Method and enzyme mixture for improved depilling of cotton goods

A method, and a specific enzyme mixture, for depilling cotton-containing goods, either as unfinished fabric or as finished garments, to create a smooth surface while minimizing the loss of fabric strength. The method consists essentially of removing less than approximately 3.0% of the initial fabric weight with a Trichoderma cellulase enzyme mixture comprising less than naturally-occuring amounts of CBHI, CBHII, EGI and EGIII protein components, and further consisting essentially of at least 80% endoglucanase II (EGII) as the protein component.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to a depilling treatment of unfinished cotton fabric 
pieces, or finished garments, with cellulase enzymes, wherein the 
treatment removes less than approximately 3.0% of the initial fabric 
weight. More specifically, this invention relates to a method for reducing 
or preventing fabric strength loss during depilling by employing certain 
specific cellulase mixtures, and preferably Trichoderma cellulase enzyme 
mixtures consisting essentially of at least 80% EGII. It has been 
discovered that with enzyme mixtures enriched in EGII, the removal of 
pills is more efficient, and fabric destruction during enzyme treatment 
can reduced by about 88%, relative to the standard commercial cellulase 
enzymes now in use. 
2. Brief Description of the Prior Art 
Cellulase enzymes widely are used to improve the appearance and softness of 
cotton-containing fabrics and garments. As used herein, the terms "cotton 
goods" denotes finished or unfinished fabrics consisting of cotton or 
blends of cotton with other fibers. 
One widespread application of cellulase enzymes is for treating 
cotton-containing fabrics so as to "stonewash" denim, in which cellulase 
enzymes largely have replaced stones for generating the soft, faded denim 
that is desired by consumers. Further details of cellulase for denim 
stonewashing can be found in Nielsen, et al, ENZYME APPLICATIONS 
(INDUSTRIAL), Encyclopedia of Chemical Technology, (Kirk-Othmer 
Publishers, 1993), vol. 9, p. 603-604 ( referred to hereafter as "Nielsen, 
et al"). 
A second widespread application of cellulase enzymes is for treating 
cotton-containing fabrics so as to remove cotton fuzz and loose surface 
fibers on or in the fabric, which categorically involves removal of less 
than approximately 3.0% of the initial fabric weight, and typically less 
than 1.0%. This process is known variously by the terms "depilling", 
"biopolishing", "biofinishing", and "reformation". The term "depilling" 
will be used herein to refer to all such fabric treatments. In depilling, 
the cellulase treatment smooths the surface of the fabric, which in turn 
imparts improved softness and appearance, thereby increasing the quality 
and value of the fabric. Cellulase treatment for depilling also helps to 
prevent the subsequent formation of fiber pills that make the garments 
appear worn, and improves the uniformity of the fabric by removing dead or 
immature cotton. Further details of cellulase for depilling further can be 
found in Nielsen, et al, at pages 595-604. Depilling of cotton-containing 
goods is the field of the present invention. 
Shear stress is applied to cotton garments during garment manufacturing and 
in repeated wearing, washing, and tumble drying, and thereby damages the 
surface. A close look reveals the presence of fibrils ranging in size from 
a few microns to a few millimeters. The damaged surface scatters light, 
giving a dull, grayish appearance with decreased color brightness and 
contrast between different colors. Dust particles also tend to adhere to 
the damaged areas, adding to the gray appearance. The damaged fibers also 
make the surface more rigid, thereby decreasing hand, or softness. 
Cellulase enzymes hydrolyze exposed beta-1,4 bonds in cellulose. This leads 
to removal of the fibrils, which are the most exposed part of the fabric. 
The removal of fibrils is believed to directly improve the softness of the 
garments and also to lead to better color and cleanliness, both by 
removing soil attached to the fibrils and by improving the penetration of 
other cleaning compounds being used. The removal of fibrils initially also 
helps to prevent a subsequent formation of fibrils. 
