Enzymes have been used in textile industry for desizing, scouring, polishing, washing, degumming, peroxide degradation in bleaching baths as well as for decolourisation of dyehouse wastewaters, bleaching of released dyestuff and inhibiting dye transfer. Most of the textile processes are heterogeneous where an auxiliary as a dye, enzyme, softener or oxidant have to be transferred from the solution to the fibre. These processes require the presence of surface-active agents, ionic force "balancers", buffers, stabilisers and others. It can be expected that an enzyme protein can interact with all chemicals in solution due to the large variety of side chains of the outer-amino-acids in the large 3D structure of the protein. In this paper various aspects are discussed where protein interactions are important for textile processing.

Backstaining during cellulase stone-washing

The production of "aged" denim garments (i.e. Jeans) with cellulases is the most successful enzyme process that emerged in the textile industry in the last decade. The aged look is obtained by the non homogenous removal of the Indigo dye trapped inside the fibers by the cooperative action of enzymatic hydrolysis and mechanical stress such as beating and friction. During enzymatic treatment of denim with cellulases the redeposition of the removed Indigo dye on the cotton fibers diminishes the desired contrast between white and blue yarns and can be easily noted on the reverse side of the fabric. Beside dye-cellulose interactions mainly dye-cellulase interactions are the major cause for Indigo backstaining. Cellulase proteins were found to have an influence on the aggregation and solubilization of the insoluble Indigo dye in the treatment baths. Protein adsorption on Indigo is much less specific than cellulase adsorption on cellulose. As in the case of the binding of CBDs to cellulosic substrates the Indigo-protein interactions are mainly due to surface hydrophobic and aromatic amino acid residues. The binding forces between Indigo and proteins are not strong and due to hydrophobic interactions and formation of hydrogen bonds. The adsorption of Indigo to cellulases and the capacity to carry microfine Indigo particles depends on the type of the enzyme and the presence and the type of the cellulose binding domain of the used enzyme. Whole enzymes were found to have more affinity to Indigo and cause more backstaining than the core enzyme without CBD (Cavaco-Paulo et al. 1998; Andreaus et al. 2000; Campos et al. 2000, Gusakov et al. 2000; Gusakov et al. 2001). In a comparative study of different cellulases no correlation was found between enzyme activity (saccharifying activity) and colour removal (topolytic activity) (Gusakov et al. 1999).

Three principally different mechanisms for backstaining were proposed. Andreaus et al. 2000 suggested that cellulase proteins interact with Indigo, reduce Indigo particle size and act as carriers of fine Indigo particles, already dispersed in the bulk solution, to the cotton fabric.

Gusakov et al. 2001 suggested that the removal of colour from denim is related to the enzymes affinity to Indigo and its capacity to transport the insoluble dye from the fabric into solution.

A third proposal for a backstaining mechanism, (Clarkson et al. 1994), refers to the presence of proteins with high Indigo affinity in the cellulase preparations, that do not desorb from the cotton fabrics after treatment. The addition of proteases to the cellulase treatment solution or the post-washing formulation was suggested and found to be efficient to reduce this kind of backstaining (Andreaus et al. 2000).

The undesired effect of backstaining may be reduced by the addition of auxiliaries during stonewashing and post-washing. Nonionic surfactants and dispersing agents were found to be the most efficient chemical reagents at low concentrations to desorb Indigo from cotton. The same products are also efficient, when applied in the denim treatment solution together with the cellulases (Andreaus et al. 2001). A better white-blue contrast may also be achieved with an after-treatment with laccases, which are able to oxidise insoluble Indigo to water soluble isatin and anthranilic acid (Campos et al. 2001).

In industry different cellulases and ageing processes for denim garments are in use. Beside Trichoderma cellulases, which still seem to be the most important cellulases, a big variety of cellulases, mainly produced by fungi, are available. For ageing of denim or other dyed textiles monocomponents are preferred over total crude mixtures. Variables such as enzyme concentration, washing time and addition of abrasive material are selected according to the desired ageing effect. Recently cellulases are also used for the ageing of sulphur or pigment dyed garments. The selection of the cellulase enzyme depends mostly on the pretended ageing effect, the level of backstaining and the price of the enzyme.

