U.S. patent application number 10/332410 was filed with the patent office on 2003-08-21 for method for enzymatic treatment of textiles such as wool.
Invention is credited to Bonnes, Philip, Cortez, Joao Marquez, Griffin, Martin.
Application Number | 20030154555 10/332410 |
Document ID | / |
Family ID | 29404265 |
Filed Date | 2003-08-21 |
United States Patent
Application |
20030154555 |
Kind Code |
A1 |
Griffin, Martin ; et
al. |
August 21, 2003 |
Method for enzymatic treatment of textiles such as wool
Abstract
The application provides a method of treating fibrous textile
goods comprising treating the fibrous textile goods with an enzyme.
This enzyme can be used to covalently link one or more active
functional compounds to the fibres and/or to trap one or more
acitve functional compound within an inter-fibre matrix and/or
within an intra-fibre matrix formed by the action of the enzyme.
Preferably, the enzyme is a traglutaminase, especially a
calcium-dependent transglutaminase. The enzyme may be used to add
primary-amine containing active agents to the textile goods and
also for the addition of proteins or peptides that have functional
groups linked to them.
Inventors: |
Griffin, Martin;
(Nottingham, GB) ; Cortez, Joao Marquez;
(Leicester, GB) ; Bonnes, Philip; (Nottingham,
GB) |
Correspondence
Address: |
JOHN S. PRATT, ESQ
KILPATRICK STOCKTON, LLP
1100 PEACHTREE STREET
SUITE 2800
ATLANTA
GA
30309
US
|
Family ID: |
29404265 |
Appl. No.: |
10/332410 |
Filed: |
January 8, 2003 |
PCT Filed: |
July 10, 2001 |
PCT NO: |
PCT/GB01/03095 |
Current U.S.
Class: |
8/115.51 |
Current CPC
Class: |
D06M 2200/20 20130101;
D06M 2200/50 20130101; D06M 2101/12 20130101; D06M 2200/45
20130101; D06M 16/003 20130101 |
Class at
Publication: |
8/115.51 |
International
Class: |
D06M 010/00 |
Claims
1. A method of treating fibrous textile goods comprising treating
the fibrous textile goods with an enzyme to either covalently or
non-covalently link one or more active functional compounds to the
fibres and/or to trap one or more active functional compounds
within an inter-fibre matrix and/or within an intra-fibre matrix
formed by the action of the enzyme.
2. A method, according to claim 1, wherein the fibrous textile
goods are derived from wool or animal hair, optionally blended with
one or more cellulosic or synthetic fibres.
3. A method, according to claim 1 or 2, wherein the enzyme is a
transglutaminase.
4. A method, according to claim 3, wherein the transglutaminase is
a calcium-dependent transglutaminase.
5. A method, according to claims 3 or 4, wherein the
transglutaminase is a tissue (type II) transglutaminase.
6. A method, according to any one of claims 3 to 5, where the
transglutaminase is a recombinant transglutaminase.
7. A method, according to any one of claims 3 to 6, additionally
comprising the step of treating the fibrous textile goods with a
protease.
8. A method, according to any preceding claim, wherein the active
agent is first bound to a protein or protein fragment, prior to
treating the fibrous textile goods.
9. A method, according to any one of claims 3 to 8, wherein the
transglutaminase is used together with a primary amine-containing
compound.
10. A method, according to claim 9, wherein the primary
amine-containing compound has a chemical moiety of general formula
--R'NH.sub.2 group, wherein R' is an aliphatic hydrocarbon chain
containing between 1 to 8 carbon atoms.
11. A method, according to claim 9 or claim 10, wherein the primary
amine-containing compound comprises at least two amine groups.
12. A method, according to claim 11, wherein the primary
amine-containing compound is putrescine.
13. A method, according to claim any one of claims 10 to 12,
wherein the --R'NH.sub.2 group is linked to an active group which
imparts further one or more functionalities to the fibres.
14. A method, according to claim 13, wherein the further
functionalities are used to bind a second active agent to the
fibres.
15. A method, according to any preceding claim, wherein the active
agent is a perfume, insect repellent, dyeing agent, softening
agent, water repellent antimicrobial agent or sunscreen.
16. A method, according to claim 15, wherein the active agent is an
intact protein, hydrolysed protein or modified hydrolysate of a
protein.
17. A method, according to claim 16, wherein the protein or
hydrolysed protein is keratin, silk, casein, fibronectin or
collagen.
18. A method, according to any one of claims 3 to 17, wherein the
transglutaminase is used as a level of 0.5-10.0 .mu.g of enzyme per
ml. of treatment liquor.
19. A method, according to any preceding claim, wherein the textile
goods are treated with a reducing or oxidated agent prior to
treatment with enzyme.
20. A method, according to claim 19, wherein the reducing agent is
sodium sulphite, dithiothreitol, 2-mercaptoethanol or
glutathione.
21. A method of protecting fibrous textile goods derived from wool
or animal hair from attack by a biological detergent comprising
treating the fibrous textile goods with an enzyme to covalently
cross-link one or more fibres of the fibrous textile goods.
22. A method according to claim 21, wherein the enzyme is a
transglutaminase.
23. A method of treating fibrous textile goods to improve
dimensional stability and/or improved yarn strength and properties
comprising treating the fibrous textile goods with a
calcium-dependent transglutaminase.
24. A method, according to claim 23, wherein the fibrous textile
goods are additionally treated wilt a protease.
25. Fibrous textile goods obtainable by a method according to any
preceding claim.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a method of treating
fibrous textile goods, such as wool, wool fibres or animal hair
with an enzyme, such as a transglutaminase, preferably either
including or in the absence of a protease enzyme. This treatment
can be used to trap one or more active functional compounds within,
by linking either covalently or non-covently such compounds onto,
the fibrous textile goods.
BACKGROUND TO THE INVENTION
[0002] The use of enzymes in the treatment of textile goods has
gained widespread acceptance and the applications of such
technology are many and diverse, including industrial processes and
household laundering. Thus, enzymes find industrial applicability
in the desizing of fabric, in enzymatic stone-washing of denim to
create an aged look, and in numerous other treatments to impart
enhanced fabric properties, such as a clean fabric surface, free of
microhairs and fibres, or improved pilling properties or fabric
hand. In domestic laundry products, enzymes are employed to assist
in the cleaning of goods, to remove soils and stains and also to
counter the formation of surface fibre, which gives a worn
appearance.
