© Color. Technol., 117 (2001) 217Web ref: 20010407

ColorationTechnology

Society of Dyers and Colourists

Laboratory- and commercial-scaleinvestigations into the action of cellulaseenzymes on TencelA Harnden,a M J Donnellyb,†,* and J Yorkb

a Tencel Technical Service, 140 Funston Avenue, San Francisco, CA 94118, USA

b Tencel Research and Technology, Acordis Fibres (Holdings) Ltd, PO Box 111,101 Lockhurst Lane, Coventry CV6 5RS, UK

The surface appearance and feel of fabrics produced from the new cellulosic fibre Tencel can beenhanced by cellulase treatment. Laboratory-scale studies have been carried out using purifiedTrichoderma reesei cellulase components to determine which enzymes provide a benefit and whichcause detrimental effects on Tencel fibre. Two enzymes were identified as beneficial and this informationwas used to produce new commercial cellulase products, using genetic engineering to remove detri-mental components and increase amounts of the beneficial enzyme components. The effects of the newcellulases on Tencel fibre (both 100% pure and blends) were then evaluated. The observed advantagesincluded improved hand-feel and reduced garment damage, especially in blends with other cellulosics.

IntroductionThe unique crystalline structure of Tencel fibres is the basisfor a property referred to as ‘fibrillation’. Fibrillation occurswhen the fibre is subjected to mechanical action in a wetstate. Swelling of the porous regions of the fibre breaks thehydrogen bonds linking the crystalline units and forcesthem apart. When this structure is subjected to mechanicalaction, the outer crystalline regions can break and peel awayfrom the main fibre. These peelings are referred to as fibrils.The fibrillation effect can be advantageous for creatingfabrics with an attractive appearance and appealing hand.

Fibrillation is most commonly used in the creation of the‘mill-wash’ or ‘peach skin’ fabrics. The process required toproduce such fabrics involves three stages: primaryfibrillation, cellulase treatment and secondary fibrillation.

Primary fibrillation occurs when the fabric is subjectedto hydro-mechanical action and the surface fibres fibrillateand are abraded. The fibrils on the long surface fibres bindtogether and the fabric can appear pilled. Hydro-mechanicalaction weakens the fibrillated fibres and makes them moresusceptible to enzymatic hydrolysis. Cellulase enzymeshydrolyse these fibres which are already weakened byhydro-mechanical action. It is the enzyme action in synergywith the mechanical action which is responsible for removalof the fibrillated fibres from the fabric surface.

Further wet processing of the fabric again leads tofibrillation. Since the upper surface of the fabric is now thecrossover point of the weave, it is here that fibrillationoccurs. In this secondary fibrillation stage, the fibrils are

† Current address: First Water Ltd, Hawkes Drive, HeathcoteIndustrial Estate, Warwick CV34 6LX, UK. Email:M.Donnelly@first-water.com

short and even and cannot, therefore, connect to formpilling. The secondary fibrillation produces two effects.Firstly, a small pile is created on the surface of the fabric.It is this pile that gives the special surface feel to the fabric,known as the peach skin effect. Further to this, since thefibrils are a fraction of the size of the fibres they appear tobe much lighter in colour even when they contain the sameamount of dye. It is this optical effect that gives the mill-wash, or dusted, appearance on the surface of the fabric.

A cellulase enzyme treatment of Tencel fabric to enhancethe surface appearance and feel of the fabric does notnecessarily require the whole cellulase system. Thesecellulolytic systems consist of several classes of cellulases,which act in a synergistic manner to degrade cellulose toglucose and other soluble sugars which is not the desiredend effect in this application. The endoglucanases (EGs) actby random cleavage of the cellulose polymer.Cellobiohydrolases (CBHs) attack the reducing or non-reducing end of the cellulose chain depending upon the typeof CBH [1], and produce primarily cellobiose. The CBHenzymes, when present, are often the dominantcomponents, sometimes representing about 50–70% of thetotal cellulase enzymes in the mixture [2]. These two classesof cellulases (EGs and CBHs) work in synergy at anefficiency that is dependent on the fraction of each in themixture [2]. Finally, cellobiase (CB), also named �-glucosidase, carries out the conversion of cellobiose toglucose and thereby eliminates end-product inhibition ofCBHs by cellobiose.

In order to carry out the hydrolysis of cellulose in avariety of environments, many microorganisms produce anarray of cellulases which differ in biochemical andfunctional properties. The actual types or classes ofcellulases produced, as well as the number and propertiesof the individual components within a class, are dependenton the microorganism in question, its growth conditions andits genetic capability. The situation is further complicated

20010407_j4500.p65 07/08/01, 12:21217

218 © Color. Technol., 117 (2001) Web ref: 20010407

by the fact that some, but not all, of the EG and CBHenzymes possess a cellulose binding domain, the functionof which is not clearly established. It has been suggestedthat this domain plays a role in concentrating or positioningthe enzyme on the substrate. It may even be involved insome type of physical modification of the cellulose such asmodification of hydrogen bonding between cellulose chains[3]. For the fungus Trichoderma reesei, at least fiveendoglucanases have been identified. In addition, at leasttwo CBH enzymes and one �-glucosidase are known [2].

