. 1985 26 (7), pp.
1291-1301 (cf. Table 1 in particular)). In WO 94/12621 other
types of enhancing agents are disclosed, e.g., phenothiazines
and phenoxazines.
= It is the object of this invention to provide a new
group of enhancing agents which are effective for enhancing
phenol oxidizing enzymes.
WO 96/10079 220 13 34 PCT/DK95/00384
2
SUMMARY OF THE INVENTION
It has now surprisingly been found that a new group
of organic chemical substances performs excellently as
enhancers of phenol oxidizing enzymes. 5 This new group of organic chemical
substances not
only make the bleaching reactions faster compared with using
the phenol oxidizing enzyme alone, but many compounds which
could not be bleached at all, may now be bleached by using
the method of the invention.
Accordingly, the invention provides a method of
oxidizing a compound with a phenol oxidizing enzyme,
characterized by the presence of an enhancing agent of the
following formula:
OH
Bo oc
0
A
in which formula A is a group such as -D, -CH=CH-D, -CH=CH-
CH=CH-D, -CH=N-D, -N=N-D, or -N=CH-D, in which D is selected
from the group consisting of -CO-E, -SOZ-E, -N-XY, and
-N+-XYZ, in which E may be -H, -OH, -R, or -OR, and X and Y
and Z may be identical or different and selected from -H
and -R; R being a C1-C16 alkyl, preferably a C1-C$ alkyl, which
alkyl may be saturated or unsaturated, branched or unbranched
and optionally substituted with a carboxy, sulfo or amino
group; and B and C may be the same or different and selected
from CmH2,+1 ; 1 5 m S 5. =
BRIEF DESCRIPTION OF THE DRAWING =
The present invention is further illustrated by
reference to Fig. 1 which shows the bleaching of gradually
added Acid Blue 45 in phosphate/borate buffer pH 10 at 35 C;
= WO 96/10079 22~/ ii 13 3/, PCT/DK95/00384
3 "~
,,.
(I): Only dye addition; (II): Dye addition in the presence of
Laccase; (III): Dye addition in the presence of Laccase +
Acetosyringone; the experiment conducted as described in
Example 8.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a method of
oxidizing a compound with a phenol oxidizing enzyme,
characterized by the presence of an enhancing agent of the
following formula:
1o OH -
BO oe
#
A
in which formula A is a group such as -D, -CH=CH-D, -CH=CH-
CH=CH-D, -CH=N-D, -N=N-D, or -N=CH-D, in which D is selected
from the group consisting of -CO-E, -S02-E, -N-XY, and
-N+-XYZ, in which E may be -H, -OH, -R, or -OR, and X and Y
and Z may be identical or different and selected from -H
and -R; R being a C1-C16 alkyl, preferably a C1-C$ alkyl, which
alkyl may be saturated or unsaturated, branched or unbranched
and optionally substituted with a carboxy, sulfo or amino
group; and B and C may be the same or different and selected
f rom CmH2,+1 ; 15 m<_ 5.
In a preferred embodiment A in the above mentioned
formula is -CO-E, in which E may be -H, -OH, -R, or -OR; R
being a C1-C16 alkyl, preferably a C1-C$ alkyl, which alkyl may
3o be saturated or unsaturated, branched or unbranched and
optionally substituted with a carboxy, sulfo or amino group;
and B and C may be the same or different and selected from
CmH2m+1 ; 1 - m <- 5.
WO 96/10079 220 13 34 PCT/DK95/00384 ~
4
In the above mentioned formula A may be placed meta
to the hydroxy group instead of being placed in the
paraposition as shown. =
In particular embodiments, the enhancing agent is
acetosyringone, syringaldehyde, methylsyringate, syringic
acid, ethylsyringate, propylsyringate, butylsyringate,
hexylsyringate, octylsyringate or ethyl 3-(4-hydroxy-3,5-
dimethoxyphenyl)acrylate.
The enhancing agent of the invention may be present
1o in concentrations of from 0.01 to 1000 M, more preferred 0.1
to 250 M, most preferred 1 to 100 M.
Preparation of Enhancing Agents
The enhancing agents described in the present
application may be prepared using methods well known to those
skilled in the art; some of the enhancing agents are also
commercially available.
We produced methylsyringate, ethylsyringate,
propylsyringate, butylsyringate, hexylsyringate and
octylsyringate by using the method disclosed in Chem. Ber.
2o 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 1H-NMR and 13C-NMR (showing
spectra as expected) and the melting point was 68-70 C.
Hydrogen peroxide/Oxygen
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
3o hydrogen peroxide, e.g., percarbonate or perborate, or a
hydrogen peroxide generating enzyme system, e.g., an oxidase
and a substrate for the oxidase, e.g., 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
W O 96110079 22ii 13 3 4 PCT/DK95100384
~,! 't
or during the process, e.g., in an amount corresponding to
levels of from 0.001-25 mM, particularly to levels of from
0.01-1 mM.
If the phenol oxidizing enzyme requires molecular
5 oxygen, molecular oxygen from the atmosphere will usually be
present in sufficient quantity. If more 02 is needed,
additional oxygen may be added.
Phenol Oxidizing Enzyme
In the context of the present invention the enzyme
lo of the phenol oxidizing enzyme may be an enzyme possessing
peroxidase activity or a laccase or a laccase related enzyme
as described below.
Peroxidases and Compounds possessinct Peroxidase Activity
Compounds possessing 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 Patent 4,077,768, EP
Patent Application 537,381, International Patent Applications
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 subdivision Deuteromycotina, class
Hyphomycetes, e.g. Fusarium, Humicola, Tricoderma,
Myrothecium, Verticillum, Arthromyces, Caldariomyces,
Ulocladium, Embellisia, Cladosporium or Dreschlera, in
particular Fusarium oxysporum (DSM 2672), Humicola insolens,
Trichoderma resii, Myrothecium verrucaria (IFO 6113),
Verticillum alboatrum, Verticillum dahlie, Arthromyces
ramosus (FERM P-7754), Caldariomyces fumaao, Ulocladium
chartarum, Embellisia allior Dreschlera halodes.
WO 96/10079 220 13 34 PCT/DK95/00384 ~
6
Other preferred fungi include strains
belonging to the subdivision Basidiomycotina, class
Basidiomycetes, e.g. Coprinus, Phanerochaete, Coriolus or
Trametes, in particular Coprinus cinereus f. microsporus (IFO
8371), Coprinus macrorhizus, 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,
1o 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
Streptoverticillum verticillium ssp. verticillium.
Other preferred bacteria include Bacillus
pumilus (ATCC 12905), Bacillus stearothermophilus,
Rhodobacter sphaeroides, Rhodomonas palustri, Streptococcus
lactis, Pseudomonas purrocinia (ATCC 15958) or Pseudomonas
fluorescens (NRRL B-11).
