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Surface modification of wool with protease extracted polypeptides
Journal of Biotechnology 156 (2011) 134–140
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Surface modification of wool with protease extracted polypeptides Edward Smith, Jinsong Shen ∗ Textile Engineering and Materials (TEAM) Research Group, De Montfort University, Leicester LE1 9BH, UK
a r t i c l e
i n f o
Article history: Received 24 June 2011 Received in revised form 4 August 2011 Accepted 11 August 2011 Available online 19 August 2011 Keywords: Polypeptides Wool Enzyme Protease Shrink-resistance
a b s t r a c t Polypeptides were extracted from wool protein fibres using the serine type protease Esperase 8.0L (EC 3.4.21.62), a subtilisin from Bacillus sp., in a reducing solution. The extracted polypeptides, in aqueous liquor, were then applied to modify the fibre surface of wool fabric with or without additional protease. The treated wool fabric was subsequently treated with the cross-linking agent, glycerol diglycidyl ether, and then underwent a curing process to affix the polypeptide to the fibre. The resulting knitted fabric showed a very high level of shrink-resistance to machine washing, without excessive fibre damage. Shrinkage of 1–2% could be achieved after 5 times 5A washes with minimal (<1%) weight loss due to washing and a burst strength of 317 kPa. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Felting shrinkage is a typical property of wool when washed and must be controlled to achieve a washable wool product. Due to the configuration of the cuticle scales on the surface of wool fibre, the mechanical action of aqueous washing causes the progressive entanglement of wool fibres leading to irreversible shrinkage of wool fabric. Smoothing or eroding the cuticle scales lowers the friction between the fibres and therefore can prevent shrinkage. Shrink-resist finishing processes often consist of an oxidation/reduction step to degrade the cuticle scales and/or an additive polymer process to mask the scales. The conventional chemical process to achieve shrink-resistant wool, which consists of a chlorination step followed by polymer deposition (Lewis, 1977, 1978), has major drawbacks with respect to chlorination causing severe ecological problems due to contamination of wastewater effluent with absorbable organic halogens (AOX) (Müller, 1992). Recent European Union legislation has imposed restrictions on AOX releases to water (Environment Agency, 2011). If the chlorination step is omitted to avoid the effluent problem, increased polymer deposition then becomes necessary, resulting in a product with a harsh handle that feels more like a synthetic fabric and less like wool. This necessitates the use of alternative, environmentally acceptable shrink-proofing processes. Extensive research has been undertaken to develop an enzyme-based shrink-resistant finishing treatment of wool (Heine and Höcker, 1995; Shen, 2009).
∗ Corresponding author. Tel.: +44 116 2577558; fax: +44 116 2577582. E-mail address: [email protected] (J. Shen). 0168-1656/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2011.08.012
Protease can promote the hydrolysis of protein compounds and would appear ideal for degrading the cuticle scales on the wool fibre surface leading to shrink proofing of the wool fibre. However proteolytic attack is not limited to the fibre surface and will penetrate into the fibre causing significant damage in terms of loss of weight and tensile strength (Nolte et al., 1996; Shen et al., 1999; Heine et al., 2000). If the molecular size of the protease is enlarged by covalent coupling with an enteric polymer the proteolytic attack is limited to the cuticle scales thus controlling the damage to the wool (Cavaco-Paulo and Silva, 2003; Silva et al., 2005, 2006a, 2006b; Shen et al., 2007; Smith et al., 2008, 2010a, 2010b). An improvement in shrink-resistance was observed, however there is extra costing involved in the modification of enzymes and commercial standards for machine washability were difficult to meet especially for knitted wool fabrics. In the current work it was considered whether commercial antishrinkage standards could be met by the attachment of a protein resin to the surface of pre-treated wool fabrics and fibres. The protein resin could be a soft protein polypeptide extracted and separated from low quality wool fibre. It was considered that treatment with a protein resin would give the treated wool fibre a softer handle than using a synthetic polymer resin. Proteins such as casein (Needles, 1970), collagen (Needles, 1970; Hesse et al., 1995) and silk sericin (Cortez et al., 2007) have been used as polymer deposition treatments in previous studies in an attempt to achieve shrink resistant wool. Several different techniques have been reported for fixing proteins on to wool using cross-linking agents. Needles (1970) used a number of different commercially available difunctional epoxides to graft commercially available proteins onto wool and found
E. Smith, J. Shen / Journal of Biotechnology 156 (2011) 134–140
that the only epoxide to give a durable protein graft onto wool fabric was glycerol diglycidyl ether. Hesse et al. (1995) used the trifunctional epoxide Araldite PT 810 (1,3,5-triglycidyl isocyanurate) to covalently fix collagen onto the fibre surface of plasma (glow discharge) pre-treated wool fabric or top and reported almost complete shrink-resistance of the treated wool could be obtained. Cortez et al. (2007) used the enzyme transglutaminase to graft silk sericin onto wool by forming cross-links with the amino acids glutamine and lysine. An improvement in the shrink-resistance, strength and perceived softness of wool was observed. However, the extent of enzymatic reaction is highly dependent on the accessibility of the target amino acids within the wool fibre proteins (Fatarella et al., 2010). In the current work, sodium sulphite was used to break down the disulphide bonds in combination with a protease to catalyse the hydrolytic cleavage of the protein molecule into smaller peptide chains. Studies show that as long as cystine disulphide bond remains intact the rate of enzyme attack on wool is relatively slow, but once some of these cross-links are broken the rate of reaction is greatly increased (Moncrieff, 1953). The extracted wool polypeptide was separated by centrifugation and the resulting supernatant liquor layer was used to treat pre-scoured knitted wool fabric. The properties of the treated knitted wool fabric were assessed.
2. Materials and methods 2.1. Materials 2.1.1. Enzyme and reagents The enzyme used was a serine type protease, Esperase 8.0L (EC 3.4.21.62), which is a subtilisin from Bacillus sp., supplied by Novozymes A/S (Bagsvaerd, Denmark). The reducing agent sodium sulphite was purchased from Fisher Scientific (Loughborough, UK). Ultravon PL, a synergetic preparation based on non-ionic surfactants, was supplied by Ciba Speciality Chemicals (Cheshire, UK). The cross-linking agent glycerol diglycidyl ether (GDE) was purchased from Sigma–Aldrich (Dorset, UK). IEC reference detergent B and sodium perborate were purchased form SDC Enterprises Ltd (Bradford, UK). The reactive dye, Lanasol Red CE used to test the dyeability of treated wool fabric was supplied by Ciba. All other chemicals used were of specified laboratory reagent grade. 2.1.2. Wool material The wool fibre used in the extraction process was clean wool top with a mean fibre diameter of 23 m and was supplied by Drummond Parkland (Huddersfield, UK). The knitted wool fabric used was supplied by Lokateks (Skofja Loka, Slovenia) and was a fine rib 1:1 knit with a mean fibre diameter of 21.3 m.