Previous work has been reported on the performance ofa small selection of nine commercial cellulase productswhich were originally designed for use in such diverseindustries as cotton processing, alcohol production andcereal processing amongst others [4]. The results showedthat only a few of the cellulase products gave usefulsoftening and defibrillation when applied to Tencel.Analysis of these products showed differences in activity,and their respective ratios, against carboxymethyl cellulose(CMCase), filter paper (FPUase) and cellobiose (CBUase).Those that worked well for Tencel were characterised by ahigh CMCase activity together with a high ratio of theCMCase to FPUase activities [4].

As the production volumes and associated enzymetreatments of Tencel expanded, the number and type ofcellulases available also increased to meet both the specificneeds of Tencel processing as well as other cellulase enzymeapplications in the textile industry as a whole. Analysis ofa wider range of commercial cellulase products, a further21, was therefore carried out.

This paper describes laboratory-scale investigationsaimed at rationalising and improving the treatment of Tencelby investigating the action of purified components of theTrichoderma reesei cellulase complex on Tencel fibre. Themajority of work was carried out on two EGs. This approachdoes not take into consideration synergistic effects amongthe enzyme components and only considers mechanicalagitation effects at the level employed within the incubationequipment used.

New engineered cellulase products, manufactured undercontrolled fermentation conditions containing increasedamounts of the beneficial components, were then tested ona large-scale under conditions which took into accountmechanical effects from textile processing equipment. Theseengineered cellulases therefore had different compositionscompared to those cellulase systems found in nature andalso to standard commercial cellulases. Several advantagesobserved for the purified components in laboratory-scalestudies relative to the whole cellulase system were alsoexhibited by the engineered cellulases in the larger-scaletrials.

appropriate, core domains (whole enzymes less the cellulosebinding domain) for each of these components were alsosupplied by Genencor International.

Tencel fibre used was 1.7 dtex from Acordis’s Grimsbyproduction unit without application of a soft finish.

Commercial-scale trials were carried out on threedifferent cellulases, Primafast 100, Primafast SGL andIndiage RFW (Genencor International). Primafast 100 is awhole cellulase containing both endoglucanase andcellobiohydrolase activities (CMCase activity, ca. 1100 units/g). Primafast SGL and Indiage RFW are both engineeredcellulases and had been enriched in a specificendoglucanase. These two cellulases had CMCase activitiesof ca. 2000 units/g.

Garments constructed from commercially producedTencel fabrics of various weights from 150–400 g/m2 wereused in the study. Tencel (100%) was assessed alongsideblends with cotton and linen, in various garmentconstructions, including shirts, pants and skirts.

MethodsTencel fibre, before enzyme treatment with pure com-ponents, was scoured with 2 g/l Zetex HPLFN non-ionicdetergent (Zeneca) for 20 min at 60 °C, rinsed in distilledwater and dried at 110 °C to about 5% w/w moisture content.

Small bundles (50 mm length, 100 mg accuratelyweighed to four decimal places) of end-tied (plastic coveredwire) fibres were treated with each of the purifiedcomponents at a dose level of 2% w/w enzyme protein ondry weight of cellulose. Enzyme and fibre were incubatedin buffer (7 ml, pH 5.0, 0.05 M citrate buffer, at a liquor tofibre ratio of 15:1) in sealed 50 ml flasks in an orbital shaker(120 rpm, Gallenkamp) at 50 °C in separate experiments for45, 80 or 120 min. The rate of reducing sugar production(as glucose equivalents) in the treatment liquor wasmonitored by the DNS method [5]. This data was used tocalculate the percentage weight of the cellulose which hadbeen solubilised. After the specified treatment time, thefibres were removed and placed on a glass filter and washedwith hot water (> 85 °C) for more than 3 min to deactivatethe enzyme. The fibres were then dried at ambienttemperature until constant weight was achieved.

Mechanical properties were then measured using anInstron machine on ten fibre samples using pretensionedsingle fibres and methods as found in the AmericanStandard Test Method D2256 (1988). An average of the tenstress–strain curves was generated using a Lotus 1-2-3spreadsheet. Conditions for the Instron tests were 20 °C anda relative humidity of 65%. Machine parameters used were:sample rate 18.21 pt/sec, cross-head speed 10 mm/min andfull-scale range 0.0001 KN. Sample gauge length was about20 mm and was measured to three decimal places andrepeated eight times for each set of ten fibre analyses. Lineardensity of fibres was measured using a Vibroscope. Themean, standard deviation and coefficient of variation werecalculated from this data for each pure component data set.A two level t-test was then carried out to determinestatistically significant differences between enzyme treatedand blank samples of fibre that had been treated under thesame conditions except for the omission of the enzyme. Datais quoted to confidence limits of > 95 and < 99.9% andwhere no statistically significant difference was found the

ExperimentalMaterialsPure samples of several components of the Trichodermareesei cellulase complex (endoglucanases coded EG A, B andC, and a cellobiohydrolase coded CBH X) were supplied byGenencor International as buffered aqueous solutions ofknown enzyme content (measured as mg protein/ml). Where

20010407_j4500.p65 07/08/01, 12:21218

© Color. Technol., 117 (2001) 219Web ref: 20010407

result was quoted as null. The percentage change in eachmechanical property after a given treatment time with eachcomponent was then corrected to take into account themolecular weight of the pure enzyme. This normalised valuewas then used to compare all pure components in terms ofthe same molar equivalence dose of enzyme.