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
transformed 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.
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
WO 96110079 220 13 3 4 PCT/DK95/00354
7
semisynthetic derivatives thereof, e.g., iron porphyrins, and
iron phthalocyanines and derivatives thereof.
Determination of Peroxidase Activity (PODU)
1 peroxidase unit (PODU) is the amount of enzyme
that catalyzes the conversion of 1 mole hydrogen peroxide
per minute at the following analytical conditions: 0.88 mM
hydrogen peroxide, 1.67 mM 2,2'-azinobis(3-ethylbenzothia-
zoline-6-sulfonate), 0.1 M phosphate buffer, pH 7.0,
incubated at 30 C, photometrically followed at 418 nm.
lo 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 1.14.99.1).
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 Asperaillus, 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. cinereus, C. comatus, C.
friesii, and C. plicatilis, Psathyrella, e.g., P.
condelleana, Panaeolus, e.g., P. papilionaceus,
Myceliophthora, e.g., M. thermophila, Schvtalidium,
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
WO 96/10079 22 13 34 PCT/DK95/00384 ~
8
culture 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 M 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 pmole syringaldazin per
minute at these conditions.
Industrial Applications
in a preferred embodiment, the method of the
invention finds application for bleaching of a textile dye or
colorant or textile dyes or colorants in solution.
Colorants and dyes are broad classes of
natural and synthetic compounds. The following description
and examples of dyes/colorants are not intended to be in any
way limiting to the scope of the invention as claimed:
Synthetic textile dyes bleachable by the
method of the invention are typically azo compounds (with one
or several azo, or diazenediyl, groups), as exemplified by
Acid Red 151, Direct Blue 1, Direct Brown 44, and Orange II,
or anthraquinone compounds, as exemplified by Acid Blue 45:
Acid Red 151
HO
NaO3S N=N -N ~ ~ .
WO 96110079 2,20 13 34 PCT/DK95100384
9
Direct Blue 1
NH OH
NaO3S 2 HO NHZ
N=N SO3Na
= I / / -
O o
SO3Na CH3 H3C SO3Na
Direct brown 44
N
=N N=N N=N S03Na
NaO3S N=N I:Z'
H2N H2 H2N NH2
Orange II
/ I \
OH
N=N
S O3Na
Acid Blue 45
OH O NH2
NaO3S
S03Na
NH2 0 HO
other structural motifs may occur together with these, as
exemplified in the formula of Reactive Blue 19:
WO 96/10079 2-20 13 34 PCT/DK95/00384 10
Reactive Blue 19
O H'-
/ S03Na
\ I \ I .
O NH
SO2CH2CH,OSO3Na
Some dyes furthermore carry groups capable of
coupling to fabric surfaces (reactive dyes), and some dyes
are complexed to metal ions. These modifications will often
not influence the applicability of the present invention.
A different structure bleachable by the method of
the invention is the indigo moiety, here exemplified by the
soluble dye indigo carmine:
Indigo Carmine
O H
O;S N
0
H S03
O
Other dyes and colorants may be of natural origin
or may be synthesized as identical to or resembling natural
structures. Examples of categories of coloured substances
extractable from vegetable sources are polyphenolic,
anthocyanine and carotenoid compounds.
A specific embodiment of the present invention is
provided by household and institutional laundering processes.
In such washing and rinsing processes, dyes and colorants
present on fabrics may leach into the washing or rinsing
liquor and discoloration of the laundry may result. Bleaching
WO 96/10079 .PCYKDK95100384
11
of the coloured compounds in solution by the method of the
invention may counteract this undesirable effect. Other
systems for dye transfer inhibition are known in the art
(e.g. WO 91/05839).
In another specific embodiment, dyes leached into
process water during textile processing may be bleached by
the method of the invention to prevent undesirable
deposition. Other systems are known in the art (e.g. WO
92/18697).
In a third embodiment, the method of the
invention finds application in bleaching of pulp for paper
production.
Accordingly, the invention provides a method
for bleaching of lignin-containing material, in particular
15.bleaching of pulp for paper production, which method
comprises treatment of the lignin or lignin containing
material with a phenol oxidizing enzyme and an enhancing
agent as described in the present invention.
In a fourth embodiment, the method of the
2o invention finds application for lignin modification, e.g., in
the manufacture of wood composites, e.g., wood fibre
materials such as chipboards, fibre boards, or particle
boards, or in the manufacture of laminated wood products,
such as laminated beams and plywood.
25 In a fifth embodiment, the method of the
invention finds application in treatment of waste water,
e.g., waste water from the chemical or pharmaceutical
industry, from dye manufacturing, from dye-works, from the
textile industry, or from pulp production (cf. e.g. US
30 4,623,465, or JP-A-2-31887).
In a more specific aspect, the invention
provides a method for treatment of waste water from dye
manufacturing, from dye-works, from textile industry, or from
pulp manufacturing, the method comprising treatment of the
35 waste water with a phenol oxidizing enzyme in the presence of
an enhancing agent of the invention.
In the above mentioned processes and in other
applications of the invention, the enhancing agent may be
220 13 34
WO 96/10079 PCT/DK95/00384
12
added at the beginning of the process or later, in one or
several additions.
According to the invention the phenol oxidizing
enzyme may be present in concentrations of from 0.001-100 mg
enzyme protein per liter.
Deteraent Compositions
According to the invention, the enhancing agent and
the phenol oxidizing enzyme may typically be a component of a
detergent composition. As such, it may be included in the
1o detergent composition in the form of a detergent additive.
Preferred detergent additive formulations are granulates, in
particular non-dusting granulates, liquids, in particular
stabilized liquids, or slurries.
Non-dusting granulates may be produced, e.g., as
disclosed in US 4,106,991 and 4,661,452 (both to Novo
Industri A/S) and may optionally be coated by methods known
in the art. Examples of waxy coating materials are
poly(ethylene oxide) products (polyethyleneglycol, PEG) with
mean molecular weights of 1000 to 20000; ethoxylated nonyl-
phenols having from 16 to 50 ethylene oxide units;
ethoxylated fatty alcohols in which the alcohol contains from
12 to 20 carbon atoms and in which there are 15 to 80
ethylene oxide units; fatty alcohols; fatty acids; and mono-
and di- and triglycerides of fatty acids. Examples of film-
forming coating materials suitable for application by fluid
bed techniques are given in patent GB 1483591. Liquid enzyme
preparations may, for instance, be stabilized by adding a
polyol such as propylene glycol, a sugar or sugar alcohol,
lactic acid or boric acid according to established methods.
Other enzyme stabilizers are well known in the art. Protected
enzymes may be prepared according to the method disclosed in
EP 238,216.