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2.3. Pre-treatment of wool fabric by alkali scour Fine rib 1:1 knitted wool fabric was pre-treated in an alkali scour solution containing 2 g/L of the non-ionic surfactant Ultravon PL and 1.6 g/L sodium carbonate at a liquor to goods ratio of 50:1 for 30 min at 60 ◦ C using a Datacolor Ahiba Nuance Top Speed II infrared dyer with the agitation set at 5 rpm. The fabric was then rinsed in deionised water with a liquor to goods ratio of 50:1 for 10 min at 60 ◦ C with 5 rpm agitation. After treatment, the wool sample was washed thoroughly with water, hydro-extracted at 2800 rpm and then left to air-dry. 2.4. Treatment of wool fabric using polypeptide extract The alkali scoured wool fabric was treated in the neat supernatant wool extract with or without additional Esperase at a liquor to goods ratio of 12:1 for up to 2 h at 60 ◦ C using a Datacolor Ahiba Nuance Top Speed II infrared dyer with the agitation set at 5 rpm. The fabric could then be transferred into a new bath set at pH 7.3 using 0.02 M phosphate buffer containing 10 g/L of the cross-linking agent glycerol diglycidyl ether (GDE) with a liquor to goods ratio of 12:1 for 30 min at 60 ◦ C with 5 rpm agitation. After the wet treatment steps, the fabric was hydro-extracted at 2800 rpm to remove excess wetness. The fabric was then cured at 140 ◦ C for 10 min in a fan assisted oven. The treated wool fabric samples were then conditioned for 24 h at 20 ◦ C and 65% relative humidity prior to property and performance testing. 2.5. Weight loss The weight loss of the wool fabric after extracted polypeptide treatment was expressed as a percentage, WL and was calculated using Eq. (1): %WL =
100 × (W1 − W2 ) W1
(1)
where W1 is the weight of conditioned wool fabric prior to extracted polypeptide treatment and W2 is the weight of conditioned wool fabric after extracted polypeptide treatment. 2.6. Bursting strength The strength of the knitted wool fabric after extracted polypeptide treatment was measured using bursting strength. A James H Heal TruBurst 610 Bursting Strength Tester was used according to ISO 13938-2:1999. A test area of 10 cm2 (35.7 mm diameter) was used and the pressure rate was set at 21 kPa/s. The mean bursting pressure and mean height (distension) at burst were recorded. 2.7. Shrinkage
2.2. Preparation of wool polypeptide extract Clean wool top was cut into snippets and placed in a 0.02 M phosphate buffer (pH 8) containing up to 12 g/L sodium sulphite, with a liquor to goods ratio of 20:1 and treated at 60 ◦ C for 30 min using a Datacolor Ahiba Nuance Top Speed II infrared dyer with the agitation set at 40 rpm. 18 activity units of the protease, Esperase 8.0L, per gram of wool fibre (u/g) was added to the mixture and mixed for a further 2 h at 65 ◦ C with an agitation of 40 rpm. The enzyme present in the mixture may be deactivated by raising the temperature to 80 ◦ C for 10 min and maintaining the agitation at 40 rpm. The resulting suspension was separated by centrifugation at 4500 rpm for 5 min using a Hettich Rotina 420 bench centrifuge with a swing out rotor. The supernatant liquid layer was collected for use in the treatment of wool.
The measurement of shrinkage due to washing of the treated knitted wool fabric was tested according to Woolmark Test Method TM31: Washing of Wool Textile Products. The samples were subjected to a 7A wash cycle for relaxation shrinkage and 5A wash cycles up to 5 times for felting shrinkage using a Miele Novotronic W980 computer controlled washing machine. Between each washing cycle the samples were flat-dried in a 50 ◦ C oven for 4 h then conditioned at 20 ◦ C and 65% relative humidity for 24 h and then weighed. Weight loss due to washing was determined and expressed as a percentage, WLW , which was calculated using Eq. (2): %WLW =
100 × (W3 − W4 ) W3
(2)
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where W3 is the weight of conditioned wool fabric before being subjected to the series of washing cycles and W4 is the weight of conditioned wool fabric after machine washing. The mean area shrinkage and weight loss due to washing were calculated.
where Abs0 is the absorbance of the original dye-bath solution at max , Abs1 is the absorbance of the exhausted dye-bath solution at max and Abs2 is the absorbance of the solution after soaping at max . Washfastness of the dyed samples was assessed according to BS EN 20105:CO1 conditions at 40 ◦ C for 30 min.
2.8. Scanning electron microscopy (SEM) of treated wool samples 3. Results and discussion To determine the extent of fibre damage and any changes to the external surface of the fibre, micrographs were taken using SEM of wool samples treated with wool polypeptide extract and compared with a wool sample which had only been alkali-scoured. The samples were prepared by attaching a double-sided adhesive carbon tab to an aluminium stub, then laying wool fibre across the sticky surface of the stub. For examination, the samples were sputter coated with gold under argon for 60 seconds using an Edwards ES150 sputter coater. Samples were examined using a Leo 430 scanning electron microscope operating with a working distance of 25 mm, an accelerating voltage of 7 kV and a magnification of 500×. 2.9. Whiteness A Datacolor SF6000 Plus CT reflectance spectrophotometer was used to determine the whiteness of the treated knitted wool fabric in terms of the CIE whiteness index. Each sample was folded into four and measured four times. All values were measured and calculated using ColorTools QC software with illuminant and observer conditions of D65 and 10◦ , respectively. 2.10. Dyeability The dyeability of the treated knitted wool fabric was tested using the reactive dye Lanasol Red CE. The fabric was processed for 30 min at 50 ◦ C in a dye-bath containing 2% owf acetic acid, 5% owf sodium sulphate and 1% owf levelling auxiliary, Albegal B (Ciba). 4% owf Lanasol Red CE was then added to the dye-bath and the temperature was raised to 100 ◦ C at a rate of 1.5 ◦ C/min and maintained at 100 ◦ C for 90 min. After dyeing the fabric was treated in a 1% owf ammonia solution (pH 8.4) at 80 ◦ C for 15 min. Dyeing took place in a Datacolor Ahiba Nuance IR dye machine at an agitation of 5 rpm and a liquor to goods ratio of 30:1. To test dye fixation, a piece of the dyed fabric was placed in a soaping solution containing 5 g/L Ultravon PL (Ciba) non-ionic surfactant and 5 g/L sodium carbonate at a liquor to goods ratio of 30:1 and treated at 100 ◦ C for 20 min using a Datacolor Ahiba Nuance IR dye machine. Dye exhaustion and total fixation efficiency were determined. Dye exhaustion (%E) is the percentage of dye absorbed on the fabric from the dye-bath solution using the exhaust method of dyeing. Total fixation efficiency (%T) is the percentage of the dye originally applied to the fabric which becomes bound covalently. The dyebath solutions sampled before and after the dyeing process and the solution after soaping were made up to the same fixed volume and absorbance measurements were taken at 510 nm, the wavelength of maximum absorption (max ) specific to Lanasol Red CE, using a Pye Unicam SP1800 UV/visible spectrophotometer. The percentage of exhaustion (%E) was calculated using Eq. (3) and the total fixation efficiency (%T) was calculated using Eq. (4). %E =
100 × (Abs0 − Abs1 ) Abs0
(3)
%T =
100 × (Abs0 − Abs1 − Abs2 ) Abs0
(4)
3.1. Shrink-resist treatment with polypeptides extracted from wool Protein polypeptide was extracted from wool fibre using sodium sulphite and Esperase. The enzyme was deactivated after extraction was complete. After centrifugation, the resulting supernatant liquid layer, containing the extracted protein polypeptide, was used neat to treat alkali scoured knitted wool fabric. The preferred fabric processing conditions were found to be 2 h at 60 ◦ C. The results in Table 1 show significant improvement in shrink resistance when the extracted wool polypeptide was used to treat knitted wool fabric; with an area shrinkage of 5.6% after 3 times 5A washes, a minimal weight loss (0.9%) during 3 times 5A washes and only minimal loss in burst strength when compared to the control samples. The effect of the components such as reducing agent, enzyme and polypeptide on the shrink-resistance of the treated knitted wool was investigated. 12 g/L sodium sulphite in a pH 8 phosphate buffer was heated to 60 ◦ C for 30 min without the presence of wool fibre snippets. The mixture was treated for a further 2 h with or without the addition of 18 u/g Esperase, then raised to 80 ◦ C to denature the enzyme. Both of these mixtures were used neat to treat alkali pre-scoured knitted wool fabric to determine whether the absence of extracted wool protein in the mixture affected the properties of the treated wool fabric. The respective area shrinkage of 10.6% and 9.8% after 3 times 5A washes show that the presence of extracted wool polypeptide with an area shrinkage of 5.6% does have an effect on shrink resistance. All the other properties, as shown in Table 1, are very similar. Wool fibre snippets were extracted in the presence of 18 u/g Esperase without the presence of sodium sulphite, following the extraction protocol, and separated by centrifugation. When the neat supernatant liquor was used to treat knitted wool fabric, the area shrinkage of the sample after 3 times 5A washes was 11.5%. This shows that sodium sulphite is required in the extraction process to extract the wool polypeptide effectively. It was considered whether fixation of the polypeptide to the fibre surface using a cross-linking agent would further improve shrink-resistance. Several cross-linking agents were considered including the difunctional epoxides; glycerol diglycidyl ether and 1,4-butandioldiglycidyl ether or the carbodiimide; 1(3-dimethylamino-propyl) 3-ethyl-carbodiimide hydrochloride. It was found that if after polypeptide extract treatment the wool fabric was transferred into a new bath containing 10 g/L glycerol diglycidyl ether (GDE) for 30 min at 60 ◦ C and cured at 140 ◦ C for 10 min, the shrink-resistance of the fabric improved to 3.6% area shrinkage after 3 times 5A washes (Table 2). Control samples show that GDE treatment has minimal effect on shrink resistance on samples that have not been treated with neat extracted polypeptide. 3.2. Shrink-resist treatment with enzyme active extracted wool polypeptides The shrink-resistance of wool treated with extracted wool polypeptides could be further improved if the enzyme used in the polypeptide extraction process was not deactivated prior to centrifugation therefore forming an enzyme active polypeptide
−0.5 (1.2) −0.1 (0.1) −1.1 (0.3) 0.9 (0.9) 9.8 (1.3) 10.6 (1.0) 11.5 (0.5) 5.6 (0.7) 406 (10) 385 (48) 423 (17) 371 (35) −0.2 (1.0) 0.5 (0.3) 0.4 (0.2) −0.2 (1.3) Treated with neat extract for 2 h at 60 ◦ C Non-ionic surfactant + Na2 CO3 30 min Without wool in 12 g/L sulphite 2 h 30 min Without wool in 12 g/L sulphite 30 min + 18 u/g Esperase 2 h Wool without sulphite 30 min + 18 u/g Esperase 2 h Wool in 12 g/L sulphite 30 min + 18 u/g Esperase 2 h
Weight loss (%)
0.6 (1.0) 0.4 (0.4) 18.4 (1.2) 13.7 (1.3)
Area shrinkage (%)
421 (15) 384 (34) Untreated No further treatment Untreated
– – Extract treatment Pre-treatment
No extract No extract
After 3× 5A washes Burst strength (kPa) Weight loss (%) Treatment of fabric Method for extraction of wool polypeptide
Table 1 The effect of wool peptide extraction and the extracted polypeptide treatment on alkali scoured wool fabric in terms of weight loss due to treatment, burst strength, area shrinkage and weight loss due to washing. Standard deviation in parentheses.