[0003] In particular, proteases have been widely employed in the
industrial treatment of wool goods to impart desirable properties.
There exists a general understanding of the mode of action of
protease on wool substrates. The enzymatic processes that are
currently being used, however, are difficult to control and can
lead to results that are not sufficiently predictable and
reproducible and cause significant damage to the fibre cuticle with
consequent strength loss. The major problems associated with wool
goods are its tendency to shrink and its handle (prickliness).
[0004] The dimensional stability of textile articles is obviously
of great significance, influencing the acceptability to the
consumer by defining the fit and comfort after repeated
launderings. A variety of methods to produce shrinkproof wool
materials are known and widely used. The most common method is a
chlorine-based process, which comprises an acid chlorination of the
wool material followed by a polymer application. Alternative
methods involve coating of the wool fibres to reduce the friction
coefficient using a polymer or monomers, which are polymerised on
the fibres. These methods achieve a significant level of
shrink-resistance to wool textiles, but they are difficult to
control, and may affect adversely the handle of wool goods, as well
as generate damaging substances that may be released into the
environment. Therefore, environmentally friendly methods such as
enzyme based processes and less aggressive chemical processes, such
as the low temperature plasma treatment have been suggested.
[0005] The scalar structure of the wool fibre is partly responsible
for the tendency of wool goods to dimensional instability. One idea
to reduce wool shrinkage is to remove or alter the scales of the
wool fibre surface using, for example, proteases. Ideally, a
commercial process would remove the surface scales to a limited
extent, reducing the fibre coefficient of friction without
significantly reducing wool fibre strength. The use of proteases
alone is not yet widely used industrially, the main reasons being
the significant losses of weight and strength that result and also
the relatively low degree of reproducibility. Many methods based on
the degradation of the structure of scales are destructive, causing
molecular degradation of the proteins, which is ultimately
responsible for the macroscopic reduction in weight and strength of
the processed wool or animal hair textiles.
[0006] Enzymatic treatments have also been suggested to improve the
handle of wool textiles as an alternative to the use of various
chemical agents, such as silicone softeners. Protease treatments
may however, if not closely controlled, cause undesirable levels of
weight and strength loss on the wool textiles.
[0007] Protease treatments of wool goods invariably lead to a
reduction, however slight, in fabric properties such as tensile or
bursting strength. There is also a measurable weight loss arising
from enzyme treatments. Such reductions must be balanced against
the enhancement of properties such as pilling performance or fabric
hand, and processing conditions and enzyme type are carefully
selected to maximise the desirable benefits whilst controlling
losses in strength and weight.
[0008] Transglutaminase is an enzyme which is found in a number of
organisms and different organs and tissues. It is responsible for
cross-linking proteins by forming covalent bonds between lysine and
glutamine residues. Transglutaminases usually have a higher
affinity for glutamine residues than lysine residues.
[0009] A variety of amines have been reported as substrates for
transglutaminase, including dansylcadaverine, methyl amine, butyl
amine, bistamine and putrescine and hydroxylamine. Hydroxylamine is
in fact the amine donor in a standard assay for transglutaminase
activity wherein hydroxylamine is covalently bound to an N-terminal
blocked peptide containing glutamine and glycine to produce
hydroxamate, which is detected by colour formation in the presence
of ferric chloride and acid. One Unit of transglutaminase is
defined as that amount which will form 1 .mu.M of hydroxamate per
minute at 37.degree. C.
[0010] Transglutaminases may be used in combination with proteases
to optimise an enzyme system that minimises the effect of such less
desirable effects like a reduction in weight and strength, whilst
achieving various desirable finishing effects, such as
shrink-resistance.
[0011] Some of the reductions in weight and strength are a
consequence of the mechanical agitation during processing, but the
protease treatments alone do contribute significantly to reductions
in weight and strength. Enzyme processing can be carried out in a
variety of machinery types, which are commonly available in the
industry. The characteristics of these machines are varied in terms
of capacity, mechanical action and agitation and liquor-to-goods
ratio, etc.
[0012] Prior to an enzymatic treatment, the fabric to be treated
preferably should be clean and free of any impurities, such as oils
and Does or softening agents, which may interfere with the action
of the enzyme. Fabrics are preferably scoured prior to the enzyme
treatment. It is of benefit, therefore to optimise both all process
parameters and enzyme system in order to minimise these losses and
to improve the quality of the finished textile goods.
[0013] A variety of enzyme-based methods have been used to process
wool textiles. U.S. Pat. No. 5,529,928 describes a method using an
initial oxidative step or an enzyme treatment (e.g. a peroxidase, a
catalase, or a lipase) followed by a protease treatment, followed
by heat treatment to obtain wool textiles with improved handle and
shrink-resistant properties.
[0014] Lorand et at. (1979) observed the specificity of the guinea
pig liver transglutaminase for synthetic primary amines. It is
revealed in this study that optimal transglutaminase activity is
achieved when compounds have alkyl amine side-chain lengths
equivalent to 5 methylene groups, no branching nor groups bulkier
than methylene along the alkyl amine chain and hydrophobic moieties
attached to the alkyl chain.
[0015] U.S. Pat. No. 5,490,980 describe a method using
transglutaminases to cross-ink beneficial substances containing an
amine moiety to glutamine residues in skin, hair or nails, but not
to fibrous textile goods.
[0016] While wool fibre (in the form of tops, yarns or fabrics) is
dead, human hair is still growing and has a living root. As a
consequence, the type of active agents that can be applied to
living hair in a scalp are different from those applicable to wool
fibres, which can be treated in industrial conditions. Treatment
temperature, pH, type and harshness of chemicals are very
different. In particular, the active agents applicable to wool and
hair are generally different, and the process of applying
transglutaminase and any active agent are also generally different
(for example, by using microbial transglutaminase, which has an
optimum temperature of 50.degree. C.).
[0017] Both human hair and wool consist mainly of keratins and have
approximately the same basic morphology. Human hair is, however,
more resistant to chemical and enzymatic attack than wool.