The detergent composition of the invention may be
in any convenient form, e.g. as powder, granules, paste or
liquid. A liquid detergent may be aqueous, typically
containing up to 70% water and 0-30% organic solvent, or
nonaqueous.
WO 96110079 22 0 13 3 4 PCT1nK95100384
13
The detergent composition comprises one or more
surfactants, each of which may be anionic, nonionic,
cationic, or zwitterionic. The detergent will usually contain
0-50% of anionic surfactant such as linear alkylbenzene-
sulfonate (LAS), alpha-olefinsulfonate (AOS), alkyl sulfate
(fatty alcohol sulfate) (AS), alcohol ethoxysulfate (AEOS or
AES), secondary alkanesulfonates (SAS), alpha-sulfo fatty
acid methyl esters, alkyl- or alkenylsuccinic acid, or soap.
It may also contain 0-40% of nonionic surfactant such as
lo alcohol ethoxylate (AEO or AE), carboxylated alcohol
ethoxylates, nonylphenol ethoxylate, alkylpolyglycoside,
alkyldimethylamine oxide, ethoxylated fatty acid monoethanol-
amide, fatty acid monoethanolamide, or polyhydroxy alkyl
fatty acid amide (e.g. as described in WO 92/06154).
The detergent composition may additionally comprise
one or more other enzymes, such as amylases, lipases,
cutinases, proteases, and cellulases.
The detergent may contain 1-65% of a detergent
builder or complexing agent such as zeolite, diphosphate,
triphosphate, phosphonate, citrate, nitrilotriacetic acid
(NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetri-
aminepentaacetic acid (DTPA), alkyl- or alkenylsuccinic acid,
soluble silicates or layered silicates (e.g. SKS-6 from
Hoechst). The detergent may also be unbuilt, i.e. essentially
free of detergent builder.
The detergent may comprise one or more polymers.
Examples are carboxymethylcellulose (CMC), poly(vinyl-
pyrrolidone) (PVP), polyethyleneglycol (PEG), poly(vinyl
alcohol) (PVA), polycarboxylates such as polyacrylates,
maleic/acrylic acid copolymers and lauryl methacrylate/-
acrylic acid copolymers.
The detergent may additionally contain other bleaching
systems which may comprise a H202 source such as perborate or
percarbonate which may be combined with a peracid-forming
bleach activator such as tetraacetylethylenediamine (TAED) or
nonanoyloxybenzenesulfonate (NOBS). Alternatively, the
bleaching system may comprise peroxyacids of, e.g., the
amide, imide, or sulfone type.
WO 96/10079 220 13 34 PCT/DK95/00384
14
The enzymes of the detergent composition of the
invention may be stabilized using conventional stabilizing
agents, e.g. a polyol such as propylene glycol or glycerol, a
sugar or sugar alcohol, lactic acid, boric acid, or a boric
acid derivative such as, e.g., an aromatic borate ester, and
the composition may be formulated as described in, e.g., WO
92/19709 and WO 92/19708.
The detergent may also contain other conventional
detergent ingredients such as, e.g., fabric conditioners
1o including clays, foam boosters, suds suppressors, anti-corro-
sion agents, soil-suspending agents, anti-soil-redeposition
agents, dyes, bactericides, optical brighteners, or perfume.
The pH (measured in aqueous solution at use con-
centration) will usually be neutral or alkaline, e.g., in the
range of 7-11.
Particular forms of detergent compositions within the
scope of the invention include:
1) A detergent composition formulated as a granulate having
a bulk density of at least 600 g/l comprising
2o Linear alkylbenzenesulfonate (cal- 7 - 12%
culated as acid)
Alcohol ethoxysulfate (e.g. C1-18
alcohol, 1-2 EO) or alkyl sulate 1 - 4%
(e.g. C _ )
Alcohol ethoxylate (e.g. C14-15 alco-
hol, 5 - 9%
7 EO)
Sodium carbonate (as Na CO ) 14 - 20%
Soluble silicate (as Na 0,2Si0 ) 2 - 6%
Zeolite (as NaAlSi0 ) 15 - 22%
Sodium sulfate (as Na SO ) 0 - 6% =
Sodium citrate/citric acid
(as CHNaO/CHO ) 0 - 15%
Sodium perborate (as NaBO.H O) 11 - 18%
TAED 2 - 6%
Carboxymethylcellulose 0 - 2% 11
WO 96/10079 220 13 34 PCT/DK95/00384
Polymers (e.g. maleic/acrylic acid
copolymer, PVP, PEG) 0 - 3%
Enzymes (calculated as pure enzyme 0.0001 - 0.1%
protein)
= 5 Minor ingredients (e.g. suds
suppressors, perfume, optical 0 - 5%
brightener, photobleach)
2) A detergent composition formulated as a granulate having
a bulk density of at least 600 g/l comprising
1o Linear alkylbenzenesulfonate (cal-
culated as acid) 6 - 11%
Alcohol ethoxysulfate (e.g. C12_1a
alcohol, 1-2 EO or alkyl sulfate 1 - 3%
(e.g. C _ )
15 Alcohol ethoxylate (e.g. C14-15 alco-
hol, 7 EO) 5 - 9%
Sodium carbonate (as Na CO ) 15 - 21%
Soluble silicate (as Na O,2Si0 ) 1 - 4%
Zeolite (as NaA1SiO) 24 - 34%
Sodium sulfate (as Na SO ) 4 - 10%
Sodium citrate/citric acid
(as CHNaO/CHO ) 0 - 15%
Carboxymethylcellulose 0 - 2%
Polymers (e.g. maleic/acrylic acid
copolymer, PVP, PEG) 1 - 6%
Enzymes (calculated as pure enzyme 0.0001 - 0.1%
protein)
Minor ingredients (e.g. suds 0 - 5%
suppressors, perfume)
3o 3) A detergent composition formulated as a granulate having
= a bulk density of at least 600 g/l comprising
Linear alkylbenzenesulfonate (cal- 5 - 9%
culated as acid)
Alcohol ethoxylate (e.g. C1z-15 alco-