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extract. It was decided that 2 h treatment on knitted wool fabric with an enzyme active wool polypeptide would be too long causing excessive fibre damage, therefore the treatment time was reduced to 30 min. Residual enzyme present on the fibre may cause fibre damage during further processing or machine washing; however curing at 140 ◦ C after the cross-linking step would denature any residual enzyme present on the treated wool fibre. This was proven in the results in Table 3 with a much higher weight loss after 3 times 5A washes if the knitted wool fabric treated with enzyme active extracted wool polypeptide was not subsequently treated with GDE and cured at 140 ◦ C in a fan-assisted oven for 10 min. Almost complete shrink resistance was achieved if alkali pre-scoured knitted wool fabric was treated for 30 min with enzyme active extracted wool polypeptide followed by cross-linking with GDE for 30 min and a 10 min cure at 140 ◦ C, with 0.9% area shrinkage after 3 times 5A washes and 1.5% area shrinkage after 5 times 5A washes (see Fig. 1) with minimal weight loss due to washing. Even though the burst strength had decreased, the burst strength of 285 kPa was above the accepted burst strength of 225 kPa for Woolmark machine wash care standards for knitted sweaters (Woolmark Company, 2000). 3.3. Shrink-resist treatment with extracted wool polypeptides in the presence of protease Better shrink resistance results were observed on treated wool fabric if the enzyme used in the extraction process was not deactivated prior to use. However, for better control of the amount of enzyme present and an improved stability of the polypeptide extract the alternative was to maintain the deactivating step in the polypeptide extraction process prior to centrifugation but Esperase would be added to the neat polypeptide extract when used as a shrink-resistance treatment on wool samples. Different concentrations of Esperase were tested. Almost complete shrink-resistance could be achieved after 3 times 5A washes if 3 u/g or greater of Esperase was added to the neat extracted polypeptide when treated on alkali pre-scoured knitted wool fabric and subsequently crosslinked and cured (Table 4). No weight loss was observed after 3 times 5A washes for these samples. Almost complete shrinkresistance (0.4%) with no weight loss (−0.5%) was observed even after 5 times 5A washes. The greater the amount of native Esperase added to the extracted polypeptide treatment liquor, the lower the burst strength (Table 4). When 3 u/g of native Esperase was present in the deactivated polypeptide extract a similar level of shrink-resistance was achieved to treatment with enzyme active extracted polypeptide (seen in Table 3). A better burst strength was observed with the former (317 kPa) than the latter (285 kPa), indicating less damage to the fibre. This shows that adding enzyme to deactivated extracted polypeptide is easier to control as a shrinkresist treatment for wool than using the enzyme active extracted polypeptide. 3.4. Fibre surface, whiteness and dyeability of wool fabric treated with extracted wool polypeptides The external fibre surfaces of wool samples treated with extracted wool polypeptide followed by cross-linking with GDE and then cured were examined using SEM and compared with a wool sample which had only been alkali-scoured (Fig. 2a). SEM images of alkali pre-scoured wool followed by treatment with extracted polypeptide (Fig. 2b), with enzyme active extracted polypeptide (Fig. 2c) or with extracted polypeptide in the presence of 3 u/g native Esperase (Fig. 2d) all showed that the wool fibres were still intact and were not severely damaged by the shrink-resist treatment.
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Table 2 The effect of the addition of cross-linking agents and high temperature curing on the extracted wool polypeptide (and control) treatment processing of alkali scoured wool fabric in terms of weight loss due to treatment, burst strength, area shrinkage and weight loss due to washing. Standard deviation in parentheses. Weight loss (%)
Extraction method
Treatment with extract (or control) on wool fabric for 2 h at 60 ◦ C
Burst strength (kPa)
After 3× 5A washes
Area shrinkage (%)
Weight loss (%)
No extraction
Without cross-link and cure With cross-link and cure
– 1.4 (0.3)
384 (34) 330 (42)
13.7 (1.3) 12.9 (1.0)
0.4 (0.4) −0.5 (0.4)
Control without wool (12 g/L sulphite 30 min + 18 u/g Esperase 2 h)
Without cross-link and cure With cross-link and cure
0.7 (0.3) 1.0 (0.3)
385 (48) 371 (1)
10.6 (1.0) 9.0 (0.6)
−0.1 (0.4) −0.3 (0.4)
Extracted wool polypeptide (12 g/L sulphite 30 min + 18 u/g Esperase 2 h)
Without cross-link and cure With cross-link and cure
−0.2 (1.3) 0.6 (0.3)
371 (35) 370 (30)
5.6 (0.7) 3.6 (1.1)
0.9 (0.9) −0.2 (0.4)
Table 3 The effect of enzyme active extracted wool polypeptide treatment on alkali scoured wool fabric in terms of weight loss due to treatment, burst strength, area shrinkage and weight loss due to washing. Standard deviation in parentheses. Weight loss (%)
Extraction method
Treatment with extract (or control) on wool fabric for 30 min at 60 ◦ C
Area shrinkage (%)
Weight loss (%)
Enzyme active without wool (12 g/L sulphite 30 min + 18 u/g Esperase 2 h) Enzyme active extracted wool polypeptide (12 g/L sulphite 30 min + 18 u/g Esperase 2 h)
With cross-link and cure
8.2 (0.1)
278 (20)
2.4 (0.9)
1.3 (0.2)
Without cross-link and cure With cross-link and cure
4.3 (0.4) 7.1 (1.6)
316 (24) 285 (30)
4.0 (0.5) 0.9 (0.5)
8.3 (1.2) 1.3 (0.6)
An improvement in whiteness was observed for wool treated with the extracted polypeptide (Table 5). A drawback of the conventional chlorine-resin methods of achieving shrink-resistance is significant yellowing of the wool fibre due the use of chlorine or the disodium salt of dichloroisocyanuric acid (DCCA) (Lewis, 1992; Millington, 2009). A higher level of whiteness was found for wool treated with the enzyme active extracted polypeptide than the comparable treatment with deactivated extracted polypeptide followed by cross-linking and curing. This would be expected due to the enzyme being active in the polypeptide, as an improvement in whiteness is usually observed when protease has been used to treat
Burst strength (kPa)
After 3× 5A washes
wool (Zhang et al., 2006; Smith et al., 2010b). A similar improved level of whiteness was observed for wool treated with deactivated extracted wool polypeptide in the presence of 3 u/g or greater of native Esperase followed by cross-link and cure as for wool treated with enzyme active extracted wool polypeptide followed by crosslink and cure. It was noticed that the greater the amount of native Esperase added to the extracted polypeptide treatment liquor, the greater the CIE whiteness (data not shown). To examine its dyeability, wool fabric which had been treated with polypeptide extract was dyed with the reactive dye Lanasol Red CE at 100 ◦ C following standard dyeing protocols. After
Fig. 1. Photograph comparing untreated knitted wool and processed knitted wool (alkali scour pre-treatment followed by 30 min treatment with enzyme active extracted wool polypeptide then followed by cross-link and curing) before and after washing.
