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Abstract: In this study, the effects of enzymes on wool yarn in the presence of different salts (ammonium sulfate, sodium tet- raborate, and di-sodium hydrogen ...
Fibers and Polymers 2009, Vol.10, No.5, 611-616

DOI 10.1007/s12221-010-0611-x

Proteolytic Enzymes Reactions on Wool Yarn and Surfactants Effects on the Enzyme Treatment Mokhtar Arami*, Firozmeher Mazaheri, and Maryam Jafar Beglou

Department of Textile Engineering, Amirkabir University of Technology, Tehran, Iran

(Received July 30, 2008; Revised April 6, 2009; Accepted April 12, 2009)

Abstract: In this study, the effects of enzymes on wool yarn in the presence of different salts (ammonium sulfate, sodium tet-

raborate, and di-sodium hydrogen phosphate) were investigated. Alcalase 2,5 LDX and Savinase 16 LEX were selected as proteolytic enzymes. In addition, the effects of surfactants (Sandozin NRW, Irgasol NA, Erkantol NR, and Sandozin EH) on the enzyme treatment of wool yarn were evaluated. The results indicated that the effects of enzymes on wool yarn were greatest in the presence of sodium tetraborate. Furthermore, the properties of wool yarn such as weight loss, strength loss, and hairiness were influenced by these surfactants. The SEM images of the treated samples confirmed the obtained results. Keywords: Proteolysis, Enzyme, Protein, Protease, Surfactant, Wool

NRW, Irgasol NA, Erkantol NR and Sandozin EH) on the enzyme treatment of wool yarn were studied.

Introduction

There is considerable interest in the use of enzymes in textile industries to achieve a variety of finishing effects on fiber such as wool [1-6]. Several kinds of enzymes such as amylase, cellulase, peroxidase, protease, pectinase, and lipase are used in enzymatic treatments of textiles [7]. It is well documented that most of fiber treatment processes in textile industries are environmentally unacceptable. In this respect, enzymatic treatments are getting strong motivation for replacement of those processes because of being less harmful to the environment. Moreover, substrate-specific enzyme reactions offer the possibility to control chemical reaction so that no undesirable side reactions take place [8-12]. Although there is currently considerable attention in the use of enzymes to achieve a variety of finishing effects on wool, it is apparent that the results of enzymatic treatments, especially with proteases, can be unpredictable and may sometimes cause unacceptable degradation of fiber [13-22]. For this purpose, some researches on water soluble-insoluble reversible polymers and non-ionic surfactants reverse micellar systems were carried out to improve protease finishing effects on wool [1-6]. Literature review showed that enzymatic hydrolysis reactions were investigated by several researchers [23-28] but the effects of those enzymes in the presence of different salts and surfactants (Sandozin NRW, Irgasol NA, Erkantol NR, and Sandozin EH) on the enzyme treatment of wool yarn were not studied. In this paper, the effects of enzymes (Alcalase 2,5 LDX and Savinase 16 LEX) on wool yarn were studied in the presence of different salts and ionic strengths for achieving more suitable stiffness without causing unacceptable weight loss, strength loss, and cuticular damage. In addition, the effects of those enzymes in the presence and absence of various surfactants (Sandozin

Materials and Methods

The wool of Merino sheep was used as starting material. Two-folded yarn of resultant linear density 20 metric was spun by Toos Co. (Iran) from wool tops of 23 µm nominal mean fiber diameters and folded in 6 turn per inch. Two serine endoproteases, Alcalase 2,5 LDX and Savinase 16 LEX, were supplied by Novo Nordisk A/S (Denmark). Sandozin NRW (nonionic surfactant, density 0.98 g/ml, supplied by Clariant Co.), Sandozin EH (anionic surfactant, density 1.07 g/ml, supplied by Clariant Co.), Irgasol NA (nonionic surfactant, density 1.00 g/ml, supplied by Ciba Co.), and Erkantol NR (nonionic surfactant, density 1.00 g/ ml, supplied by Rezin Saveh Co.) were used as surfactants. All other chemicals were purchased from Merck (Analar Grade). Hanks of yarn (2 g) were treated for 30 min at 60 oC in an Ahiba 1000 Turbomat hank sample dyeing machine at LR (liquor to fiber ratio) 40 ml/g in aqueous solutions containing Irgasol NA (1.2 ml/l) and sodium carbonate (0.2 g/l or 1.6 g/ l). Alternatively, hanks were similarly treated in solutions containing Irgasol NA (1.2 ml/l), sodium sulfite (6.3 g/l), and sodium carbonate (1.6 g/l). These pretreatments were followed by rinsing in a large volume of distilled water. Then all samples were allowed to drain, de-watered by centrifuging, and dried at ambient temperature. After the pretreatments, yarn samples were treated at LR 40 ml/g in 0.1 M ammonium sulfate (ionic strength 0.3 M) and mentioned surfactants (If required) in the presence of Alcalase 2,5 LDX or Savinase 16 LEX (0.36 ml/g wool). Then, other hanks were treated in similar solutions in the presence of sodium tetraborate (ionic strength 0.3 M) or disodium phosphate (ionic strength 0.3 M). In addition, other samples were treated under similar conditions over various