hol, 7 EO) 7 - 14%
Soap as fatty acid (e.g. C16-Z2 fatty 1 - 3%
acid)
WO 96/10079 2 0 3 3 4 PCT/DK95/00384 ~
16
Sodium carbonate (as Na CO ) 10 - 17%
Soluble silicate (as Na 0,2Si0 ) 3 - 9%
Zeolite (as NaAlSiO ) 23 - 33%
Sodium sulfate (as Na SO4) 0 - 4%
Sodium perborate (as NaBO.H O) 8 - 16%
TAED 2 - 8%
Phosphonate (e.g. EDTMPA) 0 - 1%
Carboxymethylcellulose 0 - 2%
Polymers (e.g. maleic/acrylic acid
1o copolymer, PVP, PEG) 0 - 3%
Enzymes (calculated as pure enzyme 0.0001 - 0.1%
protein)
Minor ingredients (e.g. suds
suppressors, perfume, optical 0 - 5%
brightener)
4) A detergent composition formulated as a granulate having
a bulk density of at least 600 g/1 comprising
Linear alkylbenzenesulfonate (cal- 8 - 12%
culated as acid)
2o Alcohol ethoxylate (e.g. C12-15 alco-
hol, 7 EO) 10 - 25%
Sodium carbonate (as Na CO ) 14 - 22%
Soluble silicate (as Na 0,2Si0 ) 1 - 5%
Zeolite (as NaAlSiO) 25 - 35%
Sodium sulfate (as Na SO ) 0 - 10%
Carboxymethylcellulose 0 - 2%
Polymers (e.g. maleic/acrylic acid
copolymer, PVP, PEG) 1 - 3%
Enzymes (calculated as pure enzyme 0.0001 - 0.1% =
protein)
Minor ingredients(e.g. suds 0 - 5%
suppressors, perfume)
Wo 96110079 .34 PCT/DK95/00384
17
5) An aqueous liquid detergent composition comprising
Linear alkylbenzenesulfonate (cal- 15 - 21%
culated as acid)
Alcohol ethoxylate (e.g. C12_15 alco-
hol, 7 EO or C alcohol, 5 EO) 12 - 18%
Soap as fatty acid (e.g. oleic 3 - 13%
acid)
Alkenylsuccinic acid (C _ ) o - 13%
Aminoethanol 8 - 18%
1o Citric acid 2 - 8%
Phosphonate 0 - 3%
Polymers (e.g. PVP, PEG) 0 - 3%
Borate (as B O) 0 - 2%
Ethanol 0 - 3%
Propylene glycol 8 - 14%
Enzymes (calculated as pure enzyme 0.0001 - 0.1%
protein)
Minor ingredients (e.g.
dispersants, suds suppressors, per- 0 - 5%
fume, optical brightener)
WO 96/10079 220 13 34 PCT/DK95/00384
18
6) An aqueous structured liquid detergent composition
comprising
Linear alkylbenzenesulfonate
(calculated as acid) 15 - 21%
Alcohol ethoxylate (e.g. C12_15
alcohol, 7 EO, or 3 - 9%
C alcohol, 5 EO)
Soap as fatty acid (e.g. oleic 3 - 10%
acid)
to Zeolite (as NaAlSiO ) 14 - 22%
Potassium citrate 9 - 18%
Borate (as B O) 0 - 2%
Carboxymethylcellulose 0 - 2%
Polymers (e.g. PEG, PVP) 0 - 3%
Anchoring polymers such as, e.g.,
lauryl methacrylate/acrylic acid 0 - 3%
copolymer; molar ratio 25:1; MW
3800
Glycerol 0 - 5%
2o Enzymes (calculated as pure enzyme 0.0001 - 0.1%
protein)
Minor ingredients (e.g.
dispersants, suds suppressors, 0 - 5%
perfume, optical brighteners)
7) A detergent composition formulated as a granulate having a
bulk density of at least 600 g/l comprising
Fatty alcohol sulfate 5 - 10%
Ethoxylated fatty acid monoethanol- 3 - 9%
amide
Soap as fatty acid 0 - 3%
Sodium carbonate (as Na CO ) 5 - 10%
Soluble silicate (as Na 0,2Si0 ) 1 - 4%
Zeolite (as NaAlSiO ) 20 - 40%
Sodium sulfate (as Na SO ) 2 - 8%
Sodium perborate (as NaBO.H O) 12 - 18%
TAED 2 - 7%
WO 96/10079 34 PCT/DK95/00384
19
Polymers (e.g. maleic/acrylic acid 1 - 5%
copolymer, PEG)
Enzymes (calculated as pure enzyme 0.0001 - 0.1%
protein)
Minor ingredients (e.g. optical
brightener, suds suppressors, per- 0 - 5%
fume)
8) A detergent composition formulated as a granulate
comprising
lo Linear alkylbenzenesulfonate
(calculated as acid) 8 - 14%
Ethoxylated fatty acid monoethanol- 5 - 11%
amide
Soap as fatty acid 0 - 3%
Sodium carbonate (as Na CO ) 4 - 10%
Soluble silicate (as Na O,2Si0 ) 1 - 4%
Zeolite (as NaAlSi0 ) 30 - 50%
Sodium sulfate (as Na SO ) 3 - 11%
Sodium citrate (as C H Na O) 5 - 12%
Polymers (e.g. PVP, maleic/acrylic 1 - 5%
acid copolymer, PEG)
Enzymes (calculated as pure enzyme 0.0001 - 0.1%
protein)
Minor ingredients (e.g. suds 0 - 5%
suppressors, perfume)
9) A detergent composition formulated as a granulate
comprising
Linear alkylbenzenesulfonate
(calculated as acid) 6 - 12%
3o Nonionic surfactant 1 - 4%
Soap as fatty acid 2 - 6%
Sodium carbonate (as Na CO ) 14 - 22%
Zeolite (as NaAlSiO ) 18 - 32%
Sodium sulfate (as Na SO) 5 - 20%
Sodium citrate (as C H Na O) 3 - 8%
WO 96/10079 22 0 4 PCT/DK95/00384
Sodium perborate (as NaBO.H O) 4 - 9%
Bleach activator (e.g. NOBS or 1 - 5%
TAED)
Carboxymethylcellulose 0 - 2%
5 Polymers (e.g. polycarboxylate or 1 - 5%
PEG)
Enzymes (calculated as pure enzyme 0.0001 - 0.1%
protein)
Minor ingredients (e.g. optical 0 - 5%
lo brightener, perfume)
10) An aqueous liquid detergent composition comprising
Linear alkylbenzenesulfonate
(calculated as acid) 15 - 23%
Alcohol ethoxysulfate (e.g. C12_15
15 alcohol, 2-3 EO) 8 - 15%
Alcohol ethoxylate (e.g. C12_15 al-
cohol, 7 EO, or 3 - 9%
C alcohol, 5 EO)
Soap as fatty acid (e.g. lauric 0 - 3%
2o acid)
Aminoethanol 1 - 5%
Sodium citrate 5 - 10%
Hydrotrope (e.g. sodium 2 - 6%
toluensulfonate)
Borate (as B O) 0 - 2%
Carboxymethylcellulose 0 - 1%
Ethanol 1 - 3%
Propylene glycol 2 - 5%
Enzymes (calculated as pure enzyme 0.0001 - 0.1%
protein)
Minor ingredients (e.g. polymers,
dispersants, perfume, optical 0 - 5%
brighteners)
VVO 96110079 '~ ~ 3 4
PC3YDK95100384
21
