Effect of Pre-existing Dyes and Fabric Type on Cellulase

Treatment of Cotton Fabrics

EUNKYUNGCHOE,SOONYOUNGPARK,HEECHEOLCHA,AND BYONGDAEJEON
Textile Research Center, Korea Institr~teoJZndz~strialTechnology. Szrwon, Korea

ABSTRACT
Fabric weight loss through cellulase treatment of six different cottons depends on
mercerization, yarn size, and fabric structure. Weight loss decreases in the order of
Ne = 60 mercerized > Ne = 60 nonmercerized = Ne = 40 mercerized > Ne = 40
nonmercerized knit fabrics > Ne = 20 = Ne = 10 nonmercerized woven fabrics,
indicating increased enzymatic hydrolysis on mercerized cotton knits with smaller
yarn size. The effect of pre-existing dyes on cellulase activity is examined by measuring
the difference in weight reduction for mercerize! and nonmercerized cotton knits
previously dyed with direct, reactive, and vat dyes. All six dyes in this study inhibit
the cellulase reaction, and the degree of inhibition depends on the dye class. The
concentration of dyes on fabric as estimated by KIS proves to be the most critical
factor governing resulting weight losses. The planar structure of vat dyes with larger
molecular sizes than indigo dye seems to play an important role in their inhibitory
effect. Compared with monoreactive, direct, and vat dyes, the largest inhibitory effect
is with bifunctional dyes, imply.ing that the crosslinking property of bifunctional dyes
is related to cellulase hydrolysis. This suggestion is further supported by the small
reduction in weight after cellulase treatment O~D~IDHEU-treated cotton fabric. A model
is proposed to explain the slower progress ofenzymatic cleavage on crosslinked cellulose
fibers.

One area of cellulase enzyme application includes Vat Blue 1 ) does not. They also found an inhibitory
garment dyeing and knit fabric processing. Because the effect of ionic dyes and ionic surfactants on cellulase
cellulase enzyme, being a biocatalyst, offers advantages activity in the treatment solution and proposed an
such as energy savings through lower treatment tem- electrostatic dye-enzyme interaction [9, 101. In addi-
peratures of 40-50°C, low environmental impacts by
avoiding the use of chemicals, and low cost by using
only the catalytic amount, better use of cellulase en-
zymes can,be an answer to the desire for both cleaner
and less polluting textile technology and high-grade
goods with added value.
By hydrolyzing a 1,4-/3 glycoside bond of the cellu-
lose molecule, cellulase splits the cellulose chain into
two segments, one revealing a reducing end group and
the other a nonreducing end group (Figure 1 ) [7].
Through these actions, cellulase enzymes effectively Reduclnu End Group on-~educlnu End Group
defuzz the surface of knit fabrics that have experienced
excess mechanical friction during the dyeing cycle [3, hut, cu(m)2or Ferrlcyanlde
5, 81. In this case, effects of pre-existing dyes on cel- A d , CupO or Ferrocyanlde
lulase activity need to bk studied to determine optimum
weight losses for dyed items. Koo and Ueda [ 4 ] have
revealed that direct and reactive dyes on the substrate
inhibit the cellulase catalytic reaction, while indigo (C.I.

' This paper was presented at the 3rd Asian Textile Conference,
}long Kong, September 19-21, 1995. FIGUREI. Enzymatic hydrolysis ofcotton cellulose.