*Corresponding author: [email protected] 611

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ranges of ionic strengths. The applied pH for each enzyme was selected according to the maximum activity of specific enzyme as recommended by the company (pH 8-8.5). Appropriate amount of sodium carbonate or acetic acid was used to adjust the pH. The treatment temperature was first set at 40 oC for 20 min and then raised to 57 oC (for Alcalase 2, 5 LDX) and 53 oC (for Savinase 16 LEX) at the rate of 1 oC/min and maintained for 60 min. These treatments were carried out in 40 rpm in an Ahiba Turbomat hank sample dyeing machine. Samples were washed with Irgasol NA (0.25 ml/l) at pH=5 and 40 oC. Then the hanks were allowed to drain, dewatered by centrifuging, and then dried at ambient temperature. All samples were conditioned in oven at 60±2 oC for 30 min then put them in a desiccator for 15 min and weight before and after treatment. The weight loss of each sample was calculated as: Weight loss % = 100(W1 – W2)/ W1 where W1: the conditioned weight of sample before enzymatic treatment and W2: the conditioned weight of sample after enzymatic treatment. The strength loss and elongation of wool yarns were determined using an Uster Dynamate II equipped with a load cell having a maximum capacity of 100 N. All samples were conditioned as mentioned above. Yarn gauge length was 50 cm and break time was 20 s. Thirty repeated measurements per sample were made. Hairiness (hairs/100 cm) of yarns was measured using a Shirley hairiness tester. The surface morphology of wool yarn and degradation of cuticle layer after enzymatic hydrolysis were investigated using SEM micrographs (Scanning Electron Microscope Philips XL 30). Result and Discussion

Impact of Enzymes

Figure 1 shows that weight loss sharply increased for both Alcalase and Savinase at low concentrations, while by further increasing the concentration, the effect on weight loss becomes constant (0.36 ml/g wool). In addition, Savinase shows more weight loss than Alcalase at the same condition. It seems that these differences are for specific behavior of each enzyme for hydrolysis of specified peptide units on protein chain. These results were comparable with the findings of other researchers [29-34].

The Effects of Ionic Strengths and Types of Saline Solutions

For assessment of saline ionic strength, samples were treated with Alcalase (0.36 ml/g wool) at different ionic strengths (0.03-0.60 M). The results showed that weight loss of samples decreased by increasing ionic strength. Therefore, it can be concluded that the enzymes activity is decreased by

Mokhtar Arami et al.

Comparison of different concentrations effects of Alcalase and Savinase in ammonium sulfate on weight loss of wool yarns pre-scoured with sodium carbonate (0.2 g/l). Figure

1.

The effects of different salts (ionic strength 0.3 M) on weight loss of wool yarns at different enzyme concentrations. Figure 2.

increasing ionic strength of the solution. This reduction of the enzyme activity might be attributed to decrease in the mobility of the enzyme molecules due to the presence of high concentration of ammonium sulfate. Figure 2 shows the effects of different salts (ammonium sulfate, sodium tetraborate, and di-sodium hydrogen phosphate) on weight loss of wool samples pre-scoured with sodium carbonate at various concentrations of Alcalase. The similar trend was observed with Savinase at the same condition. These remarkable activities of Alcalase and Savinase in sodium tetraborate may be attributed to the favorable change in the enzyme protein conformation when it interacts with tetraborate ions [15]. It is well known that enzyme activity depends upon the enzyme adsorption onto the substrate so that the reactive cleft in the enzyme protein fits precisely around the reaction site on the substrate [35,36]. Therefore, the particular combination of size and charge of adsorbed tetraborate ions may induce steric changes in the reactive cleft of the enzyme protein, which facilitate this “lock-andkey” adsorption-reaction mechanism [15].