11) An aqueous liquid detergent composition comprising
Linear alkylbenzenesulfonate
(calculated as acid) 20 - 32%
Alcohol ethoxylate (e.g. C12_15 alco-
hol, 6 - 12%
7 EO, or C alcohol, 5 EO)
Aminoethanol 2 - 6%
Citric acid 8 - 14%
Borate (as B O) 1 - 3%
lo Polymer (e.g. maleic/acrylic acid
copolymer, anchoring polymer such
as, e.g., lauryl 0 - 3%
methacrylate/acrylic acid
copolymer)
Glycerol 3 - 8%
Enzymes (calculated as pure enzyme 0.0001 - 0.1%
protein)
Minor ingredients (e.g. hydro-
tropes, dispersants, perfume, 0 - 5%
optical brighteners)
12) A detergent composition formulated as a granulate having
a bulk density of at least 600 g/l comprising
Anionic surfactant (linear
alkylbenzenesulfonate, alkyl sulfa-
te, alpha-olefinsulfonate, alpha- 25 - 40%
sulfo fatty acid methyl esters,
alkanesulfonates, soap)
Nonionic surfactant (e.g. alcohol 1 - 10%
ethoxylate)
Sodium carbonate (as Na CO) 8 - 25%
Soluble silicates (as Na O, 2Si0 ) 5 - 15%
Sodium sulfate (as Na SO ) 0 - 5%
Zeolite (as NaAlSi0) 15 - 28%
Sodium perborate (as NaBO .4H O) 0 - 20%
Bleach activator (TAED or NOBS) 0 - 5%
Enzymes (calculated as pure enzyme 0.0001 - 0.1%
protein)
Minor ingredients (e.g. perfume, 0 - 3%
optical brighteners)
WO 96/10079 220 13 34 PCT/DK95/00384
22
13) Detergent formulations as described in 1) - 12) wherein
all or part of the linear alkylbenzenesulfonate is replaced
by (C12-C18) alkyl sulfate.
14) A detergent composition formulated as a granulate having
a bulk density of at least 600 g/l comprising
(C -C ) alkyl sulfate 9 - 15%
Alcohol ethoxylate 3 - 6%
Polyhydroxy alkyl fatty acid amide 1 - 5%
Zeolite (as NaAlSi0 ) 10 - 20%
1o Layered disilicate (e.g. SK56 from
Hoechst) 10 - 20%
Sodium carbonate (as Na CO ) 3 - 12%
Soluble silicate (as Na 0,2Si0 ) 0 - 6%
Sodium citrate 4 - 8%
Sodium percarbonate 13 - 22%
TAED 3 - 8%
Polymers (e.g. polycarboxylates and 0 - 5%
PVP=
Enzymes (calculated as pure enzyme 0.0001 - 0.1%
protein)
Minor ingredients (e.g. optical
brightener, photo bleach, perfume, 0 - 5%
suds suppressors)
15) A detergent composition formulated as a granulate having
a bulk density of at least 600 g/l comprising
(C -C ) alkyl sulfate 4 - 8%
Alcohol ethoxylate 11 - 15%
Soap 1 - 4%
Zeolite MAP or zeolite A 35 - 45%
Sodium carbonate (as Na CO ) 2 - 8%
Soluble silicate (as Na O,2Si0 ) 0 - 4%
P Sodium percarbonate 13 - 22%
TAED 1 - 8%
W O 96110079 ~ 2 0 4 PCT/DK95100384
23 1
Carboxymethyl cellulose 0 - 3%
Polymers (e.g. polycarboxylates and 0 - 35%
PVP)
Enzymes (calculated as pure enzyme 0.0001 - 0.1%
protein)
Minor ingredients (e.g. optical o - 3%
brightener, phosphonate, perfume)
16) Detergent formulations as described in 1) - 15) which
contain a stabilized or encapsulated peracid, either as an
io additional component or as a substitute for already specified
bleach systems.
17) Detergent compositions as described in 1), 3), 7), 9) and
12) wherein perborate is replaced by percarbonate.
18) Detergent compositions as described in 1), 3), 7), 9),
12), 14) and 15) which additionally contain a manganese
catalyst. The manganese catalyst may, e.g., be one of the
compounds described in "Efficient manganese catalysts for
low-temperature bleaching", Nature 369, 1994, pp. 637-639.
19) Detergent composition formulated as a nonaqueous
2o detergent liquid comprising a liquid nonionic surfactant such
as, e.g., linear alkoxylated primary alcohol, a builder
system (e.g. phosphate), enzyme and alkali. The detergent may
also comprise anionic surfactant and/or a bleach system.
The following examples further illustrate the
present invention, and they are not intended to be in any way
limiting to the scope of the invention as claimed.
PCT/DK95/00384 ~
WO 96/10079 ~ ~ "~ ~ ~ 3 4
24
EXAMPLE 1
Bleaching of Direct Blue 1 with soybean peroxidase with and
without acetosyrinaone
A crude soy bean peroxidase (SBP), obtained from Mead
Corp., Dayton, Ohio, was purified by anion and cation chroma-
tography followed by gelfiltration to a single protein on
SDS-PAGE with an RZ-value (A404~A2$0n) of 2.2:
125 ml of crude SBP were adjusted to pH 7, diluted to
2.3 mS and filtered through 0.8 f.c filter. The sample was
1o applied to 300 ml DEAE column equilibrated with 20 mM
phosphate pH 7.0 and the peroxidase eluted with a 1 M NaCl
linear gradient in the same buffer. Fractions with peroxidase
activity were pooled.
Pooled fractions from anion exchange chromatography (190
ml) were concentrated and washed by ultrafiltration (GR61PP
membrane from Dow, Denmark). pH was adjusted to 5.3 ionic
strength to 2.3 mS in the sample before application to a 200
ml S-Sepharose column previously equilibrated with 50 mM
acetate pH 5.3. The effluent containing the peroxidase
2o activity was concentrated and washed by ultrafiltration to a
final volume of approx. 10 ml.
A 5 ml concentrated sample from cation exchange chroma-
tography was applied to a 90 cm Sephacryl S-200 column
equilibrated and eluted with 0.1 M acetate pH 6.1. Fractions
with peroxidase activity giving only one band on SDS-PAGE
were pooled.