Tt,.vrilr Res. 1.67(3), 155-162 (1997)

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tion to these pioneering works, a variety of dyes still ton knit was either mercerized or nonmercerized, while
need to be examined to clarify their effects on cellulase denim fabrics were nonmercerized. The scoured and
hydrolysis and to establish a more proper process in bleached fabrics were provided by Woosung Industry
which dyes and cellulase exist together either in solution in Korea.
or on the substrate. Denimax Acid XCL with an activity of 1500 EGU/
In this study, we have chosen six dyes to compare g was a Novo product. Fixapret TX 2386 (BASF) was
the effect of three major dye classes on cotton, to de- used as a crosslinking agent. All other chemicals were
termine the difference between monoreactive and bi- reagent grade.
functional reactive dyes, and to estimate the molecular
size effect of vat dyes on cellulase activity. The
~ h l ~ ~ ~ u - t r ecotton
a t e d fabric is also cellulase treated
Six kinds of cotton fabrics were cut into
to evaluate the intermolecular crosslinking effect on
cm simples and subjected to cellular Ne
the cellulase reaction. In addition, we have examined
= 20 denim fabric was DhIDHEu-treated using Fixapret
the influence of fabric type, mercerization, yam sizes,
TX 2386 reagent with a wet pickup of 80% and sub-
and knitted versus woven structures on the catalytic
jected to cellulase treatment.
cellulase reaction.
Ne = 40 mercerized and nonmercerized knits were
dyed with the six dyes listed in Table I according to
Experimental the procedures recommended by the dye manufactur-
ers. Dye concentrations were 0.5, I, 2,3%owf for each
Characteristics of the dyes and cotton fabrics are dye. After K / S values of dyed knit samples were mea-
summarized in Tables I and 11, respectively. Each cot- sured, they were cellulase treated.

TABLE
I. Characteristicsof dyes used.
Commercial name/
Dye class CI number Dye type/chemical structure

Direct dye Direct Chrysamine G

CI Direct Yellow 24

Reactive dye. Cibacron Blue F-R l

T (monofunctional)
Procion Red MX-G DCT (bifunctional)
Procion Red WE3B MCT-hlCT (bifunctional)
Cibacron Blue C-R FT-VS or VS-VS (bifunctional)

indigo (CI Vat Blue. 1)

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 Using Denimax Acid XCL with an activity of 1500
EGU/g (Novo products), an enzyme solution was
prepared by dissolving each portion of cellulase ac-
cording to the variables below in 1 liter of the pH 5
solution (an acetic acid/sodium acetate buffer).
A Launder-0-Meter (Atlas, LP2) was used to treat
the fabrics. Four replicates of 10.5 X 10.5 cm swatches
were treated in an individual Launder-0-Meter can
with 400 ml treating solution at 50°C. Extra sample
swatches were added to make a liquor ratio of 1:20.
Treated fabrics were placed in boiling water for 10
minutes, rinsed with distilled water, and then air dried.
The three variables were as follows: Enzyme con-
centrations including 0.5, 1, 2, 3, 4, and 8 g/l were
used with a treatment time of 1 hour to treat Ne = 40
and 60 mercerized and nonmercerized cotton knits.