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Proteolytic Enzymes Reactions on Wool Yarn and Surfactants

Effect of Pretreatments

The results showed that weight loss of samples by Alcalase treatments was negligible without pretreatment. The weight loss increased rapidly by adding a little amount of sodium carbonate in pretreatment but after 1.6 g/l concentration became constant. Figure 3 shows the effects of different concentrations of Alcalase on weight loss of wool yarn samples treated under different conditions (sodium carbonate (0.2 and 1.6 g/l) and sodium sulfite (6.3 g/l)). As it was seen in Figure 3, weight loss rose up and wool was damaged by increasing the sodium carbonate and using sodium sulfite for pretreatments. For the case of sodium sulfite pretreatment, the increasing of weight loss of samples might be attributed to the cystine (disulfide) cross-link breakage which makes wool more susceptible for enzymatic hydrolysis. In addition, similar results were obtained by using Savinase (Figure 4). The high weight loss for sodium carbonate pretreatment was attributed to the alkaline hydrolysis of protein.

The effects of different concentrations of Alcalase enzyme on weight loss of wool yarn samples treated under different conditions ( △) untreated sample, ( □ ) sodium carbonate (0.2 g/l), ( ▲) sodium carbonate (1.6 g/l), and ( ■ ) sodium sulfite (6.3 g/l). Figure

Effect of Surfactants

Figure 5 shows the surfactant effect on enzymatic treatment of samples pre-treated by sodium carbonate (0.2 g/l), then treated in enzyme (0.36 ml/g Wool) in the presence of nonionic surfactants (Sandozin NRW, Erkantol NR, and Irgasol NA) and anionic surfactant (Sandozin EH) at same concentration (1 g/l). The similar trend was obtained for Savinase enzymatic treatment of samples under the same condition. It was observed that the surfactant effect in enzymatic treatment on weight loss of samples were not significant, while their effects on other properties of wool such as hairiness and strength loss were considerable. The hairiness of wool samples was reduced by using Alcalase and Savinase in comparison to the untreated samples (Figure 6). This reduction was greater for the case of Savinase incorporated with surfactant solution. The results showed that the hairiness of samples depended on enzyme and surfactant effects. In this investigation, the parameters such as strength loss, elongation, and coefficient variant for all samples treated in several conditions were determined. Figure 7 shows that the presence of surfactants (Erkantol and Sandozin) in enzymatic

3.

The effects of different concentrations of Savinase enzyme on weight loss of wool yarn samples treated under different conditions ( ◆) untreated sample, ( ■ ) sodium carbonate (0.2 g/l), and (△) sodium carbonate (1.6 g/l). Figure

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The comparison of different concentrations effects of Alcalase on weight loss of wool yarns without Surfactant, with Sandozin NRW, Sandozin EH, Erkantol NR and Irgazol NA in ammonium sulfate. Figure 5.

Hairiness of treated wool with Alcalase or Savinase (0.36 ml/g wool) in ammonium sulfate with or without surfactant in comparison of untreated wool. Figure 6.

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Average distribution of elongation and their variation coefficient of wool treated with Alcalase (0.36 ml/g wool) in ammonium sulfate, with or without surfactants Treatment condition Elongation increase (%) CV (%) Untreated wool 0.00 14.83 Alcalase 16.30 20.63 Alcalase + Sandozin NRW 22.46 16.31 Alcalase + Irgasol NA 25.77 15.36 Alcalase + Erkantol NR 24.06 16.25 Alcalase + Sandozin EH 14.25 22.17 Table 1.

Strength loss of treated wool with Alcalase (0.36 ml/g wool) in ammonium sulfate with or without surfactants. Figure 7.

Average distribution of elongation and their variation coefficient of wool treated with Savinase (0.36 ml/g wool) in ammonium sulfate, with or without surfactants Treatment condition Elongation (%) CV (%) Untreated wool 0.00 14.83 Savinase -19.38 15.97 Savinase + Sandozin NRW -16.53 14.37 Savinase + Irgasol NA -17.90 16.68 Savinase + Erkantol NR -22.92 13.26 Savinase + Sandozin EH -16.65 21.25 Table 2.

Average distribution of elongation and their variation coefficient of wool pre-scoured in sodium carbonate (0.2 or 1.6 g/l), treated with Alcalase (0.36 ml/g wool) in ammonium sulfate CV Elonga- CV Treatment condition Strength loss (%) (%) tion (%) (%) Untreated wool 0.00 8.28 0.00 14.83 0.2 g/l sodium carbonate 5.72 17.96 26.11 28.15 1.6 g/l sodium carbonate 30.24 22.96 36.83 37.36 Alcalase treatment 5.30 8.69 16.30 20.63 0.2 g/l sodium carbonate + 9.20 8.48 28.96 25.84 Alcalase 1.6 g/l sodium carbonate + 62.92 30.60 -59.18 34.54 Alcalase Table 3.

