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Biocatalysis and Biotransformation

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Simultaneous protease and transglutaminase treatment for shrink resistance of wool

Kh. M. Gaffar Hossain a; Ascension Riva Juan b; Tzanko Tzanov a a Grup de Biotecnologia Molecular i Industrial, b Enginyeria Txtil i Paperera, Universitat Politcnica de Catalunya, Barcelona, Spain First Published on: 10 September 2008

To cite this Article Gaffar Hossain, Kh. M., Riva Juan, Ascension and Tzanov, Tzanko(2008)'Simultaneous protease and

transglutaminase treatment for shrink resistance of wool',Biocatalysis and Biotransformation,

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Biocatalysis and Biotransformation 2008, 17, iFirst article

ORIGINAL ARTICLE

Simultaneous protease and transglutaminase treatment for shrink resistance of wool

KH. M. GAFFAR HOSSAIN1, ASCENSION RIVA JUAN2, & TZANKO TZANOV1

1

Grup de Biotecnologia Molecular i Industrial and 2Enginyeria Textil i Paperera, Universitat Politecnica de Catalunya, ` ` Barcelona, Spain

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Abstract A bioprocess for machine washable wool, combining the advantages of both protease and transglutaminase in a simultaneous enzymatic treatment has been developed. This process reduced the felting tendency of woven wool fabrics by 9% at the expense of only 2% weight and tensile strength loss. In contrast to previously described protease-based processes for shrink resistant wool, the anti-felting properties achieved in the simultaneous enzymatic treatment produced insignificant fibre damage, confirmed also by scanning electron images of the fabrics.

Keywords: Bioprocessing, protease, transglutaminase, anti-felting, wool

Introduction The market value of wool is limited by the fact that consumers place increasingly high demands on machine washability and softness. Felting shrinkage is a typical property of wool due to the configuration of the scales of the wool fibre, especially during washing. The most widely used shrink-resist finishing for wool is the chlorine-Hercosett process (Holme 1993). This process, consisting of strong acid chlorine treatment followed by polymer resin application has the disadvantage of disposal of absorbable organic chlorides (AOX) in addition to the specific synthetic label of the resin-coated fabrics. Various enzymatic methods have been used to modify the properties of wool including application of proteases, lipases, protein disulphide isomerase and transglutaminase (King & Brockway 1987; Heine & Hocker 1995; Griffin et al. 2002a). Reduction of wool shrinkage was claimed with oxidases and peroxidases (Yoon 1998). A process for obtaining soft, shrink-resistant wool using a three-step process, comprising chemical oxidation, enzyme treatment (with peroxidase, catalase or lipase) followed by a protease treatment, was also reported (Ciampi et al. 1996). The enzymatic

processes, using proteases to hydrolyse the cuticle cells of the fibres and to reduce inter-fibre friction, thereby eliminating the cause for the shrinkage are difficult to control, and are not sufficiently predictable and reproducible on an industrial scale (Cortez et al. 2004). Such treatment, besides removal of the cuticle layer, can cause excessive proteolytic damage to the fibre with consequent high levels of weight and tensile strength loss due to penetration of the protease into the bulk of the fibres (Masumi et al. 1991).The application of proteases alone for shrinkproof wool could therefore not find any industrial application so far (Griffin et al. 2002a). On the other hand, proteases are now routinely used in domestic laundry detergent compositions for improved cleaning performance at low temperature. Nevertheless, the exposure of wool goods to the action of proteasebased detergents can cause irreversible damage, leading to loss of fabric strength, shape and poor colour fastness (Cortez et al. 2005). To overcome this limitation in the protease processing of wool two alternative approaches have been proposed. One is to limit the action of the proteases only to the surface of the fibres by increasing their molecular size through grafting

Correspondence: Tzanko Tzanov, Grup de Biotecnologia Molecular i Industrial, Universitat Politecnica de Catalunya, C. Colom, 11, ` 08222 Terrassa, Barcelona, Spain. Fax: '34937398225; E-mail: tzanko.tzanov@upc.edu ISSN 1024-2422 print/ISSN 1029-2446 online # 2008 Informa UK Ltd DOI: 10.1080/10242420802364940

Kh. M. Gaffar Hossain

Transglutaminase 15 Transglutaminase activity (U g )
-1

Protease 8 (U mg ) 6 4 Protease activity

-1

10 5 0 20 37 45 50 55 60 Temperature (C) 65 70

2 0

Figure 1. Temperature prole of protease and transglutaminase activity.