Cellulase enzymes have several advantages over conventional fabric 
softeners used to improve the smoothness and sheen on cotton fabrics. 
Conventional softeners, which are primarily clay or cationic surfactants, 
coat the fabric and impart a greasy feel, which is undesirable. Softeners 
also decrease the water absorbency, which is a disadvantage for towels and 
the like. The enzymes are also preferred from an environmental point of 
view. 
In a typical depilling treatment step during garment manufacturing, fabric 
(usually dyed), water, buffer, detergents, and enzyme are added to a 
rotating horizontal or vertical drum jet dyer, washing machine, or other 
device that provides agitation and shear to the fabric. The treatment is 
typically for 15 to 120 minutes at 35.degree. C. to 60.degree. C., at a pH 
of 4 to 6.5 . The ratio of liquor to fabric is usually between 2.5:1 and 
6:1, by weight . The amount of cellulase enzyme added typically 
corresponds to a cellulase activity of about 1,000 to 200,000 CMC units 
per kilogram of fabric, based on the cellulase assay method of Ghose 
(1987). After treatment, the enzyme is often destroyed by heating the 
solution to 70.degree. C. for 10 minutes. The fabric is removed from the 
machine, dried, and prepared in rolls, sometimes after additional dying. A 
summary of publications that further describe details of conventional 
cellulase treatments for depilling of cotton fabrics during manufacturing 
is found in U.S. Pat. No. 5,232,851, at Column 1. 
For a depilling treatment during a laundering step, the cellulase is 
included in a detergent mixture with the many other ingredients. The other 
ingredients might include other enzymes, such as proteases, lipases, and 
cellulases, as well as surfactants, buffers, builders, bleach, 
anti-redeposition agents, optical brighteners, anti-oxidants, and 
solubilizers. 
One conventional detergent mixture containing cellulase enzymes is further 
described by Clarkson, et al, in U.S. Pat. No. 5,290,474, (hereafter 
referred to as "Clarkson '474"). The treatment is typically for 15 to 60 
minutes at 20.degree. C. to 70.degree. C., and at a pH of 7 to 9.5. The 
ratio of liquor to fabric, by weight, is usually between 2.5:1 and 10:1. 
The amount of cellulase enzyme added typically corresponds to a cellulase 
activity of about 200 to 40,000 CMC units per kilogram of fabric, based on 
the cellulase assay method of Ghose (1987). 
Cellulase enzymes are used for the depilling of cotton fabrics and of 
blends of cotton and man-made fibers, including lyocell, rayon, polyester, 
acrylic, nylon, and cellulose acetate. Further details are illustrated in 
Clarkson '474, at column 7. Cellulase treatment is carried out on fabrics 
or sewn garments comprising material made of cotton or cotton blends, with 
or without a resinous finish. Cellulase depilling may be carried out on 
fabrics of at least 40% cotton, by weight. However, results are more 
pronounced and economical if the cotton content is more than 60% by 
weight, and the best results are obtained if the cotton content is more 
than 75% by weight. 
The cellulase enzymes from one particular genus of wood-rotting fungus, 
Trichoderma, often are used in depilling applications. Trichoderma 
cellulases are preferred in textile processing and laundering because of a 
highly potent action against cotton and other forms of cellulose. 
Trichoderma cellulase products are commercially available from Iogen 
Corporation of Ottawa, Ontario, Canada; Genencor International; Novo 
Nordisk; Enzyme Development Company, and others. Commercial cellulases 
such as Iogen Cellulase are referred to as "natural" or "complete" 
cellulases because they contain most, if not all, of the six most 
prevalent naturally occuring cellulase components: cellobiohydrolase I 
(CBHI); cellobiohydrolase II (CBHII); endoglucanase I (EGI); endoglucanase 
II (EGII); endoglucanase III (EGIII) and endoglucanase V (EGV). 
The widespread use of complete cellulases for depilling attests to the 
usefulness of these enzymes. However, one disadvantage of such complete 
cellulases in depilling treatments is that they can cause a significant 
loss of strength of the fabric. See Clarkson, et al, U.S. Pat. No. 