[0018] Wool fibres are available in large quantities and are more
flexible than hair and are therefore easy to spin into threads and
make garments. Because of the protruding scales in the wool fibre
felting shrinkage is a major problem, mainly in knitted
garments.
[0019] Differences also exist in the type of chemicals that may be
applied. Strong alkali and acids, dyes, resins, etc, can easily be
applied to wool without concern to toxicity to the wool, since it
is dead. The range of finishing compounds applicable to human hair
and wool are also different. Wool processing takes place in large
industrial machinery and allows for the use of harsh chemicals and
harsh treatment conditions.
[0020] Japanese patent JP 3213574 describes a process to treat wool
or animal hair using a calcium independent transglutaminase of
mircrobial origin by cross-linking the amino acid functional groups
of the cuticle of animal hair so as to produce hair or hair fibre
containing material having improved shrink-resistance, pilling
resistance and hydrophobic property.
[0021] The deficiencies in terms of performance, cost and
environment of the wool processing methods used currently in the
textile industry indicate that there is a need for a new process
that imparts improvements in shrink resistance, softness,
appearance, resistance to pilling without a decrease in tensile and
bursting strength.
[0022] This invention features a number of departures from normal
accepted practice in the enzymatic treatment of wool goods, which
renders it novel both in terms of application method and in the
effects achieved.
[0023] The invention provides a method of treating fibrous textile
goods, preferably derived from wool or animal hair, comprising
treating the fibrous textile goods with an enzyme to either
covalently or non-covalently bind one or more active functional
compounds to the fibres and/or to trap one or more functional
compounds within an inter-fibre matrix and/or within an intra-fibre
matrix formed by the enzyme.
[0024] A second aspect of the invention provides a method of
protecting fibrous textile goods, preferably derived from wool or
animal hair, from attack by biological detergents comprising
treating the fibrous textile goods with an enzyme to covalently
link one or more fibres of the fibrous textile goods.
[0025] A third aspect of the invention provides a method of
treating fibrous textile goods to improve dimensional stability
and/or improve yarn strength comprising treating the fibrous
textile goods with a calcium-dependent transglutaminase. Such a
treatment also may improve tensile and burst strength, shrinkage
resistance, handle, reduces pilling, improves softness, improves
dye uptake and washfastness, especially when used together with a
protease.
[0026] Preferably, the fibrous textile goods are derived from wool
fibre blended with one or more cellulosic or synthetic fibres.
[0027] Preferably, the enzyme is a transglutaminase (TGase),
especially a calcium-dependent transglutaminase such as tissue type
II transglutaminase.
[0028] The biological detergent may be one containing a protease.
The inventors have unexpectedly found that using an enzyme to
cross-link the fibres reduces the amount of damage from biological
detergents such as those containing proteases, and effectively
reduces the amount of dye released during washing.
[0029] Fibrous textile goods obtainable by the methods of the
invention are also provided.
[0030] In the context of the present invention a pretreatment with
reducing agents may be applied with the intent of breaking cystine
bonds on wool to make it more accessible to further action by
enzymes.
[0031] Alternatively, an oxidative treatment may be used.
[0032] Proteolytic enzymes may be used to break down the cuticle
structure in the fibre surface in such a way to render it more
accessible to transglutaminases, without excessive fibre damage and
loss of weight and strength. Transglutaminases form intra- and
inter-isopeptide bonds in the keratin molecule, stabilising the
protein molecules. Their use results in an increase in total fibre
and fabric strength, as well as rendering the fabrics less prone to
felt. When used in combination with proteases, transglutaminases
prevent excessive molecular breakdown associated with protease
treatments, preventing thereby a reduction in weight and strength
loss.
[0033] The proteases may be used before, during or after the use of
transglutaminase.
[0034] The processing of wool textiles with transglutaminases may
involve a treatment with calcium-dependent transglutaminases such
as tissue transglutaminase, alone or after a pretreatment with a
protease. Incubation with transglutaminases carried out prior to a
protease treatment leads to a reduction in the loss of yarn and
fabric strength compared to a protease treatment only (under the
same conditions). The same benefits may also result from processing
by pre-soaking the wool or animal hair fibres with TGases in the
absence of Ca.sup.2+to allow a better penetration of the enzyme
into the fibre. The enzyme may then be activated in a later stage
by the addition of the Ca.sup.2+ions.
[0035] The use of a calcium dependent transglutaminase to deliver
benefits such as increased tensile and burst strength, improved
shrink resistance, handle, reduction of pilling, improved softness,
improved softness and improved dye uptake and washfastness presents
the advantage of an effective control of the enzyme
activation/deactivation by the addition of either calcium or a
sequestering agent.
[0036] The selection of an optimised transglutaminases,
specifically tailored by gene manipulation, could ensure a high
degree of dimensional stability, whilst minimising the negative
effects from other enzymes (proteases) or chemicals, such as
strength loss and harsh handle. Further, the enzyme preparation may
be tailored to deliver improvements in other areas as well as
dimensional stability.
[0037] Such a recombinant enzyme may be identified or produced by
conventional recombinant technology. EP 0268772A2 describes the
expression of biologically active Factor XIII. U.S. Pat. No.
6,190,896 describes the production of an active human cellular
transglutaminase, and WO 0129187 describes a process for the
production of a microorganism-origin transglutaminase.
[0038] Transglutaminase or its optimised derivatives can be used to
improve, for example, fabric handle, pilling performance, wrinkle
resistance, the setting process, improve durable press
finishing.
[0039] The active agents may be attached to the fibrous textile
goods by means of using a transglutaminase to react a primary amine
group with a peptide bound .gamma.-glutamine or a .gamma.-glutamine
with .epsilon.-lysine residue on the fibres and form a covalent
bond. Alternatively, the active agents may be trapped within a
matrix of inter-cross-linked fibres or, indeed, within an
intra-fibre cross-linked matrix. That is, within a matrix by
cross-inking adjacent fibres or by cross-linking within the same
fibre.
