Note: Descriptions are shown in the official language in which they were submitted.
`~094/12620 21 S O ~ ~ 3 PCT~K93/00394
ENHANCEMENT OF ENZYME REACTIONS
TECHNICAL FIELD
This invention relates to activation of enzymes. More
specifically, the invention relates to peroxidase enhancing
5 agents.
The invention also relates to methods of oxidizing a
substrate with a source of hydrogen peroxide in the presence of
a peroxidase enzyme and a peroxidase enhancing agent. More
specifically, the invention relates to a method of bleaching of
10 dye in solutions, to a method of inhibiting the transfer of a
textile dye from a dyed fabric to another fabric when said
fabrics are washed together in a wash liquor, to a method of
bleaching of lignin-containing material, in particular bleach-
ing of pulp for paper production, to a method of treatment of
15 waste water from pulp manufacturing, and to a method of
enzymatic polymerization and/or modification of lignin or
lignin containing material.
R~ ~aR~uND ART
Peroxidases (E.C. 1.11.1.7) are enzymes that catalyse
20 the oxidation of a substrate (an electron or hydrogen donor)
with hydrogen peroxide. Such enzymes are known from microbial,
plant and animal origins, e.g. peroxidase from Coprinus
cinereus (cf. e.g. EP 179,486). They are typically hemopro-
teins, i.e. they contain a heme as a prosthetic group.
Use of peroxidase together with hydrogen peroxide or a
hydrogen peroxide precursor has been suggested e.g. in bleach-
ing of pulp for paper production, in treatment of waste water
from pulp production, for improved bleaching in laundry
detergents, for dye transfer inhibition during laundering, and
30 for lignin modification, e.g. in particle board production.
The compound 2,2'-azino-bis(3-ethylbenzothiazoline-6-
sulfonate), ABTS, supplied by Boehringer Mannheim, is a
chromogenic substrate, and a common peroxidase and phenol
WO94/12620 PCT~K93100394
2150~3
oxidase assay agent. These enzymes catalyse the oxidation of
ABTS by hydrogen peroxide and dioxygen, respectively, producing
a greenish-blue colour, which process may be monitored photo-
metrically.
ABTS has been found to form a stable radical cation when
oxidized by a laccase enzyme (polyphenol oxidase, EC 1.10.3.2),
and has been proposed to act as a redox mediator for oxidation
of non-phenolic lignin model compounds [Bourbonnais R, Paice M
G; FEBS Lett (1990) 267 99-102].
Studies on demethylation and delignification of kraft
pulp by a laccase enzyme in the presence of ABTS showed that
the extent of partial demethylation by laccase was increased in
the presence of ABTS [Bourbonnais, R. and Paice, M.G; Appl.
Microbiol. Biotechnol. (1992) 36 823-827].
Certain oxidizable substrates e.g. metal ions and
phenolic compounds such as 7-hydroxycoumarin (7HCm), vanillin
(VAN), and p-hydroxybenzenesulfonate (pHBS), have been de-
scribed as accelerators or enhancers, able to enhance bleaching
reactions (cf. e.g. WO 92/18683, WO 92/18687, and Kato M and
20 Shimizu S, Plant Cell Physiol. 1985 26 (7), pp. 1291-1301 (cf.
Table 1 in particular), or Saunders B C et al., Peroxidase,
London, 1964, p. 141 ff).
SUMMARY OF THE lN V ~:~. . lON
It is an object of the invention to provide an agent for
25 enhancing the activity of peroxidase enzymes, and to provide a
method of enhancing the activity of peroxidase enzymes. It has
now surprisingly been found that the activity of peroxidases
increases significantly in the presence of an azino compound as
described herein.
Accordingly, in its first aspect, the present invention
provides an agent for enhancing the activity of a peroxidase
enzyme, the agent being described by the general formula
A=N-N=B
W094/~620 21~ O ~ 6 3 PCT~K93/00394
in which formula A and B, which may be identical or
different, independently represents any of the substituents
presented in Fig. l;
in which substituents the symbols X and Y, which may be
5 identical or different, independently represents carbon,
nitrogen, which nitrogen may be unsubstituted or substituted
with a substituent group R5, sulfur, oxygen, selenium or
tellurium;
and in which substituents the substituent groups R1, R2,
lO R3, and R4, which may be identical or different, independently
represents hydrogen, halogen, a hydroxy group, a C1 -C3 alkoxy
group, a formyl group, a carboxy group, a sulfo group, a nitro
group, a C1-C5 alkyl group, which alkyl group may furthermore be
saturated or unsaturated, or an amino group, which amino group
15 may furthermore be unsubstituted or substituted once or twice
with a substituent group R5;
which substituent group R5 represents halogen, a hydroxy
group, a C1-C3 alkoxy group, a C~-C5 alkyl group, or an amino
group.