The bleaching rate of Direct Blue 1(DB1) by the
purified SBP was determined using an enhancer according to
the invention. The following conditions were used:
W O 96110079 220 13 .3/ PCT/DK95/00384
~i
Final concentration
200 .l 50 mM Britton-Robinson buffer*
pH 6, 8 and 10, respectively 10 mM
200 l DB1 - 3.0 Abs. Units (610 nm) 0.6 (A610nm)
5 200 E.cl SBP with A4o4rm = 0.0005 at pH 6
and 8 or with A4o4rm = 0.005 at pH 10 0.0001 or
0. 001 (A404nm)
200 l 50 M enhancer 10 M
200 fa, l 10 0/..cM H202 2 0 /.cM
1o * (50 mM acetic acid, 50 mM phosphoric acid, 50 mM boric
acid, pH adjusted to the value of interest with NaOH).
** corresponding to approximately to 0.04 mg/1 and 0.4 mg/l.
Reagents were mixed in a thermostated cuvette at 30 C
and the bleaching was started by addition of hydrogen
15 peroxide. The bleaching was detected spectrophotometrically
at 610 nm, which is the wavelength of the absorption peak of
DB1. Bleaching was followed for 4 minutes, and the reduction
in absorbance (100x (A610nm,start-A610nm,4min. ) /A610nm,start ) was
determined.
20 A61orn-,start was determined by replacement of hydrogen
peroxide with water.
Table 1
Bleaching of Direct Blue 1 with SBP in 4 Minutes
Enhancer % DBi bleaching in 4 min.
pH 6 pH 8 pH 10
10x[SBP]
25 No 0.7 <0.7 <0.7
acetosyringone 19.8 20.0 3.3
From the results presented in Table 1 above, it
appears that by adding an enhancer of the invention a much
faster bleaching of the dye is obtained compared to the
3o experiment without enhancer.
WO 96/10079 220 ' 3 3 4 PCT/DK95/00384
26
EXAMPLE 2
Bleaching of Direct Blue 1 with Coprinus cinereus peroxidase
with and without enhancers
A Coprinus cinereus peroxidase (CiP) obtained as
described in WO 9412621 was used.
Dilutions of CiP were made in a solution of 0.15
gram/1 of Triton X-405.
The bleaching rate of Direct Blue 1 (DB1) by
purified CiP was determined using the following conditions:
Final concentration
200 l 50 mM Britton-Robinson buffer* 10 mM
200 l DB1 - 3.0 Abs. Units (610 nm) 0.6 (A61onm)
200 l 0.40 mg/1 CiP (pH 8.5) 0.08 mg/1 (pH 8.5) or
0.80 mg/1 CiP (pH 10.5) 0.16 mg/i (pH 10.5)
200 l 25 M enhancer 5 M
200 1 10 0AM H202 20 f.t,M
* (50 mM acetic acid, 50 mM phosphoric acid, 50 mM boric
acid, pH adjusted to the value of interest with NaOH).
Reagents were mixed in a thermostated cuvette at
2o 30 C and the bleaching was started by addition of hydrogen
peroxide. The bleaching was detected spectrophotometrically
at 610 nm, which is the wavelength of the absorption peak of
DB1. Bleaching was followed for 1 minute, and the initial
reduction in absorbance, -AmAbs/minute, was determined.
WO 96110079 ~ ~ O PCT/DK95100384
2 7 ~!
Table 2
Initial Bleaching of Direct Blue 1 with CiP
Enhancer -AmAbs/minute
pH: 8.5 10.5
Acetosyringone 239 1
Syringaldehyde 151 4
Methylsyringate 245 8
No enhancer 2 0
From the results presented in Table 2 above it
1o appears that by adding an enhancer of the invention a much
faster bleaching of the dye compared to the experiment
without enhancer is obtained. Even at pH 10.5 a significant
bleaching with an enhancer of the invention is obtained,
whereas no bleaching at all can be seen without the addition
of an enhancer.
EXAMPLE 3
Bleaching of Chicago Sky Blue 6B (CSB) with Coprinus cinereus
peroxidase and enhancers
Bleaching tests were performed in exactly the same way
2o as described in Example 2 except that instead of using DB1
Chigaco Sky Blue (CSB) (obtainable from Aldrich) was used,
and the following enhancers were tested:
methylsyringate
ethylsyringate
propylsyringate
butylsyringate
hexylsyringate
octylsyringate
ethyl 3-(4-hydroxy-3,5-dimethoxyphenyl)acrylate.
The following results were obtained:
WO 96/10079 220 13 34 PCT/DK95/00384
28
Table 3
Initial Bleaching of CSB with CiP
Enhancer -AmAbs/minute
pH: 8.5 10.5
methylsyringate 211 42
ethylsyringate 240 52
propylsyringate 228 60
butylsyringate 228 48
hexylsyringate 276 36
1o octylsyringate 192 15
ethyl 3-(4-hydroxy-3,5-
dimethoxyphenyl)acrylate 48 48
No enhancer 8 6
EXAMPLE 4
Bleaching of Direct Blue 1(DB1) using various Coprinaceae
laccases and methvlsyringate at PH 5.5-8.5.
Bleaching of the dye Direct Blue 1 at various pH values
was conducted using a laccase obtained from Coprinus comatus,
Coprinus friesii, Coprinus plicatilis, Panaeolus
papilionaceus or Psathyrella condolleana and methylsyringate.
The above mentioned strains were fermented in the
following way:
The strains were inoculated on PDA agar plates (PDA: 39
g/l potato dextrose agar) and grown at 26 C for 3 days. Shake
flasks were then inoculated with 6-8 small squares ("0.5 cm x
0.5 cm) of agar containing mycelium and fermented for 3-10
days at 26 C and 200 rpm using the following medium:
WO 96/10079 220 13 34 pCT)DK95100384
29
Deposit no. Medium Growth
Coprinus comatus* CBS 631.95 A 10 days
Coprinus friesii CBS 629.95 A 3 days
Panaeolus
papilionaceus CBS 630.95 A 10 days
Psathyrella
condolleana CBS 628.95 B 7 days
Coprinus plicatilis CBS 627.95 A 8 days
* All the strains mentioned in this Example have been
1o deposited according to the Budapest Treaty on the Inter-
national Recognition of the Deposits of Microorganisms for
the Purpose of Patent Procedures, on 16 August 1995, at
Centraalbureau voor Schimmelcultures, Oosterstraat 1, Postbus
273, NL-3740 AG Baarn, Netherlands, under the above mentioned
Accession numbers.
Media:
A: soja meal 30 g/1
maltodextrin 15 g/1
bacto peptone 5 g/l
pluronic 0.2 g/l
B: potato meal 50 g/l
barley meal 25 g/1
BAN 800MG* 0.025 g/1
Na-caseinate 5 g/1
crushed soja 10 g/1
Na2HPO4, 12 H20 4.5 g/1
Pluronic 0.05 ml/l
* BAN 800MG obtainable from Novo Nordisk A/S.