Strength loss of treated wool with Savinase (0.36 ml/g wool) in ammonium sulfate with or without surfactants. Figure 8.

treatments using Alcalase decreases the strength loss except for Irgasol NRW. The same trend was observed with Savinase (Figure 8). The Savinase effect on strength loss of sample is greater than Alcalase (Figures 7 and 8). It is clear that the type of surfactant has different effect on enzymes. The presence of surfactants in Alcalase solution decreases the Alcalase activity while it increases the susceptibility of wool substrate for proteolytic reactions of Savinase. In other words, the surfactants interact with proteins of wool yarn and might modify enzyme conformation to outer enzyme activity and selectivity [37]. The addition of surfactants especially non-ionic surfactants into enzyme treatment solutions causes the increasing elongation of the wool sample (Table 1). Table 2 (the effect of Savinase) reveals that surfactants have no significant effect on the elongation of the samples. Table 3 represents the strength loss and elongation of the raw samples (untreated) are negligible. While using 0.2 g/l sodium carbonate pretreatment does not increase significantly. However, pre-treating of samples using concentrated alkaline (1.6 g/l) even without enzymatic treatment causes drastic strength loss of samples. This strength loss increasing

prevents the pre-treatment of samples with concentrated sodium carbonate solution. Microscopic Studies

The SEM (Scanning Electron Microscopy) micrographs of untreated and treated wool fibers with enzymes under various conditions (Figure 9(a)-(e)) confirmed significant differences of effectiveness of enzymes. It is evident that the fiber destruction is caused by the diffusion and the hydrolytic attack of the native proteases onto non-keratinous parts of the protein fiber [15-30]. These observations coincide with the results of strength loss measurements. Figures 9(a) and (b) indicate that, in the case of samples treated only with enzymes, the external layer of cuticle is removed slightly and protuberance in boundary of scales

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results of weight loss, strength loss, and hairiness. Thus, due to the strong interaction of Savinase enzyme, it is recommended to omit treatment with this enzyme unless its amount is reduced. The SEM micrographs of samples showed that the alkaline and reducing agents caused intensive damages on wool. The damage effects of agents could be related to the destruction of the cystine linkage in wool sample.

Conclusion

Scanning electron microscopic images (SEM) of wool: (a) untreated wool, (b) wool treated with Alcalase enzyme, (c) wool pre-scoured in sodium carbonate (1.6 g/l) and treated with Alcalase enzyme, (d) wool pre-scoured in sodium sulfite (6.3 g/l) and treated with Alcalase enzyme, and (e) wool pre-scoured in sodium carbonate (1.6 g/l) and treated with Savinase enzyme. Figure 9.

varied a little. In the case of strong pre-treatment (1.6 g/l sodium carbonate) samples, scale’s protuberances in their boundaries changed significantly and external layer of cuticle was destroyed (Figure 9(c)). As shown in Figure 9(d), with sodium sulfite as a reducing reagent pre-treated sample before enzyme treatment boundary of scales was smoothed as Figure 9(c). In addition on external layer of cuticle some grooves and pores were created and the wool fiber surface was deformed. In addition, Figure 9(e) shows that deep grooves in cuticle were produced and wool fiber surface was destroyed drastically by pretreatment using sodium carbonate solution (1.6 g/l) before treating with enzyme. Results show that the effects of enzymes are negligible without pre-treatment. Also almost all of protuberances were removed and cuticle layer was destroyed with strong alkaline pretreatment. The SEM micrographs supported the using of 0.2 g/l sodium carbonate pre-treatment of the samples prior to enzymatic treatment. In addition, results showed that Savinase with similar condition was more destructive than Alcalase. These images confirmed the

This research showed that the type of enzyme and its concentration affected the proteolytic hydrolysis of wool. So, for attaining acceptable results, proper type and dose of enzymes must be selected. With increasing the ionic strength of the solution for both enzymes, weight loss of samples and enzymes activity decreased. Sodium tetraborate has more effects on enzymes activity than di-sodium hydrogen phosphate and ammonium sulfate. However, ammonium sulfate is usually recommended in textile industries due to the unfavorable environmental effect of sodium hydrogen phosphate. A lower alkaline concentration pre-treatment is more useful with subsequent enzyme treatments, and their results are sensible such as visible characters like fabric handle, flexibility, whiteness, appearance, and measurable properties like hairiness. SEM images revealed that the wool property was changed without more damages by using lower alkaline concentration pretreatment and selection of the optimized condition for each enzyme.

Acknowledgements The authors thank the Rezin Saveh Co. for their cooperation for using their instruments and Novo Nordisk A/S for supplying enzymes.

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