Enzyme Applications in Fiber Processing - American Chemical Society

Chapter 18

Pectin-Degrading Enzymes for Scouring Cotton

1

2

M . Michelle Hartzell and You-Lo Hsieh

Downloaded by UNIV LAVAL on July 10, 2014 | http://pubs.acs.org Publication Date: March 31, 1998 | doi: 10.1021/bk-1998-0687.ch018

Division of Textiles and Clothing, University of California, Davis, CA 95616

In our previous study, a pectinase was found to improve the surface wetting properties of greige cotton fabrics following a water pretreatment at 100°C. This study further evaluated seven pectin -degrading enzymes, i.e., four pectinases, two pectinesterases, and a pectin lyase, for scouring raw cotton fabrics. Three of the pectinases significantly improved the wettability of cotton fabrics following a 100°C water pretreatment to the same extent as alkaline scouring. The other pectinase, pectinesterases and pectin lyase had no beneficial effects on improving the wettability of raw cotton fabrics. Reaction conditions for the three pectinase treatments were optimized in respect to temperature, concentration, pH, and time. The pectinase treated fabrics did not exhibit additional shrinkage, color change, nor significant strength loss from the fabrics pretreated in water at 100°C.

The majority of the non-cellulosic components in cotton are located on the fiber surfaces (the cuticles) (1-3). The cuticles contain approximately 0.6% waxes, 0.9% pectins, 1.3% proteins, 2.0% non-cellulosic polysaccharides, ash and other miscellaneous compounds, all of which protect the cells from potential environmental and pathogenic damage during cell growth and development (4). The surface waxes facilitate yarn spinning and fabric weaving by acting as a lubricant (5). Conventionally, these hydrophobic non-cellulosic compounds are removed via an alkaline scouring process to facilitate uniform dyeing and finishing. Although hot aqueous alkaline scouring is highly effective for removing the non-cellulosics from raw cotton, the process conditions are limited to high pH and high temperature. We have surveyed several enzymes on their ability to improve the wetting properties of

'Current address: Department of Human Environments, Utah State University, Logan, UT 843322-2910. Corresponding author.

212

©1998 American Chemical Society

In Enzyme Applications in Fiber Processing; Eriksson, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.


213

cotton under aqueous conditions (6). Enzymes, in general, are active under a much broader range of pH and at lower temperatures. Scouring with enzymes thus offers greater flexibility for scouring cotton. For instance, enzyme scouring enables cleaning cotton blends containing fibers which may not be suited for scouring under alkaline conditions. A variety of enzymes are capable of reacting with specific components on cotton fiber surfaces. Cellulases, pectinases, proteases, and lipases have been studied and compared to sodium hydroxide scouring (7-70). In one study, both the cellulase and pectinase treatments generated more weight loss than either the protease and lipase treatments; cellulase and pectinase were assumed to remove a greater amount of noncellulosic material with the pectinase hydrolyzing the pectin and the cellulase dislodging other non-cellulosic compounds by hydrolyzing the supporting cellulose in the primary cell wall (7-8). The fabric wettability of the cellulase treated cotton improved, but remained less wettable than the bleached fabric. Another study found that fabric wettability improved when the cotton fabrics were extracted with chloroform prior to cellulase and pectinase treatments (9-10). Cellulase reduced cotton yarn strength while pectinase alone had little effect on strength. The addition of cellulase to the pectinase treatment significantly decreased strength. We demonstrated that a pectinase following a water pretreatment at 100°C reduced the water contact angle within the range of commercially scoured fabrics (7). The pectinase treated cotton fabrics experienced some strength loss due to the small percentage of cellulase present in the pectinase formulation. Cellulase was the only enzyme to improve fabric wettability when used alone, however, more significant strength losses were observed with the cellulase treatments. The pectinase, protease and lipase studied produced only limited effects on the fabric wettability. The 100°C water pretreatment is assumed to alter the wax composition and/or organization on the fiber surface to allow greater pectinase access to the pectin (5). Pectin is composed mainly of a polygalacturonic acid backbone (85% methylated) interspersed with rhamnose and side chains of arabinose and galactose (2). Pectinases hydrolyze the polygalacturonic acid backbone into the individual D galacturonic acid monomers (77). Pectinesterases hydrolyze the ester groups from the methylated acid groups in the polygalacturonic acid backbone, and pectin lyase cleaves the -C-C- and -C-O- linkages. Among the many pectin-degrading enzymes identified, nine different pectinases have been discovered thus far from many sources with varying substrate specificity, activity and protein structure (72, 13). One fungal source, Aspergillus niger, alone produces two separate pectinases. This work aimed to further evaluate the use of pectin-degrading enzymes as scouring agents for raw cotton. Seven commercially available pectin-degrading enzymes were selected. As with our earlier study (7), completely aqueous conditions were employed for all enzyme reactions. The length of the pretreatment time in water at 100°C and the conditions of the enzyme reactions were optimized for greater pectinase effectiveness at reduced temperatures and close to neutral pH. The effects of the treatments on fabric wetting properties, fabric and yarn properties, yarn strength, and enzyme activity were measured. Experimental Materials. The substrate used in this study was a plain weave, one-hundred percent cotton fabric in the grey state (Nisshinbo California Incorporated). Fabric samples were either raveled to a dimension of 10 cm by 14 cm weighing approximately 1.5 grams, or 5 cm by 14 cm weighing approximately 0.75g. Very little starch sizing was present on the fabric as indicated by an iodine staining test, thus no attempts were