In its second aspect, the invention provides a method of
oxidizing a substrate with a peroxidase enzyme, in the presence
of a source of hydrogen peroxide, and in the presence of a
peroxidase enhancing agent of the invention.
In a specific aspect, the invention provides a method of
25 inhibiting the transfer of a textile dye from a dyed fabric to
another fabric when said fabrics are washed together in a wash
liquor, the method comprising treatment of the wash liquor with
a peroxidase enzyme in the presence of a source of hydrogen
peroxide and in presence of a peroxidase enhancing agent of the
30 invention.
In a particular aspect, the invention provides a
detergent additive capable of inhibiting the transfer of a
textile dye from a dyed fabric to another fabric when said
fabrics are washed together in a wash liquor, the detergent
35 additive comprising an enzyme exhibiting peroxidase activity,
a source of hydrogen peroxide and a peroxidase enhancing agent
of the invention.
WO94/12620 i ~ PCT~K93/0039~
2150~(i3 4
In another particular aspect, the invention provides a
detergent composition capable of inhibiting the transfer of a
textile dye from a dyed fabric to another fabric when said
fabrics are washed together in a wash liquor, the detergent
5 composition comprising an enzyme exhibiting peroxidase ac-
tivity, a source of hydrogen peroxide, and a peroxidase
enhancing agent of the invention.
In another aspect, the invention provides a method of
bleaching of lignin-containing material, in particular bleach-
10 ing of pulp for paper production, the method comprisingtreatment of the lignin or lignin containing material with a
peroxidase enzyme in the presence of a source of hydrogen
peroxide and in the presence of a peroxidase enhancing agent of
the invention.
15In a further aspect, the invention provides a method of
enzymatic polymerization and/or modification of lignin or
lignin containing material, the method comprising treatment of
the lignin or lignin containing material with a peroxidase
enzyme in the presence of a source of hydrogen peroxide and in
20 presence of a peroxidase enhancing agent of the invention.
In a yet further aspect, the invention provides a method
of treatment of waste water, e.g. waste water from the chemical
or pharmaceutical industry, the method comprising treatment of
the waste water with a peroxidase enzyme in the presence of a
25 source of hydrogen peroxide and in the presence of a peroxidase
enhancing agent of the invention.
BRIEF DESCRIPTION OF DRAWINGS
The present invention is further illustrated by refe-
rence to the accompanying drawings, in which:
30Fig. 1 shows the substituents II, III, IV, and V, of the
general formula I according to the invention;
Fig. 2 shows a comparison of a peroxidase enhancing
agent of the invention (ABTS) and pHBS, applied to bleaching of
Methyl Orange by a CoPrinus cinereus peroxidase (l: pHBS, 20 ~M
W094/~620 215 0 ~ 6 3 PCT~K93/00394
H2O2; 2: pHBS, 200 ~M H2O2; 3: ABTS, 20 ~M H2O2; 4: ABTS, 200 ~M
HzO2);
Fig. 3 shows accelerated bleaching of Methyl Orange by
a CoPrinus cinereus peroxidase in the presence of varying
5 concentrations of a peroxidase enhancing agent of the invention
(ABTS) (1: 0 ~M ABTS; 2: 1 ~M ABTS; 3: 5 ~M ABTS; and 4: 10 ~M
ABTS);
Fig. 4 shows a comparison of the initial bleaching rates
during bleaching of Direct Blue 1 (DBl) at pH 10.5 (O ABTS, 1
lO nM peroxidase; 0 VAN, lOO nM peroxidase; 7HCm, 100 nM
peroxidase; pHBS, lOO nM peroxidase); and
Fig. 5 shows a comparison of the initial bleaching rates
during bleaching of DB1 at pH 8.8 (and pH 10.5) (O ABTS pH 8.8:
O VAN pH 8.8; 7HCm pH 8.8; 0 ABTS pH 10.5; and pHBS pH
15 8.8).
