ENZYMATIC TREATMENT OF WOOL FABRICS WITH LIPASE IN THE IMPROVEMENT OF SOME PROPERTIES OF WOOL FABRICS

The enzymatic treatment of textiles signifi cantly improves some of their physicochemical properties as well as increases their aesthetic values and comfort of use. Enzymes can be used in order to develop environmentally friendly processes by reducing the concentration of chemical agents, water and energy consumption. Wool fabrics were pretreated with hydrogen peroxide in the presence of stabiliser imino disuccinic acid sodium salt (IDAS). The eff ect of after-treatment of the pre-oxidised wool fabric with commercially produced lipase enzyme on its dyeability with acid dyes, as well as on some of its physico-mechanical properties, was assessed. Wool fabrics treated with hydrogen peroxide ⁄ imino disuccinic acid sodium salt ⁄ lipase enzyme exhibit improved wettability and, hence, dyeability with acid dyes, as well as enhanced resistance to felting shrinkage and pilling.


INTRODUCTION
Textile industry use various chemicals in diff erent processes and can often have a polluting eff ect on the environment. The fi nishing of textile materials generates a large amount of wastewater, which contains organic as well as inorganic residues. Dyehouse effl uent that has acidic or alkaline pH values has also often been observed. The search for new, effi cient and eco-friendly alternatives have increased interest in using green catalysts i.e., enzymes. However, with introduction of enzymes in textile industries are ensuring eco-friendly processing of textile products. In general, the use of enzymes leads to a reduction in water and energy consumption. The enzyme treatment not only keeps the fabric looking new after repeated washings, but enhances feel, color, softness and drapeability which translates into a higher quality textile or apparel product [1,2,3,4].
Enzymes are eco-friendly natural macromolecules (biological catalysts) used in the textile industry since 1960`s. Enzymes have been used for desizing, scouring, polishing, washing, degumming and bleaching of fabrics as well as for decolouring of dyehouse wastewater. A catalyst is any substance which makes a chemical reaction go faster, without itself being changed. All enzymes are made of protein and because they are sensitive to heat, pH and heavy metal ions. Enzymes are completely biodegradable and accomplish their work quietly and effi ciently without leaving and pollutant behind [5].
Lipase enzyme has also found use as a catalyst for production of diff erent products used in cosmetic industry, pulp and paper industry, textile industry, degreasing of leather and also in pharmaceutical industry [6]. Bacterial lipases are glycoproteins, but some extracellular bacterial lipases are lipoproteins. Most of the bacterial lipases reported so far are constitutive and are non-specifi c in their substrate specifi city and a few bacterial lipases are thermostable. Among bacteria, Achromobacter sp., Alcaligenes sp., Arthrobacter sp., Pseudomonas sp., Staphylococcus, Serratia sp. and Chromobacterium spp. have been exploited for the production of lipases [7].
Environmental protection is becoming a serious concern for textile wet-processors. Many enzyme-assisted textile processes were envisaged as environmentally acceptable alternatives [8]. Lipase enzyme action concentrates on the hydrolysis of fats, especially triglycerides and fatty acids. However, lipase acts on the bonds between protein and lipids on the surface of wool, breaking its hydrophobic barrier [9]. Most of these processes focused on the use of lipases in the modifi cation of wool fi bres. The improvement in the dye ability of wool fi ber due to enzyme pretreatment may be varied according to the characteristics of wool fi ber and dye. Enzyme can digest the amorphous region and induced the dyeing rate positively and decreased the affi nity for small molecular weight dye molecule like leveling acid dye. Lipase enzyme treatment is considerably improved the dye ability of wool fi ber due to degradation of the hydrophobic F-layer and so the accessibility of the fi ber to the aqueous dye liquor increased [10].
Wool is a product of nature, it is formed in the proper skin of a sheep. Wool is a complex natural fi bre composed mainly of proteins (97%) and lipids (1%), with a heterogeneous morphological structure [11]. It is built from a protein called keratin which is formed in the process of biosynthesis of α-amino acids. Wool fibre is in fact a creation of dead cells. The wool forming cells show diverse structures, shapes and properties. Chemical modifi cation of hydrophobic surface structure of wool fi bre leads to the improvement of the functional quality of these fi bres. When wool is used as substrate for enzyme catalyzed reactions, a special type of enzyme kinetics is followed. Enzyme diff usion plays a much more decisive role in this heterogeneous system of soluble enzyme and solid substrate. The kinetics does not only depend on the concentration of the reaction partners, the temperature and the pH value of the liquor, but also on the diff usion of the enzyme to, and into, the solid phase of the substrate and the diff usion of the reaction products out of the solid phase into the liquor. Alkaline peroxide pretreatment improves enzyme diff usion inside wool fi bre [12]. Even a mild enzyme treatment of wool can improve the dye ability without causing signifi cant fi ber damage [13]. Cegarra et al. (1992) stated that the enzyme treated wool had more amine terminal groups than untreated wool, so enzyme treted wool fi ber capable of attracting the dye as well as dyed more intensely dyed than the untreated fi bers [14].
Particularly, the application of enzymatic treatments in the fi nishing of wool fabrics to improve some of their properties is a topic in which research has been intensifi ed in the last years. One of the main aims is the study of fi nishing processes more environmentally respectful than those traditionally used.
Enzymes are used to improve the softness or to improve the effi ciency of the subsequent fi nishing process either as pretreatment and enzyme-assisted fi nishing or bio-fi nishing. For wool top enzyme treatment can be given to impart shrink-resistance fi nish and for woolen fabric, it can be given to improve the dye ability. Nowadays enzymatic treatment of woolen fabric can consider as an alternative shrink proofi ng process [15]. El Sayed et al. (2002) developed an enzyme-based anti-felting process with the objective of developing an AOX-free process [16]. They also proposed the mechanism for shrink resistance imparted by this enzyme combination. The lipase removed lipids from the outer surface of wool fi ber.
The wool industry is facing many challenges, amongst others, competition from other fbres, diminishing consumption, environmental pressures, etc. Innovative methods and processes should continuously be developed to produce products which will improve the competitiveness of the industry. Particularly, the application of enzymatic treatments in the fnishing of wool fabrics to improve some of their properties is a topic in which research was intensifed in the last years. One of the main aims is the study of fnishing processes more environmentally respectful than those traditionally used [17].
The present work aims to utilise stabilised hydrogen peroxide (H 2 O 2 ) followed by lipase enzyme to improve some properties of wool, such as shrink proofing and enhanced dyeability. Sigma-Aldrich, Germany. It is a light brown powder, preparation and produced by submerged fermentation from Rhizopus oryzae, with a declared activity of 30 units/mg. One unit corresponds to the amount of enzyme which releases 1 μmol fatty acid from triglycerides per minute at pH/ 7.2 and 37°C (olive oil as substrate). Process Parameters: Optimum temperature/ 40°C; pH/ 6.5-7.5 (optimum 7.2); Time/ 30-60 min; Dosage/ 0.5%-2.0% owm (on the weight of the material). Storage temperature 2-8°C.
 All other chemicals used in this study were of analytical grade.
 Four acid dyes were used in this investigation (Table 1). They were of commercial grade.