5,246,853, (hereafter "Clarkson '853"). Loss of strength arises from the 
action of the cellulase against cellulose on the main body of the fabric, 
rather than just the desired action against fuzz or pills. Excessive 
strength loss can cause damage to the fabric, such as pin holes or overly 
worn spots, and decrease the useful life of the fabric. Decreasing the 
strength loss would overcome these problems. In addition, decreasing the 
strength loss would allow one to achieve the desired appearance and 
softness in fabrics with higher strength than presently achievable. This 
would result in valuable new products for the industry and the consumer. 
To decrease the loss of strength caused by Trichoderma cellulase, efforts 
have focused on the properties of the individual enzymes that comprise 
Trichoderma cellulase. 
Trichoderma naturally makes a mixture of about two dozen different types of 
cellulase enzymes, which are individually known as components. Several of 
the most prevalent of these components have been identified and named, 
including cellobiohydrolase I (CBHI), cellobiohydrolase II (CBHII), 
endoglucanase I (EGI), endoglucanase II (EGII), endoglucanase III (EGIII), 
and endoglucanase V (EGV). 
Each of the Trichoderma cellulase enzymes have been classified into an 
appropriate family of the more than 40 recognized families of hydrolase 
enzymes. Classification is based on the sequence of amino acids that 
comprise the enzymes and the three dimensional structure, as described by 
Claesssens and Henrissat, "SPECIFICITY MAPPING OF CELLULOLYTIC ENZYMES:

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
It has been discoverred that cotton goods treated with a Trichoderma 
cellulase mixture consisting essentially of at least 80% EGII component 
exhibit a smooth appearance and soft feel, with less loss of fabric 
strength than goods treated with other Trichoderma cellulase preparations. 
The Trichoderma cellulase mixtures of the invention offer superior 
depilling with a lower fabric strength loss than other Trichoderma 
cellulase mixtures. 
Preferred compositions of cellulase mixtures according to the present 
invention also are shown in the FIGURE. To better appreciate the scope of 
the present invention, and for purposes of enabling practice of the 
present invention, certain terms now will be explained, or more 
particularly defined. 
As used herein the term "cotton goods" refers to fabrics, either as piece 
goods or sewn into garments, comprising cotton or cotton blends, and 
either before or after dying and with or without a resinous finish. Hence, 
the term "cotton goods" is broader than "cotton fabric", as customarily 
used in in the garment industry to refers to the material or piece goods 
before sewing. The term "goods" should be viewed as a shorthand 
abbreviation for "fabric or garments, unfinished or finished" and not to 
connote any preference as to a preferred practice of the invention. 
In a preferred embodiment, the cotton goods consist of cotton or blends of 
cotton with non-cotton fibers, such as nylon, acrylic, polyester, rayon, 
or lyocell, such that the cotton content of the fabric is more than 40% by 
weight. More preferably, the cotton content is more than 60% by weight. 
Most preferably, the cotton content is more than 75% by weight. 
The term "treatment" refers to depilling treatments carried out during the 
manufacturing process or in subsequent laundering. In either case, 
treatment is carried out by adding cotton goods to a rotating horizontal 
or vertical drum jet dyer, washing machine, or other device that contains 
the fabric, water, buffer, detergents, surfactants, and cellulase enzyme 
while providing agitation and shear to the fabric. The treatment is often 
followed by a rinsing with water to remove the spent chemicals and debris 
from the fabric, including the loose fibrils. After treatment, the fabric 
is removed from the machine and dried. 
The treatment conditions used in the following examples are believed 
consistent with those generally used for depilling. When depilling takes 
place in a typical manufacturing process, treatment time is about 15 to 
about 120 minutes; treatment temperature is about 35.degree. C. to about 
60.degree. C., the ratio of liquor to fabric is between about 2.5:1 and 
about 10:1 by weight, and the pH is about 4.0 to about 6.0. When depilling 
takes place in a typical laundering, the treatment time is about 10 to 60 
minutes, the treatment temperature is about 20.degree. C. to about 
70.degree. C., the ratio of liquor to fabric is between about 2.5:1 and 
about 10:1 by weight, and the pH is about 7.0 to about 9.5. 