Dyeing in dyebaths prepared with enzymatically recycled textile effluents

The removal of hydrogen peroxide or hydrolysed dyestuff after the bleaching and dyeing processes respectively, are the two major areas where the washing liquors can be recycled by means of an appropriate enzymatic treatment with oxidoreductases. This treatment could be carried out with enzymes in both free (Tzanov et al. 2001a) and immobilised form. The main disadvantage of using free enzymes is that the protein remains in the recycled effluent, which is reused for dyeing. During dyeing the proteins undergo thermally induced denaturation, precipitate binding dye from the solution and thereby decrease its concentration (Tzanov et al. 2001b). The use of immobilized enzymes will avoid this problem and will provide resistant against inactivation catalysts.

Effect of ionic and non-ionic additives in the textile baths on enzyme activity

The dye/protein interactions in recycling dyehouse wastewater and other applications where enzymes and dyes are used together can improve enzyme stability at stress temperature and pH. Synthetic azoaromatic sulfonate dye anions frequently provide notable protection for enzymes from inactivation by acids and elevated temperature, through a co-precipitation-protection mechanism (Matulis et al. 1999). These anionic dyes bind to enzyme molecules forming ion pairs between negatively charged sulfonate groups and positively charged protein groups. The addition of increasingly large aliphatic or aromatic groups increases the ability of the sulfonate for enzyme stabilisation. Large organic non-polar groups displace water from near environment of ion pairs, preventing in such a way the denaturant action of the water.

All textile processes due to their heterogeneous character require the application of surface-active agents. Ionic surfactants show strong associative behaviour with globular proteins in aqueous solutions. The charged headgroup of a surfactant is electrostatically attracted to an oppositely charged amino acid residue of the protein. At higher concentration the surfactants forms a layer of micellar structure on the protein molecule. This surfactant layer decreases the conformational mobility of the unfolded protein thereby increasing the stability of the globular state. Some oligomeric enzymes, however, lose catalytic activity in the presence of surfactants, due to dissociation into their constituent subunits.

Most textile operations are carried out in aqueous medium. Non-polar amino acids constitute about one half of surface area of proteins and are organised as hydrophobic surface clusters (Longo and Combes, 1999). The interaction of these hydrophobic clusters with water is responsible for the heat-induced denaturation of biocatalysts. Water could be considered as a reactant in inactivation reactions and as a lubrificant in conformational changes associated with protein unfolding. Stabilisation with hydrophilic compound near to the hydrophobic cluster will trap the surrounding water and create a protecting shield to the hydrophobic regions. Surfactants may facilitate the transfer of hydrophobic substrate through the layer of water molecules to the enzyme-binding site (Khalaf et al. 1996).

The salts take part in any dyebath composition. In the presence of salts enzyme activation and stabilisation has been observed. Salts stabilize the proteins in very concentrated urea solutions (Dötsch et al. 1995). Urea is the most widely used hydrotropic agent for textile dyes. The high amount of salt presented in the dyeing effluents increases the ionic strength and enhances the electrostatic coupling of the anionic dyes and the positively charged proteins, thereby forming more stable dye/enzyme aggregates.

The effect of the stabilizing compounds has been attributed mainly to their exclusion from the protein surface (Lee and Teemasheff, 1981; Gekko and Ito, 1990). One major difference between the protein stabilizing and destabilizing excluded compounds is in their chemical nature. The first class of compounds is very polar and has almost no hydrophobic groups in the molecule, whereas the second class has hydrophobic character.

Effect of cross-linking and immobilization on enzyme stability

Cross-linking with bi-functional reagents normally stabilises the enzymes. However, excessive cross-linking may lead to aggregation, precipitation, loss of activity and distortion of the 3D enzyme structure. The immobilization of enzyme on insoluble supports provides stabilisation effect at elevated temperature and pH. The stabilisation depends on the position of the support attachment to the protein molecule. In general, unfolding of soluble proteins is initiated at their most labile site. The stabilisation is most successful when this unfolding region is strengthened through immobilisation or cross-linking (Ulbrich-Hofmann and Mensfeld, 1999). For the conservation of the functional state of biocatalysts at extreme conditions, a balance between stability and flexibility should be found. When using enzymes immobilized on insoluble supports the interaction of the support with the textile baths chemicals must also to be taken into consideration.

Concluding Remarks

The application of biocatalysts in textile practice requires a careful investigation of the possible protein-textile chemicals interactions. The operational stability of the enzymes depends on the slight balance of stabilizing and destabilizing interactions. In general, the formation of a protecting shell around the protein, the cross-linking and immobilization are some guidelines for preservation of the globular state of the proteins and the favorable microenvironment for enzyme catalysis.

