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  Biopolishing enzymes & their influence on cotton

Acid cellulase, when used in biopolishing, offers a number of benefits such as improvement in pill resistance, cooler feel, brighter luminosity of colours and softness, and at the same time the treatment results in certain adverse effects like loss in weight and strength, infer K J Vishnu Vardhini and N Selvakumar.

In the textile industry especially in the apparel sector, cotton, the king of fibres, is widely used because of its superior properties. Cotton and cotton-blended fabrics are subjected to various wet processing treatments to enhance its value. The conventional methods of wet processing of cotton lead to a number of pollution hazards. A number of environmental regulations are to be fulfilled to safeguard the natural resources and this has led to growth in the use of enzymes in the textile industry for greener processing of textiles.

Enzymes are high molecular weight biological catalysts that mediate all biochemical reactions and are derived from fungal and bacterial sources for industrial use. These enzymes are classified into six major classes, based on the reactions they catalyse, and most of the enzymes used in the textile industry belong to the class ‘hydrolases’(1). These enzymes catalyse hydrolysis reactions where water insoluble material is converted into soluble products, which can be washed away. Enzymes called with a specific name always represent a group of enzymes, which has the same catalytic property.

The first use of enzymes in textile industry is in desizing cotton fabric with amylase, wherein the starch is hydrolysed, and this is still used extensively(2). Bioscouring carried out with a mixture of proteases, pectinases, lipases and cellulases has also proved to enhance the properties of cotton material(3). In the bleaching process, glucose oxidase enzyme is used to achieve controlled production of hydrogen peroxide from oxidation of glucose released during enzyme desizing(1). In denim washing, neutral cellulases are used instead of stones or along with stones to give faded effect(4). Laccases also find application in denim processing to decolour indigo with the help of a mediator thus giving a bleached effect(5,6).

In flax retting, pectinases and hemicellulases are used for hydrolysing pectin and lignin(6). Shrink-proofing and deprickling of wool are carried out using protease enzymes, which modifies the scales on the fibre(7). A mixture of cellulases and pectinases is used in the carbonisation of wool(7,8). Degumming of silk is carried out using serine protease, which degrades sericin, which is a protein leaving the fibroin, also a protein intact in the fibre(1,6). Polyester hydrophilisation is done with lipases, which hydrolyses fatty acid esters and other carboxylic acid esters in the fibre(6,8). Cellulase enzymes are also found to be useful in laundry detergents as an alternative to household fabric softeners(9). Biopolishing is an important finishing treatment carried out on cellulosic fabrics using acid cellulases to achieve improvement in gloss, luminosity of colours and resistance to pilling, cooler feel and clear surface(10-12). This article deals with acid cellulases used for biopolishing of cotton fabrics, and the effect of their application on cotton fibre and products made from it, namely, yarns and fabrics.

Cellulases

Cellulases are derived from both fungal and bacterial sources. They find extensive application on cellulosic materials, and about 10% of the finishing of these materials is estimated to be performed by these enzymes to achieve various effects(13). They also find application in food, pharma and paper industries(14). Cellulases used in biofinishing of cellulosic fabrics are derived from more than ten different fungal species, which vary in their component composition, application pH and special effects produced(13). Cellulases derived from the fungus, Trichoderma reesei, is widely used in textile finishing, since it gives higher yield in industrial production. In addition to cellulases originating from the above fungus, those originating from Humicola insolens can also degrade cotton cellulose efficiently, and they find extensive application in biostoning of denim fabric(15-18).

Components of cellulase and mechanism of its action on cellulose

Cellulase derived from Trichoderma reesei contains a group of enzymes namely, endoglucanases (EG), cellobiohydrolases (CBH) or exoglucanases and b-glucosidases and they act synergistically to hydrolyse cellulose. It has been found that this fungus secretes atleast five types of EGs and two types of CBHs. It is estimated that the secreted enzyme constitutes 60% of CBHI and 10% EGI and EGII. The structures of EG and CBH shown in Figure 1 reveal the presence of cleft in the EG and a tunnel in the CBH. It is reported that due to the presence of tunnel, CBH has a more pronounced effect on the rate of hydrolysis than the EG(13,19,20). Cellulases can be used for biopolishing as derived from their sources or after enriching the EG content in the mixture. The mixture of endoglucanases, exoglucanases and b-glucosidases are called whole cellulases or total cellulases. Using advances in biotechnology new strains are being developed for producing novel cellulase compositions(21).