The amount of cellulase mixture used to depill depends on the concentration 
of active protein in the cellulase mixture, the amount of cotton goods 
being treated, and the desired amount of depilling effect, the time of 
treatment and other parameters well-known to those skilled in the art. 
When used for depilling in a typical manufacturing process, the preferred 
amount of Trichoderma cellulase mixture is generally between about 2,000 
and about 100,000 CMC units of enzyme per kg of fabric and more preferably 
between about 10,000 and about 40,000 CMC units per kg of fabric. When 
used for depilling in a typical laundering, the preferred amount of 
Trichoderma cellulase mixture is generally between about 200 and about 
40,000 CMC units of enzyme per kg of fabric and more preferably between 
about 1,000 and about 10,000 CMC units per kg of fabric. 
One option for controlling the action of the enzyme, which is recommended 
but not required, is to destroy the enzyme after treatment by heating the 
solution to about 70.degree. C. for 10 minutes, by adding chemicals that 
destroy enzyme activity, or by immediately drying the fabric. 
The terms "CBHI", "CBHII", "EGI", "EGII", "EGIII", and "EGV" refer to the 
most prevalent protein components known to be made by Trichoderma 
naturally, and are classified as described hereinbefore, within TABLE 1. 
It is contemplated that the modified Trichoderma cellulase mixtures of the 
present invention also may be generated as cellulase mixtures which are 
obtained from a Trichoderma sp. that has been genetically modified so as 
to overproduce, underproduce or not produce one or more of the CBH or EG 
components, using techniques generally known, as suggested by Bjork '463 
and Clarkson '853. 
Hence, these endoglucanases and cellobiohydrolases may include not only 
enzymes that are a part of the natural Trichoderma cellulase enzyme 
mixture, but also such modified cellulase mixtures as truncated cellulase 
proteins comprising either the binding domain or the core domain of the 
CBHs or EGs, or a portion or derivative thereof. See, generally, Fowler 
'782. 
Other contemplated techniques for creating modified cellulase mixtures may 
include alterations in the degree of glycosylation, or substitution(s) of 
amino acid(s) in the primary structure of the cellulases or truncated 
cellulases. It is also contemplated that any natural or modified 
components of Trichoderma cellulases, such as those outlined above, shall 
be considered as Trichoderma cellulase components, even if they are 
produced in a genetically modified host microorganism, other than 
Trichoderma. 
The term "total cellulase protein" refers to the sum total of CBHI, CBHII, 
EGI, EGII, EGIII, EGV, and other active Trichoderma cellulase protein 
components. This term is meant to exclude non-cellulase enzymes, such as 
amylase, protease, hemicellulase, and lipase. This term is also meant to 
exclude cellulase protein that may still be present, but was inactivated 
by heat, chemical, or other means. 
The term "natural cellulase preparation" refers to Trichoderma cellulase 
compositions that are typically produced in a submerged culture by the 
fungus Trichoderma. Methods for their production and recovery are well 
documented in the literature and widely known to those skilled in the art. 
Commercial sources for these enzymes include Iogen Corporation, Genencor 
International, Novo Nordisk, Sigma Chemicals, and Enzyme Development 
Corporation. 
In practicing the invention, at least 80% of the total cellulase protein in 
the cellulase mixtures will be the EGII component. In a preferred 
embodiment, at least 90% of the total cellulase protein is the EGII 
component. In a more preferred embodiment, at least 95% of the total 
cellulase protein is the EGII component. 