Using a constant amount of cellulase ( 3 g/l), six fabric
specimens in Table I1 and the DMD~~EU-finished Ne
= 20 cotton denim were treated for different times of
30, 60, 90, 120, 150, and 240 minutes. Dyed cotton
knit samples Ne = 40 mercerized and nonmercerized
were treated with an enzyme concentration of 8 g/l
for 2 hours.
TABLE
11. Construction characteristics of cotton fabrics used.
- --
Yarn Fabric Fabric count, ends
number, weight, Xpicks (/cm), wales
Fabrics Ne dm2 X courses (/ern)
311 Twill denim 10 311.8 26 X
31 1 Twill denim 20 176.0 35 X
Single jersey knit 40 284.0 16 X
Single jersey knit 60 162.4 18 X
PROPERTY
MEASUREMENTS
The conditioned weights (65% RH, 21°C) were
measured before and after cellulase treatment to de-
termine weight loss. Tear strengths of four replicates
per each cellulase treatment of the DMDHEU-treated
denim fabric were measured in the warp direction with
an Elmendorf tearing tester. Tear strengths of undyed
and bifunctional dyed cotton knits were also measured
in the wale direction after cellulase treatment.
Results and Discussion
We investigated the cellulase treatment of cottons
using six different fabrics, including Ne = 40 and 60
single jersey knits (mercerized and nonmercerized, re-
spectively) and Ne = 10 and 20 31 1 twill denims, to
see the effects of mercerization, yam size, and fabric
structure (knitted versus woven) on weight loss.
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When weight loss values after cellulase treatment of
cotton knits were plotted versus different enzyme con-
centrations (Figure 2 ) and treatment times (Figure 3),
we observed that the first 30 minutes of treatment or
the treatment with a low enzyme concentration pro-
duced similar weight losses regardless of fabric type,
indicating that cellulase acis primarily on protruding
surface hairs at the beginning. There was a proportional
relationship between weight loss and enzyme concen-
tration, as well as between weight loss and treatment
time. Weight loss increased in an additive way as en-
zyme concentration or treatment time increased by
multiples. In both Figures 2 and 3, fabric weight loss
by cellulase treatment of cotton depended on mercer-
ization and yam size. Weight loss decreased in the order
ofNe = 60 mercerized > Ne = 60 nonmercerized = Ne
= 40 mercerized > Ne = 40 nonmercerized knit fabrics,
indicating increased enzymatic hydrolysis on mercer-
ized cotton with smaller yarn size. These results are
probably due to the easier accessibility of cellulase en-
a
zymes to larger surface area of an equal weight sample
with smaller yam size and to the smooth, circular cot-
ton fiber resulting from morphological changes after
mercerization.
20
22
13
17
- b
rn
&
0
8 0
I
8 at PH 5 and at
.
Enzyme Concentration (g/l)
: N e e mercenzed single jersey kn~t
: Ne=W nonmercerized single jersey kn~t
: N e 4 mercerized sing!e jersey kn~t
0 : N e - 4 nonmercerized single jersey kn~t
FIGURE2. Effects of mercerization and yarn size
on weight loss at different enzyme concentrations.
We compared the weight losses of cellulase-treated
Ne = 20 and Ne = 10 nonmercerized denim fabrics
 al pH 5 and 50C

