ects of Lipase and Cutinase Enzyme Surface Treatments on Light Re " ectance and Colour Changes in Non-Circular Cross-Sectional Polyester Fibres

Lipase and cutinase enzymes were applied to non-circular cross-sectional polyester fi bres. Refl ectance and colour changes of the fi bres were investigated under specifi c treatment conditions. The results indicated that lipase L0777 did not aff ect these fi bres, regardless of time of treatment or changes in pH. With cutinase, pits on the surfaces of the fi bres occurred when cutinase was applied at 55oC and pH of 7.00 and 8.50, respectively, for 24 hours. This was demonstrated by refl ectance and colour changes, as well as by SEM images. The wide-angled x-ray diff raction (WAXD) curves of the cutinase-treated fabrics were ambiguous in that the small changes may have been the result of heat rather than enzyme treatment. Diff erential scanning calorimetry (DCS) results for both untreated and cutinase-treated polyester fi bres showed obvious changes. The peak at 250oC did not change but that at 265oC increased in area, indicating re-crystallisation.


Introduction
Historical applications of enzyme treatments in natural textiles are the "dew retting" of ax using enzymes secreted from micro-organisms in soils; amylases for removing starch sizing from cotton fabrics [1]; cellulases for the bio-polishing of fabrics and incorporation within detergents for removing surface fuzz, reducing the scattering of light and the "brightening" of cotton fabrics [2]; and scouring [3][4][5] which uses cellulases [6][7][8][9], pectinases [10][11][12][13] and pectate lyases [14][15][16].Other enzymes have been used for changing the chemical and physical surface properties of common polyester, polyethylene terephthalate (PET).Interest in modifying the surfaces of PET initially focused on alkaline treatments.Figure 1 [17] shows the hydrolysis reaction of PET with sodium hydroxide, in which the electron-de cient carbonyl of PET is attacked by hydroxyl ions in aqueous sodium hydroxide, resulting in chain scission and the formations of carboxylate and hydroxyl end groups.It is believed that this reaction is limited to the surface and it was concluded that most of the resulting PET oligomer le the bre surface and went into the solution.It was observed that a er the reactions the turbid sodium hydroxide solution gradually cleared and a white layer of sediments was found in the bottom of the solution.
e development of biotechnologies with speci c applications to bres has presented new possibilities.Lipases [18,19,20], with the capability of catalysing hydrolysis and the synthesis of esters formed from glycerol and long-chain fatty acids, have created applications [21,22] that have led to possibilities in bioprocess engineering [23].Early studies by Tokiwa and Suzuki [24,25] demonstrated that lipases could hydrolyse certain polyesters, and that the rate of hydrolysis was strongly related to the polyester melting point, the chemical structure of the polyester, or the number of polyester molecules within the reactive mixture [26].Figure 2 illustrates the model.e time-dependent degradation pro les of PET were strongly in uenced by the material's surfaces and by the addition of surfactants [27], producing monomeric-like materials.Cross-linked heterochain aromatic polyesters resisted microbial degradation [28].e temperature di erence between the melting point of the polymer and where polyester degradation by lipase took place turned out to be the primary controlling parameter for aliphatic polyesters [29].Selectivity of lipases with regard to aliphatic or aromatic environments near the ester bonds did not occur, but the lengths of the aliphatic domains and the speci c inter-structure were factors [30][31][32].Compared to alkaline treatments, ve of six lipases were more e ective in improving the wetting and absorbent properties of PET fabrics [33].Full strength was retained.is was con rmed by Chaya and Kitano [34].Peeling and strati cation were observed on surface layers of the bres with the formations of hydroxyl and carboxylic groups and ester derivatives [35].Increased