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Fig. 2. (a–d) Scanning electron micrographs (SEM) of wool fibres (500× magnification) from treated knitted wool fabric samples: (a) alkali scoured only; (b) alkali scour pre-treatment followed by 2 h treatment with extracted wool polypeptide then followed by cross-link and curing; (c) alkali scour pre-treatment followed by 30 min treatment with enzyme active extracted wool polypeptide then followed by cross-link and curing; and (d) alkali scour pre-treatment followed by 30 min treatment of extracted wool polypeptide in the presence of 3 u/g Esperase then followed by cross-link and curing.
Table 4 The effect of the addition of different concentrations of Esperase on the extracted wool polypeptide (and sodium sulphite control) treatment processing of alkali scoured wool fabric in terms of weight loss due to treatment, burst strength, area shrinkage and weight loss due to washing. Standard deviation in parentheses. Extraction method
Treatment with extract (or control) on wool fabric with or without additional Esperase for 30 min at 60 ◦ C, followed by cross-link and cure
Weight loss (%)
Burst strength (kPa)
Control without wool (12 g/L sulphite 2 h 30 min)
Without Esperase With 3 u/g Esperase With 6 u/g Esperase
−0.3 (0.3) 9.3 (0.7) 12.7 (1.0)
377 (13) 246 (21) 167 (13)
11.7 (0.1) 2.4 (0.6) 2.4 (0.5)
0.9 (0.1) 2.1 (0.2) 4.9 (0.3)
Enzyme active extracted wool polypeptide (12 g/L sulphite 30 min + 18 u/g Esperase 2 h) deactivated
Without Esperase With 1.5 u/g Esperase With 3 u/g Esperase With 6 u/g Esperase
−0.1 (0.5) 3.7 (0.2) 5.5 (0.2) 7.6 (0.2)
379 (13) 345 (30) 317 (11) 272 (19)
7.8 (0.5) 3.6 (1.4) 1.1 (0.4) 0.8 (0.9)
−0.8 (0.2) −0.7 (0.6) −0.2 (0.2) 0.8 (0.5)
After 3× 5A washes
Area shrinkage (%)
dyeing the sample treated with the polypeptide extract but not cross-linked was less shrink-resistant with an area shrinkage of 11% compared to an area shrinkage of 5.6% for the corresponding undyed sample, both after 3 times 5A washes (data not shown). It would appear that the high temperature the sample was subjected to during dyeing caused the loss of polypeptide from the fibre surface therefore losing the shrink-resistant effect. When the sam-
Weight loss (%)
ple treated with polypeptide extract followed by cross-linking with GDE and curing at 140 ◦ C was subsequently dyed with the reactive dye Lanasol Red CE the shrink-resistance only slightly decreased to an area shrinkage of 5.5% compared to an area shrinkage of 3.6% for the corresponding undyed sample, both after 3 times 5A washes (data not shown). This shows that cross-linking is required to fix the polypeptide onto the fibre surface to ensure shrink-resistance
Table 5 CIE whiteness index values and dyeability with 4% (owf) Lanasol Red CE of alkali scoured wool fabric treated with extracted wool polypeptide in terms of dye exhaustion and fixation. Standard deviation in parentheses. Extract treatment on alkali scoured wool fabrica
CIE whiteness index
No further treatment 2 h treatment with extracted polypeptide (deactivated) 30 min treatment with extracted wool polypeptide (enzyme active) 30 min treatment with extracted wool polypeptide (deactivated) and 3 u/g Esperase
−0.23 (3.41) 4.83 (0.68) 13.43 (1.56) 13.17 (2.91)
%E = dye exhaustion, %T = total fixation efficiency. a All samples treated with extracted polypeptide were subsequently cross-linked with GDE then cured at 140 ◦ C.
Dyeability %E
%T
99.5 99.1 99.4 99.3
88.8 89.6 91.8 86.1
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properties are maintained after dyeing. When the sample treated with extracted wool polypeptide in the presence of 3 u/g of native Esperase followed by cross-link and cure was subsequently dyed with 4% owf Lanasol Red CE at 100 ◦ C, there was only minimal loss in shrink resistance after 3 times 5A washes, from 1.1% for the undyed sample to 1.5% for the dyed sample. Treatment of alkali pre-scoured wool with either extracted polypeptide, enzyme active extracted polypeptide or extracted polypeptide in the presence of 3 u/g native Esperase does not affect the dyeability of wool as the results in Table 5 show very similar results with respect to dye exhaustion and fixation as for alkali scoured wool fabric after dyeing with 4% owf Lanasol Red CE. The excellent wash-fastness properties to BS EN 20105:CO1 conditions of the dyed control sample were maintained after dyeing of alkali pre-scoured wool which had been treated with either extracted polypeptide, enzyme active extracted polypeptide or extracted polypeptide in the presence of 3 u/g native Esperase followed by cross-linking and curing, with no staining (greyscale rating of 5) on the multifibre strip. 4. Conclusions Almost complete shrink-resistance can be imparted on knitted wool fabric when treated with protease extracted wool polypeptide followed by cross-linking with glycerol diglycidyl ether and a high temperature cure. An area shrinkage of less than 2% can be achieved after 5 times 5A washes with minimal weight loss during washing. Knitted wool fabric treated by this shrink-resist method meets burst strength standards for knitted wool garments, has an improved whiteness and a soft handle. The treated wool fabric is dyeable with reactive dye and as long as the polypeptide extract is affixed to the wool fibre, a high level of shrink-resistance can be maintained after dyeing. Acknowledgements We would like to thank the East Midland Development Agency (EMDA) and the European Regional Development Fund (ERDF) for funding the Higher Education Innovation and Regional Fellowships (HIRF 385 and HIRF 447F) to undertake this research. References Cavaco-Paulo, A., Silva, C.J.S.M., 2003. Treatment of animal hair fibres with modified proteases. World Patent WO03097927. Cortez, J., Anghieri, A., Bonner, P.L.R., Griffin, M., Freddi, G., 2007. Transglutaminase mediated grafting of silk proteins onto wool fabrics leading to improved physical and mechanical properties. Enzyme Microb. Technol. 40, 1698–1704.
