Publicação
Enzymatic treatment of wool with modified proteases
| Resumo: | The tendency of wool to felt and shrink is mainly due to its scaly structure. The chlorine-Hercosett is the most widespread process used to modify the scales of wool fibres with the purpose of providing resistance to felting and shrinkage. There have been many attempts to replace this chlorine process by an environmental friendly enzymatic process that wouid similarly degrade the scales. However, although proteases are large molecuies, their attack is not oniy Iimited to the scales; they penetrate inside the fibre causing unacceptabie weight and strength ioss. lt is believed that if the proteases are chemicaliy modified in order to increase their molecular weight, then they will act just on the surface of the fibres, thus providing wool with anti-shrinking behaviour, which is the main idea of this research. In this work the screening of the attack of the proteoiytic enzymes inside the fibre was made by means of several techniques. Among them, special attention was paid to the study of the protein adsorption inside the wool fibres. It was demonstrated that the penetration of protein (measured as the maximum adsorption capacity in g protein/g wool) was higher when the wool was previously subjected to a surfactant washing and bleaching. Furthermore, it was observed that the diffusion of the proteases into wool was dependent on their size. The free enzyme penetrated into wool fibre cortex while the modified enzyme, with a bigger size, was retained at the surface, in the cuticle layer. lt was also confirmed that the diffusion of proteases was facilitated by the hydrolytic action. Scanning electron microphotographs were also used to observe the intensity of the proteolytic attack. Some techniques of increasing the proteases molecular weight were attempted, namely the covalent crosslinking method using the bifunctional reagent glutaraldehyde. It was observed that the Iow amount of free Iysine residues available in the protease for crosslinking was affecting the process A more successful technique was attained by covalently coupling the enzyme to a soluble-insoluble polymer of high molecular weight. An enzyme conjugated to such a carrier may be used as a catalyst in its soluble form and then be recovered via the insoluble state. Moreover, this system overcomes the problem of the non accessibility of the enzyme to the macromolecular substrate, wool, whilst in the soluble state. When comparing to the native enzyme, the immobilized form presented a lower specific activity towards high molecular weight substrates but a higher thermal stability at ali temperatures tested. lt also exhibited a good storage stability and reusability, which makes this enzyme conjugate quite interesting from an industrial point of view. Wool fabrics were treated with the immobilized serine protease using harsh conditions and subjected subsequently to several machine washings, after which they presented a significant Iower weight loss than wool treated with the native enzyme, in the same conditions. Using a moderate enzymatic treatment, a reduction to about half of the initial area shrinkage was attained, both for free and immobilized enzymes. However, the immobilized Esperase presented 92% of the original tensile strength resistance while native Esperase kept only 75% of its initial resistance. The coupling of the protease to the polymer, Eudragit S-100, was optimized by using experimental design techniques. This optimization strategy allowed for an enzyme conjugate wherein the enzyme was covalently crosslinked to the polymer, with high activity yield and high operational stability at 6000. Ali these results prove that modified proteases attained by this immobilization method, using a soluble-insoluble polymer of high molecular weight, can be a promising alternative for wool bio-finishing processes at an industrial levei, since it is an effective way of removing wool scales and can be an environmental friendly option to the conventional chiorine treatments. This process needs to be further characterized for its complete understanding and optimization. |
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| Autores principais: | Silva, Carla J. S. M. |
| Ano: | 2005 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | The tendency of wool to felt and shrink is mainly due to its scaly structure. The chlorine-Hercosett is the most widespread process used to modify the scales of wool fibres with the purpose of providing resistance to felting and shrinkage. There have been many attempts to replace this chlorine process by an environmental friendly enzymatic process that wouid similarly degrade the scales. However, although proteases are large molecuies, their attack is not oniy Iimited to the scales; they penetrate inside the fibre causing unacceptabie weight and strength ioss. lt is believed that if the proteases are chemicaliy modified in order to increase their molecular weight, then they will act just on the surface of the fibres, thus providing wool with anti-shrinking behaviour, which is the main idea of this research. In this work the screening of the attack of the proteoiytic enzymes inside the fibre was made by means of several techniques. Among them, special attention was paid to the study of the protein adsorption inside the wool fibres. It was demonstrated that the penetration of protein (measured as the maximum adsorption capacity in g protein/g wool) was higher when the wool was previously subjected to a surfactant washing and bleaching. Furthermore, it was observed that the diffusion of the proteases into wool was dependent on their size. The free enzyme penetrated into wool fibre cortex while the modified enzyme, with a bigger size, was retained at the surface, in the cuticle layer. lt was also confirmed that the diffusion of proteases was facilitated by the hydrolytic action. Scanning electron microphotographs were also used to observe the intensity of the proteolytic attack. Some techniques of increasing the proteases molecular weight were attempted, namely the covalent crosslinking method using the bifunctional reagent glutaraldehyde. It was observed that the Iow amount of free Iysine residues available in the protease for crosslinking was affecting the process A more successful technique was attained by covalently coupling the enzyme to a soluble-insoluble polymer of high molecular weight. An enzyme conjugated to such a carrier may be used as a catalyst in its soluble form and then be recovered via the insoluble state. Moreover, this system overcomes the problem of the non accessibility of the enzyme to the macromolecular substrate, wool, whilst in the soluble state. When comparing to the native enzyme, the immobilized form presented a lower specific activity towards high molecular weight substrates but a higher thermal stability at ali temperatures tested. lt also exhibited a good storage stability and reusability, which makes this enzyme conjugate quite interesting from an industrial point of view. Wool fabrics were treated with the immobilized serine protease using harsh conditions and subjected subsequently to several machine washings, after which they presented a significant Iower weight loss than wool treated with the native enzyme, in the same conditions. Using a moderate enzymatic treatment, a reduction to about half of the initial area shrinkage was attained, both for free and immobilized enzymes. However, the immobilized Esperase presented 92% of the original tensile strength resistance while native Esperase kept only 75% of its initial resistance. The coupling of the protease to the polymer, Eudragit S-100, was optimized by using experimental design techniques. This optimization strategy allowed for an enzyme conjugate wherein the enzyme was covalently crosslinked to the polymer, with high activity yield and high operational stability at 6000. Ali these results prove that modified proteases attained by this immobilization method, using a soluble-insoluble polymer of high molecular weight, can be a promising alternative for wool bio-finishing processes at an industrial levei, since it is an effective way of removing wool scales and can be an environmental friendly option to the conventional chiorine treatments. This process needs to be further characterized for its complete understanding and optimization. |
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