Several methods described in the literature are useful for producing the 
cellulase enzyme mixtures of the present invention. For example, 
Trichoderma strains in theory may be genetically modified to delete the 
production of the CBHI, CBHII, EGI, EGIII, and EGV components. See 
Clarkson '474 and Clarkson '428. Alternatively, the components may be 
removed or purified from a natural cellulase preparation by using ion 
exchange chromatography to produce the mixtures desired. This last 
technique in particular has been illustrated in EXAMPLES 1 and 2. 
To determine the relative amounts of each of the cellulase components in a 
cellulase mixture, the method that would most commonly be used would be to 
run a standard isoelectric focusing (IEF) gel and comparing the protein 
profile with that for purified standards of each component. A description 
of this method is found in EXAMPLE 2. This method is sufficient for the 
routine analysis of cellulase preparations, but does not unequivocally 
identify the proteins. The preferred, 95% EGII mixture taught herein 
reveals a single cellulase protein band, with an isoelectric point that is 
between approximately 5.0 and 5.6, depending upon the precision of the 
measuring equipment, with the isoelectric point typically 5.3. 
The definitive procedure is to determine the amino acid sequence of each of 
the proteins to verify that they match those previously published for the 
whole or truncated Trichoderma cellulase components, as listed in the 
references to TABLE 1. The determination of amino acid sequences is 
described by several references familiar to those skilled in the art, 
including P. Matsudaira, SEQUENCE FROM PICOMOLE QUANTITIES OF PROTEINS 
ELECTROBLOTTED ONTO POLYVINYLIDENE DIFLUORIDE MEMBRANES, Journal of 
Biological Chemistry, vol. 262, p.10035-10038 (1987), and K. L. Stone and 
K. R. Williams, HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC PEPTIDE MAPPING 
AND AMINO ACID ANALYSIS IN THE SUBNANOMOLE RANGE, Journal of 
Chromatography, vol. 359, p.203-212 (1986). 
The cellulase mixtures of this invention may be combined with various 
adjuvants as known to those skilled in the art. For example, a surfactant 
(anionic or nonionic) compatible with these cellulase components may be 
useful. Other materials possibly useful with these cellulase mixtures 
include fillers, solvents, buffers, enzyme stabilizers, pH control agents, 
enzyme activators, builders, other anti-redeposition agents and the like. 
The enzyme composition may be formulated as a solid product wherein the 
solid may be granular, spray dried or agglomerated. Alternatively, the 
enzyme composition may be formulated as a liquid, gel, or a paste product. 
A liquid preparation is preferred herein. 
EXAMPLES OF THE PRESENT INVENTION 
The above detailed description discloses the compositions of the invention 
and methods of making and using the compositions in the "depilling" of 
fabric clothing items. Other choices of wash conditions such as 
concentration, measurement, pH, temperature, and the like, will be evident 
to those skilled in the art based on the teachings herein. The following 
specific examples further illustrate benefits and advantages from the 
present invention. 
EXAMPLE 1 
Enrichment of EGII from a Trichoderma cellulase composition 
Approximately 10 liters of a commercial Trichoderma cellulase preparation 
known as Iogen Cellulase (commercially available from Iogen Corporation, 
Ottawa, Ontario, Canada), was adjusted to pH 7 with sodium hydroxide, 
dialysed across an Amicon 10,000 molecular weight cutoff membrane to a 
conductivity of 580 microsiemens and diluted to a protein concentration of 
8 mg/ml with a pH of 7. The cellulase was fed onto a 3 liter column of 
Q-Sepharose anion exchange resin (commercially available from Pharmacia 
Biotech, of Uppsala, Sweden). A total of 9 liters of feed was added to the 
column. At these conditions, those components with low isoelectric points, 
(including CBHI, EGI, and EGV) bind to the column more tightly than the 
other components. CBHI and EGI are, therefore, the most prominent bound 
components. At this point, the column was washed with 9 liters of 2 mM 
sodium phosphate buffer solution, pH 7, conductivity 370 microsiemens. The 
eluent was collected, adjusted to pH 4 with hydrochloric acid, and 
dialyzed to 200 microsiemens conductivity across an Amicon 10,000 
molecular weight cutoff membrane. 