Environment Agency, 2011. http://www.environment-agency.gov.uk/business/ topics/pollution/3921.aspx. Fatarella, E., Ciabatti, I., Cortez, J., 2010. Plasma and electron-beam processes as pre-treatments for enzymatic processes. Enzyme Microb. Technol. 46, 100–106. Heine, E., Höcker, H., 1995. Enzyme treatments for wool and cotton. Rev. Prog. Color. 25, 57–63. Heine, E., Hollfelder, B., Shen, J., Bishop, D., 2000. Parameters effecting the morphology of wool using enzyme treatment. In: Proceedings of the 10th International Wool Textile Research Conference, Aachen, November 26–December 1, 2000, pp. EN-3, 1–12. Hesse, A., Thomas, H., Höcker, H., 1995. Zero-AOX shrinkproofing treatment of wool top and fabric part II: collagen resin application. Text. Res. J. 65, 371–378. Lewis, D.M., 1992. Ancillary processes in wool dyeing. In: Lewis, D.M. (Ed.), Wool Dyeing. Society of Dyers and Colourists, Bradford, pp. 111–136. Lewis, J., 1977. Superwash wool part 1: a review of the development of Superwash technology. Wool Sci. Rev. 54, 2–29. Lewis, J., 1978. Superwash wool part 2: continuous shrinkproofing processes. Wool Sci. Rev. 55, 23–42. Millington, K.R., 2009. Improving the whiteness and photostability of wool. In: Johnson, N.A.G., Russell, I.M. (Eds.), Advances in Wool Technology. Woodhead Publishing, Cambridge, pp. 217–247. Moncrieff, R.W., 1953. Wool Shrinkage and its Prevention. The National Trade Press Ltd., London. Müller, B.M., 1992. Absorbable organic halogens in textile effluents. Rev. Prog. Color. 22, 14–21. Needles, H.L., 1970. Grafting commercially available proteins onto wool using crosslinking agents. Text. Res. J. 40, 771–775. Nolte, H., Bishop, D.P., Höcker, H., 1996. Effects of proteolytic and lipolytic enzymes on untreated and shrink-resist treated wool. J. Text. Inst. 87, 212–226. Shen, J., 2009. Wool finishing development of novel finishes. In: Johnson, N.A.G., Russell, I.M. (Eds.), Advances in Wool Technology. Woodhead Publishing, Cambridge, pp. 147–182. Shen, J., Bishop, D., Heine, E., Hollfelder, B., 1999. Some factors affecting the control of proteolytic enzyme reactions on wool. J. Text. Inst. 90, 404–411. Shen, J., Rushforth, M., Cavaco-Paulo, A., Guebitz, G., Lenting, H., 2007. Development and industrialisation of enzymatic shrink-resist process based on modified proteases for wool machine washability. Enzyme Microb. Technol. 40, 1656–1661. Silva, C.J.S.M., Gübitz, G., Cavaco-Paulo, A., 2006a. Optimisation of a serine protease coupling to Eudragit S-100 by experimental design techniques. J. Chem. Technol. Biotechnol. 81, 8–16. Silva, C.J.S.M., Prabaharan, M., Gübitz, G., Cavaco-Paulo, A., 2005. Treatment of wool fibres with subtilisin and subtilisin-PEG. Enzyme Microb. Technol. 36, 917–922. Silva, C.J.S.M., Zhang, Q., Shen, J., Cavaco-Paulo, A., 2006b. Immobilization of proteases with a water soluble-insoluble reversible polymer for the treatment of wool. Enzyme Microb. Technol. 39, 634–640. Smith, E., Farrand, B., Shen, J., 2010a. The removal of lipid from the surface of wool to promote the subsequent enzymatic process with modified protease for wool shrink resistance. Biocatal. Biotransform. 28, 329–338. Smith, E., Schroeder, M., Guebitz, G., Shen, J., 2010b. Covalent bonding of protease to different sized enteric polymers and their potential use in wool processing. Enzyme Microb. Technol. 47, 105–111. Smith, E., Zhang, Q., Shen, J., Schroeder, M., Silva, C., 2008. Modification of Esperase by covalent bonding to Eudragit polymers L100 and S100 for wool fibre surface treatment. Biocatal. Biotransform. 26, 391–398. Woolmark Company, 2000. Product Specifications for Knitted Apparel Products, K1. Zhang, Q., Smith, E., Shen, J., Bishop, D., 2006. An ethoxylated alkyl phosphate (anionic surfactant) for the promotion of activities of proteases and its potential use in the enzymatic processing of wool. Biotechnol. Lett. 28, 717–723.
Contents lists available at SciVerse ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Surface modification of wool with protease extracted polypeptides Edward Smith, Jinsong Shen ∗ Textile Engineering and Materials (TEAM) Research Group, De Montfort University, Leicester LE1 9BH, UK
a r t i c l e
i n f o
Article history: Received 24 June 2011 Received in revised form 4 August 2011 Accepted 11 August 2011 Available online 19 August 2011 Keywords: Polypeptides Wool Enzyme Protease Shrink-resistance
a b s t r a c t Polypeptides were extracted from wool protein fibres using the serine type protease Esperase 8.0L (EC 3.4.21.62), a subtilisin from Bacillus sp., in a reducing solution. The extracted polypeptides, in aqueous liquor, were then applied to modify the fibre surface of wool fabric with or without additional protease. The treated wool fabric was subsequently treated with the cross-linking agent, glycerol diglycidyl ether, and then underwent a curing process to affix the polypeptide to the fibre. The resulting knitted fabric showed a very high level of shrink-resistance to machine washing, without excessive fibre damage. Shrinkage of 1–2% could be achieved after 5 times 5A washes with minimal (<1%) weight loss due to washing and a burst strength of 317 kPa. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Felting shrinkage is a typical property of wool when washed and must be controlled to achieve a washable wool product. Due to the configuration of the cuticle scales on the surface of wool fibre, the mechanical action of aqueous washing causes the progressive entanglement of wool fibres leading to irreversible shrinkage of wool fabric. Smoothing or eroding the cuticle scales lowers the friction between the fibres and therefore can prevent shrinkage. Shrink-resist finishing processes often consist of an oxidation/reduction step to degrade the cuticle scales and/or an additive polymer process to mask the scales. The conventional chemical process to achieve shrink-resistant wool, which consists of a chlorination step followed by polymer deposition (Lewis, 1977, 1978), has major drawbacks with respect to chlorination causing severe ecological problems due to contamination of wastewater effluent with absorbable organic halogens (AOX) (Müller, 1992). Recent European Union legislation has imposed restrictions on AOX releases to water (Environment Agency, 2011). If the chlorination step is omitted to avoid the effluent problem, increased polymer deposition then becomes necessary, resulting in a product with a harsh handle that feels more like a synthetic fabric and less like wool. This necessitates the use of alternative, environmentally acceptable shrink-proofing processes. Extensive research has been undertaken to develop an enzyme-based shrink-resistant finishing treatment of wool (Heine and Höcker, 1995; Shen, 2009).
∗ Corresponding author. Tel.: +44 116 2577558; fax: +44 116 2577582. E-mail address: [email protected] (J. Shen). 0168-1656/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2011.08.012
Protease can promote the hydrolysis of protein compounds and would appear ideal for degrading the cuticle scales on the wool fibre surface leading to shrink proofing of the wool fibre. However proteolytic attack is not limited to the fibre surface and will penetrate into the fibre causing significant damage in terms of loss of weight and tensile strength (Nolte et al., 1996; Shen et al., 1999; Heine et al., 2000). If the molecular size of the protease is enlarged by covalent coupling with an enteric polymer the proteolytic attack is limited to the cuticle scales thus controlling the damage to the wool (Cavaco-Paulo and Silva, 2003; Silva et al., 2005, 2006a, 2006b; Shen et al., 2007; Smith et al., 2008, 2010a, 2010b). An improvement in shrink-resistance was observed, however there is extra costing involved in the modification of enzymes and commercial standards for machine washability were difficult to meet especially for knitted wool fabrics. In the current work it was considered whether commercial antishrinkage standards could be met by the attachment of a protein resin to the surface of pre-treated wool fabrics and fibres. The protein resin could be a soft protein polypeptide extracted and separated from low quality wool fibre. It was considered that treatment with a protein resin would give the treated wool fibre a softer handle than using a synthetic polymer resin. Proteins such as casein (Needles, 1970), collagen (Needles, 1970; Hesse et al., 1995) and silk sericin (Cortez et al., 2007) have been used as polymer deposition treatments in previous studies in an attempt to achieve shrink resistant wool. Several different techniques have been reported for fixing proteins on to wool using cross-linking agents. Needles (1970) used a number of different commercially available difunctional epoxides to graft commercially available proteins onto wool and found
E. Smith, J. Shen / Journal of Biotechnology 156 (2011) 134–140
that the only epoxide to give a durable protein graft onto wool fabric was glycerol diglycidyl ether. Hesse et al. (1995) used the trifunctional epoxide Araldite PT 810 (1,3,5-triglycidyl isocyanurate) to covalently fix collagen onto the fibre surface of plasma (glow discharge) pre-treated wool fabric or top and reported almost complete shrink-resistance of the treated wool could be obtained. Cortez et al. (2007) used the enzyme transglutaminase to graft silk sericin onto wool by forming cross-links with the amino acids glutamine and lysine. An improvement in the shrink-resistance, strength and perceived softness of wool was observed. However, the extent of enzymatic reaction is highly dependent on the accessibility of the target amino acids within the wool fibre proteins (Fatarella et al., 2010). In the current work, sodium sulphite was used to break down the disulphide bonds in combination with a protease to catalyse the hydrolytic cleavage of the protein molecule into smaller peptide chains. Studies show that as long as cystine disulphide bond remains intact the rate of enzyme attack on wool is relatively slow, but once some of these cross-links are broken the rate of reaction is greatly increased (Moncrieff, 1953). The extracted wool polypeptide was separated by centrifugation and the resulting supernatant liquor layer was used to treat pre-scoured knitted wool fabric. The properties of the treated knitted wool fabric were assessed.