DET~TT~n DI8CLOS~RB OF THB lNv~ lON
The Peroxidase Enhancinq Agent
The present invention relates to the use of a known
chemical compound for enhancing the activity of peroxidase
20 enzymes. Accordingly, the invention provides an agent capable
of enhancing the effect of a peroxidase enzyme.
The agent is an azino compound described by the general
formula I:
A=N-N=B
in which formula the symbols A and B, which may be
identical or different, independently represents any of the
substituents II, III, IV, and V, presented in Fig. l;
in which substituents the symbols X and Y, which may be
identical or different, independently represents carbon,
30 nitrogen, which nitrogen may be unsubstituted or substituted
with a substituent group Rs, sulfur, oxygen, selenium or
tellurium;
WO94/12620 PCT~K93/00394
21~0~63
and in which substituents the substituent groups R1, R2,
R3, and R4, which may be identical or different, independently
represents hydrogen, halogen, a hydroxy group, a C1-C3 alkoxy
group, a formyl group, a carboxy group, a sulfo group, a nitro
5 group, a C1-C5 alkyl group, which alkyl group may furthermore be
saturated or unsaturated, linear or branched, or an amino
group, which amino group may furthermore be unsubstituted or
substituted once or twice with a substituent group R5;
which substituent group R5 represents halogen, a hydroxy
lO group, a C1-C3 alkoxy group, a C1-C5 alkyl group, or an amino
group.
The peroxidase enhancing agent of the invention may be
in free form or in the form of an addition salt.
In preferred embodiments, the substituent groups R1, R2,
15 R3, and R4, which may be identical or different, independently
represents hydrogen, halogen, a hydroxy group, a C1-C3 alkyl
group, or a sulfo group. Preferably, the halogen is fluoro,
chloro, or bromo. Preferably, the C1-C3 alkyl group is methyl,
ethyl, propyl, or isopropyl.
In preferred embodiments, the substituent group R5
represents halogen, a hydroxy group, a C1-C3 alkoxy group, a C1-
C3 alkyl group, or an amino group.
In a most preferred embodiment, a peroxidase enhancing
agent of the invention is 2,2'-azino-bis(3-ethyl-
25 benzothiazoline-6-sulfonate). This compound, abbreviated ABTS,
is a chromogenic substrate, and a common peroxidase and phenol
oxidase assay agent.
It has, moreover, been demonstrated that ABTS, contrary
to the enhancers known and described above, is capable of
30 acting as a peroxidase enhancing agent at highly alkaline
conditions, i.e. above pH 9. This feature allows ABTS to be
implemented into e.g. detergent compositions, intended for
performance in the range pH 7-13, particularly the range pH 8-
12, preferably the range pH 9-ll.
WO94/12620 21~ 6 3 PCT~K93/00394
Methods of Oxidizing in the Presence of Peroxidases
In another aspect, the invention provides a method of
oxidizing a substrate with a source of hydrogen peroxide in the
presence of a peroxidase enzyme, the method being characterized
5 by the presence of a peroxidase enhancing agent of the general
formula
A=N-N=B
in which formula the symbols A and B, which may be
identical or different, independently represents any of the
10 substituents II, III, IV, and V, presented in Fig. l;
in which substituents the symbols X and Y, which may be
identical or different, independently represents carbon,
nitrogen, which nitrogen may be unsubstituted or substituted
with a substituent group R5, sulfur, oxygen, selenium or
15 tellurium;
and in which substituents the substituent groups R1, R2,
R3, and R4, which may be identical or different, independently
represents hydrogen, halogen, a hydroxy group, a Cl-C3 alkoxy
group, a formyl group, a carboxy group, a sulfo group, a nitro
20 group, a C~-C5 alkyl group, which alkyl group may furthermore be
saturated or unsaturated, or an amino group, which amino group
may furthermore be unsubstituted or substituted once or twice
with a substituent group R5;
which substituent group R5 represents halogen, a hydroxy
25 group, a C1-C3 alkoxy group, a C1-C5 alkyl group, or an amino
group.
The peroxidase enhancing agent of the invention may be
in free form or in the form of an addition salt.