The resulting solution had most of the CBHI and EGI removed from the 
initial cellulase mixture, as verified by the diminished intensity of 
these bands on IEF gels. The EGV was probably also removed, although the 
concentration of EGV is so low in the initial cellulase mixture as to make 
a quantitative assessment of its concentration difficult. 
At this point, the dialyzed eluent (10 g/L protein) was fed to a 60 ml 
column of S-Sepharose cation exchange resin, (commercially available from 
Pharmacia Biotech). A total of 60 ml of feed was added to the column, 
followed by washing with 180 ml of 2 mM sodium acetate buffer, pH 4. This 
resin binds most tightly to the components with lower isoelectric points, 
which would include EGII. The wash eluent contained primarily CBHII as 
well as EGIII and was discarded. 
A solution of 10 mM sodium acetate buffer, pH 4, was then fed to the column 
to desorb the EGII. The first 300 ml of eluent corresponding to this feed 
was collected in 10 ml fractions and then analyzed to determine the exact 
components present, by the steps of EXAMPLE 2. 
EXAMPLE 2 
Analysis And Identification Of Cellulase Components By IEF 
A standard polyacrylamide gel isoelectric focusing (IEF) technique was used 
to analyze the composition of cellulase components. This method is 
described in ISOELECTRIC FOCUSING PRINCIPLES AND METHODS, (Pharmacia Fine 
Chemicals, 1982). The gels were 5% polyacrylamide and were run at pH 3 to 
10. The proteins were stained with Coomassie blue and destained with a 
mixture of methanol and acetic acid. 
The samples analyzed included aliquots of the 10 ml fractions collected in 
the elution of EXAMPLE 1. These aliquots were diluted to 2 to 5 mg/ml of 
protein. A 50 microliter sample of Iogen Cellulase was also analyzed, as 
were enriched samples of CBHI, CBHII, EGI, and EGII, and isoelectric point 
markers at several isoelectric points. 
Iogen Cellulase has protein bands present corresponding to all of the 
single components, plus several other proteins. The fractions from the 
eluent from EXAMPLE 1 were deficient in CBHI, CBHII, EGI, EGIII, and EGV, 
as indicated by the absence of bands corresponding to these proteins at 
isoelectric points of 4.3, 6.0, 4.6, 7.7, and 3.7, respectively. In the 
fractions from the eluent from EXAMPLE 1, only one protein band was 
visible, at an isoelectric point of 5.3. This band corresponds to EGII, as 
indicated by the isoelectric point and subsequent observation of a high 
activity against carboxymethylcellulose and a low activity against filter 
paper. 
EGII is the only major band visible in these fractions. It accounts for 95% 
of the total cellulase protein present with the balance consisting of 
CBHI, CBHII, EGI, and EGIII. Quantification of protein concentration is 
carried out by scanning laser densitometry, such as by using a Sharp JX 
330 scanner with ImageMaster software, (commercially available from 
Pharmacia Biotech). 
The 10 ml fractions consisting primarily of EGII were combined and 
concentrated by ultrafiltration to 6.5 g/L, then stored frozen. This 
enzyme was denoted as "enriched EGII" and used for further experiments. 
EXAMPLE 3 
Improved Fines Removal By Enriched EGII 
Four Trichoderma cellulase enzyme preparations were evalauted for 
performance in depilling applications, as follows: 
1. The enriched EGII cellulase of EXAMPLE 2. 
2. A cellulase preparation of 50% EGI, 37% EGII, and 13% EGIII. This 
preparation is free of CBHI. This preparation matches the best mixture of 
Clarkson '853, as indicated in the FIGURE. The proportions of 
endoglucanases in this mixture were determined, as follows: 
In Clarkson '853, at Example 13, the proportions of cellulase components in 
the natural mixture are listed as being CBHI 45-55%; CBHII 13-15%; EGI 
11-13%; EGII 8-11%; EGIII 1-4%. At Example 16, the best and preferred 
enzyme is said to have all CBHI and CBHII deleted. If CBHI and CBHII are 
removed from the total mixture, and the average concentrations of the 
remaining enzymes are normalized to total 100%, the result will be as 
stated above. 