214

made to desize the fabrics to avoid preliminary disruptions to the organization of the cotton fiber surface. A 0.33:1 (L/g) liquonfabric ratio was used for enzyme, buffer, and water solutions. Seven commercially available enzymes, four pectinases, two pectinesterases, and a pectin lyase, were used in this study (ICN, Costa Mesa, C A and Sigma, St. Louis, MO) (Table I). A l l chemicals were certified ACS grade except for the certified grade hexadecane (Fisher Scientific, Pittsburgh, PA), and the D-galacturonic acid (ICN), apple pectin (ICN), citrus pectin (ICN), and carbazole (Sigma). A Millipore, Mill-Q Water System was used for water purification. Water (72.6 dynes/cm ) and hexadecane (26.7 dynes/cm) were used as the wetting liquids for this study. Table I : Enzyme S purees and Stable Conditions Source Activity Ë . C J Conditions: Temp,°C p H u An g pro 778 Aspergillus niger 3.2.1.15 40 4.0-7.0 pectinase (AN 1)


Enzyme(ID)

pectinase (AN2)

Aspergillus niger

3.2.1.15

24-37

4.0-5.0

365

pectinase (AN3)

Aspergillus niger

3.2.1.15

40-70

4.5-5.5

11.8

pectinase (Rl)

Rhizopus species

3.2.1.15

25

4

350

Aspergillus japonicus pectinesterase(PEl) orange peel

4.2.2.10

25

5.5

100,000

3.1.1.11

45-50

4.0-8.5

10Ô

pectinesterase(PE2) orange peel

3.1.1.11

50-60

4.5-5.5

62 solid

pectin lyase(PL1)

Methods. The greige cotton fabrics were subjected to a water pretreatment at 100°C prior to enzyme treatment. The fabrics were immersed in 100°C water for varying lengths and replications. Three minutes of centrifugation followed the final immersion before drying at 65% relative humidity and 70°F until constant weight was obtained (7). A l l enzyme treatments were performed in a phosphate buffer solution, but varied in time, temperature, concentration and/or pH. Treatments affecting the fabric wettability were duplicated. The initial enzyme concentration used was approximately 3650 units per gram cotton fabric. After achieving constant temperature, the fabric was added to the enzyme solution for the desired time duration. The enzyme treatments were performed at constant temperatures controlled by a water-bath (Dubroff Metabolic Shaking Incubator, Precision) set to 50 revolutions per minute. Enzyme activity was ceased by immersion into a pH 8.5 phosphate buffer, then rinsed in water. The fabric was centrifuged for three minutes (International Clinical Centrifuge), then dried at 65% relative humidity and 70°C for four days or until constant weight was obtained (7). Water contact angles (WCAs) were calculated from the wetting force (F ) measured on a tensiometer apparatus described earlier (6, 14). This method decouples the wetting force (F ) from the absorbed liquid: w

w

F =py cos0 w

LV


(1)

215

(where y

LV

is the surface tension of the wetting liquid, ρ is the perimeter of the fabric

sample, and θ is the water contact angle). Assuming a zero contact angle, the perimeter of the sample was calculated from wetting force in hexadecane (F ) obtained in the second measurement: heM

p=i ^

(2)