soluble polymers adducts, e.g. PVA or PEG on the proteins (Cavaco Paulo & Silva 2003). Normally increased molecular size of the enzymes translates into lower activity and thus longer process time to achieve the desired shrink-resist effect. Besides this, the process for enzyme modification is rather complicated and not easily reproducible, requiring expensive cross-linking agents and subsequent purification. The other, recently developed approach is to apply transglutaminases (TG) as a pre- or post-treatment to prevent or compensate the reduction in tensile strength and degradation of wool in protease treatment (Lorand et al. 1978; McDevitt & Winkler 2000; Gembeh et al. 2005; Cortez et al. 2004; Du et al. 2007). Transglutaminase is able to cross-link proteins, as well as peptides and various primary amines through acyl transfer reactions (Aeschlimann & Paulsson 1994; Griffin et al. 2002b). The strength improvement was attributed to the formation of TG-mediated intra- and inter-isopeptide cross-links [o-(g-Glu)Lys], grafting the o-amino group of a lysine to the g-carboxyl group of a glutamic acid residue. Though this two-step process appears to be

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efficient, its major drawback is increased time and energy consumption. The target of this work was to design a simultaneous process using transglutaminase and protease exploiting the benefits provided by each of these enzymes. Proteolysis of the cuticle scales of the fibres would bring about shrink-resistance, but would also allow for penetration of the transglutaminase and improved dimensional stability and mechanical characteristics of the fibres due to (glutamyl)lysine cross-linking of wool proteins. Materials and methods Textile material and enzymes Woven 100% wool fabric supplied by Lokateks (Slovenia) was washed, prior to enzymatic treatment, with 1 g L(1 non-ionic surfactant Cotemol NI (Color Center, Spain) in a laboratory winch machine (1:20 in 0.1 M Na2CO3, NaHCO3 buffer pH 9.0) at 408C for 30 min. Thereafter, the fabric was bleached at the same bath ratio with 0.1 mL L(1 of 30% H2O2 (0.1 M Na2CO3, NaHCO3 buffer pH 9.0) at 558C for 1 h (Silva et al. 2005). The bleached textile
Protease 12 10 Protease activity

Transglutaminase 40 Transglutaminase activity (U g-1) 30

20 10

6 4 2

0 5 6 7 7.5 8 8.5 pH 9 9.5 10 10.

Figure 2. pH prole of protease and transglutaminase activity.

(U mg-1)

Simultaneous protease and transglutaminase treatment

P2.5+TG0.1 P2.5+TG0.05 P2.5+TG0.025 P2.5+TG0.01 P2.5 TG0.1 C 0 5 10 15 20 Tensile strength loss (%) 25 30

Figure 3. Tensile strength loss of wool fabrics after biotreatment in 50 mM Tris-HCl buffer, pH 8, 508C for 60 min of samples. C: Control sample treated only with buffer; TG0.1: 0.1 U mL (1 TG; P 2.5: 2.5 U mL (1 protease; P2.5'TG0.01: 2.5 U mL (1 protease and 0.01 U mL (1 TG; P2.5 ' TG0.025: 2.5 U mL (1 protease and 0.025 U mL (1 TG; P2.5 ' TG0.05: protease 2.5 U mL (1 and 0.05 U mL (1 TG; P2.5'TG0.1: 2.5 U mL (1 protease and 0.1 U mL (1 TG.
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material was further treated with commercial protease Esperase 6.0T (EC 3.4.21.14) 1.01 mg prot. mL(1 from Novozymes A/S, Denmark, and microbial transglutaminase (EC 2.3.2.13) 4.86 mg prot. mL(1 from BDf, Spain. Enzyme characterization The proteolytic activity of Esperase was measured on soluble casein according to a Sigma enzymatic assay by incubating 1 mL of enzyme with 5 mL of 0.65% casein solution in 50 mM phosphate buffer

pH 7.5 for 10 min at 378C. The reaction was stopped by addition of 5 mL of 110 mM trichloroacetic acid. The precipitate was removed by filtration and centrifugation. Then 2 mL of the filtrate were mixed with 5 mL of 500 mM Na2CO3 solution and 1 mL of four-fold diluted Folins reagent. After mixing, the colour was allowed to develop for 30 min at 378C. The absorbance due to the amino acids produced was measured spectrophotometrically at 660 nm, using DL-tyrosine as a standard. One unit of activity is defined as the amount of enzyme that hydrolyses casein to produce colour equivalent

P2.5+TG0.1 P2.5+TG0.05 P2.5+TG0.025 P2.5+TG0.01 P2.5 TG0.1 C 0 2 4 6 Weight loss (%) 8 10

Figure 4. Weight loss of wool fabrics after biotreatment in 50 mM Tris-HCl buffer, pH 8, 508C for 60 min; sample description as in Figure 3.