In preferred embodiments, the substituent groups R1, R2,
30 R3, and R4, which may be identical or different, independently
represents hydrogen, halogen, a hydroxy group, a C1-C3 alkyl
group, or a sulfo group. Preferably, the halogen is fluoro,
chloro, or bromo. Preferably, the C1-C3 alkyl group is methyl,
ethyl, propyl, or isopropyl.
WO94/12620 ~ PCT~K93/00394
2150563 -- 8
In preferred embodiments, the substituent group R5
represents halogen, a hydroxy group, a C1 -C3 alkoxy group, a C1-
C3 alkyl group, or an amino group.
In a further preferred embodiment, the peroxidase
5 enhancing agent of the invention is 2,2'-azino-bis(3-ethyl-
benzothiazoline-6-sulfonate) (ABTS).
The enzyme employed in the method of the invention may
be any peroxidase enzyme comprised by the enzyme classification
EC 1.11.1.7. Such enzymes are known from microbial, plant and
10 animal origins.
Preferably, the peroxidase employed in the method of the
invention is producible by plants (e.g. horseradish peroxidase)
or microorganisms such as fungi or bacteria. In a further
preferred embodiment, the peroxidase is derived from Coprinus,
15 e.g. C. cinereus or C. macrorhizus, or from Bacillus, e.g. B.
pumilus, particularly a peroxidase according to PCT/DK
90/00260.
The peroxidase may furthermore be one which is produc-
ible by a method comprising cultivating a host cell transformed
20 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
25 peroxidase from the culture.
Particularly, a recombinantly produced peroxidase is a
peroxidase derived from a Coprinus sp., in particular Coprinus
macrorhizus or cinereus according to WO 92/16634.
The peroxidase enhancing agent of the invention may be
30 present in concentrations of from 0.01 to 100 ~M, more prefer-
red 0.1 to 50 ~M, most preferred 1 to 10 ~M.
The source of hydrogen peroxide may be hydrogen peroxide
or a hydrogen peroxide precursor, e.g. percarbonate or per-
borate, or a hydrogen peroxide generating enzyme system, e.g.
35 an oxidase and a substrate for the oxidase. Hydrogen peroxide
may be added at the beginning or during the process, e.g. in an
amount of 0.001-5 mM, particularly 0.01-1 mM. When using
W094/~620 21 ~ O ~ 6 3 PCT~K93/00394
Coprinus peroxidase, 0.01-0.25 mM H2O2 is preferred, and with B.
pumilus peroxidase 0.1-1 mM H202.
Industrial Applications
Methods according to the invention of oxidizing a
5 substrate with a source of hydrogen peroxide in the presence of
a peroxidase enzyme find various industrial applications.
In a preferred embodiment, the method of the invention
finds application for bleaching of dye in solutions.
In another embodiment, the method of the invention finds
10 application for dye transfer inhibition, e.g. for treatment of
dyed textiles (cf. e.g. W0 92/18687) or during laundering (cf.
e.g. WO 91/05839).
Accordingly, in a specific embodiment, the invention
provides a method for inhibiting the transfer of a textile dye
15 from a dyed fabric to another fabric when said fabrics are
washed together in a wash liquor, the method comprising
treatment of the wash liquor with a peroxidase enzyme in the
presence of a source of hydrogen peroxide, and the presence of
a peroxidase enhancing agent of the invention. The textile dye
20 may be a synthetic dye such as an azo dye, or a natural or na-
ture-identical dye.
In a third embodiment, the method of the invention finds
application in bleaching of pulp for paper production. The use
of a peroxidase together with hydrogen peroxide or a hydrogen
25 peroxide precursor in bleaching of paper pulp has been de-
scribed in e.g. SE 88/0673 and US 4,690,895.
Accordingly, the invention provides a method for
bleaching of lignin-containing material, in particular bleach-
ing of pulp for paper production, which method comprises
30 treatment of the lignin or lignin containing material with a
peroxidase enzyme in the presence of a source of hydrogen
peroxide and in the presence of a peroxidase enhancing agent of
the invention.
In a fourth embodiment, the method of the invention
35 finds application for lignin modification, e.g. in particle
board production. Binders for producing wood composites such as
WO94/12620 PCT~K93/00394
2150~G 3
. 10
fibre boards and particle boards can be made from peroxidase
treated lignin (cf. US 4,432,921).
Accordingly, the invention provides a method for
enzymatic polymerization and/or modification of lignin or
5 lignin containing material, which method comprises treatment of
the lignin or lignin containing material with a peroxidase
enzyme in the presence of a source of hydrogen peroxide, and
the presence of a peroxidase enhancing agent of the invention.