Ylv

With ρ known, the water contact angle may be determined from the wetting force in water (F ) using equation 1. Vertical liquid retention capacity (C , μΐ/mg) and water retention (C , μΐ/mg) values were derived from the weight of the total liquid retained (B ) in hexadecane and water, respectively. When deriving C or Cm, the liquid and water retention values were converted to volume and normalized by the weight of the specimen (W ): w

v

m


sp

v

s

C = ^ / p

(3)

A minimum of three measurements were taken for both water contact angles and water retention. Yarn breaking strength (ASTM method 2256), fabric count and fabric thickness (ASTM 1910), weight loss and CIE L*a*b* spectrophotometer measurements were used for fabric characterization. Test methods and procedures were described earlier in The activity ^ g pectin hydrolyzed into galacturonic acid monomers per minute) of the pectinases at their optimum conditions were derived by using the galacturonic acid assay modified by Taylor and Buchanan-Smith (75). This assay utilizes carbazole in acidic conditions to measure the quantity of D-galacturonic acid liberated by pectinase hydrolysis. The three replicates of pectinase treated pectin was further hydrolyzed by the presence of concentrated sulfuric acid, and incubated with a 0.1% (w/v) carbazole reagent for four hours at 40°C. The carbazole reacts with the acid group of the D-galacturonic acid to form a pink color measured via visible absorbance (Figure 1). The concentration of D-galacturonic acid was determined by the absorbance at 525nm (Hitachi U-2000 Spectrophotometer). Results and Discussion Initial Survey of Enzymes. The effects of the seven pectin-degrading enzymes were surveyed using the mid-range temperatures and pHs within those specified by the manufacturers (Table Π). The fabrics were immersed in three fresh boiling (100°C) water baths, each for two-minutes (3x2m). To facilitate the detection of the enzymatic effects, relatively high enzyme concentrations were employed. The same enzyme activity level per mass of cotton, i.e., 3633 to 3687 u/g, was maintained to differentiate the effectiveness of these enzymes. The untreated cotton fabric has a W C A of 93.9° (±3.3). Three pectinases, i.e., A N 1 , AN2, and AN3, substantially reduce the water wetting contact angles of the cotton fabrics into the range of 45° to 60° (Figure 2). Their effects on cotton fabric wettability are similar to commercial scouring (6). The two pectinesterases (PE1 and PE2) are much less effective on



216

Figure 1. Reaction of Carbazole reagent with D-galacturonic acid detected in pectinase assay.

100-

a ο u

$

90 H 80·

g> 70ce « s ο υ

Τ 1

60 50· 40·

I I

g

ζ

ζ,

ι rH

Γ

* τΗ £ rH ^ (S£ Ρη ^

^

Figure 2. Water contact angles of pectin-degrading enzyme treated fabrics (3x2m 100°C water pretreatment).


217

improving the water wettability of cotton fabrics. The pectin lyase (PL1) and the Rhizopus pectinase (Rl) show no effect (Figure 2).

Enzyme ID

Table Π: Survey Conditions Survey Conditions: Activity Concentration u/g cotton Temp., C pH 40 3684 5.0 14.2 g/L 30 5.0 30.0 ml/L 3650 50 5.0 30.0 ml/L 3650 25 4.0 31.6 mg/L 3687 25 110.5mg/L 5.0 3683 47 7.0 10.0 ml/L 3633 50 236.0 mg/L 3658 5.0