0 30 60 90 120 150 180 210 240

Treatment Time (minutes)

: Ne- rnercenzed single jersey b,t Treatment Time(minutes)
: Ne=60 nonrnercerizedsingle jersey knit
Kn:l:ed Fabncs A : Ne.60 sing!e jersey knil
: Ne* rnercenzed s~ng!ejersey knit
: Ne4O single jersey
0 : Ne-4 nonmercenzed single iersey.kn~t

Woven F a b r a : Ne=lO 31 E#:ll denim

FIGURE 3. Effects of mercerization and yarn size 0 : Ne.20 ?I1 Iv;i!I denim
on weight loss at different treatment times.

4. Weight loss of knitted fabrics versus woven fabrics.

FIGURE
with nonmercerized knitted fabric (Ne = 40 and Ne
= 60) in Figure 4; the knitted fabrics showed higher
weight loss values than the woven fabrics: Ne = 60 cerized fabric was always higher than the nonmercer-
nonmercerized knits > Ne = 40 nonmercerized knits ized fabric at the same owf for all dye types used (Figure
> Ne = 20 = Ne = 10 nonmercerized woven fabrics. 5). Therefore, there exist two factors that affect enzy-
The yam size dependency on weight loss observed in matic hydrolysis in opposite ways. Higher dye uptake
the knitted fabric was not apparent in the woven fabric, of mercerized fabrics is likely to result in more efficient
probably because the loose structure of the knits facil- blocking of the access of cellulase enzymes to the 1,4-
itated enzyme attacks on their surface area, while the 3!, glycoside bonds ofthe cellulose chains by dyes present
much denser and tighter woven fabric could not effec- on cellulose fiber. On the other hand, mercerized cotton
tively differentiate the larger surface areas of yams with knits have a greater tendency to attack by cellulase, as
smaller yam diameters. discussed earlier.
To determine the optimum weight loss for a cotton Weight reduction decreased after cellulase treatment
fabric, we needed to establish an approximate guide to of cotton knits dyed with a direct dye (Figure 6), a vat
how much weight loss would take place on dyed fabrics dye (Figure 7), and reactive dyes (Figure 8). For all
compared with corresponding undyed fabrics. With this three major dyes on cotton fibers, weight reduction less-
in mind, we examined the effect of pre-existing dyes ened as the concentration of dyes on fabrics increased.
on cellulase activity by measuring differences in weight Mercerization, however, did not aid cellulase hydrolysis
reduction on cotton knits previously dyed with direct, significantly on dyed items as expected. The amount of
reactive, and vat dyes. For undyed cotton samples, the dyes on fabric as estimated by measuring K / S seemed
weight loss ofthe mercerized knit was higher than that to play a more important role in determining weight
of the nonmercerizedknit, as seen at the starting point loss resulting from cellulase reactions. Figure 6 shows
in Figures 6, 7, and 8, as well as in Figures 2 and 3. the inhibitory effect of direct dye on enzymatic hydrol-
Cellulase is more active for mercerized cotton than ysis. The decrease in weight loss after dyeing with CI
nonmercerized cotton, resulting in the higher weight Direct yellow 24 was about 2% for mercerized cotton
losses of the former [2,6]. When mercerized and non- knit and about 1% for nonmercerized cotton knit. This
mercerized cotton fabrics were dyed at four different phenomenon can be easily understood based on earlier
owf levels-0.5, 1, 2, 3%-the dye uptake of the mer- research reporting that compounds such as anionic and

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 C. I. Direct Ycllow 24

:t C. L Vat Orange 11

Dya Concentntion(#. o.wL) Dye Concmtratian(%, 0 . ~ 1 . )

9 9
8 8
7 7
6 6
5
8: 4

3 3
2 2
F I G ~ J 7.
RE Weight loss of mercerized ( 0 ) and nonrnercerized ( 0 )
cotton knits (Ne = 40) previously dyed with C1 Vat Black 25.
1 1
0 0
0.0 .5 1.0 1.5 2.0 2.5 3.0 3.5 0.0 .5 1.0 1.5 2.0 2.5 3.0 3.5
Dye Cononvation(%.0 . ~ 1 ) Dye Concentration(%, 0.w.f.) (a) Cibacron Blur F-R (b) Cibrcron Blue C-R
7
( : mercerized 0: nonrnercerized) 7~

FIGURE 5. Higher dye uptake of mercerized cotton versus

nonmercerized cotton at the same dye bath concentration.
4

0 1 2 3 4 5 6 7 8 8 1 0
KIS KIS
(c) Proclon Rml M X G (d) Procion Rml H-E3B
7

FIGURE 6. Weight loss of mercerized ( 0 ) and nonrnercerized ( 0 ) .

cotton knits ( N e = 40) previously dyed with Direct yellow 24. F~GURE 8. Weight loss of mercerized ( 0 ) and nonmercerized ( 0 )
cotton knits ( N e = 40) previously dyed with monoreactive dye ( a )
and bifunctional dyes (b, c, d ) .

cationic surfactants or anionic dyes inhibit the cellulase

reaction, probably due to their electrostatic interaction
with cellulase [4,9, 101. The anionic character ofdirect
or reactive dyes resulted from the sulphonate group in
this study (Table I).
1n contrast to earlier work reporting that indigo dye
did not cause changes in weight loss after cellulase
treatment between undyed and indigo-dyed cotton
fabrics (41, a vat dye in our experiment suppressed the
enzyme reaction, resulting in a significant decrease in
weight loss (about 4% for mercerized knit and about
2% for nonmercerized knit when dyed at 3% owf, re-
spectively) (Figure 7). Since nonionic vat dyes are not