Surface appearance of fibres was examined usingscanning electron microscopy (SEM) at about 500�magnification (fibrillation appearance of about a dozenfibres) and at 7000� magnification (surface appearance of asingle fibre). Approximately a dozen photographs of eachsituation were taken for later examination.

Degree of polymerisation (DP) changes to fibres weremeasured by placing about 50 mg of enzyme treated fibre(equivalent molar dose of each enzyme) after either 1, 2 or4 h treatments in anhydrous dimethylsulphoxide (DMSO,10 ml) in a sealed bottle. These were allowed to stand formore than 1 h. After replacing the DMSO with a freshaliquot (10 ml) the swollen cellulose was treated withphenylisocyanate (1 ml), shaken and allowed to stand atambient temperature. Solubilisation was completed byheating at 70 °C for a few hours with periodic shaking thenreacted to completion over 24 h. After cooling excessphenylisocyanate was destroyed by addition of drymethanol (2 ml) followed by reaction at ambient temper-ature for ca. 1 h. The resulting cellulose tricarbanilate (CTC)was then precipitated by pouring into a methanol–watermixture (70:30, 100 ml) and standing for a few hours. Theslurry was then filtered through a nylon 6.6 membrane (0.45micron pore size, 47 mm diameter, Anachem) using amembrane filter unit under vacuum.

After removal of the vacuum the solid was slurried withca. 10 ml of 70 % v/v aqueous methanol then the vacuumwas reapplied. This procedure was repeated with 10 mlaliquots of water then 70% aqueous methanol. The solidobtained was removed from the filter and placed inpreweighed bottles and dried in a vacuum oven at 80 °C.The samples were then analysed for molecular weightdistribution by gas permeation chromatography (GPC)following dissolution of the CTC samples in HPLC gradetetrahydrofuran (THF) via periodic shaking and overnightincubation to produce a concentration of about 2 mg/ml. Foreach sample solution an aliquot of 100 �l was injected intothe GPC column and eluted at a flow rate of 1 ml/min andusing a temperature of 40 °C through a polystyrene (PS-DVB)guard column (50 � 7.5 mm) followed by a set of threeporous gel columns (PS-DVB, 300 � 7.5 mm) rated at 106,105 and 104 Å (Polymer Labs, UK). Baselines and peak limitswere set manually. A minimum of two injections was carriedout for each sample solution to check reproducibility. Theconcentration of the eluent was measured by refractiveindex (RI) using an Optilab interferometric refractometer.On-line light scattering measurements were made using aMini-Dawn, three angle laser light scattering detector (WyattTechnology). The specific RI increment for pure CTC (aschecked by FTIR) in THF at 40 °C was dn/dc = 0.165 ml/g.Molecular weight results (number, weight and z-average)were calculated using version 4.2 of Astra software and theDP were obtained by dividing by the molecular weight ofthe repeat unit (519).

Defibrillation efficiency of pure enzymes was determinedby prefibrillating small bundles of fibres (5 mm staple

lengths) in a glass screw top jar (60 ml) containing 32 mldistilled water and 16 g glass micro-spheres (Croxton andGarry, code 2024 CP03). The jar was sealed and shaken(Stewart Scientific flask shaker at 1800–1900 rpm) for 30min. The fibrillated fibre was removed, washed withdistilled water and filtered on a glass no. 1 filter then driedat ambient temperature for 24 h. On an in-house scale oflevel of fibrillation these fibres were 4.5 (scale from 0–10where 0 represents no fibrillation and 10 a high level).Samples of prefibrillated fibres were treated with purecellulase components in the same manner as unfibrillatedfibres except reaction conditions were 1, 2 or 3 h andenzyme dose was 1 or 2% w/w on dry weight of fibre. Afterdrying overnight three or four fibres were removed from thebundle and examined using a microscope (40�magnification). A photograph of two areas of each of twodifferent fibres were then taken and each was scored witha fibrillation index (FI) by comparison with the photographsof each fibrillation index on the in-house range of 0–10. Anaverage FI was calculated from the photographs of eachenzymatic treatment.