hydrophilicity of PET fabrics lipase treatment has also been shown by Alisch-Mark et al. [36], Kim and Song [37][38][39] using di erent surfactants in the experiments [40].Khoddami et al. [41] implied that esterase hydrolysis was limited to surfaces and more a ected by any increase in surface area than changes in the internal structure from drawing.Billing et al. [42] found that an esterase showed high speci city towards short and middle chain-length fatty acyl esters of p-nitrophenol.Donelli et al. [43] indicated that crystallinity increased and amorphous content decreased but The Eff ects of Lipase and Cutinase Enzyme Surface Treatments on Light Refl ectance and Colour Changes in Non-Circular Cross-Sectional Polyester Fibres their enzyme had higher activity on amorphous PET but minor changes in crystalline PET.Cutin is part of the cutical, the waxy polymeric coating on all plant surfaces.Cutin consists of omega hydroxyl acids and their derivatives, which are interlinked via ester bonds, thus forming a large polyester polymer.Cutinase studies have been active.Masaki et al. [44] demonstrated that a cutinase could degrade high molecular weight polylactic acid (PLA) and other "biodegradable" plastics.Vertommen et al. [45] showed the e ect of crystallinity.For Donelli et al. [46] both alkaline and enzyme treatments increased hydrolysis in amorphous and crystalline lms.Crystalline PET was modi ed more strongly by alkali than by cutinase, whilst the opposite occurred for the amorphous lm. is implied that alkali was more e ective than cutinase in enhancing the hydrophilicity of PET lms, with the effect stronger on amorphous than crystalline lms.A genetically modi ed bacterial cutinase [47] provided valuable insight as to how enzymes can be improved by molecular engineering for synthetic bre changes.e modi ed cutinase [48] hydrolysed fatty acid monoesters with varied acyl chain lengths and had preference for short-chain substrates.e activity was higher than cutinases from bacteria and fungi.Cutinases from Humilica insolens (HiC), Pseudomonas mendocina (PmC) and Fusarium solani (FsC) on PET lms [49] used lms with a low-crystallinity of 7% (lc) and biaxially oriented (bo) poly(ethylene terephthalate) (PET) with a crystallinity of 35% as model substrates.e cutinases had a 10-fold higher activity for lcPET than for the bo-PET.For all three cutinases, the aqueous soluble degradation products were exclusively terephthalic acid (TPA) and ethylene glycol (EG).Aqueous, insoluble oligoesters, particularly cyclic trimers commonly extracted from PET bres during dyeing, are o en blamed for the greying of PET fabrics.ese can be removed by enzyme-catalysed hydrolysis under mild conditions, which cleans the dyeing machine and improves the lustres of fabrics [50].Recili and Gorensek [51] the in uence of treatments of PET on the quantity of extracted oligomers and their compositons.Alisch-Mark et al. [36] demonstrated that the colour in the fabrics became more intense, corresponding to an increase in hydroxyl groups on the surfaces of hydrolase-treated bres.Wang et al. [52] stated that bis (2-hydroxyethyl) terephthalate (BHT)-induced extracellular lipase catalyses the hydrolysis of the PET model substrate diethyl p-phthalate (DP).ere was an increase in K/S values of dyed PET fabrics a er enzymatic treatment, as well as increased moisture regain and weight loss.e water contact angle and static half decay time decreased slightly.Eberl et al.
[53] conrmed that a lipase from ermomyces lanuginosus and cutinases from ermobi da fusca and Fusarium solani hydrolyse PET.Lipases and cutinases are both EC 3.1.1,hydrolases that could hydrolyse the ester bond in PET.Lipases speci cally attack the ester bond in lipids (fats).Cutinases are speci c for the hydrolysis of primary alcohol esters contained in cutin, the protective covering of plants.Given the extensive work mentioned above on the surface modi cation of PET with enzymes such as lipases and cutinases, it is postulated that lipases and/or cutinases could change the surfaces of PET bres, thus creating changes in the light re ected, and the colours of the PET fabrics.