e

AN1 AN2 AN3 Rl PL1 PE1 PE2

Water Pretreatment We have shown that three consecutive two-minute rinses in water at 100°C (3x2m) improve the effectiveness of pectinase and cellulase on cotton fabric (7). The 3x2m pretreatment in water at 100°C reduces the W C A to 77.6° (±16.7) and increases water retention from 0.15 μΐ/mg (±0.10) to 1.20pJ/mg (±1.08) (Figure 3). Both fabric thickness and lightness increase. The effects on fabric wettability, shrinkage, and lightness are lessened when the exposure to water is reduced to two minutes (Figure 3a; Table III). Shorter pretreatment times of 30 and 10 seconds, however, improve fabric wettability and water retention more so than the fabrics pretreated for two minutes (Figure 3 a and 3b). Both shrinkage and lightness of these fabrics are comparable to those fabrics treated either once or three times for two minutes (Table ΙΠ). Therefore, the duration of the pretreatment in 100°C water was further reduced to two seconds. Both the water contact angle and water retention values of the fabric treated for 2 seconds surpass those treated for 30 and 10 seconds (Figure 3a and 3b). The shorter pretreatment times of 2, 10, and 30 seconds seemingly allow the waxes to melt, exposing more hydrophilic components. However, subsequent pectinase treatments on the two-second (2s) pretreated fabrics do not produce water wetting properties equal to those on the 3x2m pretreated fabrics (Figure 4). It is likely that waxes melt and bead up from the short 2s exposure to water at 100°C, but do not disperse into the water. Prolonged exposure (one hour) to the enzyme-buffer solution at 40°C may cause softening and spreading of the waxes over the fiber surface preventing enzyme access to the pectin. The 2s pretreatments in 100°C water were repeated twice (2x2s) and four-times (4x2s). Although higher repetitions of the 2s pretreatment cause the fabrics to be less wettable and absorbent (Figure 3), the subsequent pectinase reactions on these cotton fabrics produce similar wetting properties as the scoured fabrics (Figure 4) (6). Therefore, 2x2s in 100°C water is the shortest pretreatment conditions for pectinase A N 1 to take effect. The cotton fabrics lose 4.3% to 5.8% weight following all water pretreatments at 100°C, irrespective of the lengths of time or the number of repetitions. The raw cotton fabric has a thickness of 320 μπι (Table III). Exposures to water at 100°C


218

et

Β 1.


0J

χ

ε

Figure 3. Water contact angles and retention of cotton fabric after pretreatments in water at 100°C. 100 90ο

§> 80 ftp g> 70-

I

6o^

Β Ο

υ 5040-

π Ο) fi ο fi

fi Ε ri Χ

ri χ χ, ri Figure 4. Water contact angles of Aspergillus niger pectinase (AN 1, 40°C, pH 5.0,14.2 g/L) treated fabric after varying pretreatment times.


219

substantially increase the fabric thickness to between 400 and 495 μπι. The increases in fabric thickness are similar among fabrics treated for 10 seconds to 2 minutes, but the highest increase in fabric shrinkage occurs with the 3x2m pretreated fabrics. The short 2-second exposure substantially increases fabric thickness, exceeding those treated for 10s to 2m. A high standard deviation accompanies this thickness value suggesting non-uniform yarn relaxation during the short boiling period. Fabric shrinkage from the 2x2s and 4x2s pretreatments is less than the 2-second pretreatment. Table III: Characteristics* of Cotton Fabrics Treated in Water at 100 C Time Weight Loss Thickness Lightness L* (μπι) (*) 85.1 (0.1) none 0.0 320 (9) 86.5 (0.8) 3 X 2 min. -5.5 495 (28) 85.5 (0.2) 2 min -5.8 405 (13) 85.4 (0.1) 30 sec -5.6 411(7) 10 sec 84.9 (0.1) -4.9 400(12) 84.6 (0.2) 2 sec -4.5 441 (21) 85.1 (0.3) 2X2sec -5.3 425 (4) 85.5 (0.1) 4 X 2 sec -4.3 426 (4)


e

Λ

number in () denotes the standard deviation

The raw cotton fabric has a lightness L * value of 85.1. The 2m, 3x2m, and 4x2s pretreatments at 100°C produce slightly lighter fabric colors (Table ΙΠ). The slight improvement in fabric lightness indicates some removal of the non-cellulosic compounds during each successive immersion. Optimization of Enzyme Reactions Generally, the advantages of enzyme reaction conditions over alkaline scouring include the lower reaction temperatures and a wider range of pH. For these pectindegrading enzymes, optimizing their temperature and pH as well as concentration and time may offer additional advantages. The 2x2s pretreatment in water at 100°C was used for all the following reactions because it was the shortest pretreatment for the effective reaction of pectinase AN1. This pretreatment causes the cotton fabric to lose 5.3% weight and increase thickness (425 μπι) while retaining the fabric lightness (L* = 85.1). A l l fabric characteristics except for weight loss of the enzyme treated fabrics are compared with those pretreated 2x2s in water at 100°C. Temperature. Pectinases are generally active and stable between 40°C and 70°C (77). Pectinases A N 2 and AN3 improve the wettability of cotton substantially at 30°C and 40°C (Figure 5a). Pectinase AN2 has no effect on cotton fabric wettability at 25°C, 55°C or 70°C. Pectinase AN3 has very little effect on fabric wettability at 55°C, and fabric wettability remains completely unchanged when incubated for one hour at 70°C. The effects of pectinase AN1 are similar to AN2, except that its effects on wettability are less at and below 35°C (Figure 5a). The improved water wettability