Treatment methods
Scouring. Before any fnishing treatment, the wool fabric must be scoured in order to remove the natural and any oily lubricants added before the carding and combing processes that can hinder the successful operation of application of enzyme. Pretreatment of wool fabric with sodium carbonate was carried out in the bath (the material liquor ratio 1:30) containing 1.8 g/L sodium carbonate and 1.5 g/L Sandozin NRW (nonionic agent) at 60°C for 30 min, followed by thorough rinsing with cold water and fnally air-drying at an ambient temperature.
Bleaching. Wool fabric was treated with 40 ml/ lLhydrogen peroxide solution (30% w ⁄w) at 65°C and pH/8 (adjusted by sodium carbonate) for 90 min. Treatment of wool fabric with lipase. After the pretreatments, wool fabric samples were treated with the enzyme Lipase F-AP 15 in the bath (the material liquor ratio 1:30) containing varying amounts of lipase (0.5, 1 and 2% owm). The treatment was carried out at 50°C and pH/ 7 for 1 h; the liquor ratio was 30:1. Treated samples were washed at 40°C and then treated at 80°C for 10 min to deactivate the enzyme. This was followed by thorough rinsing with distilled water and, fi nally, air-drying at room temperature.
Dyeing procedures. The dyeing of untreated wool, as well as that treated with hydrogen peroxide/ IDAS or hydrogen peroxide/IDAS/lipase, was carried out using the exhaustion technique.
Dyeing of wool fabric with acid dyes. The dyeing of untreated as well as treated wool fabrics was carried out using 1.5% owm concentration of acid dyes 1, 2, 3 or 4, using a liquor ratio of 30:1, in the presence of 10-15% Glaubers salt, 1.5 g/L Sandozin NRW (nonionic agent) with pH value of the bath 4.5-5.5, at 90 °C for 100 min. For adjusting the pH in bath, appropriate amount of sodium carbonate or acetic acid were used. The conditions of dyeing are illustrated in Table 2. Wool fabric was immersed in the dye bath containing the above recipe and was stirred for 15 miutes. The dye bath was heated to 90°C, was added sodium sulphate (Glaubers salt) and the temperature was maintained for another 100 minutes with stirring. The dyed wool fabric was rinsed thoroughly with running water and air-dried.