3. A cellulase preparation with 96% CBHI, 2% EGI, and 2% EGII. This 
preparation matches the best of the mixtures reported by Bjork '463. The 
proportions of enzymes in the mixture were determined, as follows: 
In Bjork '463, at Example 3, Table III, it is taught that the best mixture 
of enzymes is 500 ppm CBHI and 20 ppm of EGI plus EGII, and those being in 
equal amounts (see line 45). Such a mixture of 500 ppm CBHI, 10 ppm EGI, 
and 10 ppm EGII will have proportions, as stated above. 
4. Iogen Cellulase, a commercial cellulase product that has the natural set 
of cellulase enzymes in the proportions described in TABLE 1 and shown in 
the FIGURE. 
The evaluation phase for these four compositions consisted of two 
measurements: (1) Removal of fines from fabric, which is desirable, and 
(2) Destruction of fabric, which is undesirable. EXAMPLE 3 discusses fines 
removal and EXAMPLE 4 discusses destruction of fabric. 
The depilling evaluation was carried out as follows. The fabric consisted 
of an undyed blend of 60% Tencel.RTM. and 40% cotton. Tencel is a 
trademark of Courtaulds Ltd for its brand of lyocell fabric. The fabric 
surface was highly pilled in a manner typical of such fabric in the 
intermediate stages of manufacturing. A circular piece of fabric of 
diameter 7.8 cm, weight 1 gram, was placed on the bottom of a 250 ml 
Erlenmeyer flat-bottomed flask. A total of 145 steel balls of diameter 
4.76 millimeters (total weight 63 g) were placed on the fabric. The 
enzymes were diluted in 50 mM citrate buffer (pH 4.8) such that 7.5 mg of 
protein was added to 6 grams of buffer. The enzyme/buffer solution was 
preheated to 50.degree. C. in a water bath, then added to the fabric. The 
flasks were shaken at 225 RPM for 1 hour in a New Brunswick gyrotory 
shaker. At this point, the contents of the flask were filtered over 
pre-weighed glass microfiber filter paper. The steel balls were removed, 
and the flask and filter paper were washed three times with deionized 
water. The filter paper was then dried for 90 minutes at 100.degree. C. in 
an oven. The amount of fines collected was determined by subtracting the 
initial weight of filter paper from the final weight and then expressing 
the result as a percentage of the initial weight of fabric. 
The results are shown in TABLE 2. The EGII enriched cellulase released more 
fines from the fabric than did the cellulases of either Clarkson '853, 
Clarkson '428 or the Iogen commercial enzyme. The advantage in fines 
removal by enriched EGII over the other enzymes also was evident from a 
visual inspection of the fabric. By removing more fines from the fabric, 
the enriched EHII produces a smoother, more acceptable appearance that the 
other enzymes tested. Alternatively, a given level of fines removal can be 
achieved with less enriched EGII than the other enzymes, which can result 
in a more economical depilling treatment. 
TABLE 2 
______________________________________ 
Depilling Results By Enriched EGII And Other Enzymes. 
Enzyme Fines removed 
(@7.5 mg/gram fabric) 
(% of initial fabric weight) 
______________________________________ 
Enriched EGII 0.80 
(95% EGII) 
Clarkson '853 0.45 
(50% EGI, 37% EGII, 13% EGIII) 
Clarkson '428 0.05 
(90% CBHI, 5% EGI, 5% EGII) 
Iogen Cellulase 0.65 
(45-55% CBHI; 13-15% CBHII; 
11-13% EGI; 8-11% EGII; 1-4%. EGIII) 
______________________________________ 
EXAMPLE 4 
Decreased Fiber Destruction By Enriched EGII Cellulase 
The second part of the enzyme evaluation is the measurement of fabric 
destruction during depilling. This evaluation is carried out using that 
amount of enzyme (mg. per gram of fabric)--for each of the four test 
enzymes mixtures--that will achieve a "total" fines removal (usually about 
0.6% by weight of initial fabric weight) and then measuring the amount of 
glucose sugar produced to deduce how much fabric fiber also was destroyed. 