Rate of enzyme adsorption to fibre was measured byplacing bundles of fibres of about 50 mm length andweighing 100–200 mg (accurately weighed using a fourdecimal place balance) in each of a set of 100 ml baffledshake flasks containing 7 ml of 0.05 M citrate buffer, pH 5.0.Agitation (100 rpm) and temperature (5 °C) were providedusing a Haake SWB20 shaker. After sealing the flasks werepreincubated for 5 min to reduce the temperature to therequired value. A sample of one of each of the selectedenzymes was then added to a flask using a steriliseddisposable pipette at 2% w/w protein on dry weight of fibre.The flask was resealed and incubated for a set period (7.5,15, 30, 45, 60, 120 or 180 min). A blank was carried out foreach incubation time by omission of enzyme to the fibre/buffer. At the end of each incubation period each flask wasremoved from the incubator and a 1 ml sample was removedfrom the treatment liquor and placed in a plastic graduatedcentrifuge tube for determination of protein concentrationas described in the commercial enzyme analysis section.Protein adsorbed (mg bound/g of fibre) was calculated bysubtraction of the determined protein concentration after aspecific incubation time from the original proteinconcentration of the treatment solution. Amount of enzymeadsorbed by the fibre (nm/g) versus incubation time wasthen plotted. Statistical data for this analysis was generatedby a six-fold repetition of the determination of the amountof endoglucanase B which was adsorbed after a 2 htreatment time. This data was then assumed to apply to allother adsorption analyses.

Analysis of commercial cellulases (coded A–U) wascarried out using the IUPAC methods [5] for CMCase (units/g or ml), FPUase (units/g) or CBUase (units/g) activities. Totalprotein contents were measured using the Sigma diagnostickit (P5656) involving protein precipitation with trichloro-acetic acid. A protein calibration curve was constructedusing bovine serum albumin (fraction V ex Sigma).

Large-scale investigationsThe performance of the three commercially availablecellulase products (Primafast 100, Primafast SGL andIndiage RFW) was assessed by application to Tencel fabrics

20010407_j4500.p65 07/08/01, 12:21219

220 © Color. Technol., 117 (2001) Web ref: 20010407

in garment form. The cellulases were dosed in the range of2–4 g/l at their optimal pH and temperature. The relativedosages of each enzyme composition were based on equalfibrillation removal efficacy. Dosage selection was based onprevious experience in laboratory and field trials. Processingroutes as detailed in Table 1 were used, further details ofwhich are given in reference 6. The timing of the cellulasetreatment was the major difference between these tworoutes, i.e. whether it occurred before or after the dyeingprocedure. Route B was also one step shorter due to thecombination of the dyeing and prefibrillation processes.

Table 1 Processing routes used in the testing of cellulases on alarge-scale

Route A Route B

Step Process Step Process

1 Desize 1 Desize2 Prefibrillation 2 Dyeing/prefibrillation3 Cellulase treatment 3 Cellulase treatment4 Dyeing 4 Soften/secondary fibrillation5 Soften/secondary 5 Tumble drying

fibrillation6 Tumble drying

Results and DiscussionLaboratory-scale investigationsEnzyme analysisThe results of the enzyme activity and protein contentanalysis of commercial cellulase products emphasise theproblem of selecting the most suitable ones for Tencelprocessing. For example, CMCase activities ranged betweenabout 0.09 and 2027.00 units/ml whilst FPUase activitieswere between 0.54 and 86 units/ml. CBUase activities variedbetween none detected and 66 units/ml. There were alsolarge differences in total protein content for the products(ranging between about 0.09 and 37% w/w) and the ratiosof CMCase to FPUase activities varied between about 0.15and 3220.00. For a particular cellulase product highactivities did not always correlate with a high ratio or withhigh protein content which underlines the remarkablevariability in enzyme types and concentrations present incommercial cellulases. Since all of these features have costand performance implications, studies were carried out onthe pure components of the Trichoderma cellulase complexin order to determine which components were mostbeneficial for the treatment of Tencel.

Effect of cellulase components on fibre propertiesCharacterisation of the effect of the Trichoderma cellulasesystem components on Tencel fibre showed some diversefeatures. For example, EGs A and B, the principal enzymesinvestigated, were shown by SEM to smooth the surface offibres as well as the channels from which fibrillationoriginates. All core enzymes investigated also smoothed thefibre surface. In a fabric this effect is likely to ease themovement of fibres over one another and this could assistin producing a ‘soft’ feel. In contrast the CBH X, and its core,

roughened the surface by a pitting action. As this enzymeremoves cellobiose from the end of the cellulose chain, itis not expected to smooth the fibre. Not all endoglucanasesbehave in a similar manner since EG C did not produce ameasurable effect under these conditions by SEM analysis.

The action of the pure individual components on themechanical properties of fibres, the degree of fibrillation andthe amount of cellulose which is converted to soluble sugarsis given in Table 2. Only data which have a confidence limitof at least 95% are quoted although the general trendsobserved can be supported when additional information isgenerated by reduction of the confidence limits to ca. 90%.