In a fifth embodiment, the method of the invention finds
lO application in treatment of waste water e.g. waste water from
the chemical or pharmaceutical industry, from dye manufac-
turing, from the textile industry, or from pulp production (cf.
e.g. US 4,623,465, or JP-A 2-31887).
In a more specific aspect, the invention provides a
15 method for treatment of waste water from dye manufacturing,
from textile industry, or from pulp manufacturing, the method
comprising treatment of the waste water with a peroxidase
enzyme in the presence of a source of hydrogen peroxide and in
the presence of a peroxidase enhancing agent of the invention.
20 Deterqent ComPositions
According to the invention, the peroxidase enhancing
agent may be added as a component of a detergent composition.
In a specific aspect, the invention provides a detergent
additive capable of inhibiting the transfer of a textile dye
25 from a dyed fabric to another fabric when said fabrics are
washed together in a wash liquor, the detergent additive
comprising an enzyme exhibiting peroxidase activity, a source
of hydrogen peroxide, and a peroxidase enhancing agent of the
invention. The detergent additive may additionally comprise one
30 or more other enzymes conventionally used in detergents, such
as proteases, lipases, amylases, or cellulases.
Preferably, the detergent additive is provided in the
form of a granulate, preferably a non-dusting granulate, a
liquid, in particular a stabilized liquid, a slurry, or in a
35 protected form.
WO94/12620 21 5 0 ~ 6 3 PCT~K93/00394
-
In another specific aspect, the invention provides a
detergent composition capable of inhibiting the transfer of a
textile dye from a dyed fabric to another fabric when said
fabrics are washed together in a wash liquor, the detergent
5 composition comprising an enzyme exhibiting peroxidase ac-
tivity, a source of hydrogen peroxide and a peroxidase enhanc-
ing agent of the invention.
The peroxidase enhancing agent of the invention may be
included in the detergent as a part of a peroxidase system,
10 comprising a peroxidase enzyme, a source of hydrogen peroxide,
and the peroxidase enhancing agent of the invention.
The peroxidase system may be included in the detergent
composition in the form of a non-dusting granulate, a liquid,
in particular a stabilized liquid, or in a protected form. Non-
15 dusting granulates may be produced, e.g. as disclosed in US4,106,991 and 4,661,452 (both to Novo Industri A/S) and may
optionally be coated by methods known in the art. Liquid enzyme
preparations may, for instance, be stabilized by adding a
polyol such as propylene glycol, a sugar or sugar alcohol,
20 lactic acid or boric acid according to established methods.
Other enzyme stabilizers are well known in the art. A protected
form of the peroxidase system may be prepared according to the
method disclosed in EP 238,216.
The detergent composition of the invention may be in any
25 convenient form, e.g. as powder, granules or liquid. A liquid
detergent may be aqueous, typically containing up to 90% water
and 0-20% organic solvent.
The detergent composition comprises surfactants which
may be anionic, nonionic, cationic, amphoteric or a mixture of
30 these types. The detergent will usually contain 0-50% anionic
~ surfactant such as linear alkylbenzenesulfonate (LAS), alpha-
olefinsulfonate (AOS), alkyl sulfate (AS), alcohol ether
sulfate (AES) or soap. It may also contain 0-40% nonionic
surfactant such as nonyl phenol ethoxylate or alcohol ethoxy-
35 late. Furthermore, it may contain a polyhydroxy fatty acid
amide surfactant (e.g. as described in WO 92/06154).
W094/~620 PCT~K93/00394
2150S63
12
The pH (measured in aqueous detergent solution) will
usually be neutral or alkaline, e.g. 7-11.
The detergent may contain 1-40% of a detergent builder
such as zeolite, phosphate, phosphonate, citrate, NTA, EDTA or
5 DTPA, alkenyl succinic anhydride, or silicate, or it may be
unbuilt (i.e. essentially free of a detergent builder). It may
also contain other conventional detergent ingredients, e.g.
fabric conditioners, foam boosters, bleaching agents, e.g.
perborate, percarbonate, tetraacetyl ethylene diamine (TAED),
10 or nonanoyloxybenzenesulfonate (NOBS), anti-corrosion agents,
soil-suspending agents, sequestering agents, anti-soil re-
deposition agents, stabilizing agents for enzymes, foam
depressors, dyes, bactericides, optical brighteners or
perfumes.