2. Materials and methods 2.1. Materials 2.1.1. Enzyme and reagents The enzyme used was a serine type protease, Esperase 8.0L (EC 3.4.21.62), which is a subtilisin from Bacillus sp., supplied by Novozymes A/S (Bagsvaerd, Denmark). The reducing agent sodium sulphite was purchased from Fisher Scientific (Loughborough, UK). Ultravon PL, a synergetic preparation based on non-ionic surfactants, was supplied by Ciba Speciality Chemicals (Cheshire, UK). The cross-linking agent glycerol diglycidyl ether (GDE) was purchased from Sigma–Aldrich (Dorset, UK). IEC reference detergent B and sodium perborate were purchased form SDC Enterprises Ltd (Bradford, UK). The reactive dye, Lanasol Red CE used to test the dyeability of treated wool fabric was supplied by Ciba. All other chemicals used were of specified laboratory reagent grade. 2.1.2. Wool material The wool fibre used in the extraction process was clean wool top with a mean fibre diameter of 23 m and was supplied by Drummond Parkland (Huddersfield, UK). The knitted wool fabric used was supplied by Lokateks (Skofja Loka, Slovenia) and was a fine rib 1:1 knit with a mean fibre diameter of 21.3 m.
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2.3. Pre-treatment of wool fabric by alkali scour Fine rib 1:1 knitted wool fabric was pre-treated in an alkali scour solution containing 2 g/L of the non-ionic surfactant Ultravon PL and 1.6 g/L sodium carbonate at a liquor to goods ratio of 50:1 for 30 min at 60 ◦ C using a Datacolor Ahiba Nuance Top Speed II infrared dyer with the agitation set at 5 rpm. The fabric was then rinsed in deionised water with a liquor to goods ratio of 50:1 for 10 min at 60 ◦ C with 5 rpm agitation. After treatment, the wool sample was washed thoroughly with water, hydro-extracted at 2800 rpm and then left to air-dry. 2.4. Treatment of wool fabric using polypeptide extract The alkali scoured wool fabric was treated in the neat supernatant wool extract with or without additional Esperase at a liquor to goods ratio of 12:1 for up to 2 h at 60 ◦ C using a Datacolor Ahiba Nuance Top Speed II infrared dyer with the agitation set at 5 rpm. The fabric could then be transferred into a new bath set at pH 7.3 using 0.02 M phosphate buffer containing 10 g/L of the cross-linking agent glycerol diglycidyl ether (GDE) with a liquor to goods ratio of 12:1 for 30 min at 60 ◦ C with 5 rpm agitation. After the wet treatment steps, the fabric was hydro-extracted at 2800 rpm to remove excess wetness. The fabric was then cured at 140 ◦ C for 10 min in a fan assisted oven. The treated wool fabric samples were then conditioned for 24 h at 20 ◦ C and 65% relative humidity prior to property and performance testing. 2.5. Weight loss The weight loss of the wool fabric after extracted polypeptide treatment was expressed as a percentage, WL and was calculated using Eq. (1): %WL =
100 × (W1 − W2 ) W1
(1)
where W1 is the weight of conditioned wool fabric prior to extracted polypeptide treatment and W2 is the weight of conditioned wool fabric after extracted polypeptide treatment. 2.6. Bursting strength The strength of the knitted wool fabric after extracted polypeptide treatment was measured using bursting strength. A James H Heal TruBurst 610 Bursting Strength Tester was used according to ISO 13938-2:1999. A test area of 10 cm2 (35.7 mm diameter) was used and the pressure rate was set at 21 kPa/s. The mean bursting pressure and mean height (distension) at burst were recorded. 2.7. Shrinkage
2.2. Preparation of wool polypeptide extract Clean wool top was cut into snippets and placed in a 0.02 M phosphate buffer (pH 8) containing up to 12 g/L sodium sulphite, with a liquor to goods ratio of 20:1 and treated at 60 ◦ C for 30 min using a Datacolor Ahiba Nuance Top Speed II infrared dyer with the agitation set at 40 rpm. 18 activity units of the protease, Esperase 8.0L, per gram of wool fibre (u/g) was added to the mixture and mixed for a further 2 h at 65 ◦ C with an agitation of 40 rpm. The enzyme present in the mixture may be deactivated by raising the temperature to 80 ◦ C for 10 min and maintaining the agitation at 40 rpm. The resulting suspension was separated by centrifugation at 4500 rpm for 5 min using a Hettich Rotina 420 bench centrifuge with a swing out rotor. The supernatant liquid layer was collected for use in the treatment of wool.
The measurement of shrinkage due to washing of the treated knitted wool fabric was tested according to Woolmark Test Method TM31: Washing of Wool Textile Products. The samples were subjected to a 7A wash cycle for relaxation shrinkage and 5A wash cycles up to 5 times for felting shrinkage using a Miele Novotronic W980 computer controlled washing machine. Between each washing cycle the samples were flat-dried in a 50 ◦ C oven for 4 h then conditioned at 20 ◦ C and 65% relative humidity for 24 h and then weighed. Weight loss due to washing was determined and expressed as a percentage, WLW , which was calculated using Eq. (2): %WLW =
100 × (W3 − W4 ) W3
(2)
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where W3 is the weight of conditioned wool fabric before being subjected to the series of washing cycles and W4 is the weight of conditioned wool fabric after machine washing. The mean area shrinkage and weight loss due to washing were calculated.
where Abs0 is the absorbance of the original dye-bath solution at max , Abs1 is the absorbance of the exhausted dye-bath solution at max and Abs2 is the absorbance of the solution after soaping at max . Washfastness of the dyed samples was assessed according to BS EN 20105:CO1 conditions at 40 ◦ C for 30 min.
2.8. Scanning electron microscopy (SEM) of treated wool samples 3. Results and discussion To determine the extent of fibre damage and any changes to the external surface of the fibre, micrographs were taken using SEM of wool samples treated with wool polypeptide extract and compared with a wool sample which had only been alkali-scoured. The samples were prepared by attaching a double-sided adhesive carbon tab to an aluminium stub, then laying wool fibre across the sticky surface of the stub. For examination, the samples were sputter coated with gold under argon for 60 seconds using an Edwards ES150 sputter coater. Samples were examined using a Leo 430 scanning electron microscope operating with a working distance of 25 mm, an accelerating voltage of 7 kV and a magnification of 500×. 2.9. Whiteness A Datacolor SF6000 Plus CT reflectance spectrophotometer was used to determine the whiteness of the treated knitted wool fabric in terms of the CIE whiteness index. Each sample was folded into four and measured four times. All values were measured and calculated using ColorTools QC software with illuminant and observer conditions of D65 and 10◦ , respectively. 2.10. Dyeability The dyeability of the treated knitted wool fabric was tested using the reactive dye Lanasol Red CE. The fabric was processed for 30 min at 50 ◦ C in a dye-bath containing 2% owf acetic acid, 5% owf sodium sulphate and 1% owf levelling auxiliary, Albegal B (Ciba). 4% owf Lanasol Red CE was then added to the dye-bath and the temperature was raised to 100 ◦ C at a rate of 1.5 ◦ C/min and maintained at 100 ◦ C for 90 min. After dyeing the fabric was treated in a 1% owf ammonia solution (pH 8.4) at 80 ◦ C for 15 min. Dyeing took place in a Datacolor Ahiba Nuance IR dye machine at an agitation of 5 rpm and a liquor to goods ratio of 30:1. To test dye fixation, a piece of the dyed fabric was placed in a soaping solution containing 5 g/L Ultravon PL (Ciba) non-ionic surfactant and 5 g/L sodium carbonate at a liquor to goods ratio of 30:1 and treated at 100 ◦ C for 20 min using a Datacolor Ahiba Nuance IR dye machine. Dye exhaustion and total fixation efficiency were determined. Dye exhaustion (%E) is the percentage of dye absorbed on the fabric from the dye-bath solution using the exhaust method of dyeing. Total fixation efficiency (%T) is the percentage of the dye originally applied to the fabric which becomes bound covalently. The dyebath solutions sampled before and after the dyeing process and the solution after soaping were made up to the same fixed volume and absorbance measurements were taken at 510 nm, the wavelength of maximum absorption (max ) specific to Lanasol Red CE, using a Pye Unicam SP1800 UV/visible spectrophotometer. The percentage of exhaustion (%E) was calculated using Eq. (3) and the total fixation efficiency (%T) was calculated using Eq. (4). %E =