[0040] The active agents may be modified by addition of a primary
amine to the active agent. Preferably, the active agent comprises
the group --R'NH.sub.2 where --R' is an aliphatic branched or
unbranched hydrocarbon chain containing 1 to 8, preferably 2 to 6,
more preferably at least 5 carbon atoms. Preferably the R' is
unbranched.
[0041] Alternatively, the primary amine of general formula of
--R'NH.sub.2 may be linked to a different functional group which
imparts further functionalities to the fibrous goods. These may, in
turn, be used to link further active agents, such as commercial
polymers for improving shrink and improving softness, to the
fibrous goods.
[0042] The active agent may have several alkylamine moieties. These
may be used to cross-link fibres or to bond the fibres to further
active agents.
[0043] Putrescine (1,4-diaminobutane) may be used as the active
agent.
[0044] Transglutaminase or its optimised derivatives can also be
used to incorporate either covalently or non-covalently active
agents that, once attached into the fibre surface, improve the
binding and consequently the performance of active agents (e.g.
commercial polymers used for shrink resistance--such as silicone
oils and cationic polymers--and improving softness--such as amino
silicones).
[0045] Suitable active compounds include but are not limited to
perfumes, insect repellents, dyeing agents, softening agents, water
repellents, antimicrobial agents, sunscreens and mixtures thereof.
The active agents include but are not limited to intact proteins,
hydrolysed proteins and modified hydrolysates, such as peptides and
peptide derivatives, keratin, silk, casein, fibronectin and
hydrolysed collagen.
[0046] Preferred fragrances include vanillin, thymol and
menthylsalicylate.
[0047] Preferred antimicrobials include phenol, cresol,
hydroxybenzoates, triclosan and cinnamic acid.
[0048] The active compounds may, in a first step, be linked to a
protein or a protein fragment chemically, and in a second step, the
protein or a protein fragment containing the active agent is
crosslinked to the fibres using transglutaminase. The protein or
protein fragment may be casein, for example. The compound may be a
shrinkage prevention compound.
[0049] TGase cross-linking of polyamines to wool also provides
additional amine groups that may be used as a platform to link
other compounds (using e.g. carbodimides--B. F. Erlanger, 1980,
Preparation of Antigenic Hapten--Carrier Conjugates, Methods in
Enzymology, 70, 85-104). The ability of TGase to give wool specific
and desired functions using these methods presents considerable
advantages.
[0050] All compounds that produce a beneficial finishing effect on
wool or animal hair textiles can also be used in the present
invention given that the compound can be modified to contain at
least one primary amine.
[0051] The proteolytic enzyme to be used in the context of the
present invention may be from plant, animal, bacterial or fungal
origin. The proteases used are most preferably subtilisins, such as
Savinase 16L (ex. Novo Nordisk).
[0052] Examples of the transglutaminase types suited for this
application include the following: guinea pig liver, human origin,
maize, alfalfa (Medicago saliva), slime mould (Physarum
polycephalum), Phytophtora cactorum and bacteria (Bacillus
subtilus, Streptoverticillium mobaraense). Ajinomoto Inc. patented
a method for production of a commercial transglutaminase by a batch
fermentation process using bacteria containing genes from
Streptoverticillium sp. Preferably a Ca.sup.2+activated tissue
transglutaminase should be used. This list is not intended to be
exhaustive, and omission from this list should not be taken as an
indication that particular types are more suited that others.
Indeed, the ideal transglutaminase preparation may be derived from
genetic manipulation of one of any number of naturally occurring
sources. Further suitable transglutaminases may be derived from
mammals, insects, crustaceans, plants and microorganisms.
[0053] As stated above, it has been found that a significant degree
of dimensional control of wool and wool blend fabrics may be
achieved if a transglutaminase is used. The transglutaminase enzyme
used for such a treatment may be chosen from mammalian, plant or
microbial source, but to optimise the properties of the treated
fabric, it may be advantageous to employ an enzyme system
specifically manufactured to achieve good dimensional stability and
whose activity is easily controllable.
[0054] Among the existing transglutaminases, a calcium dependent
transglutaminase, such as tissue (type II) transglutaminase,
presents several advantages. This enzyme is activated by the
presence of calcium ions, which renders it easily controllable. It
is active at room temperatures, being most active at 37.degree. C.,
which allows a wide range of processing methods to be used, as well
as significant energy savings to be made. It is also readily
inactivated by heating to 60.degree. C. or by removal of
Ca.sup.2+either by washing or with addition of chelating agents for
divalent metal ions, e.g. EDTA.
[0055] It is known that enzymes require specific and controlled
treatment conditions in order to achieve optimum and reproducible
end-effects during processing of wool goods. Typically, the type of
electrolyte used, temperature, liquor pH and agitation are all
controlled to a high degree to ensure an effective and even
treatment over the goods as a whole.
[0056] The treatment liquor may contain suitable pH-buffering
agents to maintain a constant pH in the range appropriate for
optimum activity of the enzyme being employed. In the case of
transglutaminases, the solution may also contain a suitable
reducing agent and an appropriate concentration of calcium ions if
the mammalian transglutaminase is to be used. Other auxiliaries may
be present in the treatment liquor--for example surfactants,
provided that their presence does not interfere with the action of
the enzyme. In this regard, the co-application of the enzyme
treatment with other finishes from die same liquor is not to be
excluded, provided that the enzyme treatment and any other
co-applied treatment(s) are mutually compatible.
[0057] The impregnation of the wool goods with transglutaminase may
be carried out at a temperature of 15-70.degree. C., especially
15-60.degree. C., most preferably 30-40.degree. C. The enzymes may
be dissolved in water at concentrations between 0.5-10.0 .mu.m of
enzyme per ml of treatment liquor, most preferably 1.0-5.0 .mu.g of
enzyme per ml of liquor. The incubation time should be from at
least 30 minutes up to 18 hours, depending on the enzyme
concentration and treatment temperature.
[0058] If a proteolytic enzyme is to be used, it is most preferably
applied at temperatures between 45-55.degree. C. during 15 to 60
minutes. The process can, however, be carried out at lower
temperatures for a longer treatment time.
[0059] Enzyme processing can be carried out in a variety of
machinery types, which are commonly available in the industry.
[0060] Fabrics derived from wool fibres are suited to this process.