The detergent composition may additionally comprise one
or more other enzymes conventionally used in detergents such as
proteases, lipases, amylases, and cellulases.
Particular forms of detergent composition within the
scope of the invention include:
a) A detergent composition formulated as a detergent
powder containing phosphate builder, anionic surfactant,
nonionic surfactant, silicate, alkali to adjust to desired pH
in use, and neutral inorganic salt.
b) A detergent composition formulated as a detergent
25 powder containing zeolite builder, anionic surfactant, nonionic
surfactant, acrylic or equivalent polymer, silicate, alkali to
adjust to desired pH in use, and neutral inorganic salt.
c) A detergent composition formulated as an aqueous
detergent liquid comprising anionic surfactant, nonionic
30 surfactant, humectant, organic acid, alkali, with a pH in use
adjusted to a value between 7 and 10.5.
d) A detergent composition formulated as a nonaqueous
detergent liquid comprising a liquid nonionic surfactant
consisting essentially of linear alkoxylated primary alcohol,
35 phosphate builder, alkali, with a pH in use adjusted to a value
between about 7 and 10.5.
W094/~620 215 0 ~ 6 3 PCT~K93/00394
e) A detergent composition formulated as a detergent
powder in the form of a granulate having a bulk density of at
least 600 g/l, containing anionic surfactant and nonionic
surfactant, phosphate builder, sodium silicate, and little or
5 substantially no neutral inorganic salt.
f) A detergent composition formulated as a detergent
powder in the form of a granulate having a bulk density of at
least 600 g/l, containing anionic surfactant and nonionic
surfactant, zeolite builder, sodium silicate, and little or
10 substantially no neutral inorganic salt.
g) A detergent composition formulated as a detergent
powder containing anionic surfactant, nonionic surfactant,
acrylic polymer, fatty acid soap, sodium carbonate, sodium
sulfate, clay particles, and sodium silicate.
h) A liquid compact detergent comprising 5-65% by weight
of surfactant, 0-50% by weight of builder and 0-30% by weight
of electrolyte.
The following examples further illustrate the present
invention, and they are not intended to be in any way limiting
20 to the scope of the invention as claimed.
EXAMPLE 1
Bleachinq of MethYl Oranqe
Accelerated bleaching of Methyl Orange (Merck) catalysed
by a recombinantly produced Coprinus cinereus peroxidase
25 (rCiP), obtained according to WO 92/16634, and hydrogen
peroxide in the presence of 2,2'-azino-bis(3-ethylb-
enzothiazoline-6-sulfonate) (ABTS, supplied by Boehringer
Mannheim) or para-hydroxybenzene sulfonate (pHBS, supplied by
Sigma) is shown in Fig. 2. The following conditions were used:
10 nM rCiP
25 ~M Methyl Orange
50 ~M ABTS or para-hydroxybenzene sulfonate
20 and 200 ~M hydrogen peroxide
W094/~620 PCT~K93/0039~
~150S63
14
50 mM Britton & Robinson buffer, pH 8.8
30C thermostat
Reagents were mixed in a 1 cm cuvette, and the bleaching
was started by addition of hydrogen peroxide. The bleaching was
5 detected spectrophotometrically at 465 nm, which is the ab-
sorption peak of this dye. Bleaching was followed with respect
to time over a span of 10 min.
EXANP~B 2
Bleaching of MethYl Orange
Accelerated bleaching of Methyl Orange (Merck) catalysed
by a recombinantly produced CoPrinus cinereus peroxidase
(rCiP), obtained according to WO 92/16634, and hydrogen
peroxide in the presence of varying concentrations of 2,2'-
azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS, supplied
15 by Boehringer Mannheim) is shown in Fig. 3. The following
conditions were used:
10 nM rCiP
25 ~M Methyl Orange
0, 1, 5 and 10 ~M ABTS
200 ~M hydrogen peroxide
50 mM Britton h Robinson buffer, pH 8.8
30C thermostat
Mixture, start and detection of the bleaching are as
described in Example 1.