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 likely to interact with cellulase [4,9], the planar struc- 6
ture of CI vat Black 25, with larger molecular sizes
than indigo dye (Table I), seems to cover the surface 5 -
of cellulose fibers more compactly and plays an im-
()
portant role in obstructing the enzyme approach.
Figure 8 shows the inhibitory effect of reactive dyes
-
0
on enzyme activity. Weight loss after cellulase treat- 2;' 0
ment decreased as the concentration of dyes on the -
g 3 0
cotton fabric increased. Compared with monoreactive
dyes (Figure 8a), the weight loss was smaller for both
2
mercerized and nonmercerized fabrics after dyeing with .F 2 2-
0
$
bifunctional dyes (Figure 8b, c, d). Prior to our work,
- l l a l 1
Betrabet el al. observed the high resistance of cotton 1
dyed with bifunctional dyes to enzyme action under
an electron microscope using the cellulase-dissolution 06 I 1 1 I

technique, where an ultra-thin cross section of dry o 30 60 90 120 150

embedded fiber was treated with a highly active cel-
lulase [I]. In their experiment, they used cellulase as Treatment Time (minutes)
an anil$ical tool to find evidence-for the formation
FIGURE 9. Weight loss of DX~DIIEU-treated
(0)
of crosslinks in cotton dyed with various bifunctional versus untreated ( 0 )Nc = 20 cotton denim fabrics.
dyes.
Lower weight losses of bifunctional-dyed cotton

-, loock1
fabric can be ascribed to two facts. First, the higher
.s
concentration of dye on the substrate makes enzyme a
access to the reaction site more difficult, resulting in
decreased weight loss. The concentration of bifunc-
5
t;
a 4

tional reactive dyes on the substrate is higher than .-: -

monoreactive dyes when dyed in the same owf, since b
the former have higher fixation properties than the lat-
ter (Figure 5 ) [6]. Therefore, cotton fabric dyed with
..- 60 -
0

a bifunctional dye will produce much lower weight !.2

losses than a monoreactive dye at the same dye bath
concentration. Second, the crosslinking property of bi-
functional dyes seems to be related to cellulase hydro-
lysis, because the degree ofcellulase enzyme inhibition
by bifunctional dyes tends to be bigger than mono-
reactive and any other dye types, even at the same K /
S values (Figure 8). This suggestion was further sup-
ported by the small reduction in weight after cellulase
treatment of DMDHEU-treated cotton fabric. As enzyme
treatment time increased, the weight loss of Ne = 20
cotton denim fabric increased up to 4.5% after 150
minutes oftreatment (Figure 9). However, weight loss
for the ~ ~ ~ ~ E U - t r eNe a t e=d20 cotton denim was
only about 1% after the first 30 minutes of cellulase
treatment, and leveled out even during further treat-
ment. Figure 10 plots the tearing strength retention of
the ~ h l ~ ~ ~ u - t r edenim
a t e dfabric after ce~lulasetreat-
ment. Starting with a tearing strength of 325 g, the
DMDHEU-treateddenim had a strength reduction of
10% after 90-1 50 minutes of cellulase treatment, im-
plying that the DMDHEU-treatedcellulose fiber was
damaged gradually, even though weight loss stayed the
same.
40 -
2
20 -
f
2 ,,
0
0 30 60 90 120 150
T r e a t m e n t Time ( m i n u t e s )
FIGURE10. Tearing strength retention of DXtDtcEu-treated
cotton denim fabric with Ne = 20 after cellulase treatment.
Based on the weight loss data (Figure 9) and strength
reduction (Figure 10) of the DhtDHEU-treated cotton
fabric, we propose a model to explain the slower pro-
gress of enzymatic cleavage on crosslinked cellulose
fibers in Figure I I. While enzymes attack cellulose
chains without difficulty in a noncrosslinked cellulose
molecule, oligomers and dimes resulting from enzyme
hydrolytic cleavage are still able to cling to the next
crosslinked cellulose chain. These broken pendant short
chains make the enzyme reaction much slower by
blocking subsequent enzyme attacks. Thus, weight

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 sur lace

Non-Crosslinkod Cal lulose Crosslinked Colluloso

0
& 'I 0.