Further, fabrics constructed from wool/synthetic blends or
wool/cellulosic fibre blends (such as cotton/wool) are also
suitable for treatment by this process.
[0061] In the present invention the textile samples may be
submitted, for example, to a pretreatment with a reducing agent
prior to the application of transglutaminase. Textile samples may
also be pretreated with a proteolytic enzyme before applying the
transglutaminase enzyme.
[0062] For example, the guinea pig liver transglutaminase (a tissue
transglutaminase, which is commercially available from Sigma) may
be applied to a wool yarn by immersion in a solution containing the
enzyme. The reaction may be carried out in a media with or without
a reducing agent, such as dithiothreitol, 2-mercaptoethanol, and
glutathione.
[0063] This enzyme may be activated by the presence of calcium
ions, and is most active at 37.degree. C., and it is readily
inactivated by washing with chelating agents or heating to
60.degree. C.
[0064] Other transglutaminases may be used, the treatment
parameters depending on which specific enzyme is to be applied. For
example, microbial transglutaminase obtainable from Ajinomoto Inc.
may be used.
[0065] The invention will now be further illustrated by reference
to a series of examples but the invention is not limited thereto.
In the examples, TGase refers to tissue transglutaminase from
guinea pig liver. The use of microbial transglutaminase is denoted
by mTGase obtained from Streptoverticillum by Ajinomoto Inc.
[0066] FIG. 1a shows that yarn strength change (from control) of
samples treated with TGase/Ca for several treatment times (control
treated in Tris-HCI buffer without TGase (-.sup..box-solid.-)). One
set of samples was pretreated with Savinase 16L
(-.sup..quadrature.-) and a second set in buffer alone with no
Savinase added (Savinase control).
[0067] FIG. 1b shows yarn elongation change (from control) of
samples treated with TGase/Ca for several treatment times (control
treated in Tris-HCI buffer without TGase). One set of samples was
pretreated with Savinase 16 L (-.sup..quadrature.-) and a second
set in buffer alone with no Savinase added (Savinase control
(-.sup..box-solid.-)).
[0068] FIG. 2 shows yarn strength change from control of samples
treated during 6 hours with a range of concentrations of TGase
(-.sup.570 -) (controls -.sup..diamond-solid.- were treated in the
same manner except without adding TGase). The samples were
pre-treated with Savinase 16 L and buffer only control.
[0069] FIG. 3a shows yarn strength change from control of samples
treated with TGase/Ca.sup.2+during 18 hours (controls were treated
in the same manner except without adding TGase). The samples were
pre-treated with sodium carbonate (Carb) and sodium sulphite (Sul).
The samples were also treated with Savinase 16 L (Sav) with
respective buffer only controls (Ct).
[0070] FIG. 3b shows percentage strength gain from control versus
percentage elongation gain from control of yarn samples treated
with 1.0 and 5.0 .mu.g/ml of TGase (corresponding to 1 and 5 in the
graphite) for samples pretreated with sulphite, chlorine and PMS
(controls were treated in the same manner except without adding
TGase).
[0071] FIG. 3c shows percentage strength gain from control versus
percentage elongation gain from control of yarn samples treated
with 10.0, 100.0 and 1000.0 .mu.g/ml of mTGase (corresponding to
10, 100 and 1000 in thc graphic) for samples pretreated with
sulphite, chlorine and PMS (controls were treated in the same
manner except without adding TGase).
[0072] FIG. 4 shows yarn strength loss caused by a protease
treatment (change from control samples). Samples were treated with
Savinase prior (SavCtTG and SavTG--red) and after (CtTGSav and
TGSav--blue) an 18-hour tTG treatment (control--Ct, Sav--Sav).
Savinase controls were treated in the same manner except without
Savinase 16 L and tTG controls without tTG),
[0073] FIG. 5a shows absorption at 511 nm of the washing liquor
after each cycle of detergent washes of samples submitted to
different TGase treatments after several washes with a biodetergent
(the percentage reduction in absorbance relative to the control is
shown as percentage values). Cycles of detergent wash followed by a
transglutaminase treatment were repeated 3 times (tTG5--tTG at 5.0
.mu./ml; tTG5 tryptone--tTG at 5.0 .mu.g/ml, with 1.0 mg/ml of
tryptone (casein digest); mTG100--mTG100 at 100.0 .mu.g/ml.).
Controls were treated in buffer without adding TGase.
[0074] FIG. 5b shows tensile strength of yarns unraveled from
fabrics submitted to different TGase treatments for cycles 1 and 3
(the percentage strength gain relative to the control is shown as
percentage values). The yarn strength of the control samples is
2.19 N (black line).
[0075] FIG. 6 shows a table indicating felting shrinkage after
three 5A washes of samples treated with transglutaminase and an
active agent followed by a treatment with a commercial polymer.
[0076] FIG. 7 shows softness of wool samples after a treatment with
tissue and microbial transglutaminase and an active agent followed
by a treatment with a commercial softener.
[0077] FIG. 8 shows subjective analysis of residual scent after a
treatment with 5.0 and 20.0 .mu.g of tTGase per ml. of liquor and
an added scent by a panel of 12 judges (2 and 5 days after
treatment). The panel graded the samples from the least to the most
intense residual scent. A control was treated in the same manner
but without tTGase.
[0078] FIG. 9a shows wool fibres treated with transglutaminase in
the presence of calcium ions and flourescine cadaverine.
[0079] FIG. 9b shows wool fibres treated with transglutaminase in
the presence of EDTA and flourescine cadaverine.
EXAMPLE 1
Transglutaminase Cross-Linking of Wool for Different Treatment
Times
[0080] Samples of 100% superfine lambswool yarn {fraction (1/13)}nm
(ex. Patons) were washed in a solution containing a reducing agent,
5.0 g/l of sodium sulphite, and 1 g/l of a non-ionic detergent at a
liquor to fibre ratio of 250 ml/g for 30 minutes at 60.degree. C.,
and subsequently rinsed in water. The yarn samples were then
treated in a shaker rotating at 100 rpm for 60 minutes at
37.degree. C., in a 0.05 M TRIS buffer solution (the pH was
adjusted to 8.5 with hydrochloric acid) containing 1% of Savinase
16 L (ex. Novo Nordisk) on the weight of wool yarn. The treated
samples were washed with a non-ionic detergent at pH 5, and then in
boiling water for 15 minutes to deactivate the proteolytic enzyme.