Fabrics and fi bres
Polyethylene terephthalate (PET) fabrics incorporating melt spun non-circular cross-sectional (NCCS) lament yarns in the lling (we ) direction were used during all experiments.e warp direction consisted of fully drawn PET lament yarns (FDY).Figure 3 shows optical images of the fabrics.Sample C01 had round cross-sectional bres with crystallinity of 25.60% and an orientation degree of 76.1%.Sample C11 had a crystallinity of 20.85% and an orientation degree of 79.20%.Sample B2 had a crystallinity of 21.90% and an orientation degree of 81.20%.All of these were non-circular cross-section bres, as shown in Figure 4.In order to observe the di erent cross-sections clearly using the optical apparatus, the NCCS PET bres were dyed black, and the bres with circular cross-sections were red.

Hydrolysis Treatments
e fabrics were treated with sodium hydroxide (NaOH), and with lipase and cutinase enzymes in order to compare the e ects of the enzymes on the known e ects of strong NaOH treatment on polyester bres.Sodium hydroxide treatments Fabrics were treated with 2% w/v NaOH solution at 95°C for 0.5, 1, 3, 5 and 6 hours, at a liquor ratio of 50:1.Enzyme treatments Two enzymes were used for treating the PET fabrics, a lipase and a cutinase.
ese are both subsets of hydrolases (EC 3.1.1)that act on carboxylic ester bonds.Lipase treatments A tris(hydroxymethyl)aminomethane (TRIS) bu er at pH 8 was used for all treatments, with the pH value adjusted by using either 1NHCl or 0.1NNaOH.e ratio of PET fabric mass (g) to volume of TRIS bu er solution (ml) was 1:80.e treatment times were 90 minutes at 40°C in a shaking water bath at 150rpm with lipase at a concentration of 6.25ml/l in solution.
e lipase used was L0777 (EC 3.1.1.3-a triacylglycerol lipase), obtained from Sigma Aldrich.It was isolated from ermocmyces lanuginousus with an activity of more than 100,000 units/g.Cutinase treatments e lipase used in this experiment was 'Stickaway' from Novozymes at a concentration of 6.25ml/l in solution.e treatment conditions are listed in Table 1.

Surface Morphology
e surfaces of the treated fabrics were examined using an FEI Quanta FEG scanning electron microscope (SEM) with accelerating voltage of 5kV.

Wide Angle X-Ray Diff raction
Wide angle X-ray di raction (WAXD) was carried out on the untreated and enzyme treated samples in order to investigate the crystallographic structure before and a er the treatments were applied.Wide-angle X-ray di raction data were collected using a Philips Analytical X-ray Instrument, X' Pert-MPD (PWD 3020 vertical goniometer and PW 3710 control unit) employing Bragg-Brentano para-focusing optics.e WAXD patterns were recorded over step sizes of 0.05° within a 10-80° range with a scanning rate of 2°/min.Line focus Ni-ltered CuK-radiation The Eff ects of Lipase and Cutinase Enzyme Surface Treatments on Light Refl ectance and Colour Changes in Non-Circular Cross-Sectional Polyester Fibres from an X-ray tube (operated at 40kV and 45mA) was collimated through Soller slits of 0.04 radians, a xed divergence slit of 1° and a mask before applying the X-rays to the samples.Figure 6a indicates that a er lipase treatment with specular component included there was a signi cant di erence in visible light re ectance for the C11 samples, less than one for the CO1 samples and virtually none for the B2 samples. is was con rmed by the CIE L* di erences as shown in Figure 6b.

Treatment with Lipase for 24 hours
In order to ascertain whether longer treatment might create more change in the bres, the white samples were treated in a TRIS bu er adjusted to pH 9.0 at 40 o C for 24 hours at the same concentration of lipase.A er that the re ectance and colour indexes were measured in both the SCI and SCE modes.e comparisons between 90min and 24 hour treatments are shown in Figures 7-9.e data in these gures show that treatment by the lipase L0777 for 24 hours had little e ect on the re ectance and colour index values in neither the SCE nor SCI modes.

SEM Images of the Fibres after Lipase Treatments
e SEM images of the samples treated with lipase for 90 minutes and 24 hours are shown in Table 2.As can be seen from the SEM images, there were no pits or cracks induced by the lipase L0777 treatment on the surfaces of the PET bres. is correlates with the small di erences in re ectance and colour changes shown in the previous gures.e solution to this question might either be by using another more e ective enzyme, or changing the treatment conditions.

Refl ectance and CIE L*a*b* values before and after cutinase treatments
e di erences in cutinase treatments for all the fabrics made of bres with cross-section of C11 are listed in Table 2. e re ectance R% in the visible light region of the cutinase treated non-circular cross-section bre fabrics was tested to explore the e ects of di erent cutinase treatments on the fabrics.
e re ectance within the visible region is shown in Figure 10(a).e primary di erences within the re ectance spectra are in the 540-620nm region.e re ectances of treated sample 2# and 4#, as well that of sample 3#, were lower than that of untreated 1# and over-treated 5# within the wavelength range of 540 to 620nm.In the 510 to 640nm range, the re ectance of the over-treated sample was higher than that of the untreated sample over the entire visible light region.is means that careful attention should be paid to the enzymatic treatment conditions and any unexpected by-e ects noted.e re ectance levels for sample 2# were lower than those of sample 1# across the entire wavelength region, which means that the cutinase would be e ective during the surface modi cation of non-circular cross-section polyester fabric under the relatively modest conditions shown in Table 3. is trend was further con rmed by the re ectances of sample #3, which was incubated at pH 8.50.e re ectance of #3 within the wavelength region from 510 to 640nm was the lowest for all the samples, indicating that the variance in pH values had a signi cant e ect.When the treatment temperature was increased, sample 4# had almost the same re ectance values as those of sample 2#. is suggests that temperature might have less signi cance.However, the re ectance values of sample 5# were the highest for all samples, even than that of the untreated sample #1.
ere might be two reasons for this.Firstly, either the higher temperature or the longer treatment time might have created a rough surface of bre, even more than in the solution at pH 8.50.Secondly, the longer treatment time may have had a synergistic effect with both the higher temperature and pH value.
e CIE L*a*b* values of the cutinase treated NCCS PET fabrics were also measured to determine whether the colours of these samples changed a er applying the treatments.ese results are shown in Figure 10b.e CIE L* value represents the whiteness or brightness of the samples, and ΔL* implies the di erence between the treated samples and the