Kh. M. Gaffar Hossain

P2.5+TG0.1 P2.5+TG0.05 P2.5+TG0.025 P2.5+TG0.01 P2.5 TG0.1 C 0 5 10 Shrinkage (%) 15 20

Figure 5. Shrinkage of wool fabrics after biotreatment in 50 mM Tris-HCl buffer, pH 8, 508C for 60 min; sample description as in Figure 3.

to 1 mmol of tyrosine per minute at pH 7.5 and 378C. The total protein concentration was determined by the Bradford method using bovine serum albumin (BSA) as a standard (Bradford 1976). The transglutaminase (TG) activity was determined according to a Sigma colorimetric procedure (Folk & Cole 1966), in which carboxybenzoyl-Lglutaminyl-glycine (NCBZGlnGly) was used as a substrate. A mixture of 12 mg mL(1 CBZ-Gln-Gly 200 mM hydroxylamine 20 mM glutathione, and 1.0 M CaCl2 was prepared in 1.0 M Tris buffer pH 6 at 378C. Then 30 ml of enzyme (2 units mL(1, prepared in cold deionized water immediately before use) were incubated in 200 ml of the mixed reagent for exactly 10 min. The reaction was stopped by addition of 500 ml of 12% (v/v) trichloroacetic acid. Finally 500 ml of 5% (w/v) FeCl3 prepared in 100 mM hydrochloric acid were added in the solution to produce colour detected spectrophotometrically at 525 nm. A calibration curve was prepared using 10 mM L-glutamic acid g-monohydroxamate. One unit of transglutaminase was defined as the amount of enzyme required to form 1.0 mmol Lglutamic acid g-monohydroxamate per minute at pH 6, 378C. The pH and temperature optima of protease and TG were determined following the assays described above by varying the temperature and pH of incubation of enzyme with substrate from, respectively, 20 to 708C, and 5 to 10.5. HPLC analysis of TG and protease The enzymes (20 ml sample) were studied after simultaneous treatment by size exclusion chromatography (SEC) using an Agilent Series 1200 HPLC system, equipped with a Zorbax GF- 450 analytical,

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6 m, 9.4 )250 mm column for proteins. The mobile phase was 0.2 M Na2HPO4 buffer pH 7.5 with a flow rate 1.0 mL min (1. Enzymatic treatment of wool The bleached wool fabric was treated simultaneously with 2.5 U mL(1 protease and 0.010.1 U mL(1 TG in 50 mM Tris-HCl buffer pH 8 at 508C for 60 min, in an Ahiba (Datacolor) laboratory dyeing machine at 30 rpm. After the biotreatment the samples were washed extensively and dried in an oven for 2 h at 508C. Fabric shrinkage Fabric shrinkage after washing was assessed according to ISO 6330 as described in IWS Test Method 31. The fabrics were washed in a Wascator washing machine (Wascator FOM71 special, Electroluxwascator, Sweden) in one cycle of wash program 7A for relaxation and three cycles program 5A for felt shrinkage, both at 408C with a load (polyester fabric) and standard detergent. All samples were tumble-dried after washing and conditioned at room temperature before measuring the area shrinkage. The results were expressed as percentage of area shrinkage and are the mean values of shrinkage measured on three different samples. Tensile strength and weight loss The samples were conditioned at 238C, 60% relative humidity for 24 h prior to evaluation. Tensile strength was determined using a tensile test machine PT-250 (Investigacion Sistemas Papeleros, S.L. Spain) in a standard procedure with 2 Kgf maximum

Simultaneous protease and transglutaminase treatment

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Figure 6. Size-exclusion chromatography elution patterns of (a) 1.5 U mL (1 protease, (b) 0.0035 U mL (1 TG, (c) 1.5 U mL (1 protease '0.0035 U mL (1 TG and (d) 1.5 U mL (1 protease'0.007 U mL (1 TG after incubation for 60 min in 0.2 M Na2HPO4 buffer pH 8 at 508C.

capacity load and 115 mm min(1 speed. The tensile resistance values are given as the mean of nine samples tested. The percentage of weight loss was calculated based on the weight of the fabric prior and after enzymatic treatment as ((W1 (W2)/W1))100, where W1 is the weight of the sample before and W2 after the enzymatic treatment. Three measurements were carried out for each sample. Surface morphology Microscopic photographs (magnification )1500 and )150) of the surface of the biotreated fabrics were obtained using a JSM 5610 scanning electron microscope (JEOL Ltd, Japan).