Finally, the samples were rinsed before submitted to further
treatment. As a control to the protease treatment, sulphite washed
yarn samples were also treated with buffer only. The guinea pig
liver transglutaminase (ex. Sigma) was then applied to the Savinase
treated and respective buffer control samples at 1.0 .mu.g of
transglutaminase per ml of liquor in a buffered solution with 0.05
M TRIS buffer (the pH was adjusted to 8.5 with hydrochloric acid).
The liquor also contained 5 mM dithiothreitol (DTT) and 5 mM of
calcium ion. The liquor to yarn ratio applied was 1:12. The yarn
samples were then incubated in a shaker rotating at 100 rpm for a
period of time between 2 to 18 hours 8 hours at approximately
37.degree. C. As a control to the transglutaminase treatments yarn
samples were treated under exactly the same parameters in a
solution containing buffer, 5 mM DTT and 5 mM calcium ion.
[0081] The enzymatic reaction was stopped after the specified time
and the yarn samples were washed in a buffered phosphate saline
solution (PBS) pH 7.4 and 1.0% of Tween 80 detergent. The yarn
samples were washed in three consecutive cycles with this solution.
The yarn samples were then rinsed in water in three consecutive
cycles and then dried and conditioned at the standard temperature
and humidity.
[0082] The tensile strength or breaking load of the yarn samples
and elongation at break were determined by the method BS EN ISO
2062:1995, and the transglutaminase treatments were compared to a
buffer alone treated control (no TGase added) for both the Savinase
treated samples and the Savinase controls (no Savinase added).
[0083] The greater improvements in strength were obtained with
transglutaminase treatments following a protease treatment (see
FIG. 1a). There was also a significant increase in elongation at
break for die transglutaminase treated samples, in particular for
those submitted to a Savinase pretreatment (see FIG. 1b).
EXAMPLE 2
Transglutaminase Crosslinking of Wool--Optimisation of Delivery and
Concentration of Enzyme Used
[0084] Samples of 100% wool yarn previously treated with sodium
sulphite (as described in Example 1) were treated with Savinase 16L
in exactly the same manner as described above. All samples were
treated with 5.0, 20.0 and 100.0 .mu.g of guinea pig liver
transglutaminase per ml of liquor, and incubated for 6 hours at
37.degree. C. All other treatment parameters were the same as in
Example 1. To a second set of samples pretreated exactly in the
same manner 1/3of the total transglutaminase was added to the
treatment bath every two hours (all other treatment parameters were
the same). The samples were washed and dried as described in
Example 1.
[0085] There was a significant improvement in the strength of all
transglutaminase treated yarns compared to the buffer treated
controls. These improvements were significantly greater for yarn
pretreated with Savinase. In particular, the treatment with 5.0
.mu.g of transglutaminase per mL of liquor produced a gain of
strength of about 34% compared to the respective control, while the
Savinase control samples treated with transglutaminase resulted in
a gain of 15% (see FIG. 2). Adding enzyme in three separated
aliquots on the same incubation time gave a significant improvement
in strength gain compared with the samples treated with one batch
of enzyme at the start of the process.
EXAMPLE 3
Effect of Different Pretreatments on the Transglutaminase
Crosslinking of Wool
[0086] Pretreatment with sodium sulphite and sodium carbonate
[0087] In this example two batches of yarn samples were first
treated with a) 0.5 g/L of sodium carbonate and 1 g/l of a
non-ionic detergent and b) 5.0 g/l of sodium sulphite and 1 g/L of
a non-ionic detergent both at a liquor to fibre ratio of 250 ml/g
for 30 minutes at 60.degree. C. Samples from a) and b) were then
submitted to a Savinase treatment as described in Example 1. The
proteolytic reaction was stopped by washing the samples with a
non-ionic detergent at pH 5 and then in hot water at 80.degree. C.
for 15 minutes. Finally, the samples were rinsed before submitted
to further treatment. As a control to the protease treatment, yarn
samples from a) and b) were also treated with buffer only.
[0088] The four resulting sets of yarn samples were then treated
with 1.0 .mu.g of transglutaminase per ml of liquor, and incubated
for a period of time between 2 to 18 hours at ambient temperature.
All other treatment parameters were the same as in Example 1. As a
control to the transglutaminase treatments yarn samples were
treated as in Example 1.
[0089] The results indicate that the transglutaminase treatment for
the samples pre-treated with a reducing agent such as sodium
sulphite result in a greater increase in yarn strength than those
treated with sodium carbonate (for both Savinase treatment and
respective buffer control--FIG. 3). In particular, the sulphite
treatment followed by Savinase and finally by transglutaminase
results in an increase in strength of 37%.
[0090] Pretreatment with chlorine and permonosulphuric acid
(PMS)
[0091] In this example samples of 100% wool yarn samples treated
with sodium sulphite (as described in Example 1) and 100% wool
knitted fabric samples (supplied by Cooper & Roe, UK)
pretreated with chlorine and permonosulphuric acid (PMS) were
compared in terms of type of pretreatment on transglutaminase
cross-linking. All samples were pretreated with Savinase 16 L in
exactly the same manner as described in Example 1. Samples from
each type of pretreatment were treated with 1.0 and 5.0 .mu.g of
guinea pig liver transglutaminase per ml of liquor. All other
treatment parameters were the same as in Example 1.
[0092] A second set of samples was treated with microbial
transglutaminase from Ajinomoto Inc. Samples from each type of
pretreatment were treated with 10.0, 100.0 and 1000.0 .mu.g of
microbial transglutaminase per ml of liquor. The treatments were
carried out in a TRIS-HCl buffered solution pH 7.0 at 50.degree. C.
for 2 hours. The liquor also contained 5 mM DTT. The liquor to yarn
ratio applied was 1:12.
[0093] The chlorine and PMS treated samples were compared with
samples treated with sodium sulphite and sodium carbonate incubated
(as described above) with 5 .mu.g of guinea pig liver
transglutaminase per ml of liquor and 100 .quadrature.g of
microbial transglutaminase per ml of liquor. Tensile strength and
elongation at break were determined for all samples (FIGS. 3b and
3c) and all transglutaminase treatments were compared to buffer
only treated controls.