References

ANDREAUS, J.; ZILZ, L.; BUDAG, N.; SCHARF, M.andCAVACO-PAULO, A. Influência de surfactantes não-iônicos e dispersantes no processamento de tecidos de algodão com celulases. In: I Congresso da Sociedade Brasileira de Biotecnologia (12^th - 14^th November, 2001a, Sao Paulo, Country, Brazil). CD dos Anais, paper BIO 035, p. 1-5.

ANDREAUS, J.; CAMPOS, R.; GÜBITZ, G. andCAVACO-PAULO, A. Influence of cellulases on Indigo Backstaining. Textile Research Journal, 2000, vol. 70, p. 628-632.

CAMPOS, R.; CAVACO-PAULO, A.; ROBRA, K.H.; SCHNEIDER, M. andGÜBITZ, G. Indigo degradation with laccases from Polyporus sp. and Sclerotium rolfsii. Textile Research Journal, 2001, vol. 71, no. 5, p. 420-424.

CAMPOS, R.; ANDREAUS, J.; GÜBITZ, G. andCAVACO-PAULO, A. Indigo-Cellulase Interactions. Textile Research Journal, 2000, vol. 70, no. 6, p. 532-536.

CAVACO-PAULO, A.; MORGADO, J.; ALMEIDA, L. andKILBURN, D. Indigo Backstaining during cellulase washing. Textile Research Journal, 1998, vol. 68, no. 6, p. 398-401.

CLARKSON, K.; LAD, P.; MULLINS, M.; SIMPSON, C.; WEISS, G. and JACOBS, L. Enzymatic compositions and methods for producing stonewashed look on indigo-dyed denim fabric, World Patent PCT WO94/29426, 1994.

DÖTSCH, V.; WIDER, G.; SIEGAL, G. and WÜTHRICH, K. Salt-stabilized globular protein structure in 7M aqueous urea solution. FEBS Letters, 1995, vol. 372, p. 288-290.

GEKKO, K. and ITO, H. Competing solvent effects of polyols and guanidine hydrochloride on protein stability. Journal of Biochemistry, 1990, vol. 107, p. 572-577.

GUSAKOV, A.; SINITSYN, A.; MARKOV, A.; SINITSYNA, O.; ANKUDIMOVA, N. and BERLIN, A. Study of protein adsorption on indigo particles confirms the existence of enzyme-indigo interaction sites in cellulase molecules. Journal of Biotechnology, 2001, vol. 87, p. 83-90.

GUSAKOV, A.; SINITSYN, A.; BERLIN, A.; MARKOV, A. and ANKUDIMOVA, N. Surface hydrophobic amino acid residues in cellulase molecules as a structural factor responsible for their high denim-washing performance. Enzyme and Microbial Technology, 2000, vol. 27, p. 664-671.

GUSAKOV, A.; POPOVA, N.; BERLIN, A. and SINITSYN, A. Comparison of saccharifying and topolytic activities of various cellulase preparations. Applied Biochemistry and Microbiology,1999, vol.35, no. 2, p.120-122.

KHALAF, N.; GOVARDHAN, C.P.; LALONDE, J.J.; PERSICHETTI, R.A.; WANG, Y.F. and MARGOLIN, A. Cross-linked enzyme crystals as highly active catalysts in organic solvents. Journal of the American Chemical Society, 1996, vol. 118, p. 5494-5495.

LEE, J.C. and TIMASHEFF, S.N. The stabilization of proteins by sucrose. Journal of Biological Chemistry, 1981, vol. 256, p. 7193-7201.

LONGO, M. A. and COMBES, D. Thermostability of modified enzymes: a detail ed study. Journal of Chemical Technology and Biotechnology, 1999, vol. 74, p. 25-32.

MATULIS, D.; WU, C.; VAN PHAM, T.; GUY, C. and LOVRIEN, R. Protection of enzymes by aromatic sulfonates from inactivation by acid and elevated temperatures. Journal of Molecular Catalysis B: Enzymatic, 1999, vol. 7, p. 21-36.

TZANOV, T.Z.; COSTA, S.; GUBITZ, G. and CAVACO-PAULO, A. Dyeing with catalase treated bleaching baths. Coloration Technology, 2001a, vol. 117, p. 1-5.

TZANOV, T.Z.; COSTA, S.; GUBITZ, G. and CAVACO-PAULO, A. Effect of the temperature and bath composition on the dyeing with catalase treated bleaching effluents. Coloration Technology, 2001b, vol.117, p. 28-32.

ULBRICH-HOFMANN, A.U. and MENSFELD, J. The concept of the unfolding region for approaching the mechanism of enzyme stabilization. Journal of Molecular Catalysis B: Enzymatic, 1999, vol. 7, p. 125-131.