100 × (Abs0 − Abs1 ) Abs0
(3)
%T =
100 × (Abs0 − Abs1 − Abs2 ) Abs0
(4)
3.1. Shrink-resist treatment with polypeptides extracted from wool Protein polypeptide was extracted from wool fibre using sodium sulphite and Esperase. The enzyme was deactivated after extraction was complete. After centrifugation, the resulting supernatant liquid layer, containing the extracted protein polypeptide, was used neat to treat alkali scoured knitted wool fabric. The preferred fabric processing conditions were found to be 2 h at 60 ◦ C. The results in Table 1 show significant improvement in shrink resistance when the extracted wool polypeptide was used to treat knitted wool fabric; with an area shrinkage of 5.6% after 3 times 5A washes, a minimal weight loss (0.9%) during 3 times 5A washes and only minimal loss in burst strength when compared to the control samples. The effect of the components such as reducing agent, enzyme and polypeptide on the shrink-resistance of the treated knitted wool was investigated. 12 g/L sodium sulphite in a pH 8 phosphate buffer was heated to 60 ◦ C for 30 min without the presence of wool fibre snippets. The mixture was treated for a further 2 h with or without the addition of 18 u/g Esperase, then raised to 80 ◦ C to denature the enzyme. Both of these mixtures were used neat to treat alkali pre-scoured knitted wool fabric to determine whether the absence of extracted wool protein in the mixture affected the properties of the treated wool fabric. The respective area shrinkage of 10.6% and 9.8% after 3 times 5A washes show that the presence of extracted wool polypeptide with an area shrinkage of 5.6% does have an effect on shrink resistance. All the other properties, as shown in Table 1, are very similar. Wool fibre snippets were extracted in the presence of 18 u/g Esperase without the presence of sodium sulphite, following the extraction protocol, and separated by centrifugation. When the neat supernatant liquor was used to treat knitted wool fabric, the area shrinkage of the sample after 3 times 5A washes was 11.5%. This shows that sodium sulphite is required in the extraction process to extract the wool polypeptide effectively. It was considered whether fixation of the polypeptide to the fibre surface using a cross-linking agent would further improve shrink-resistance. Several cross-linking agents were considered including the difunctional epoxides; glycerol diglycidyl ether and 1,4-butandioldiglycidyl ether or the carbodiimide; 1(3-dimethylamino-propyl) 3-ethyl-carbodiimide hydrochloride. It was found that if after polypeptide extract treatment the wool fabric was transferred into a new bath containing 10 g/L glycerol diglycidyl ether (GDE) for 30 min at 60 ◦ C and cured at 140 ◦ C for 10 min, the shrink-resistance of the fabric improved to 3.6% area shrinkage after 3 times 5A washes (Table 2). Control samples show that GDE treatment has minimal effect on shrink resistance on samples that have not been treated with neat extracted polypeptide. 3.2. Shrink-resist treatment with enzyme active extracted wool polypeptides The shrink-resistance of wool treated with extracted wool polypeptides could be further improved if the enzyme used in the polypeptide extraction process was not deactivated prior to centrifugation therefore forming an enzyme active polypeptide
−0.5 (1.2) −0.1 (0.1) −1.1 (0.3) 0.9 (0.9) 9.8 (1.3) 10.6 (1.0) 11.5 (0.5) 5.6 (0.7) 406 (10) 385 (48) 423 (17) 371 (35) −0.2 (1.0) 0.5 (0.3) 0.4 (0.2) −0.2 (1.3) Treated with neat extract for 2 h at 60 ◦ C Non-ionic surfactant + Na2 CO3 30 min Without wool in 12 g/L sulphite 2 h 30 min Without wool in 12 g/L sulphite 30 min + 18 u/g Esperase 2 h Wool without sulphite 30 min + 18 u/g Esperase 2 h Wool in 12 g/L sulphite 30 min + 18 u/g Esperase 2 h
Weight loss (%)
0.6 (1.0) 0.4 (0.4) 18.4 (1.2) 13.7 (1.3)
Area shrinkage (%)
421 (15) 384 (34) Untreated No further treatment Untreated
– – Extract treatment Pre-treatment
No extract No extract
After 3× 5A washes Burst strength (kPa) Weight loss (%) Treatment of fabric Method for extraction of wool polypeptide
Table 1 The effect of wool peptide extraction and the extracted polypeptide treatment on alkali scoured wool fabric in terms of weight loss due to treatment, burst strength, area shrinkage and weight loss due to washing. Standard deviation in parentheses.
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extract. It was decided that 2 h treatment on knitted wool fabric with an enzyme active wool polypeptide would be too long causing excessive fibre damage, therefore the treatment time was reduced to 30 min. Residual enzyme present on the fibre may cause fibre damage during further processing or machine washing; however curing at 140 ◦ C after the cross-linking step would denature any residual enzyme present on the treated wool fibre. This was proven in the results in Table 3 with a much higher weight loss after 3 times 5A washes if the knitted wool fabric treated with enzyme active extracted wool polypeptide was not subsequently treated with GDE and cured at 140 ◦ C in a fan-assisted oven for 10 min. Almost complete shrink resistance was achieved if alkali pre-scoured knitted wool fabric was treated for 30 min with enzyme active extracted wool polypeptide followed by cross-linking with GDE for 30 min and a 10 min cure at 140 ◦ C, with 0.9% area shrinkage after 3 times 5A washes and 1.5% area shrinkage after 5 times 5A washes (see Fig. 1) with minimal weight loss due to washing. Even though the burst strength had decreased, the burst strength of 285 kPa was above the accepted burst strength of 225 kPa for Woolmark machine wash care standards for knitted sweaters (Woolmark Company, 2000). 3.3. Shrink-resist treatment with extracted wool polypeptides in the presence of protease Better shrink resistance results were observed on treated wool fabric if the enzyme used in the extraction process was not deactivated prior to use. However, for better control of the amount of enzyme present and an improved stability of the polypeptide extract the alternative was to maintain the deactivating step in the polypeptide extraction process prior to centrifugation but Esperase would be added to the neat polypeptide extract when used as a shrink-resistance treatment on wool samples. Different concentrations of Esperase were tested. Almost complete shrink-resistance could be achieved after 3 times 5A washes if 3 u/g or greater of Esperase was added to the neat extracted polypeptide when treated on alkali pre-scoured knitted wool fabric and subsequently crosslinked and cured (Table 4). No weight loss was observed after 3 times 5A washes for these samples. Almost complete shrinkresistance (0.4%) with no weight loss (−0.5%) was observed even after 5 times 5A washes. The greater the amount of native Esperase added to the extracted polypeptide treatment liquor, the lower the burst strength (Table 4). When 3 u/g of native Esperase was present in the deactivated polypeptide extract a similar level of shrink-resistance was achieved to treatment with enzyme active extracted polypeptide (seen in Table 3). A better burst strength was observed with the former (317 kPa) than the latter (285 kPa), indicating less damage to the fibre. This shows that adding enzyme to deactivated extracted polypeptide is easier to control as a shrinkresist treatment for wool than using the enzyme active extracted polypeptide. 3.4. Fibre surface, whiteness and dyeability of wool fabric treated with extracted wool polypeptides The external fibre surfaces of wool samples treated with extracted wool polypeptide followed by cross-linking with GDE and then cured were examined using SEM and compared with a wool sample which had only been alkali-scoured (Fig. 2a). SEM images of alkali pre-scoured wool followed by treatment with extracted polypeptide (Fig. 2b), with enzyme active extracted polypeptide (Fig. 2c) or with extracted polypeptide in the presence of 3 u/g native Esperase (Fig. 2d) all showed that the wool fibres were still intact and were not severely damaged by the shrink-resist treatment.