EXAMPLE 3
Bleachinq of Direct Blue 1
The initial bleaching of Direct Blue 1 (DB1) by recom-
binantly produced Coprinus cinereus peroxidase (rCiP), obtained
according to WO 92/16634, using ABTS as accelerator, was
30 compared to the best of the hitherto known accelerators: 7-
~094/~620 215 0 S 6 3 PCT~K93/00394
-
hydroxycoumarin (7HCm), vanillin (VAN), and p-hydroxybenzene
sulfonate (pHBS). The following conditions were used:
1 nM rCiP or 100 nM rCiP (at pH 10.5)
0, 10, 25, 50, or 75 ~M accelerator, respectively
50 mM Britton & Robinson buffer, pH 8.8 or 10.5,
respectively
20 ~M hydrogen peroxide
Reagents were mixed in a 1 cm cuvette, and the bleaching
was started by addition of hydrogen peroxide. The bleaching was
10 detected spectrophotometrically at 610 nm, which is the ab-
sorption peak of this dye. Bleaching was followed for 10
minutes, and the bleaching rates (-~mAbs/min) were determined
from the initial (linear) reduction in absorbance.
At pH 10.5 the bleaching using 100 nm rCiP and ABTS as
15 accelerator was so fast that bleaching was already completed
before the cuvette could be placed in the spectrophotometer,
the reason why the dosage of rCiP at pH 10.5 was reduced to 1
nM when used in combination with ABTS, although a dosage near
100 nM rCiP was necessary for all other (hitherto known)
20 accelerators in order to see a significant reduction in
absorbance.
The results of initial bleaching rate per minute have
been illustrated in Figs. 4 and 5 as function of accelerator
concentration.
EXANPLE 4
Enhanced DYe Transfer Inhibition bY ABTS
A washing trial was carried out in a Terg-o-tometer to
investigate the effect of ABTS on peroxidase based dye transfer
inhibition. For a comparison, also the established enhancer
30 pHBS was tested.
Clean white tracer test pieces (cotton, Style#400 from
Testfabrics, Inc., USA; bleached, but unbrightened) were washed
together with nylon test pieces dyed with the azo dye Acid Red
W094/~620 - PCT~K93/00394
2150S63 16
151 (C.I. 26900; available, e.g. from Aldrich Chemical Co.).
Reference test pieces were cut out of the same cotton cloth and
washed in the absence of dyed fabric. The dye transfer in a
given Terg-o-tometer pot was measured as the Hunter color
5 difference
~E = ~(~L) +(~a) +(^b)
between the tracer pieces in that pot and the above reference
pieces, the Hunter L, a, and b values being evaluated from
10 remission data obtained with an unfiltered daylight source on
a Datacolor Elrephometer 2000.
The detergent solution for the washing trial was made up
using 4.5 g/l of a commercially available European high-pH
powder detergent containing no bleach and no optical bright-
15 ener. The water used was tap water mixed with demineralizedwater in the ratio 1:2; the mixture had a hardness equivalent
to approx. 1.1 mM Ca2'.
The detailed experimental conditions were:
Duration of wash: 15 min.
20 Terg-o-tometer agitation: 70 rotations/min.
Temperature: 35C
pH: Adjusted to 10.5 with NaOH prior
to addition of peroxidase system
Textile load: Approx. 6 g nylon dyed with acid
Red 151 and 1 g white cotton per
litre washing liquor
Peroxide source: In all cases, 50 ~M H2Oz was present
together with the peroxidase
Peroxidase: Recombinantly produced Coprinus
cinereus peroxidase, obtained ac-
cording to WO 92/16634, at 5 nM
After washing, the test pieces were rinsed thoroughly in
cold tap water and dried in the dark overnight, after which the
remission measurements were performed.
WO94/12620 215 0 S 6 ~ PCT~K93/00394
,
17
Treatments with various concentrations of the two
enhancers yielded the following results:
Hunter ~E with respect to white,
washed fabric
l~M ABTS 34.9
5~M ABTS 32.3
20~M ABTS 23.7
l~M pHBS 34.8
105~M pHBS 34.5
20~M pHBS 30.8
Differences of 22 units of Hunter ^E were statistically
significant.
In both cases, the peroxidase system with 1 ~M enhancer
15 provided no significant dye transfer inhibition (reference
without peroxidase system not included here). However, as is
seen, the ABTS system has an effect already at 5 ~M of enhan-
cer, whereas the pHBS system does not; and at 20 ~M enhancer,
the ABTS system has a much larger effect than the pHBS system.