Result and discussion The activity of protease and TG were determined in the range of 2070 8C and pH 5 to 10.5 (Figures 1 and 2). The overlap in the temperature and pH profiles of protease and TG allow for their simultaneous co-application. Based on these data the compromise conditions of pH 8 and 508C were chosen for the simultaneous bioprocess. Tensile strength, weight loss and shrinkage of the biotreated fabrics Fabric samples were treated with protease and TG separately, and in a simultaneous process with increasing amount of TG. Protease treatment alone was able to reduce fabric shrinkage after washing by

Kh. M. Gaffar Hossain

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Figure 7. SEM images [magnication )150 and )1500 (insert)] of wool fabrics after biotreatment in 50 mM Tris-HCl buffer, pH 8, 508C for 60 min with: (a) 2.5 U mL (1 protease, (b) 2.5 U mL (1 protease and 0.1 U mL (1 TG simultaneously.

5%, but also caused about 25% strength decrease and 7% weight loss in comparison to the untreated sample (Figures 35). TG alone did not significantly influence the shrinkage behaviour and the mechanical properties of the fabric. The results showed significant improvement in fabric strength as well as reduction of shrinkage and weight loss with the increase in TG concentration when compared with the untreated and protease-treated fabrics. Carrying out the process with 2.5 U mL(1 protease and 0.1 U mL(1 TG resulted in only 2% weight and tensile strength loss, combined with 9% reduction in shrinkage. In the one-bath bioprocess, however, interaction between the enzymes might be expected in terms of digesting of TG by protease or cross-linking of protease by TG. Indeed, a decrease of protease activity was observed with the increase of TG concentration (data not shown). This might be due to either cross-linking of protease by TG or TG acting as a competing substrate in the protease enzymatic activity assay. SDS-PAGE electrophoresis and HPLC experiments showed that the molecular mass of TG and protease is about 58 and 20 kDa, respectively. Under simultaneous treatment conditions the band/peak of TG (SDS-PAGE/HPLC) disappeared and smaller molecular weight fragments appeared showing digestion of TG by protease. However, under the optimum TG concentration conditions for the simultaneous process, TG was still present in the

mixture, while there was no increase in molecular size of the protease due to cross-linking (Figure 6). Therefore, enhancement of wool properties obtained in the combined bioprocess was most probably due to proteolytic removal of the cuticle scale, creating conditions for penetration of TG beyond the cuticle layer into the cortex of the fibre (Masumi et al., 1991; Cortez et al. 2004), where the number of glutamine residues is higher (Church et al. 1997), catalysing o-(g-Glu)Lys cross-linking. Surface morphology of the biotreated fabrics Surface SEM images of the enzymatically treated fabrics (Figure 7a) showed significant proteolytic damage of the fibres, however, this was not uniform due to the heterogeneity of the wool itself (Rippon 1992). Some proteolytic damage and less defined cuticle scales can also be observed on the fibres treated in the simultaneous bio-process in Figure 7b. Conclusion The enzymatic process developed in this work combines the ability of protease to impart antifelting properties to wool fibres, hydrolysing their cuticle scales, with fibre stabilisation provided by TG-catalysed cross-linking of wool proteins. The shrink resistance of woven wool fabrics achieved in this one step, mild approach was superior to the shrink-resistance achieved with a single protease treatment. Weight and tensile strength loss of 2%

Simultaneous protease and transglutaminase treatment in the simultaneous process are insignificant compared with the 25% strength deterioration and 7% loss of protein material with the protease treatment. Fibre damage due to the simultaneous protease/TG treatment was not observed in scanning electron micrographs of the fabric surface. Besides the simplicity of the simultaneous method, the relatively short treatment time (60 min) to obtain the desired shrink-resistance properties is another advantage. Acknowledgements We gratefully acknowledge the EU project Contract No. 032877-ENZUP for the nancial support to this research. Declaration of interest: The authors report no conicts of interest. The authors alone are responsible for the content and writing of the paper.
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