Introduction

By-products from the textile industry of natural fibres (wool, silk, cotton, etc.) consist of fibrous polymers which are unfit for spinning because of their unsuitable physical properties. These materials could be recovered, dissolved and transformed into filaments by chemical spinning processes. In the first decade of the 20^th century, fibres based on casein, soybean, peanut, zein, etc. received considerable attention in the United States, Europe and Japan as a cheaper substitute for wool and silk.

Today, the exploitation of these renewable resources, together with the possibility of producing fibres with intermediate or innovative properties by combining different materials (keratin, cellulose, chitosan, silk fibroin), has great importance.

Keratin wastes are a potential renewable starting material, but in their native state they are insoluble in most of the solvents used for fibre spinning, due to the presence of disulphide bonds which form crosslinks between the protein chains. Another element of stability is the close network of inter-and intra-molecular hydrogen bonds [2].

Keratin dissolution may take place only after cleavage of the disulphide bonds; this can be achieved by reduction or oxidation. Reducing agents are always used in combination with a protein denaturing agent, like urea, that breaks hydrogen bonds. The reducing agents often used are thiols, for example thioglycollic acid [18,13,9], dithiothreitol (DTT), and 2-mercaptoethanol [13]. Disulphide bonds can also be broken up by simple reducing agents such as sodium sulphite (sulphitolysis) [14a].

The range of oxidative agents able to break S-S bonds consists of peracids (performic and peracetic acid), hydrogen peroxide and chlorine [7, 17, 16].

The present work deals with the preparation and characterisation of pure keratin and cellulose films and keratin/cellulose blend films, prepared by regeneration from cuprammonium solution.

Among the wide range of solvents available, the cupric tetramine hydroxide solvent system (Cu(NH3)4(OH)2) was used because it is employed in the industrial Bemberg Process for the manufacture of rayon cupro. [11]. Moreover, it has been reported that this solvent is able to dissolve silk by-products, leading to the preparation of silk-cellulose blend solutions which are suitable for fibre spinning [4].

Experimental

Materials

Wool fibres (WF) were cleaned by the sohxlet extraction with petroleum ether to remove fatty matters, washed with distilled water and dried at 50°C for 4 hours. The cleaned and dried wool (0.2g) was dissolved in a solution (10ml) containing 0.3 M sodium disulphite and 10 M urea, adjusted to pH 7 with NaOH 5 N, for 4 hours at 65°C under intermittent shaking. Subsequently, the suspension was passed through metal sieves (250 µm) and filtered by centrifugation with centrifugal-driven Millipore filters (pore size 5 µm), at 12,000 rpm for 15 minutes, to remove the residual undissolved material. Proteins in solution were precipitated by the addition of HCl 5N up to pH<4. The keratinous material obtained was washed three times with distilled water (5 ml each) and dried at 50°C for 4 hours. This sample was identified as the mB/U wool extract.

WF, cleaned as described above, were also subjected to extraction with a DTT/Urea (Sigma-Aldrich) solution, containing Tris/HCl (Sigma-Aldrich) 550 mM, pH 8.6; DTT 140 mM, EDTA (Sigma-Aldrich) 5mM, urea 8 M [15]. This sample was referred to as the DTT/U wool extract, and was used as a reference sample for electrophoresis studies. Cellulose powder from cotton linters purchased from Fluka was used without any preliminary treatment.

The mB/U extract and cellulose powder were dissolved in the cuprammonium hydroxide solvent (Cu solvent), a blue solution in which a strong bivalent base exists, i.e. cupric tetramine hydroxide ([Cu(NH3)4](OH)2). This metal complex was obtained from basic copper sulphate (CuSO4•3H2O). The cuprammonium solution of pure cellulose (6% w/w) was prepared according to the standards of the Bemberg Process [11].

The cuprammonium solution of pure keratin was prepared in the following way: 700 µl of ammonia solution (28%), cooled in the freezer, was added to a flask containing a suspension of copper basic sulphate (50 mg), water (100 µl), keratins (70 mg) and NaOH 8% w/w (110 µl). Dissolution was performed in a flask plunged in a bath of water and ice, under intermittent shaking, for 7 hours. Pure and blend films were prepared by dissolving the polymers separately and by mixing them in the following keratin/cellulose (w/w%) proportions: 80/20, 60/40, 40/60, 20/80.

Solutions were spread over a glass plate and coagulated in a bath of 0.5 M sulphuric acid. The films prepared as described were washed with glycerine/water (50/50 vol%), then rinsed with distilled water and dried at room temperature.