The mechanism of cellulase action on cellulose (Figure 2)(14) is as follows: (i) The endoglucanases degrades cellulose by selectively cleaving through the amorphous sites and breaking long polymer chains into shorter chains (ii) Cellobiohydrolases degrades cellulose sequentially from the ends of glucose chains, thus producing cellobiose as the major product and it plays a mediator role in degrading cellulose and (iii) b-glucosidases complete the hydrolysis reaction by converting cellobiose into glucose (4,15,22,23).

Activity of cellulase

Activity of an enzyme, expressed as U/g or U/ml, is a measure of conversion of substrate molecules into products by a g or ml of an enzyme in a unit time. Cellulases form glucose as product. The substrates commonly used for characterisation of cellulases are carboxy methyl cellulose (CMC), phosphoric acid swollen Avicel and filter paper (FP). Among these CMC is amorphous cellulose, whereas FP has both amorphous and crystalline cellulose. Screening of whole cellulase preparations is done predominantly using FP activity(24) involving Whatman No:1 filter paper. This type of characterisation of cellulase using activity is not much useful in textile applications since the activity determined does not have any correlation with weight loss and strength loss obtained in fabrics(25)

Effect of total cellulase and their components on cotton 

Since cellulase attacks the 1, 4 - b-glucosidic bonds of cellulose and forms glucose, cotton products treated with this enzyme experience weight loss. Moreover, the treatment results in change in many other properties also and the extent of these changes depends on various factors. Literature pertaining to studies carried out with total cellulases and total cellulases with some components enriched in it are given below. 

Effect on supramolecular structure 

Karen Kleman – Leyer et al(22) studied the molecular size distributions of cotton cellulose treated with EGI, CBHII and their combination for periods varying from 12 to 192 hours. The treatment carried out for 12 hours with EGI and its combination with CBHII has found to increase polydispersity by two fold whereas the treatment for longer duration resulted in decreased polydispersity values. The above actions reveal that the EGI continuously degrades cellulose. Molecular size distributions of cotton treated with CBHII was found to be unaffected. The fibre crystallinity index after cellulase treatment on cotton substrates was found to be unchanged regardless of the extent of agitation and also the nature of the enzyme used ie, monocomponent EG and total cellulase(21,26)

Marie – Alice et al(20) studied the effect of time of treatment of whole cellulase on supramolecular structure of cotton. The SEM photographs of the samples revealed that the treatments have caused damage to the primary wall. The results namely increase in number average molecular weight and a reduction in polydispersity obtained in the study confirmed the removal of primary wall and this damage increased with treatment time. It was also found that there was no change in pore size distribution and disordered and highly ordered regions. 

Effect on fibre and yarn properties 

Melissa Ann Stewart(27) reported that treatment with biopolishing enzyme results in strength loss in cotton fibres. An attempt on yarns spun using fibres treated with monocomponent cellulases CBHI, CBHII, EGI, EGII and commercial enzyme revealed that the tenacity of the yarns spun using the above fibres were lower than that of yarns spun with untreated fibres. The EGI, EGII and commercial enzyme treated yarns show higher yarn hairiness and it is suggested that such a behaviour is due to the endo-activity of these enzymes. All the enzymes used in the study were found to have insignificant effect on yarn evenness(28)

100% cotton and cotton/polyester yarns made from different spinning systems were evaluated by Radhakrishnaiah et al(29,30) after treating them with a commercial cellulase enzyme. The cotton yarns were spun from ring, rotor and OE friction systems while the cotton/polyester yarns were spun from ring, rotor and air-jet systems. It was reported that cotton and cotton/polyester yarns spun from various spinning systems and cotton/polyester bicomponent yarns suffered a significant loss in strength and breaking elongation on treatment with cellulase. The only exception found in this case was the friction spun cotton yarn. Changes due to cellulase enzyme hydrolysis of cellulosic fabrics have been studied by Buschle-Diller et al(31). They found that strength loss in yarn increased with increased weight loss in cotton fabrics. 