A representative example of the changes of fibrillationlevel with treatment time is given in Figure 1 for the enzymeEG A (1 and 2% doses). This type of analysis was carriedout for each of the Trichoderma components. The data showvery complex relationships which are time-dependent andare likely to be influenced by variation in the fibreproperties, such as differences in fibrillation rate along fibrelength which might explain the difficulty in reproducing theblank experiments. However, the main emphasis ininterpreting these results is at the longer treatment timesfor the more effective 2% dose level where the differencesbetween blank and enzyme-treated fibre tends to be greatest.

In addition, the observed effects are probably dependenton the rate of adsorption of the enzyme onto specific regionsof the fibre. For example, EG C which exhibited the greatesteffect on strength and extension loss (ca. 20 and 40%,respectively) appears to have no effect on linear density andonly marginally reduced the initial modulus. Although thisenzyme adsorbs to the highest level of all theendoglucanases, in terms of amount and percentage ofprotein present in the treatment liquor (see Table 3), it ispoor at defibrillation especially over extended treatmenttimes of 3 h. It does, however, perform slightly better after1 or 2 h reaction time, in that the FI is reduced below thatfor the prefibrillated fibre, although this result is still inferiorto the other two endoglucanases tested.

These results again suggest that all endoglucanases donot behave the same and could indicate that EG C adsorbswith the least specificity of all of the endoglucanases in thatmore of the surface and surface fibrils are acceptable, oraccessible, to this enzyme. In contrast EG A and EG B adsorbto much lower levels but do have better defibrillationcapabilities, especially for EG A. This could suggest thatthese two endoglucanases are more specific in theiradsorption characteristics. One could speculate that theymight for example have a preference for lower crystallineregions such as that presumed to be present at kink-bandsor at the surface of the fibre fibrils as opposed to the mainbody of the fibre.

In addition, EG A and B produce lower strength lossesand have little effect on extension at break (Table 2).Furthermore, both of these EGs reduce the initial modulusto a greater extent than EG C which could contribute to asofter feel in that the fibres would be less stiff to the touch.EG A also shows an early increase in linear density whichcan be explained as arising from a swelling of the fibrediameter. This could arise during the early stages of enzymeadsorption in that the binding mechanism involves aninteraction with the surface to allow penetration of waterinto the fibre so that a plasticising effect takes place. This

20010407_j4500.p65 07/08/01, 12:21220

© Color. Technol., 117 (2001) 221Web ref: 20010407

Table 2 Specific defibrillation efficiencies and effects on fibre mechanical properties fordifferent enzyme treatments

Testa,b

LinearTreatment density/ Break Initial Defibrillation Cellulosetime swelling tenacity modulus efficiencyc solubilisedd

Enzyme (min) (%) (%) (%) (FI units) (%)

EG A 45 +13.5 Null –18.080 Null Null Null

120 Null –11.6 –12.0180 1.0240 0.087

EG B 45 Null –9.7 –8.380 Null –11.0 Null

120 Null –12.5 Null180 6.0240 0.042

EG C 45 Null –10.2 Null80 Null –12.1 Null

120 Null –19.0 –3.6180 8.0240 0.022

CBH X 45 Null Null –6.280 Null Null Null

120 +9.5 Null +7.0180 Not determined240 0.137

a 2% w/w enzymeb A plus symbol indicates an increase in value, a minus symbol a decrease in value, null indicates no

statistical difference in value between test and blankc Initial FI for all treatments (including blank) = 4.5 units; final FI value for blank treatment = 8.0 unitsd Cellulose solubilised to reducing sugars measured in equivalents of glucose

0 1 2 30

4

8

Enzyme treatment time, h

FI

1% EG ABlank2% EG ABlank

Figure 1 Relationship between treatment time and defibrillationefficiency for EG A; pH 5; temp., 50 °C

Table 3 Data for adsorption of endoglucanases to fibre (obtained at 5 °C)

Time to reach Removal of Onset ofmaximum Enzyme protein in enzymeabsorption adsorbed treatment desorption

Enzyme on fibrea (min) (nmol/g dry fibre) liquorb (%) from fibre (min)

EG A 105 (80–113) 90 22 150EG B 35 (24–45) 270 62 100EG C 110 (100–120) 525 69 120

a Range of time at maximum absorption in parenthesesb Measured at maximum value of enzyme absorption on fibre

could also be an explanation for the reduction of initialmodulus for this enzyme, and EG B, after 45 min treatment.

It is interesting to note that a similar transient increasein linear density and decrease in initial modulus was alsoobserved after ca. 1 h when Tencel fabrics were treated onthe commercial-scale with Primafast 100 cellulase. Thismight have contributed to the increased fabric bulk that wasobserved [7]. CBH X also increases the fibre diameter butonly after extended reaction times (Table 2). Since thisenzyme roughens the fibre surface by the formation of pits,it decreases the initial modulus after 45 min treatment butincreases it after 180 min hence overall produces negativeeffects.