Methods

SDS-PAGE was performed using Xcell SureLock Mini-Cell (Invitrogen), on 12% polyacrilamide gels capable of separating proteins with molecular weights included between 100,000 and 14,000 Da. Myosin, bovine serum albumin, glutamic dehydrogenase, alcohol dehydrogenase, carbonic anhydrase, myoglobin and lysozyme were used as the molecular weight markers. FT-IR spectroscopy analyses were performed with a FT-IR Nicolet NEXUS spectrometer equipped with an ATR diamond cell (SPECAC). Measurements were taken directly on dried samples. Microscopic investigations were performed with an LEO (Leica Electron Optics) 435 VP SEM, using an acceleration voltage of 15 kV and 30 mm working distance. The films were mounted onto aluminium stubs by means of double-sided adhesive tape and sputter-coated with a 20 nm thick gold layer in rarefied argon, using an Emitech K 550 Sputter Coater, with a current of 20 mA for 180 s.

Results and Discussion

Extraction and dissolution of wool keratins

Extraction and dissolution of wool proteins takes place effectively after cleavage of the disulphide bonds, which can be achieved by oxidation, reduction or sulphitolysis. However, because almost all oxidizing and reducing agents are harmful, we preferred to use sulphitolysis with m-bisulphite/urea as the extraction system. During sulphitolysis, cystine disulphide bonds are cleaved by sulphite to give cysteine thiol and cysteine-S-sulphfonate (Bunte salt) residues, according to the following reaction:

1. WSSW + SO3^2- WS^-+ WSSO3^-(Bunte Salt)

where WSSW is the cross-linked wool keratin in the fibre, SO3^2- is the sulphite, WS^- is the reduced

-

keratin, and WSSO3 is the Bunte salt. Bisulphite (HSO3^-) and disulphide (S2O5^2-) may also be used because, along with sulphite, they exist together in aqueous solution at the equilibrium [14a].

The biochemical characterisation of keratins extracted from wool fibres by sulphitolysis (the mB/U extract) was performed by SDS-PAGE. Figure 1 shows the electrophoresis separation pattern of the mB/U extract, compared with keratins extracted with the conventional DTT/urea solvent system (the DTT/U extract) used as a reference.

Figure 1. Electrophoretic separation patterns (SDS-PAGE) samples; column 1: MW standard, column 2: DTT/U wool extract, column 3: mbisulphite/urea extract (mB/U), column 4: keratin proteins regenerated from the Cu solvent (regK)

The DTT/U extract (lane 2) shows the two important keratin fractions, that is, the low sulphur proteins (LS) of the intermediate filaments whose molecular weight falls in the range of 67-43 KDa and the high sulphur proteins (HS) of the matrix, whose molecular weight falls in the range of 28-11 KDa, (LS). The mB/U extract shows only the signals related to proteins with the highest molecular weight; in fact, the precipitation of the protein fraction lacking in disulphide bonds takes place at its isoelectric point (pH 4.4), whereas the protein fraction rich in disulphide bonds, with an isoelectric point at 2.9, does not precipitate at pH 4.4 [5, 6, 1].

The presence of the intermediate filaments proteins in the mB/U extract (line 3) suggests that the reducing conditions of sulphitolysis do not cause the degradation of these proteins.

The mB/U extracted keratins were then dissolved in the cuprammonium hydroxide solvent (Cu solvent). The latter is a strongly alkaline solvent; dissolution was therefore carried out in a cold ice bath to avoid triggering hydrolytic degradation phenomena [4]. The electrophoresis pattern of keratin proteins recovered after dissolution with the cuprammonium solvent and regeneration in a 0.5 M sulphuric acid bath is shown in Figure 1 (regKP sample, lane 4). The strong bands of LS proteins falling in the high molecular weight range which characterise the mB/U extract were hardly detectable; regions of higher protein density appeared in the medium-to-low fractionation range at around 30-14 KDa. The loss of high molecular weight keratins indicates that the chemical structure of proteins was significantly affected by the strongly alkaline environment of the cuprammonium solvent, despite the careful control of the treatment temperature. Degradation probably occurred because of the alkaline attack on sensitive amino acids, which resulted in main chain fission [14b].