SEM Images of Fabrics after Cutinase Treatments
e SEM images of the cutinase treated samples are shown in Figure 11.Some pits on the surface of the treated PET bres can be seen in Figure 11.A number of cracks appeared as the pH values of the TRIS bu er increased up to 8.50 with the presence of cutinase.ese data matched well with those of Kim et al. [37,39], although there was some di erence in the details of the cutinase used and conditions applied.It appears that the pH value of the TRIS bu er has a signi cant e ect on cutinase activities, especially on its hydrolysis of PET bres.
Figure 12 shows the surface of the treated bres as the treatment temperature increased to 55 o C with TRIS bu er pH at 8.50.As can be seen in Figure 12, the surface of the PET bres seems to have been uniformly degraded, thus not forming individual pits on the surface.

SEM images of NaOH-treated fabrics
Sodium hydroxide attacks the surface of the PETbres.e SEM images shown in Figure 13 indicate that pits occurred on the surface of the PET bres a er 0.5 hours, which matched the results in literature [52].e numbers and area covered by the pits increased as the treatment time increased.planes.e Gaussian double peaks-tting was used to analyse the WAXD curves in Figures 14 and 15. e results are shown in Table 3 and Figure 16.Table 3 indicates that there were sharp decreases in the intensity, area and width of the main peak (P1) between samples 1# and 2#.e intensity decreased whilst the area increased as the pH increased when comparing sample 2# with 3#. e inverse trend occurred as the treatment temperature increased, when comparing samples 3# and 4#.

Diff erential Scanning Calorimetry Results
e di erential scanning calorimetry (DSC) curves are shown in Figure 17.ese show that the crystallinities of the treated samples decreased as cutinase treatments were applied; there are two peaks in the DCS curves of both untreated and treated PETbres.e obvious change is that the peak at 250°C did not change but the peak at 265°C increased in area.e trend follows the changes in pH, temperature and time of treatment.e two peaks at 250 and 265°C in the DSC curves might be ascribed to the core-shell crystallisation characteristics of melt spun PET laments with circulated ambient air cooling; 250°C would represent the core part and 265°C the shell.Similarly to the reaction of PET with sodium hydroxide [52], lipase and cutinase also attacked the surfaces of the substrates used [33][34][35][36][37][38]40], especially within the amorphous region [41,44,45,48]. is caused the crystallinity percentage of the outer layer surface to increase [48], leading to the corresponding increase in melting   e application of 2 % w/v NaOH creates surface pits on PET bres.e use of lipase L0777, however, was ine ective during surface modi cation to those fabrics under speci c treatment conditions.
ere were no minor improvements a er long hours of treatment and changes in pH values.Whether or not the specular factor was included (SCI) or excluded (SCE) in re ectance measurements it had insigni cant e ects on the re ectance spectrum and colour changes of the untreated and lipase treated white NCCS PET fabrics.However, surface pits on the bres resulted when cutinase treatments were applied at a temperature of 55 o C and pH values of 7.00 and 8.50, respectively, for 24 hours.ese results were con rmed by the re ectance and colour changes as well as in the SEM images.ese results were comparable to those obtained in NaOH treated bres.
e internal structural di erences between the untreated and cutinase treated PET fabrics were con rmed by wide-angle x-ray di raction (WAXD) and di erential scanning calorimetry (DSC) analysis.WAXD patterns showed that the most prominent peak at 2θ = 21.3 o disappeared after cutinase treatment, as well as a small peak shi from 2θ = 22.9 o to 2θ = 22.5 o (110).ese changes in WAXD were not dependent on the X-ray incident angles (0 o , 45 o and 90 o ).ese internal changes are undoubtedly the result of the heat treatment involved rather than the enzymes themselves.e results from cutinase suggest the need for extensive investigation into this class of enzymes for application when modifying PET surfaces, both for changes in re ectance properties but also for creating more reactive surfaces for further functional changes.