Effect on fabric properties 

Cellulase treatment of fabric results in a number of changes in their properties. On treating with whole cellulases, loss in breaking strength was observed and it was found to have non-linear relationship with weight loss(30). The knitted fabrics were found to show reduction in bursting strength and improvement in pilling resistance(31). Ramkumar and Gus Abdalah(32) reported that the cellulase enzyme treatment significantly improved the fabric smoothness, which is measured in terms of frictional parameters. The cellulase treated cotton woven fabrics show reduction in bending rigidity and hysteresis of shear force(33). Joao et al(34), reported that the dimensional stability of cotton woven and knitted fabrics improved on cellulase treatment. The low stress mechanical properties of cellulase treated fabrics were found to improve. The fabrics became smoother, softer and fuller and offered less resistance to bending and stretching. Comparison between the properties of cellulase treated cotton fabrics and alkali treated polyester fabrics showed that the former undergoes a reduction in residual curvature and residual shear strain to a lesser extent than the latter(33)

Effect on dyeing 

Trarore(36) and Buschle – Diller reported that the colour yield of cotton increases on cellulase pretreatment. Ibrahim et al(37) found increase in colour yield for direct and reactive dyes with increasing weight loss on cellulase treatment on cotton. In contrast to this, Koo et al(38) reported reduction in colour yield with increase in weight loss. Investigation by Buschle – Diller et al(39) showed no significant change in colour yield for medium weight loss. They further reported that there is an improvement in colour yield for reactive dyes and it further increased with increase in weight loss. Vat dyes also showed an improvement in colour yield but it decreased with increase in weight loss. Anand Kanchagar(40) found that there were no changes in colour yield for reactive dyes for smaller weight losses. The results of the above studies reveal that there is a need for further work in order to understand the behaviour of various classes of dyes on the colour yield obtained on cellulase treated cotton materials. 

Factors affecting cellulase action on cotton Pretreatment 

Mercerised fabrics subjected to cellulase enzyme treatment were found to increase rate of hydrolysis due to increased available adsorption sites. Further cellulase treatment results in higher strength loss in mercerised fabrics compared to unmercerised fabrics(41). Moika Nicolai and Axel Nechwatal(42) reported that pretreatments with ammonia and NaOH enhanced the effect of enzymatic treatment on cotton yarns. 

Kier boiled cotton yarn samples subjected to various pretreatments were considered for evaluating the rate of hydrolysis on treatment with cellulase enzyme. Pretreatments namely mercerisation with 24% NaOH, decrystallisation using liquid ethylamine at icebath temperature and decrystallisation using liquid ethylamine under ambient conditions were used. The rate of hydrolysis obtained for all the pretreated samples were higher than that of the sample not subjected to pretreatment. Among the pretreated samples decrystallisation treatment carried out using liquid ethylamine under ambient conditions was found to give highest rate of hydrolysis. The same trend was observed with respect to degree of polymerisation and % crystallinity of the pretreated samples(43)

The effect of pretreatments such as steaming, oxidation with Fenton’s reagent and washing with mild and strong alkalis on the accessibility of cotton fibres for cellulase was determined. It was found that all the pretreatments improved accessibility of cotton towards cellulase. The order of accessibility of cellulases was in the following order of steaming < oxidation < mild alkaline wash < strong alkaline wash(28)

Predyeing 

Studies conduced on enzymatic action of cellulase enzyme on dyed cotton using direct, reactive and vat dyes reveal that the cellulase action is retarded either by the presence of dye molecules or interactions between dye and cellulose molecules(36-38, 44-47). Factors, namely, dye class and size, substantivity and functionality of the dye molecule, which influence the retardation of cellulase action also have been investigated(37,44,46-48). It is proposed that the blockage caused by the dye molecules prevents cellulase approaching 1, 4-b glucoside linkages in cellulose resulting in the retardation of the action of cellulase. 

Agitation 

Agitation is an important factor in cellulase treatment of cotton fabrics. A number of studies have been conducted to understand the effect of agitation on fabric properties. Especially, for the depilling of fabrics agitation plays an important role as it helps in cellulase adsorption followed by cutting of fibres and fibrils which were weakened by cellulase action(15). SEM photographs of cellulase treated fabrics showed that higher level of agitation used for the treatment affect the fibre surface in a short treatment time itself(26)

It was found that when treatment was given in jet and winch machine using same type of enzyme and similar process conditions, the level of effects produced on fabrics were different which is due to the difference in agitation provided by these machines(49). Jim Liu et al(25) based on study conducted involving enzymes namely monocomponent endoglucanases, endoenriched cellulases and total cellulases at different levels of agitation suggested that when selecting cellulases for biopolishing, the machine to be used has to be given due importance. 