FT-IR analysis

To study the effect of the sulphitolysis and dissolution of wool proteins in the Cu solvent on the chemical structure and molecular conformation of the mB/U and regKP keratins, the samples were investigated by FT-IR spectroscopy (Figure 2, spectra b and c, respectively). For comparison purposes, the spectrum of wool fibres (WF) is presented in Figure 2 (spectrum a). The latter displays the characteristic absorption bands of proteins at 1630 cm^-1 (Amide I), 1515 cm^-1 (Amide II) and 1230 cm^-1 (Amide III). Amide I is primarily a C=O stretching vibration, while amide II and III are heavily mixed vibrational modes. In particular, amide II results from N-H bending and C-N stretching vibrations. Amide III is a very complex range resulting from in-phase combination of C-N stretching and N-H in-plane-bending, with some contribution from C-C stretching and C=O bending vibrations

Figure 2. FT-IR spectra of WF(a), mB/U (b), regK (c) samples

Amide I, II, and III are very important bands because, from their position and shape, information about protein structure and conformation can be derived. In fact, hydrogen bonding, steric situations and environmental properties are known to influence the frequencies of the amide vibrations. However amide II and III are complex vibrational modes, and correlations between band shape and the structure of proteins may become rather questionable. For this reason, it has been decided to study the shifts and shapes of amide I in more detail [8].

Figure 3 shows an enlarged plot of the amide I range of the WF, mB/U, and regKP samples. While the bands of WF and mB/U (spectra a and b, respectively) almost overlapped, indicating that the reductive extraction did not significantly affect the physico-chemical structure of wool keratins, the regKP sample exhibited a blue shift of the amide I peak (spectrum c). This behaviour can be explained by taking into account that the frequency of the C=O vibration is related to the strength of the hydrogen bond formed by the carbonyl group. The stronger the hydrogen bond, the lower the energy and the frequency of the amide I vibration. Therefore, the blue shift observed suggests that the energy of the C=O bond was higher in the regKP than in the WF and mB/U keratins. This feature could be related to the less complex steric situation of the protein chains in the regKP sample. It is likely that the alkaline degradation to which keratins were subjected during dissolution in the Cu solvent caused disordering and disruption of the native structure, and made the resulting material more amorphous.

Figure 3. FT-IR spectra of WF(a), mB/U (b), regK(c) keratin samples: close-up of the amide I range

Following reductive extraction, a new peak appeared at 1022 cm^-1 in the mB/U sample (Figure 2, spectrum b) and in the regK sample (Figure 2, spectrum c). This peak is attributed to the formation of Bunte salts [8, 3].

The infrared spectra taken for regenerated pure keratin and cellulose films and keratin/cellulose blend films are shown in Figure 4.

Figure 4 . FT-IR spectra of Cellulose(a), Ker/Cell 20/80 (b), Ker/Cell 40/60 (c), Ker/Cell 60/40 (d), Ker/Cell 80/20 (e), regK (f) samples

The IR spectrum of the regenerated cellulose film shows the characteristic absorptions typical of cellulose [12].

The IR spectra of keratin/cellulose blend films (Figure 4) were characterised by the presence of absorptions bands typical of pure components. The intensity ratio between the keratin’s amide II band (1540 cm^-1) and the cellulose band at 895 cm^-1, shown in Table 1, well fits the blend composition of the relevant samples.

Table 1. Intensity of IR bands at 1540cm^-1 and 895 cm^-1 and I1540/I895 intensity ratio

Surface Morphology

The SEM investigation of the blend films showed the differences between regenerated pure cellulose (Figure 5a), regenerated pure keratin (Figure 5b) and the keratin/cellulose blends. Figure 5c-d shows the 40/60 and 80/20 keratin/cellulose blends as an example.

The air surface of pure films appeared more homogeneous in comparison with the blend films. Nevertheless the morphology of blend films still appeared coherent, with surface wrinkling, but without cracks.

Figure 5. Scanning Electron Microscopy photographs of regenerated pure cellulose(a), regenerated pure keratin (b), Ker/Cell 40/60 film (c), Ker/Cell 80/20 (d); 1.04KX, 15 KV

Conclusions

The results reported in this study show that keratins extracted from wool by m-bisulphite/urea are soluble in the cuprammonium hydroxide solvent system.

SDS-PAGE analysis shows that the alkaline environment brought about a certain degree of protein degradation, which appeared as a decrease in molecular weight.

The FT-IR spectra show the amide I blue shift of the regenerated keratin, suggesting a less complex steric assembling of protein chains.

The intrinsic physical and chemical properties of regenerated keratin proteins probably make them unsuitable as starting material alone for fibre spinning.

However, blending with cellulose could open new opportunities for recycling and exploitation of keratin by-products and other valuable wastes produced during textile processing.

Several solutions of keratin and cellulose resulted in homogenous films with a wrinkled surface but without cracks.

Studies in progress indicate that this approach is highly promising, and may lead to the production of a new range of textile fibres with innovative properties and based on renewable natural resources.

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