-0
, y
aP%--C?coc
0 0
%
d 1 .-o
-a? ,XE\ 4. Weight Loss (%)
4
-

FIGURE12. Tearing strength retention after cellulase treatment of

Ne = 40 mercerized cotton knits undyed (0)and previously dyed
* Roduclng End Group with bifunctional dy:s: Cibacron Blue C-R ( 0 ) .Procion Red MX-
t Nonfleducing End Group
G (V),and Procion Red H-E3B (m).

FIGUREI I. A schematic diagram showing the slower progress

of enzymatic cleavage on crosslinked cellulose fibers.
losses for crosslinked fibers are much smaller than
noncrosslinked fibers. Even though the weight loss of
a crosslinked fiber is small, strength reduction may oc-
cur significantly, since crosslinked fibers are damaged.
In Figure 12, the tear strength of cotton knits previously
dyed with bifunctional dyes was lower than an undyed
sample (2.3 Kg for Ne = 40 mercerized single jersey
knit). Although this result also agrees with our finding,
further study, including strength reduction data after
cellulase treatment ofcotton samples dyed with various
dyes capable of noncrosslinking and crosslinking
properties, is needed to clarify that the suggested model
is as suited to cotton samples dyed with bifunctional
dyes as to DMDHEU-treatedcotton fibers.
Finally, we compared the decreased weight losses
caused by each dye class in Figure 13. Our results are
summarized by listing the decreasing order of weight
loss after cellulase trkatment of cotton specimens with
the following dye classes: for mercerized cotton knit
(Figure 13a), undyed > direct = monoreactive > vat
> bifunctional, and for nonmercerized cotton knit
(Figure 13b), undyed > direct > vat > monoreactive
> bifunctional.
Conclusions
Several finishing effects obtained by cellulase treat-
ment of cotton knitted fabrics are laundry proof and
permanent, and cannot be achieved with the usual
chemical applications. When knitted cotton fabrics and

(a) mercerized (b) nonmercerized

7 7

6 - 6

-
n G U R E 13. Comparison of inhibitory ef-
-
3 51
G
5

! -
fects of direct, mono, bireactive, and vat 0
dyes in enzymatic hydrolysis: A = Direct 4
Yellow 24, = monoreactive Cibacron l tpl
Blue F R 0 = Vat Black 25, l = bifunc-
tional Cibacron Blue C-R, Procion Red
53 l M-
4
3

hIX-G, and Protion Red H-E3B. 2

1 - l

1 1 1 1 1 1 1 1 1
0 0
1 3 1 2 3 4 5 6 7 8 9 1 0 0 1 2 3 4 5 6 7 8 9 1 0
KIS KIS

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garments are treated with cellulase enzymes, the degree
of fabric weight loss determines fabric properties. T o
modify the surface optimally by bio-finishing, an op-
timum weigh loss must be obtained on the garments.
Among many factors affecting the cellulase reaction,
we examined the fabric type dependency and effect of
dyes pre-existing on the substrate, including mercerized
and nonmercerized cotton fabrics.
Fabric weight loss by cellulase treatment of cotton
decreased in the order Ne = 60 mercerized > Ne = 60
nonmercerized x Ne = 40 mercerized > Ne = 40
nonmercerized knit fabrics > Ne = 20 --
Ne = 10
nonmercerized woven fabrics, indicating increased en-
zymatic hydrolysis on mercerized cotton knits with
smaller yarn size. All of the six dyes selected in this
study inhibited the cellulase reaction, resulting in de-
creased weight losses. The degree of inhibition de-
pended on dye class and dye concentration. We have
explained the inhibitory effects ofdyes in different ways
according to the dye structure-the ionic interaction
between cellulase and anionic dyes with sulphonate
groups, the crosslinking ability of bifunctional dyes, o r
the large planar structure of vat dyes.
10. Ueda, M., Ohgoh, I., Yoshimura, Y., and Wakida, T.,
Literature Cited
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93 (9). 338 (1977).
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