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Table 2 The effect of the addition of cross-linking agents and high temperature curing on the extracted wool polypeptide (and control) treatment processing of alkali scoured wool fabric in terms of weight loss due to treatment, burst strength, area shrinkage and weight loss due to washing. Standard deviation in parentheses. Weight loss (%)
Extraction method
Treatment with extract (or control) on wool fabric for 2 h at 60 ◦ C
Burst strength (kPa)
After 3× 5A washes
Area shrinkage (%)
Weight loss (%)
No extraction
Without cross-link and cure With cross-link and cure
– 1.4 (0.3)
384 (34) 330 (42)
13.7 (1.3) 12.9 (1.0)
0.4 (0.4) −0.5 (0.4)
Control without wool (12 g/L sulphite 30 min + 18 u/g Esperase 2 h)
Without cross-link and cure With cross-link and cure
0.7 (0.3) 1.0 (0.3)
385 (48) 371 (1)
10.6 (1.0) 9.0 (0.6)
−0.1 (0.4) −0.3 (0.4)
Extracted wool polypeptide (12 g/L sulphite 30 min + 18 u/g Esperase 2 h)
Without cross-link and cure With cross-link and cure
−0.2 (1.3) 0.6 (0.3)
371 (35) 370 (30)
5.6 (0.7) 3.6 (1.1)
0.9 (0.9) −0.2 (0.4)
Table 3 The effect of enzyme active extracted wool polypeptide treatment on alkali scoured wool fabric in terms of weight loss due to treatment, burst strength, area shrinkage and weight loss due to washing. Standard deviation in parentheses. Weight loss (%)
Extraction method
Treatment with extract (or control) on wool fabric for 30 min at 60 ◦ C
Area shrinkage (%)
Weight loss (%)
Enzyme active without wool (12 g/L sulphite 30 min + 18 u/g Esperase 2 h) Enzyme active extracted wool polypeptide (12 g/L sulphite 30 min + 18 u/g Esperase 2 h)
With cross-link and cure
8.2 (0.1)
278 (20)
2.4 (0.9)
1.3 (0.2)
Without cross-link and cure With cross-link and cure
4.3 (0.4) 7.1 (1.6)
316 (24) 285 (30)
4.0 (0.5) 0.9 (0.5)
8.3 (1.2) 1.3 (0.6)
An improvement in whiteness was observed for wool treated with the extracted polypeptide (Table 5). A drawback of the conventional chlorine-resin methods of achieving shrink-resistance is significant yellowing of the wool fibre due the use of chlorine or the disodium salt of dichloroisocyanuric acid (DCCA) (Lewis, 1992; Millington, 2009). A higher level of whiteness was found for wool treated with the enzyme active extracted polypeptide than the comparable treatment with deactivated extracted polypeptide followed by cross-linking and curing. This would be expected due to the enzyme being active in the polypeptide, as an improvement in whiteness is usually observed when protease has been used to treat
Burst strength (kPa)
After 3× 5A washes
wool (Zhang et al., 2006; Smith et al., 2010b). A similar improved level of whiteness was observed for wool treated with deactivated extracted wool polypeptide in the presence of 3 u/g or greater of native Esperase followed by cross-link and cure as for wool treated with enzyme active extracted wool polypeptide followed by crosslink and cure. It was noticed that the greater the amount of native Esperase added to the extracted polypeptide treatment liquor, the greater the CIE whiteness (data not shown). To examine its dyeability, wool fabric which had been treated with polypeptide extract was dyed with the reactive dye Lanasol Red CE at 100 ◦ C following standard dyeing protocols. After
Fig. 1. Photograph comparing untreated knitted wool and processed knitted wool (alkali scour pre-treatment followed by 30 min treatment with enzyme active extracted wool polypeptide then followed by cross-link and curing) before and after washing.
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139
Fig. 2. (a–d) Scanning electron micrographs (SEM) of wool fibres (500× magnification) from treated knitted wool fabric samples: (a) alkali scoured only; (b) alkali scour pre-treatment followed by 2 h treatment with extracted wool polypeptide then followed by cross-link and curing; (c) alkali scour pre-treatment followed by 30 min treatment with enzyme active extracted wool polypeptide then followed by cross-link and curing; and (d) alkali scour pre-treatment followed by 30 min treatment of extracted wool polypeptide in the presence of 3 u/g Esperase then followed by cross-link and curing.
Table 4 The effect of the addition of different concentrations of Esperase on the extracted wool polypeptide (and sodium sulphite control) treatment processing of alkali scoured wool fabric in terms of weight loss due to treatment, burst strength, area shrinkage and weight loss due to washing. Standard deviation in parentheses. Extraction method
Treatment with extract (or control) on wool fabric with or without additional Esperase for 30 min at 60 ◦ C, followed by cross-link and cure
Weight loss (%)
Burst strength (kPa)
Control without wool (12 g/L sulphite 2 h 30 min)
Without Esperase With 3 u/g Esperase With 6 u/g Esperase
−0.3 (0.3) 9.3 (0.7) 12.7 (1.0)
377 (13) 246 (21) 167 (13)
11.7 (0.1) 2.4 (0.6) 2.4 (0.5)
0.9 (0.1) 2.1 (0.2) 4.9 (0.3)
Enzyme active extracted wool polypeptide (12 g/L sulphite 30 min + 18 u/g Esperase 2 h) deactivated
Without Esperase With 1.5 u/g Esperase With 3 u/g Esperase With 6 u/g Esperase
−0.1 (0.5) 3.7 (0.2) 5.5 (0.2) 7.6 (0.2)
379 (13) 345 (30) 317 (11) 272 (19)
7.8 (0.5) 3.6 (1.4) 1.1 (0.4) 0.8 (0.9)
−0.8 (0.2) −0.7 (0.6) −0.2 (0.2) 0.8 (0.5)
After 3× 5A washes
Area shrinkage (%)
dyeing the sample treated with the polypeptide extract but not cross-linked was less shrink-resistant with an area shrinkage of 11% compared to an area shrinkage of 5.6% for the corresponding undyed sample, both after 3 times 5A washes (data not shown). It would appear that the high temperature the sample was subjected to during dyeing caused the loss of polypeptide from the fibre surface therefore losing the shrink-resistant effect. When the sam-
Weight loss (%)
ple treated with polypeptide extract followed by cross-linking with GDE and curing at 140 ◦ C was subsequently dyed with the reactive dye Lanasol Red CE the shrink-resistance only slightly decreased to an area shrinkage of 5.5% compared to an area shrinkage of 3.6% for the corresponding undyed sample, both after 3 times 5A washes (data not shown). This shows that cross-linking is required to fix the polypeptide onto the fibre surface to ensure shrink-resistance
Table 5 CIE whiteness index values and dyeability with 4% (owf) Lanasol Red CE of alkali scoured wool fabric treated with extracted wool polypeptide in terms of dye exhaustion and fixation. Standard deviation in parentheses. Extract treatment on alkali scoured wool fabrica
CIE whiteness index
No further treatment 2 h treatment with extracted polypeptide (deactivated) 30 min treatment with extracted wool polypeptide (enzyme active) 30 min treatment with extracted wool polypeptide (deactivated) and 3 u/g Esperase
−0.23 (3.41) 4.83 (0.68) 13.43 (1.56) 13.17 (2.91)
%E = dye exhaustion, %T = total fixation efficiency. a All samples treated with extracted polypeptide were subsequently cross-linked with GDE then cured at 140 ◦ C.
Dyeability %E
%T
99.5 99.1 99.4 99.3
88.8 89.6 91.8 86.1
140
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properties are maintained after dyeing. When the sample treated with extracted wool polypeptide in the presence of 3 u/g of native Esperase followed by cross-link and cure was subsequently dyed with 4% owf Lanasol Red CE at 100 ◦ C, there was only minimal loss in shrink resistance after 3 times 5A washes, from 1.1% for the undyed sample to 1.5% for the dyed sample. Treatment of alkali pre-scoured wool with either extracted polypeptide, enzyme active extracted polypeptide or extracted polypeptide in the presence of 3 u/g native Esperase does not affect the dyeability of wool as the results in Table 5 show very similar results with respect to dye exhaustion and fixation as for alkali scoured wool fabric after dyeing with 4% owf Lanasol Red CE. The excellent wash-fastness properties to BS EN 20105:CO1 conditions of the dyed control sample were maintained after dyeing of alkali pre-scoured wool which had been treated with either extracted polypeptide, enzyme active extracted polypeptide or extracted polypeptide in the presence of 3 u/g native Esperase followed by cross-linking and curing, with no staining (greyscale rating of 5) on the multifibre strip. 4. Conclusions Almost complete shrink-resistance can be imparted on knitted wool fabric when treated with protease extracted wool polypeptide followed by cross-linking with glycerol diglycidyl ether and a high temperature cure. An area shrinkage of less than 2% can be achieved after 5 times 5A washes with minimal weight loss during washing. Knitted wool fabric treated by this shrink-resist method meets burst strength standards for knitted wool garments, has an improved whiteness and a soft handle. The treated wool fabric is dyeable with reactive dye and as long as the polypeptide extract is affixed to the wool fibre, a high level of shrink-resistance can be maintained after dyeing. Acknowledgements We would like to thank the East Midland Development Agency (EMDA) and the European Regional Development Fund (ERDF) for funding the Higher Education Innovation and Regional Fellowships (HIRF 385 and HIRF 447F) to undertake this research. References Cavaco-Paulo, A., Silva, C.J.S.M., 2003. Treatment of animal hair fibres with modified proteases. World Patent WO03097927. Cortez, J., Anghieri, A., Bonner, P.L.R., Griffin, M., Freddi, G., 2007. Transglutaminase mediated grafting of silk proteins onto wool fabrics leading to improved physical and mechanical properties. Enzyme Microb. Technol. 40, 1698–1704.
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