Figure 2 :
Figure 2: Process of chemical reaction between PET and enzymes

Figure 3 :
Figure 3: Optical images of fabrics

Figure 4 :
Figure 4: Cross-sectional images of bres e fabrics for enzymatic experiments were rst scoured to remove any dust and/or oil that might have remained on the fabric a er weaving.All the fabric samples were subjected to a solution of 1g/l of sodium hydroxide (NaOH) and 3g/L sodium hydrosulphite (Na 2 SO 4 ) at a liquor to fabric ratio of 20:1 for 20 minutes at 80°C.ese were then washed thoroughly in cold distilled water and then conditioned at 26°C and 65% relative humidity for 24 hours.
Re ectance (R%) was measured within the visible light regions of 360 to 750nm.e CIE L*a*b*C* values were monitored using a Colour-Eye 7000A Spectrophotometer (Macbeth, USA) with specular component included (SCI) and with specular excluded (SCE).

e
treated bres were changed into powder in a Wiley Mill, and 4 milligram samples weighed out.e temperature range for di erential scanning calorimetry (DSC) was from 95 to 290°C, heating at 10°C/ min within a owing 40cc/min nitrogen atmosphere.3Results and Discussion3.1 Lipase Treatment for Ninety Minutes e re ectance of the specimens and the CIE L*a*b*C* values measured changed a er the lipase treatments were applied to the fabrics for 90 minutes.Figure 5 shows the colour index values and reectance of specimens before enzyme treatments.

Figure 5 :
Figure 5: CIE L*a*b* values (a) and re ectance R% (b) of samples with specular component included (SCI) and excluded (SCE) before lipase treatments

Figure 6 :
Figure 6: Re ectance R% (a) and CIE L* values (b) of samples before and a er lipase treatment for 90 minutes with specular component included (SCI) mode

Figure 7 :
Figure 7: Re ectance R% of the samples treated with lipase for 90min (a) and 24h (b) with specular component included (SCI) and excluded (SCE)

Figure 9 :
Figure 9: Re ectance R% (a) and CIE L*a*b* values (b) of the lipase treated white samples with TRIS buffer at pH 9 for 24h under SCI and SCE modes

Figure 8 :
Figure 8: CIE L*a*b* values of the lipase treated white samples for 90min (a) and 24h (b) with specular components included (SCI) and excluded (SCE)

Table 2 :
SEM images (5000 ×) of PET bres a er lipase treatments letn.58(1), 33−46 The Eff ects of Lipase and Cutinase Enzyme Surface Treatments on Light Refl ectance and Colour Changes in Non-Circular Cross-Sectional Polyester Fibres standard/untreated one.e results shown in the gure demonstrate that the ΔL* values of all the treated samples had decreased, which means that the treated samples became less bright when compared to the untreated sample.e exception was sample #5, which sharply increased in CIE L* value a er the cutinase treatment.is was contrary to what was expected.

Figure 10 :
Figure 10: Re ectance (a) and CIE L*a*b* values (b) of the untreated and treated samples in the visible region

The
Eff ects of Lipase and Cutinase Enzyme Surface Treatments on Light Refl ectance and Colour Changes in Non-Circular Cross-Sectional Polyester Fibres3.7 Wide Angle X-Ray Diff ractione results from wide angle X-Ray di raction (WAXD) of the untreated and cutinase treated samples are shown in Figure14.ere is a major peak with high intensity (110) and minor peaks (010 and 100) for the untreated sample.e 110 peak disappears during the cutinase treatment although it is unclear as to whether the crystallinity changes between the untreated and cutinase treated samples resulted from the cutinase only, or the water heat-treatment.

Figure 14 :
Figure 14: WAXD Results of untreated and cutinase treated samples

Figure 16 :
Figure 16: Analysis of WAXD curves of cutinase treated PET fabrics: (a) peak position & width; (b) areas & height energy, re ected by the increase in the area under the peak at 265°C within the DSC pro le.

Figure 17 :
Figure 17: DSC results for untreated and cutinase treated samples

Table 3 :
Gaussian Fit Analysis of WAXD curves *Note: P1-peak 1, P2-peak 2 derived by using Gaussian double-peak tting Tekstilec 2015, letn.58(1), 33−46 The Eff ects of Lipase and Cutinase Enzyme Surface Treatments on Light Refl ectance and Colour Changes in Non-Circular Cross-Sectional Polyester Fibres The Eff ects of Lipase and Cutinase Enzyme Surface Treatments on Light Refl ectance and Colour Changes in Non-Circular Cross-Sectional Polyester Fibres