Analytical methods
All the samples (control-untreated fabric, pretreated and enzyme-treated) were subjected to various testing for physicochemical properties in order to check the effect of treatment on these properties. Prior to the measurements, samples were conditioned for 24 h at 20°C and 65% relative humidity. The tests included: Whiteness index -The degree of whiteness of the wool fabrics was measured with a spectrophotometer (Datacolor) using the CIE method according to EN ISO 105-J02:1997(E) standard. The degree of whiteness of the wool fabrics was given as W-CIE. Each value is an average of three measures determined on diff erent positions on the wool sample.
Weight loss -The weight loss as result of the oxidative and enzymatic treatments was determined by weighing the fabric samples before and after treatment and was expressed in percent. All samples with approximate dimensions of 10 × 10 cm were left in oven at 105 ± 2°C for 2 h then placed in a desiccator for balancing and weighted until constant weight. Assays were performed in duplicate. The weight loss of each sample was calculated as follows, shown in Eqn 1:

Weight loss (%) =
(1) where W 1 : the dry weight of fabric sample before treatment and W 2 : the dry weight of the same fabric sample after treatment.
Alkali solubility -The alkali solubility of the untreated as well as some of the treated fabrics was evaluated using the standard method ASTM D1283-85. One gram of wool sample afer being dried in an oven at 110 °C for 15 min was weighed and then was put into a beaker containing 100 ml of sodium hydroxide of 0.1 N at 65 °C for 1 h. Then the contents were fl tered and rinsed with distilled water for 5 min and dried in an oven at 110 °C for 1 h. Afer being desiccated, the alkali solubility of the samples was calculated as a percentage of the original weight according to the following relation, shown in Eqn 2: Alkali solubility (%) = (2) where S 1 : the dry weight of sample before sodium hydroxide treatment and S 2 : the dry weight of sample afer sodium hydroxide treatment. Values are the average of three replicates.
Water absorbency -The absorbency of the treated as well as the untreated wool fabrics was estimated according to AATCC Test Method 79-2010 standard test conditions. This test method is designed to measure the water absorbency of textiles by measuring the time it takes a drop of water placed on the fabric surface to be completely absorbed into the fabric. A drop of water was allowed to fall from a fi xed height onto the taut surface of a test specimen. A measured drop of water is placed on the fabric 1 cm from the surface. Time is recorded until the water drop absorbs completely. Values are given as the mean of three replicates.
Felting shrinkage -Fabrics of wool and related animal fbres will shrink when subjected to alternating compression and relaxation in aqueous medium. This contraction which is known as milling or felting shrinkage is due to the migration of the fbres, their unique scale structure and elasticity. To do this, the wool fabric samples were frst marked 10 × 10 cm. Then they were subjected to milling under laboratory conditions in a soap bath of 1% (the material liquor ratio 1:40) for 45 min at 50°C. Te milling solution was prepared as follows: 10-g soap flakes are dissolved in hot water and diluted to 1 L using a graduated cylinder. Afer this treatment, the samples were removed from the soap solution, rinsed in cold water and dried, and then they were laid flat on the bench and measured, with a steel ruler. If the length is a 1 (cm) and the breadth b 1 (cm), before treatment, Initial area (I 1 ) = a 1 × b 1 (cm²). If the length is a 2 (cm) and the breadth b 2 (cm), afer treatment, fnal area (I 2 ) = a 2 × b 2 (cm²) [17]. The felting shrinkage was assessed using the following relation, shown in Eqn 3:

Felting shrinkage (%) = (3)
Pilling resistance -Pilling resistance of fabric samples was determined according to BS EN ISO 12945-2:2000 (Modifed Martindale method) using a pilling tester. This method is intended for the determination of the resistance to pilling and surface change of textile fabrics. The standard atmosphere for conditioning and testing textilesas directed in ISO 139 was used, i.e. 65 ± 2% relative humidity and 20 ± 2 °C temperature. The samples are visual assessment on a scale from A, B to C where: sample A is best (No change, for zero pills); B -for three to fi ve pills and sample C for more than fi ve pills.
Tensile strength loss -Tensile strength of a woven fabric is one of the most important mechanical properties. Tensile strength is the ratio between the maximum load that a material can support without fracture and the original cross-sectional area of the material. The tensile strength of the fabric samples was determined according to ASTM D5035-90 (strip test) with an Instron Tensile Tester Model 4411 (Instro Inc., USA) under standard atmospheric condition (65 ± 2% relative humidity and 21 ± 1°C temperature). Sample size had a width of 5 cm and length 18 cm. Each specimen was mounted centrally in the pneumatic flat surface clamps of the tensile testing machine and tested. The tensile strength loss is then calculated as percentage of the original sample tensile strength. Te tensile strength loss values are given as the mean of 10 replicates.

Tensil strength loss (%) =
where T 1 : tensile strength of untreated sample and T 2 : tensile strength of sample afer treatment.
Colour fastness to laundering -The colour fastness of the dyed wool fabrics to washing was determined according to AATCC test method 61-2007 (Color fastness to laundering: accelerated) using a laboratory laundrometer. Evaluate the colour change of the test specimens as directed in ATTCC Evaluation Procedure using the Gray Scale for Colour Change.

Eff ect of treatment of wool fabric with the hydrogen peroxide/IDAS/lipase system
Wool fabrics were treated with hydrogen peroxide in the presence of stabiliser IDAS. Pretreatment of wool fabric with hydrogen peroxide is essential for improving its degree of whiteness and for preparing the fabric for subsequent enzymatic treatment. The improvement in the degree of whiteness of the oxidised wool fabrics, compared with the untreated fabric, was used as a measure of the effi ciency of the bleaching bath in the oxidation of the wool surface. The whiteness index of the treated fabrics was then assessed. The degree of whiteness of the wool fabrics treated with hydrogen peroxide in the presence of IDAS was 44.3 compared with 42.3 and 30.1 for those samples treated with hydrogen peroxide alone and untreated, respectively. Wool fabrics, pre-oxidised by hydrogen peroxide in the presence of IDAS, were after-treated with lipase enzyme at diff erent concentrations (0.5%, 1% and 2% owm) at optimum pH and temperature (pH 7 and 50°C) for 1 h. The eff ect of these treatments on the degree of whiteness, felting shrinkage and weight loss were assessed. Results in Table 3 show that the treatment of wool fabric with the hydrogen peroixde/IDAS/lipase system increased its degree of whiteness compared with the untreated sample. The treatment of wool with the hydrogen peroxide/IDAS/ lipase enzyme increases its resistance to felting shrinkage as the enzyme concentration increases up to 1% owm. A further increase in the enzyme concentration has no eff ect on the felting resistance of wool fabrics.
The eff ect of treatment of wool with hydrogen peroxide/IDAS/lipase (1% owm) on some physicochemical properties was assessed and is summarised in Table 4.
The data in Table 4 show that the alkali solubility of the enzyme-treated wool fabrics increased to 13.2%, compared with 10.8% for the untreated sample, and reached 17.9% in the case of enzyme-treated fabrics. Values are within the acceptable limit. Also data from table show that the water absorbency of the wool fabrics is highly enhanced by treatment with the hydrogen peroxide/IDAS/lipase system (23 s) compared with 2300 s and 1420 s in the cases of the untreated and the hydrogen peroxide-treated wool fabric, respectively. Treatment of the wool fabric with the hydrogen peroxide/IDAS/lipase enzyme system lowered its tendency to form pills. Treatment of wool  with the hydrogen peroxide/IDAS/lipase system resulted in a remarkable decrease in the tensile strength of the fabric.

Eff ect of treatment of wool fabric with the hydrogen peroxide/IDAS/lipase system on wool dyeing with acid dyes
The wool fabrics were dyed with four diff erent acid dyes. Untreated and treated wool fabrics were dyed in the same bath for 45 min. The dyed samples were removed from the dyeing bath and washed with cold water, squeezed and dried. The colour fastness to laundering of the dyed untreated wool fabrics as well as the treated wool fabrics were assessed and are shown in Table 5. The data in this table illustrate that wool fabrics treated with the hydrogen peroxide/IDAS/lipase system and dyed with acid dyes have slightly better colourfastness to laundering.