Defibrillation efficiency graphs for enzyme dose levels of1% w/w were also plotted for each of the purifiedcomponents (data not shown). Compared to the equivalent

20010407_j4500.p65 07/08/01, 12:21221

222 © Color. Technol., 117 (2001) Web ref: 20010407

data for a 2% enzyme dosage less effective removal of fibrilswas noted. This suggests that at least the latter amount ofenzyme is required to obtain beneficial effects. Fibrillationgeneration in the blanks arising from mechanical actionduring the incubation is consistent in increasing the FI fromthe initial value of 4.5 to a final value of about 8.0 after 3h. However, the shapes of the graphs for different enzymetreatments again suggest complex relationships are inoperation. For example EG A and EG B show an increase infibrillation over the first 1–2 h of treatment in the samemanner, but not as extensive, as the blanks. Only afterreaching a peak does the FI decrease below the blanks inthe later stages of treatment. This may support the abovearguments that the binding domain reduces hydrogenbonding hence making the fibre more susceptible tofibrillation. In contrast, EG C does not show an increase inFI over the early stages instead FI is reduced relative to theblank. Eventually, however, the overall treatment becomesno better than the effects seen for the blanks. This mayrelate to the possible lack of specificity of this enzyme forthe fibrils as discussed earlier.

It is interesting to note that EG A requires about 60–90min to reach maximal adsorption (Table 3). It is possible thatonly after this has occurred does the enzymatic action ofdefibrillation overtake that of fibrillation generation arisingfrom mechanical action effects and the FI begin to bereduced after about 2 h as observed in Figure 1 (for 2% EGA). Products high in CBH enzymes have previously beenshown to be poor at defibrillation and thus were not furtherinvestigated for this property in the present study [4]. Allcores tested also showed an increase in fibrillation over thefirst 1–2 h treatment and then a reduction below that seenfor the blanks at the later stages of treatment.

The adsorption data in Table 3 were generated at 5 °Cwhich is a temperature designed to minimise reaction anddesorption [8]. After reaching a maximal adsorption, eachendoglucanase does show a plateau region in which theamount of enzyme adsorbed is constant but eventually thereare indications of desorption in that this amount decreasesafter about 100–150 min incubation. A possible explanationhere is that the surface of the fibres may have changed eitherdue to fibrillation generation or swelling so that the bindingdomains are less effective at retaining the enzyme on thesurface. The six-fold repetition of the determination of theamount adsorbed after a 2 h treatment of EG B suggests thatthe procedure is reasonably reproducible (average proteinadsorbed, 60.4%; range, 57.8–62.5%; standard deviation,1.83; coefficient of variance, 3.00; 95% confidence limits,60.4±1.92) with data obtained for each endoglucanase beingdifferent.

The figures for the amount of cellulose which isconverted to reducing sugars (expressed as glucoseequivalents) are all extremely low. Given that some wholecellulases are optimised for conversion of a large proportionof certain types of cellulose to glucose, this analysisconfirms that the proportion of the overall reaction ofcellulases on cellulose that is being investigated in thepresent paper is also quite small but contains many subtleand complex effects.

The data from the Instron analyses can also berepresented in the form of a stress–strain diagram. Figure 2provides a representative example of an averaged curve for

the ten analyses of the effects of EG A treatment over 2 h.This enzyme reduces the modulus over the whole of therange of the test, which could contribute to a softeningeffect, but only causes a small loss in strength and has littleeffect on elongation (the averaged break extension is 10.3%for EG A treated fibre and 10.8% for the blank fibre). Theslight oscillation in the curve at high extensions, and theapparent break at about 12% extension (Figure 2) is due tothe fact that at this stage there are fewer data points sincesome of the test data are for samples which have broken.The overall affect on the shape of the stress–strain curvefor EG A treatment contrasts with EG C treatment (data notshown) which causes a high loss of strength and extensionand does not effect the modulus up to and including theyield point.

0 4 8 120

10

20

30

40

Strain, %

Str

ess,

CN

/tex

(a)(b)

Figure 2 Stress-strain curve for EG A; treatment time, 2 h;(a) blank; (b) EG A treated

Effect of cellulose components on fibre degree ofpolymerisationFigure 3 gives the change of the degree of polymerisation(DP) of the fibre following enzyme treatment for CBH X orEGs A, B or C. The core enzymes did not produce ameasurable difference from the curve generated for theblank samples. This could suggest that the binding domainis required for a high degree of enzyme action to occur. Thismight arise because this domain concentrates the enzymeat specific points on the cellulose surface or because theCBD assists in making the surface more accessible toenzyme attack for example by reducing hydrogen bondingbetween cellulose chains as discussed earlier. Alternativelyfor the enzymes without binding domains then a size effectmay predominate in which the small enzyme is able topenetrate more of the cellulose pores and hence have agreater relative action.