Ideally, a removal of 0.6% of initial fabric weight will correspond to 
removing only the undesirable fines, with little if any additional fiber 
destruction inside the fabric structure. 
This example used the same enzymes as EXAMPLE 3. The depilling treatments 
and collections of filtrates were carried out using the same techniques as 
in EXAMPLE 3, except the dosage of each enzyme was chosen so that upon 
examination all existing fines (about 0.6% of the initial fabric weight in 
the test samples) are shown to be removed. 
The amount of fabric--beyond the fines--also destroyed to glucose was 
determined as follows. The filtrates had sulfuric acid added to a 
concentration of 20 grams per liter and were heated to 121.degree. C. in a 
steam autoclave for 1 hour. The flasks were then cooled to ambient 
temperature and adjusted to pH 5 with sodium citrate buffer solution. The 
glucose concentration of the filtrates was then measured on a Dionex 
pulsed amperometric HPLC (Dionex Co., San Jose, Calif.). The glucose 
concentration was related to the initial weight of fabric to determine the 
percentage conversion to glucose. 
The procedures were carried out with several levels of cellulase to 
establish the level required to remove fines from four fabric samples, 
each measured to have about 0.6% of initial fabric weight as fines. The 
level required for Enriched EGII was only 6.0 milligrams of enzyme per 
gram of fabric. The enzyme of Clarkson '853 required 9.0 mg/gram. The 
enzyme of Clarkson '428 required 45.0 mg/gram. The Iogen cellulase 
required 7.2 mg/gram. Once a level of cellase for a complete depilling was 
identified, the amount of fabric also destroyed to glucose by that level 
of cellulase was derivable, since in each test the glucose attributable to 
fines removal alone is a constant. The results are shown in TABLE 3. 
The enriched EGII cellulase caused far less undesirable fabric destruction 
in the complete depilling tests than any of the other three enzyme test 
mixtures evaluated: the commercial cellulase, the enzyme of Clarkson '853, 
and the enzyme of Clarkson '428. The decrease in fabric destruction by the 
enriched EGII cellulase indicates that a stronger fabric results from 
enriched EGII than from treatment with any other known mixtures. 
For a given depilling treatment, the enriched EGII cellulase causes 63% 
less destruction of fabric than the cellulase mixtures taught by Clarkson 
'853. 
For a given depilling treatment, the enriched EGII cellulase causes 80% 
less destruction of fabric than the cellulase mixtures taught by Clarkson 
'428. 
For a given depilling treatment, the enriched EGII cellulase causes 88% 
less destruction of fabric than the standard cellulase mixtures used for 
commercial depilling treatments. 
TABLE 3 
______________________________________ 
Depilling Results By Depleted Cellulase And Other Enzymes. 
Cellulase Level 
Fabric destruction* 
Enzyme (mg./gram fabric) 
(% of initial fabric weight) 
______________________________________ 
Enriched EGII 
6.0 0.30 
(95% EGII) 
Clarkson '853 
9.0 0.80 
(50% EGI, 37% EGII, 
13% EGIII) 
Clarkson '428 
45.0 1.50 
(90% CBHI, 5% EGI, 
5% EGII) 
Iogen Cellulase 
7.2 2.50 
______________________________________ 
*after removing all fines (about 0.6% of initial fabric weight) 
While preferred embodiments of our invention have been shown and described, 
the invention is to be defined solely by the scope of the appended claims, 
including any equivalent for each recited claim element that would occur 
to one of ordinary skill and would not be precluded by prior art 
considerations.