After fermentation the culture broths were centrifugated
so and the supernatants were used in the tests described below.
The bleaching rate of DB1 was determined using the
following conditions:
220 1334
WO 96/10079 PCT/DK95/00384
Final concentration
400 l 50 mM Britton-Robinson buffer*,
(pH 5.5, 7.0, and 8.5 respectively), 20 mM
200 l DB1 - 3.0 Abs. Units (610 nm) 0. 6 (A67orm)
5 200 l 50 M methylsyringate 10 M
200 l laccase at pH 5 and 7: 4 LACU/1
at pH 8.5: 20 LACU/1
*(50 mM acetic acid, 50 mM phosphoric acid, 50 mM boric
acid, pH adjusted to the value of interest with NaOH).
10 Reagents were mixed in a 1 ml thermostated cuvette at
30 C and the bleaching was started by addition of the
laccase.
The bleaching was followed spectrophotometrically at
610 nm, which is the wavelength of the absorption peak of
15 DB1, with readings every 5 sec. for a period of 5 minutes.
The initial bleaching rate was determined from the first
linear part of the absorbance curve.
The following results were obtained with methyl-
syringate:
20 -AmAbs/minute
Laccase:
pH: 5.5 7.0 8.5
C. comatus 33 23 2
25 C. friesii 40 55 61
Pan. papilionaceus 16 19 18
Ps. condolleana 45 54 43
C. plicalitis 42 39 14
The following results were obtained with no
30 enhancer:
WO 96/10079 220 13 34 PCT/DK95100384
31
-AmAbs/minute
Laccase:
pH: 5.5 7.0 8.5
s C. comatus 0 0 0
C. friesii .0 0 0
Ps. condolleana 0 0 0
C. plicalitis 0 0 0
EXAMPLE 5
Bleaching of Direct Blue 1 (DB1) using Coprinus cinereus
laccase with/without enhancing agents at pH 5.5-8.5.
Bleaching of the dye Direct Blue 1 at various pH
values was conducted using Conrinus cinereus laccase and one
of the following enhancing agents:
None
acetosyringone
syringaldehyde
methylsyringate.
The laccase was 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 in Example
3). 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 a preparation with a laccase activity of 3.6
LACU/ml. The estimated purity was >80% on a protein basis.
WO 96/10079 220 13 34 PCT/DK95/00384
32
The bleaching rate of DB1 was determined using the
following conditions:
Final concentration
400 l 50 mM Britton-Robinson buffer*,
5(pH 5.5, 7.0 and 8.5 respectively), 20 mM
200 l DB1 - 3.0 Abs. Units (610 nm) 0.6 (A61orm)
200 l 50 M enhancing agent 10 M
200 l C. cinereus laccase 1 mg/1
* (50 mM acetic acid, 50 mM phosphoric acid, 50 mM boric
1o acid, pH adjusted to the value of interest with NaOH).
Reagents were mixed in a 1 ml thermostated cuvette at
30 C and the bleaching was started by addition of the
laccase.
The bleaching was followed spectrophotometrically at
15 610 nm, which is the wavelength of the absorption peak of
DB1, with readings every 5 sec. for a period of 5 minutes.
The initial bleaching rate was determined from the first
linear part of the absorbance curve.
The following results were obtained:
2o Enhancing agent -AmAbs/minute
pH: 5.5 7.01 8.5
none 13 5 3
aceto-
25 syringone 28 94 50
syring-
aldehyde 29 79 28
methyl-
syringate 20 94 57
WO 96110079 220 13 34 PCTmK95/00384
33
EXAMPLE 6
Bleaching of Direct Blue 1 (DB1) using Coprinus cinereus
laccase and acetosvringone
Bleaching of the dye Direct Blue 1 at various pH
values was conducted using Coorinus cinereus laccase and the
enhancing agent acetosyringone.
The laccase was obtained as described in Example 5.
The bleaching rate of DB1 was determined using the
following conditions:
Final concentration
400 l 50 mM Britton-Robinson buffer*,
(pH 4, 5, 6, 7, and 8 respectively), 20 mM
200 l DB1 - 3.0 Abs. Units (610 nm) 0.6 (A61 on.)
200 l 50 M acetosyringone 10 M
200 l C. cinereus laccase 3.2 mg/l
* (50 mM acetic acid, 50 mM phosphoric acid, 50 mM boric
acid, pH adjusted to the value of interest with NaOH).
Reagents were mixed in a 1 cm thermostated cuvette at
30 C and the bleaching was started by addition of the
laccase.
The bleaching was detected spectrophotometrically at
610 nm, which is the wavelength of the absorption peak of
DB1. After 5 sec. bleaching was followed for 4 minutes.
The.following results were obtained:
Initial DB1 bleaching
(-AmAbs/ min)
pH (% of pH 7-value)
4 18 ~
3o 5 13 0
6 35 0
7 100 ~
8 69 %
220 13 34
WO 96/10079 PCT/DK95/00384
34
It can be seen from the results given above that the
optimum bleaching is achieved at pH around 7, but the system
also shows an effective bleaching at pH 8.
EXAMPLE 7
Bleaching of Direct Blue 1 with Trametes villosa laccase with
and without enhancing agents
Laccase obtained from Trametes villosa: 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
lo 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
NaC1 gradient around 0.25 M. Fractions with laccase activity
from 10 runs were pooled and concentrated by ultrafiltration
to an activity of 500 LACU/ml.
The following conditions were used:
Final concentration
400 l 50 mM Britton-Robinson buffer*,
pH 5.5 and pH 7.0 respectively, 20 mM
200 l DB1 - 3.0 Abs. Units (610 nm) 0.6 (A61orm)
200 l 50 M enhancer 10 M
200 l Enzyme dilution
*(50 mM acetic acid, 50 mM phosphoric acid, 50 mM
boric acid, pH adjusted to the value of interest with NaOH).
Reagents were mixed in a 1 cm thermostated cuvette at 30 C
and the bleaching was started by addition of enzyme.
The bleaching was detected spectrophotometrically
at 610 nm, which is the absorption peak of DB1. After 5 sec.
3o bleaching was followed for 4 minutes.
From the results presented below, it appears that
adding enhancers of the invention a much faster bleaching of
WQ 96(10079 220 13 34 PCT/DK95/00384
the dye can be obtained compared to the experiment without
enhancer. Enzyme dosages given are in the final incubation
mixture.
