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Kh. M. Gaffar Hossain a; Ascension Riva Juan b; Tzanko Tzanov aa Grup de Biotecnologia Molecular i Industrial, b Enginyeria Txtil i Paperera, Universitat Politcnica deCatalunya, Barcelona, Spain

First Published on: 10 September 2008

Gaffar Hossain, Kh. M., Riva Juan, Ascension and Tzanov, Tzanko(2008)'Simultaneous protease andtransglutaminase treatment for shrink resistance of wool',Biocatalysis and Biotransformation,

10.1080/10242420802364940

http://dx.doi.org/10.1080/10242420802364940

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ORIGINAL ARTICLE

Simultaneous protease and transglutaminase treatment for shrink

resistance of wool

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

1Grup de Biotecnologia Molecular i Industrial and

2Enginyeria Textil i Paperera, Universitat Politecnica de Catalunya,

Barcelona, Spain

Abstract

A bioprocess for machine washable wool, combining the advantages of both protease and transglutaminase in asimultaneous enzymatic treatment has been developed. This process reduced the felting tendency of woven wool fabricsby 9% at the expense of only 2% weight and tensile strength loss. In contrast to previously described protease-basedprocesses for shrink resistant wool, the anti-felting properties achieved in the simultaneous enzymatic treatment producedinsignificant 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 finish-

ing 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 applica-

tion of proteases, lipases, protein disulphide isomer-

ase and transglutaminase (King & Brockway 1987;

Heine & Hocker 1995; Griffin et al. 2002a).

Reduction of wool shrinkage was claimed withoxidases 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 predict-

able and reproducible on an industrial scale (Cortez

et al. 2004). Such treatment, besides removal of the

cuticle layer, can cause excessive proteolytic damageto 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 shrink-

proof 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 clean-

ing performance at low temperature. Nevertheless,

the exposure of wool goods to the action of protease-

based detergents can cause irreversible damage,

leading to loss of fabric strength, shape and poorcolour 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: [email protected]

Biocatalysis and Biotransformation

2008, 17, iFirst article

ISSN 1024-2422 print/ISSN 1029-2446 online # 2008 Informa UK Ltd

DOI: 10.1080/10242420802364940


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to 1 mmol of tyrosine per minute at pH 7.5 and

378

C. The total protein concentration was deter-mined by the Bradford method using bovine serum

albumin (BSA) as a standard (Bradford 1976).

The transglutaminase (TG) activity was deter-

mined according to a Sigma colorimetric procedure

(Folk & Cole 1966), in which carboxybenzoyl-L-

glutaminyl-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 byaddition 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 solu-

tion to produce colour detected spectrophotometri-

cally 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 L-

glutamic 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 temperatureand 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 chromato-

graphy (SEC) using an Agilent Series 1200 HPLC

system, equipped with a Zorbax GF- 450 analytical,

6 m, 9.4)250 mm column for proteins. The mobile

phase was 0.2 M Na2HPO4 buffer pH 7.5 with aflow 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 accord-

ing to ISO 6330 as described in IWS Test Method

31. The fabrics were washed in a Wascator washing

machine (Wascator FOM71 special, Electrolux-

wascator,

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 shrinkagemeasured 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

0 5 10 15 20

C

TG0.1

P2.5

P2.5+TG0.01

P2.5+TG0.025

P2.5+TG0.05

P2.5+TG0.1

Shrinkage (%)

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.

4 Kh. M. Gaffar Hossain


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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 measure-

ments 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 simulta-

neous co-application. Based on these data the

compromise conditions of pH 8 and 508

C werechosen 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

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.

Simultaneous protease and transglutaminase treatment 5


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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 mechan-

ical 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 UmL(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 experi-

ments 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 anti-

felting 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%

Figure 7. SEM images [magnification )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.

6 Kh. M. Gaffar Hossain


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in the simultaneous process are insignificant com-

pared 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 financial support to this

research.

Declaration of interest: The authors report no

conflicts of interest. The authors alone are respon-

sible for the content and writing of the paper.

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