[0094] It can be seen from FIGS. 3a and 3b that the effect of a
transglutaminase treatment varies for the three types of
pretreatment. The gain in strength is greater for PMS treated
fabric and the percentage elongation gain is greater for the
sulphite treated yarns, for both tissue and microbial
transglutaminases. Strength gains as high as 30% comparing to the
control can be achieved for PMS treated fabrics, and elongation
gain of up to 35% comparing to the control were obtained for the
sulphite pretreatment using the microbial TG.
EXAMPLE 4
Transglutaminase Treatments Prior to Protease Treatment
[0095] This experiment was carded using the same procedure as
described in Example 1, except that in one batch of samples the
protease treatment was carried out prior to the transglutaminase
treatment and on the second batch the protease treatment was
carried out after the transglutaminase treatment. In both yarn
sample batches the concentration of Savinase 16 L were carried out
as described in Example 1. All samples were treated with 1.0 .mu.g
transglutaminase was per ml of liquor, and incubated for 18 hours
at 37.degree. C. All other treatment parameters were the same as
described in Example 1. The samples were washed and dried as
described in Example 1.
[0096] The results (see FIG. 4) indicate that the transglutaminase
treatment prior to the Savinase treatment not only reduced the loss
of strength caused by the subsequent protease treatment (comparing
to the Savinase/protease treatment) but also increased the strength
of the yarns in a greater extent comparing to the transglutaminase
buffer control (comparing to die Savinase/protease treatment).
EXAMPLE 5
Protection of Wool Fibres and Garments from Attack with Domestic
Biodetergents Using Transglutaminase
[0097] In this example 10 g samples from a 100% wool knitted fabric
(dyed with a reactive red dye) were washed with 10 ml/l of Persil
Performance (commercial biodetergent containing proteases) for 30
minutes at 40.degree. C. with a LR of 1:15 in 250 ml beakers in a
shaker at 100 rpm and then flat dried.
[0098] One set of the washed samples was then treated with 5.0 and
20.0 .mu.g/mL of tTGase, 5.0 .mu.g/mL of tTGase and 1.0 mg/mL of a
casein enzymatic digest (tryptone) for 2 hours at 37.degree. C., pH
8.5 (compared with a control treated only with buffer under the
same conditions). Another set of samples was incubated with 100
.mu.g/mL of mTGase at 55.degree. C., pH 7.0 for 1 hour (compared to
a buffer only control treated under the same conditions). Samples
were then washed again as described above and treated with
transglutaminase and the procedure repeated three times.
[0099] The absorbance of the washing solutions was used as a
measure of the amount of dye released into the washing bath after
each washing cycle (FIG. 5a). The percentage values shown in FIG.
5a illustrate the reduction in absorbance at 511 nm, which was
calculated as the percentage difference in the measured absorbance
between control samples treated only with buffer and the
transglutaminase treated samples. After 3 detergent
wash/transglutaminase treatment cycles the treated fabrics still
release significantly less dye than the control treated only with
buffer.
[0100] Strength loss and elongation was also measured, both after
the transglutaminase treatment and after each detergent wash. The
results in FIG. 5b show an increasing retention of strength of the
transglutaminase treated samples after each detergent wash
(compared to samples treated with buffer alone).
EXAMPLE 6
Effect of Transglutaminase Mediated Incorporation of Active
Compounds into Wool Fibres on Felting Shrinkage
[0101] In this example samples of a 100% wool knitted fabric
(supplied by Cooper & Roe, UK) were treated with Savinase 16 L
using the same procedure as described in Example 1. A pancreatic
digest of milk casein (Tryptone) and putrescine were incorporated
into wool fibres using transglutaminase. Commercial polymers used
for wool shrinkage prevention (supplied by Precision Processes
Textiles, UK) were added to fabric samples treated only with
Savinase 16 L and to fabrics treated with transglutaminase and an
active agent (polymers used included MRSM, XM and TM--see Table 6).
Control samples treated only with buffer and treated only with the
commercial polymers were included (treatments 4, 5 and 6). All tTG
treatments were carried out as in Example 1 using 5.0 .mu.g of
transglutaminase per mil of liquor, and were incubated for 2 hours
at a liquor ratio of 1:10. All other treatment parameters were the
same as in Example 1. All mTG treatments were carried out using 100
.mu.g of transglutaminase per ml of liquors, and were incubated for
1 hour at a liquor ratio of 1:10. All other treatment parameters
were the same as in Example 3. In treatments 2, 7, 9 and 11 (see
Table 6) the casein digest (0.5 mg/ml) was incorporated into the
wool fibres using tTG (5.0 .mu.g/ml) prior to polymer treatments.
In treatments 3, 8, 10 and 12 (see Table 6) putrescine (2.0 mM) was
incorporated into the wool fibres using tTG (5.0 .mu.g/ml) prior to
polymer treatments.
[0102] The polymer treatments were carried out by exhaustion
subsequently to the TG-casein digest/putreascine treatments at pH
5.5 and 40.degree. C., according to manufacturer
recommendations.
[0103] In treatments 14 and 15 microbial transglutaminase was
incubated together with the polymer at pH 5.5, 40.degree. C. for 1
hour.
[0104] The samples were tested for felting shrinkage according to
the IWS TM 31, except that the dimension of the samples tested was
reduced to 120.times.100 mm. After drying, all samples were sewn
around the edges. Three fabric samples of each experiment were
tested for felting shrinkage by washing in an Electrolux Wascator
washing machine with ECE standard detergent at 40.degree. C.,
according to the ISO 5A programme.