Study conducted by Lenting and Warmoeskerken(50) reveal that a minimum level of agitation is to be employed for optimum performance of cellulase towards biopolishing. Also when suggesting for minimizing tensile strength loss on cellulase treatment to fabrics agitation is considered as an important factor(44) and on measuring the tactile properties of fabrics treated with and without mechanical agitation, it was found that they were highly influenced by agitation and positive effects were found due to agitation(35)

Time 

Treatment time has a greater influence on cellulase action on cotton. Fibre damage increases dramatically when longer treatment durations are used(15). Longer treatment times with no agitation were found to affect the internal structure, whereas short treatment times with higher agitation affect the fibre surface(25)

Generally, increase in treatment time results in greater weight loss and strength loss. The relationship between time of treatment with these properties were found to be non linear(15,25,43). The enzymatic hydrolysis for longer treatment time leading to higher weight loss results only in a slight decrease in degree of polymerisation(44). An attempt was made by Ajoy and Nolan(51) to model the enzymatic hydrolysis involving initial substrate concentrations, flow rate and treatment time. The following empirical equation was suggested. Fractional conversion, Pt/So = where, Pt is the product concentration (mg/ml) at time t, So is the initial substrate concentration (mg/ml), x, y, z are flow rate dependant constants. 

Yarn and fabric structure 

Yarns produced with different structural features subjected to cellulase treatment were evaluated for their hand related mechanical behaviour. Cotton yarns spun from ring, rotor and OE friction systems and cotton/polyester yarns spun from ring, rotor and air-jet systems were considered. Also polyester cotton bicomponent yarns that exhibit systematic differences in fibre arrangement within the yarn were considered. It was found that hand related properties of all the above yarns improved on cellulase treatment. 

Among the cotton and P/C yarns, OE friction spun cotton yarn and air-jet spun P/C yarn showed maximum improvement in hand related properties. The treatment altered the properties of blended P/C ring spun yarn more than those of the P/C bicomponent yarn. The fibre arrangements in the bicomponent yarn was found to influence the weight loss suffered by the cellulase treatment(29,30). Nilgun Ozdil et al(32) reported that knitted fabrics, produced from yarns spun using spinning routes such as OE rotor, combed ring and carded ring, on treatment with cellulase give difference in weight loss, strength loss and pilling rate showing that the type of yarn used for fabric production has influence on cellulase treatment. 

The fabric structure also influences the weight loss on cellulase treatment. It was found that weight loss was more in knitted fabrics than the woven fabrics due to structural differences. When poplin and flannelette fabrics of different EPI and PPI were treated with different compositions of enzyme at low and higher agitations, its effect on weight loss, ratio of breaking load to weight loss and pilling level which reveal that fabric structure influences the cellulase treatment(15,21)

Synergistic effects of cellulase action on cotton 

Lea Heikinheimo and Johanna Buchert(19) studied the synergistic effect of T reesei cellulases namely CBHI, CBHII, EGI and EGII alone and in different combinations on knitted fabrics. Results obtained on the properties evaluated reveal that there are clear differences between the action of individual enzymes and their defined mixtures. No correlation was found to exist between high weight loss and good pilling results as well as weight loss and strength loss. It is suggested that the reduced pilling tendency can be obtained with lower strength loss by tailoring different cellulases in the enzyme mixture. 

Jim Liu et al(25) carried out studies with monocomponent endoglucanase, endoenriched cellulase and total cellulase and found that there is a correlation between weight loss and pilling note for each of the above enzymes but among enzymes there is no concurrence in correlation obtained. 

Cavaco Paulo and Almedia(15) have conducted studies on different fabrics and found that the ratio of breaking load to weight loss differ for different compositions of enzymes used. The activity of total cellulase was found to be affected by the level of agitation used in the treatment. At high agitation levels, EG activity in the total cellulase was found to increase as against the reduction in the CBH activity(26). Further EG treatment at high level of agitation makes fabrics feel harsher whereas total cellulase treatment makes fabrics feel softer. In order to minimise tensile strength loss in cellulase application, EG enriched cellulase on even, monocomponent EG is found to be suitable(52)

Conclusion 

It is clear from the number of studies carried out pertaining to acid cellulases and its application on cotton materials that the use of this enzyme results in both beneficial and adverse effects. Research findings bring out the facts regarding the enzyme as well as the effect of pretreatment, material parameters and process variables on cellulase action. Findings also give an understanding on the effect of cellulase action on post treatments. 

Yet, there are areas that require the attention of researchers in order to gain better understanding on acid cellulase and its action on cotton fibre and its products having varying degrees of structural complications as well as associated process conditions used. It is hoped that this review would certainly be of use to those who are involved in the research and application of this enzyme, ‘Acid cellulase’. 

References 

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K J Vishnu Vardhini Department of Textile Technology, A C College of Technology, Anna University, Chennai 600 025. N Selvakumar. Department of Textile Technology, A C College of Technology, Anna University, Chennai 600 025 Asst Professor.

published March , 2009
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