EG C produced the greatest shift of distribution towardslower molecular weight species. This is indicated by thereduction in high molecular weight species and also theloss, via the washing stage of the isolation procedure, of lowmolecular weight species compared to the blank experiment.This level of change of DP distribution is sufficient to expecta change in the mechanical properties of the fibre hencecorrelates with the large reduction in strength and extensionobserved during fibre treatment with this enzyme. EG B hasa much smaller effect and EG A did not show a significanteffect on molecular weight distribution. CBH X also did notproduce a significant difference from the blank except fora slight increase at the high molecular weight region. This

20010407_j4500.p65 07/08/01, 12:21222

© Color. Technol., 117 (2001) 223Web ref: 20010407

1040

0.4

0.8

1.2

105 106 107

Blank/EG ACBH XEG BEG C

Figure 3 Change of the degree of polymerisation of the fibre(measured by GPC) following enzyme treatment with CBH X orEGs A, B or C

could be due to a small amount of reversion ortransglycosylation reaction by this enzyme in whichglycosidic bonds are formed not hydrolysed [9]. If thisinvolves a cross-link reaction then this may explain theslight increase in initial modulus (stiffness) of fibresobserved after a 2 h treatment with this enzyme (Table 2).

Correlation of all of the above data suggests that CBH Xis not a desirable enzyme for the treatment of Tencel dueto its poor defibrillation ability, and its propensity toroughen the fibre surface as well as the possibility ofincreasing fibre modulus (stiffness). Similarly EG C causeshigh DP changes and strength and extension losses, seemsless specific in its action, is poor at defibrillation and doesnot reduce initial modulus to the extent shown for EGs Aand B. EGs A and B have a number of favourable propertiessuch as little or a low effect on DP, modest strength losscombined with little effect on extension, are effective atdefibrillation and cause a reduction in initial moduluswhich might contribute to a softening effect.

Large-scale investigationsThe results described here summarise the experience gainedon the cellulase treatment of Tencel garments during anumber of trials on pilot and full-scale equipment [6,7].Cellulases with different compositions were used in thesestudies, namely Primafast 100 (whole cellulase containingEG and CBH), Primafast SGL and Indiage RFW (engineeredcellulases containing a specific EG). Fibrillation removal,tensile strength loss, fabric hand, and fabric weight werethe tests carried out on garments following these trials.Cellulase treatment after dyeing (Route B in Table 1)produced a more significant wash down effect particularlyon garment seams. However, no other significant physicaldifferences were seen between the two possible processroutes A and B.

Fibrillation removal efficacyEach of the enzymes used was found to be effective inproviding acceptable fibrillation removal at dosages in therange 2–4 g/l. Following this level of enzyme treatmentgarments did not show any significant appearance changeon subsequent washings. Earlier experience in theprocessing of piece goods had shown an aggressive enzyme

to be needed for complete fibrillation removal. For exampleIndiage RFW, an engineered cellulase, had previously beenconsidered a gentle enzyme with regard to fibrillationremoval in the treatment of Tencel fabrics. However, ingarment processing, this cellulase resulted in a high degreeof defibrillation over a similar processing time-scale as thewhole cellulase product Primafast 100. It is thought that thehigh level of mechanical action in garment processing incombination with enzymatic action removes fibrils moreeffectively.

Strength lossSince 100% Tencel is a very strong fibre, fabric strength lossresulting from controlled cellulase treatment is usually wellwithin acceptable limits. However, when Tencel is blendedwith other weaker cellulosic fibres like cotton, or thoseeasily hydrolysed by cellulase enzymes such as linen, thenfabric strength loss may be larger and hence more of aconcern.

In fabric processing trials, the engineered componentcellulases had produced significantly reduced strengthlosses compared to whole cellulases. In garment processingtrials also, the engineered component cellulases resulted inlower strength loss, however, the differences betweencellulases used were small and the results were notconsistent in all trials. Strength loss was more dependenton enzyme dosage than on enzyme type. The highmechanical action of garment processing may again be afactor, overriding the influence of the enzyme compositions.

In contrast, the use of Tencel blends showed up muchgreater differences in action between the cellulases tested.On Tencel/cotton and Tencel/linen blended fabrics, theengineered component cellulases typically resulted in up to15% lower fabric strength loss versus the whole cellulase.

Damage on garmentsProcess damage from cellulase treatment such as fabricbreak down on seams, pockets and elasticised waists is animportant consideration in garment processing. In severecases, if the enzymatic treatment process is not carefullycontrolled, fabric deterioration can occur to such an extentthat the material becomes unsaleable. The appearance offrayed seams or holes for woven garments and damaged ormultiple loose threads for knitted garments must thereforebe avoided as far as possible.

Production trials on fabrics and monitoring in garmentprocessing facilities have shown that fabric breakdown issignificantly reduced when engineered componentcellulases are used compared to conventional wholecellulases. This is especially evident on delicate fabrics andTencel blends.