[0105] There was a significant improvement in the felting shrinkage
on all transglutaminase treated fabrics compared to the buffer only
treated control (no TGase added). In particular, the treatment with
5.0 .mu.g of transglutaminase per ml of liquor delivered an
improvement of a gain of strength about 29% compared to the
respective control (see Table 6). Treatments 8 (tTG 5 .mu.g/ml
together with 2.0 mM of putrescine followed by the polymer MRSM)
and 15 (mTG 100 .mu.g/ml together with polymer TM) resulted in the
greatest improvements in dimensional stability Treatments 8 and 15
shrunk by 12.2% and 11.6%, respectively, and the control treated in
buffer alone (no TG, additive or polymer added) shrunk by as much
as 49.5%. There is a significant difference between the effects of
incorporated putrescine alone (31%) or polymer MRSM alone (25%) and
the added effect of applying the polymer after incorporation of
putrescine (12.2%). Similarly, there is a significant difference
between the effects of mTG alone (36%) or polymer TM alone (26.5%)
and the added effect of applying the polymer together with mTG
(12.2%).
EXAMPLE 7
Effect of Transglutaminase Mediated Incorporation of Active
Compounds into Wool Fibres on the Handle of Wool Fabrics
[0106] Samples of 100% wool single jersey fabric supplied by Cooper
& Roe, UK (5 g), were used in this example. A pancreatic digest
of milk casein (Tryptone, 1.0 mg/ml) and putrescine (2.0 m) were
incorporated into wool fibres using 5.0 .mu.g/ml of tTGase as in
Example 6. A commercial softener (an amino silicone, supplied by
Precision Processes Textiles, UK) was added to untreated fabric
samples and to samples treated with transglutaminase and an active
agent (tryptone and putrescine--see FIG. 7) according to the
manufacturers recommendations. Control samples treated only with
buffer (without adding TGase) under exactly the same conditions of
the tGase treatments (Example 1) and controls treated under the
conditions of application of the softener were included. A control
treated only with the commercial softener was also included.
[0107] The mTGase treatment was carried out using 100 .mu.g of
microbial transglutaminase per ml of liquor, for 1 hour, 55.degree.
C. and pH 5.5, together with the commercial softener. All other
treatment parameters were the same as in Example 3.
[0108] A panel of 15 judges assessed the subjective softness of the
wool samples (FIG. 7). All panellists assessed the samples
according to the same regime. All judges used the same surface of
the fabric (they were asked to assess softness by gently rubbing
the face of the fabric between the fingers and thumb, trying to use
the same pressure on all samples). The softer the fabric would
feel, the higher the score given. Panellists were asked to score
the fabrics from 1 (not soft) to 10 (extremely soft), comparing to
the control fabric (treated in buffer alone without adding TGase
additive or polymer), which was assigned a score of 5. The results
shown in FIG. 7 are the mean scores and respective standard
deviations.
[0109] The score obtained for the sample treated only with the
softener was 7.26, very similar to the score of the sample treated
with tTGase and tryptone followed by the softener (7.23), and was
significantly softer than the control sample treated with buffer
alone without adding TGase additive or polymer (5.0). The sample
treated with tTGase and putrescine followed by the softener was
given a score of 8.39, which was formed to be significantly
different from the sample treated only with softener (with 95%
confidence level). The sample treated with mTGase together with the
softener was found to be the softest, with a score of 9.2 (the
difference to the significant sample treated only with softener was
significant for a 95% confidence level).
EXAMPLE 8
The Use of Transglutaminase to Extend the Life of a Desired Scent
on Wool Fibres
[0110] Samples of 100% wool knitted fabric (supplied by Cooper
& Roe, UK) were treated with 5.0 and 20.0 .mu.g/ml of tTGase as
described in Example 1. Controls were treated in a similar manner
but without adding tTGase. A commercially available scent (4 ml/l)
was also added to each treatment.
[0111] A panel of 12 judges assessed the samples according to the
level of residual smell 2 and 5 days after the treatment (FIG. 8).
After 2 days, and particularly after 5 days the samples treated
with 20.0 .mu.g/ml of tTGase and the scent were attributed the most
intense level of residual smell (after 5 days 11 of the 12 judges
classified the treatment with 20.0 .mu.g/ml of tTGase as being the
one with most intense residual smell.). This indicates that TGase
extends the life of a desired scent applied to wool fibres.
EXAMPLE 9
Transglutaminase Crosslinking of Fluorescein Cadaverine to Wool
Fibres
[0112] Wool fibres with approximately 21 .mu.m diameter were washed
it a solution with 0.5 g/L sodium carbonate and 1.0 g/L non-ionic
detergent. The fibres were subsequently treated in a 0.05 M TRIS
buffer solution (the pH was adjusted to 8.0 with hydrochloric acid)
containing 20 mg of proteinase VIII per g of fibre in a shaker
rotating at 100 rpm for 60 minutes at 37.degree. C. The proteolytic
reaction was stopped by washing the samples with a non-ionic
detergent at pH 5 and then in hot water at 80.degree. C. for 15
minutes. Finally, the samples were rinsed before submitted to
further treatment. As a control to the protease treatment, fibre
samples were also treated with buffer alone.
[0113] The guinea pig liver TGase was then applied to the Savinase
treated and respective buffer control samples at 1.0 .mu.g of TGase
per ml of liquor in a TRIS buffered solution pH 8.5. The liquor
also contained 0.5 mM fluorescein cadaverine, 5 mM DTT and 5 mM of
calcium ion. The liquor to yarn ratio applied was 1:250. The fibre
samples were incubated in a shaker rotating at 100 rpm for 18 hours
at approximately 37.degree. C. As a control to the
transglutaminase/calcium treatments, fibre samples were treated
under exactly the same manner in a solution containing buffer, 5 mM
DTT and 5 mM EDTA, as a negative control.
[0114] After treatment the fibre samples were first washed in PBS
pH 7.4 and 1.0% of Tween 80. Further washing with methanol was
carried out to remove non-bound fluorescein cadaverine from the
surface of the wool fibres. The samples were then air-dried.
[0115] The treated fibre samples were mounted in 70% glycerol and
examined under a confocal microscope. FIGS. 9a and 9b illustrate
the pictures obtained from fibre samples submitted to a protease
treatment followed by a transglutaminase treatment. It is clear
from. FIGS. 9a and 9b that there is a significant difference in the
amount of fluorescein cadaverine incorporated by the
transglutaminase between positive and negative controls.
[0116] This indicates it is effective to cross-link primary amines
with beneficial active groups to wool using tissue
transglutaminases.
* * * * *