Bleaching of Direct Blue 1 with Trametes villosa laccase,
5 obtained as described above, at pH 5.5 (1.6 mg/1) and pH 7.0
(16 mg/1):
Enhancer DB1 bleaching in 4 minutes
(-AmAbs/4 min)
pH 5.5 pH 7.0
No enhancer 0 0
Acetosyringone 447 242
Syringaldehyde 438 112
EXAMPLE 8
Bleachina of gradually added Acid Blue 45 with Coprinus cine-
reus laccase with and without enhancing agent
Ideally, dye transfer inhibition systems for
laundry applications should be tested in a real wash where
dyed fabrics give off dyes to the wash solution as a result
of the combined action of the detergent, temperature and
mechanical agitation taking place.
To simulate such a process, however, a magnetically
stirred beaker was used as the reaction vessel and dye was
added gradually from a stock solution (using a Metrohm 725
dosimat). The solution was monitored spectrophotometrically
using a Zeiss multichannel spectrometer (MCS) equipped with a
fibre-optics immersion probe.
Stock solutions of acetosyringone was prepared in a
suitable water/ethanol mixture. Stock solutions of the
3o anthraquinone dye Acid Blue 45 were made with water.
WO 96/10079 220 13 34 PCT/DK95/00384 ~
36
The laccase was recovered from a 10-liter
fermentation of Coprinus cinereus (IFO 30116) as described in
Example 4.
The following conditions were used in the
experiment:
Temperature: 35 C
Medium and pH: 50 mM/50 mM phosphate/borate buffer at pH 10
Acetosyringone (when applicable): 10 M
Laccase: 10 mg/1
io Dye addition program: linear addition at a rate of ca 0.34
abs/40 min, referring to the absorbance of Acid Blue 45 at
its maximum absorbance wavelength (590 nm for Acid Blue 45).
Fig. 1 shows the results of the bleaching tests.
The following symbols are used: (I): Only dye addition; (II):
Dye addition in the presence of Laccase; (III): Dye addition
in the presence of Laccase + acetosyringone.
It can be seen from Fig. 1 that the bleaching
effect is enhanced by acetosyringone.
EXAMPLE 9
2o Dye Transfer Inhibition Using Coprinus cinereus Laccase
A small-scale experiment was carried out in which
clean cotton test pieces were washed together with dyed
fabrics bleeding dye into the wash solution, the experiment
conducted in the absence and in the presence of laccase and
enhancing agent. =
After wash, the Hunter colour difference between
the above mentioned cotton pieces and clean cotton pieces
(washed in the absence of bleeding fabrics) was measured and
taken as a measure of the degree of dye transfer resulting
f rom the wash.
Materials used:
Bleeding fabrics dyed with Acid Red 151 (AR 151) or
Direct Blue 1 (DB1).
WO 96110079 220 13 34 PCT/DK95/00354
37
Clean white cotton (bleached, no optical brightener
added).
Liquid detergent and powder detergent as typically
met in the North American market place; both detergents
contained no bleaching system.
Coprinus cinereus laccase, obtained as described in
Example 4.
Washing tUrocedure :
The washing processes were carried out in beakers
1o with magnetical stirring at 35 C for 15 min., after which the
test fabrics were rinsed thoroughly in tap water and air-
dried overnight in the dark before the Hunter readings were
taken by using a Datacolor Elrephometer 2000 reflectance
spectrometer.
Laccase system: Laccase at a level of 10 mg/1 with the
enhancing agent acetosyringone at a level of 10 M.
The following results were obtained:
Wash in liquid detergent solution (2 g/l, water hardness
6 dH) at pH 8.5:
Hunter colour difference (delta E)
with respect to white, washed cotton
Cotton washed Cotton washed
with AR 151 with DB 1
bleeders bleeders
-------------------------------------------------------------
Wash with no laccase
system 12 26
Wash with laccase
system 1 7
2201334
WO 96/10079 PCT/DK95/00384
38
Wash in powder detergent solution (1 g/l, water hardness
6 dH) at pH 10.0:
Hunter colour difference (delta E)
with respect to white, washed cotton
Cotton washed Cotton washed
with AR 151 with DB 1
bleeders bleeders
-------------- ---------------------------------------------
Wash with no laccase
lo system 21 29
Wash with laccase
system 4 8
Typical significant differences in the delta E readings are
2-3 units, so the data reflect significant reduction of dye
transfer with the laccase treatments relative to the treat-
ment with no laccase system.
EXAMPLE 10
Dye Transfer Inhibition Using MYceliophthora thermophila
Laccase
A small-scale experiment was carried out in which
clean cotton test pieces were washed together with dyed
fabrics bleeding dye into the wash solution, the experiment
conducted in the absence and in the presence of laccase and
enhancing agent.
After wash, the Hunter colour difference between
the above mentioned cotton pieces and clean cotton pieces
(washed in the absence.of bleeding fabrics) was measured and
taken as a measure of the degree of dye transfer resulting
from the wash.
WO 96/10079 220 13 3 4 PC7Y7)K95100384
39
Materials used:
Bleeding fabrics dyed with Acid Red 151 (AR 151) or
Direct Blue 1 (DB1).
Clean white cotton (bleached, no optical brightener
added).
Liquid detergent (No. 1) as typically met in the
European market place; liquid detergent (No. 2) as typically
met in the North American market place.
Mviceliophthora thermonhila laccase, produced as
io described in PCT/US95/06815).
Washina procedure:
The washing processes were carried out in beakers
with magnetical stirring at 35 C for 15 min., after which the
test fabrics were rinsed thoroughly in tap water and air-
dried overnight in the dark before the Hunter readings were
taken by using a Datacolor Elrephometer 2000 reflectance
spectrometer.
Laccase systems: M. thermophila laccase at a level of 0.87
mg/1 with the enhancing agent acetosyringone (AS) or the
zo enhancing agent methyisyringate (MS) at a level of 10 M.
The following results were obtained:
Wash in solution of liquid detergent No. 1 (7 g/l, water
hardness 12 dH) at an initial pH of 7.0:
Hunter colour difference (delta E)
with respect to white, washed cotton
Cotton washed Cotton washed
with AR 151 with DB 1
bleeders bleeders
-------------------------------------------------------------
so Wash with no laccase
system 7 27
WO 96/10079 220 13 3 4 PCT/DK95/00384
Wash with AS-based laccase
system 5 13
Wash with MS-based laccase
system 4 12
5 Wash in solution of liquid detergent No. 2 (2 g/l, water
hardness 6 dH) at nH 8.5:
Hunter colour difference (delta E)
with respect to white, washed cotton
Cotton washed Cotton washed
10 with AR 151 with DB 1
bleeders bleeders
------------------------------------------------------------
Wash with no laccase
system 14 29
15 Wash with AS-based laccase
system 5 10
Wash with MS-based laccase
system 3 8
Typical significant differences in the delta E readings are
2o 2-3 units, so the data reflect significant reduction of dye
transfer with the laccase treatments relative to the treat-
ment with no laccase system.