Fabric handA panel of thirty experts from within the textile industrywere used to assess fabric hand. Features examined weresoftness and bulk of the fabric. A scale of 1–3 was usedwhere a rating of 1 designated worst hand and 3 representedbest hand. Figure 4 shows the aggregate of panel scoreratings [6]

On 100% Tencel, the handle attributes of the engineeredcomponent cellulases treatments were assessed higher thanthe whole cellulase, but the differences were small at the

20010407_j4500.p65 07/08/01, 12:21223

224 © Color. Technol., 117 (2001) Web ref: 20010407

100% Tencel Tencel/cotton Tencel/linen0

40

80Fa

bric

han

d pa

nel s

core

Primafast 100Indiage RFWPrimafast SGL

Figure 4 Aggregate of fabric hand panel scores

dosages tested. Larger differences were seen in the Tencel/cotton and Tencel/linen blends. Panellists consistently ratedtreatments by engineered component cellulases as softer andbulkier compared to the whole cellulase, irrespective of thedosages. This suggests that hand preference may be directlyrelated to the composition of the enzymes and not to enzymedosage. Possibly the three enzyme compositions testedinfluenced the fabric hand properties differently due todifferences in their mode of action on cellulose as describedin the introduction.

Further to subjective hand evaluation, more objectivetests using the ‘Kawabata Evaluation System’ [10] wereconducted on a small group of 100% Tencel garments.Analysis of surface, compression, shear, bending and tensileproperties correlated well with the overall results of thesubjective evaluations. The fabric handle attributes weregraded higher for fabrics treated with engineered componentcellulases. Tensile properties of fabrics treated with wholecellulase were poor, with low recovery to applied force, afactor that can negatively influence consumer judgement offabric hand.

Fabric mass/unit areaThe mass per unit area of the finished garments wasmeasured to assess its influence on fabric hand. Resultsshow that there were no significant differences in the massper unit area at equal degree of fibrillation removal. Thissuggests that hand preferences may be directly related tothe composition of the enzymes.

ConclusionsIn the laboratory-scale investigations, complex relationshipswhich are time and dose dependent can be determinedwhen pure components of the Trichoderma complex actupon Tencel fibre. In the absence of considerations whichinclude synergistic effects and the level of mechanicalagitation other than that used in the incubation, statisticallysignificant differences in effects of the pure components onTencel fibre can be determined. This allows the componentsto be classified as more or less desirable for the treatment

of Tencel when judged by their effect on fibre mechanicalproperties, their specificity in mode of action and theirdefibrillation efficiency. Endoglucanase A and B appearmore preferable than endoglucanase C or cellobiohydrolaseX.

Some but not all effects seen in the laboratoryinvestigations carried over into the large-scale tests,presumably due to agitation differences. Mechanical actionhas previously been shown to be an important factor in thecellulase processing of cotton cellulose [11] and was felt tohave a large effect in these trials as compared to those seenpreviously in the treatment of Tencel fabrics. Engineeredcellulases were shown to be at least as effective atfibrillation removal as whole cellulases. Lower strengthlosses were observed especially when Tencel was blendedwith other more delicate cellulosics. When dosed optimally,engineered component cellulases also produce superiorfabric handle with better bulk and drapeability, andminimised seam damage. For the future, it would beinteresting to determine the effect of pure cellulasecomponents, rather than enriched cellulases, on themechanical properties of Tencel fabric after treatment underthe various mechanical agitation conditions encountered inlarge-scale fabric processing equipment. Further work oncharacterising the action of the cellulose binding domainwould also be useful in understanding the action ofcellulases on celluloses in general and on Tencel inparticular.

* * *

Peter Laity and John Horsler are thanked for help with theGPC analysis and Dr John Meredith for contributions toenzyme analyses. Genencor International are thanked forsupply of the purified enzymes.

References 1. T T Teerei, Trends Biotechnol., 115 (1997) 160. 2. R Bhamidimarri, A G Hashimoto and P Hobson, Bioresour.

Technol., 36 (1991) 42, 67. 3. L Ruohonen, T Alwyn-Jones, J K C Knowles, T T Teerei and T

Reinikainen, J. Biotechnol., 24 (1992) 169. 4. M J Donnelly and P Goode, Action of selected commercial

cellulases on the new cellulosic fibre Tencel, 10th AnnualMeeting of Warwick Biotransformation Club, Warwick, UK(1997).

5. T K Ghose, Pure Appl. Chem., 59 (1987) 257. 6. A Kumar and A Harnden, Book of Papers, AATCC Int. Conf.,

Philadelphia (1998) 482. 7. Commercial-scale trials at Wardles, UK (1997). 8. E Hoshino, M Nomura, M Takai, M Ozaki, K Nisizawa and

T Kanda, J. Ferm. Bioeng., 77 (1994) 496. 9. B Tousaint and M R Vignon, Biotechnol. Lett., 12 (1990) 587.10. K Ito and S Kawabata, Conception of automated tailoring

controlled by fabric objective measurement data (Osaka: TextileMachinery Society of Japan, 1986).

11. A Cavaco-Paulo and L Almeida, Biocatal., 11 (1994) 353.

20010407_j4500.p65 07/08/01, 12:21224