220

50 60 Temperature, °C -5.0 α ϋΧ) G ce

-7.5-10.0-

'S -12.5-15.0 Temperature, °C Figure 5. Temperature effects of Aspergillus niger pectinases on fabrics (2x2s 100°C water pretreatment): • AN1 (14.2 g/L,pH 5.0) • AN2 (30 ml/L, pH 5.0) • AN3 (30 ml/L, pH 5.0)


221


with A N 1 , AN2 and AN3 is coupled with increased water retention of the cotton fabrics (Figure 5b). Compared to the 2x2s 100°C water pretreated fabric, no changes in fabric thickness nor lightness are detected for these enzyme treated fabrics at varying temperatures. Weight loss remains greater for fabrics treated at 30°C and 40°C than treatments at higher temperatures (Figure 5c). Nitrogen contents of the fabrics treated with pectinases AN1, AN2, and AN3 are much lower than that of the raw cotton (Table IV). The very low amounts of nitrogen indicate very low amounts of proteinacious materials on the fibers. These materials are likely residual cotton cell wall proteins. At such low levels, absorption of enzyme proteins from the pectinase reactions appears unlikely. Table IV: Nitrogen Contents of Pectinase Treated Fabrics Temperature Nitrogen Content Enzyme Concentration CC) (%) na none 0.41 na AN1 40 0.10 14.2 g/L AN2 30 0.14 30.0 ml/L AN3 40 0.18 30.0 ml/L Λ

A

possibly none

Among the pectinases studied, three are from Aspergillus niger which is known to produce two forms of pectinases (13). Because the effects of pectinases AN2 and AN3 on cotton are similar as a function of temperature, it is likely that they are the same form of Aspergillus niger pectinase from two separate commerical sources. Therefore, either AN2 or AN3, but not both, were evaluated to represent this form of Aspergillus niger pectinase. The different effects of pectinase AN1 on cotton fabrics suggest that AN1 is the other Aspergillus niger pectinase. pH. Information provided by the manufacturers shows that the stable and active pH ranges for pectinases A N 1 , A N 2 , and A N 3 are 4.0-7.0, 4.0-5.0, and 4.5-5.5, respectively. In addition to the pH 5.0 used in the initial survey, the effects of pectinases AN1 and A N 2 on cotton fabric wettability and retention were also examined at higher pH of 6.0 and 7.0 (Figure 6a and 6b). No changes in fabric wetting are observed for either pectinases AN1 and A N 2 at these p H values. Pectinases AN1 and AN2 were also used without the buffer. Pectinase AN1 in water is ineffective whereas pectinase AN2 in water reduces the cotton fabric wettability to the same level as in the pH 5.0 buffer. The pH of pectinase AN1 in water remains to be 7.0 whereas AN2 in water reduces the pH to 5.0, indicating inclusion of buffer in the A N 2 formulation. Both AN1 and AN2 require pH at 5.0 to be effective in improving cotton fabric wettability. Similar behavior is expected from AN3 as the two enzymes are assumed to be the same pectinase enzyme. The effects of these two pectinases on fabric thickness and shrinkage are less at pH 6.0 and 7.0 and are consistent with the effects on water wettability. There is no change in lightness nor weight loss (Figure 6c) for these enzyme treated fabrics at varying pHs.


222


ΒΑ Ό υ

90.0-

ω 80.0*Ξο β 70.0-

ce

ο

60.0-

Βα ο

50.0-

U

40.020

30

40

50


Time, minute

Time, minute

Figure 8. Reaction time effects on Aspergillus niger pectinase AN1 on fabrics (2x2s 100°C water pretreatment): Β 3x2min.inH Oatl00°C • 2x2sec.inH OatlOO°C 2

2

•

Apple pectin Citrus pectin

Β

ε m

m υ

Enzyme Applications in Fiber Processing - American Chemical Society

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