Enzymatic treatment of polyamide 6.6 fibres

The controlled hydrolysis of polyamide fibres surfaces leads to the formation of amino and carboxylic end groups. The presence of these groups improves hydrophilicity and creates further starting points for covalent bonding of certain compounds. The coupling of flame retardants, proteins and other compounds bring extra added value to polyamide fibres. Chemical hydrolysis, tradicionally used to modify the surface of these fibres, is an “all-or-nothing” event leading always to yellowing and loss of fibres resistance. Given that enzymes are large globular proteins, their catalytic action remains at the surface of the fibres, therefore preserving their intrinsic structure. This dissertation successfully presents the use of enzymes to functionalise the surface of polyamide 6.6 fibres. The ability of a cutinase from Fusarium solani pisi and a protease, a Subtilisin from Bacillus sp. to hydrolyse polyamide 6.6 fibres was evaluated. Different methodologies were developed in order to monitor the formation of the products resulting from enzymatic hydrolysis. The first one is based on the reaction of the compound 2,4,6-trinitrobenzenesulfonic acid (TNBS) with the amino groups released to the bath treatment and the other one is based on the reaction of a specific wool reactive dye with the terminal amino groups at the surface of the treated fabrics. In this work it was proved that the enzymatic hydrolysis with cutinase or protease leads only to surface modifications, being the reaction selective. Studies were performed in order to reduce the treatment time and increase enzyme adsorption, hence increasing hydrolysis efficiency towards polyamide 6.6 fibres, by incubating polyamide 6.6 fabrics with cutinase in the presence of organic solvents. The organic solvents used were benzyl alcohol (BA) and dimethylacetamide (DMA), which are commonly used in polyamide processing as dyeing assistants. The stability of cutinase in the presence of these solvents was evaluated. It was observed that cutinase activity is preserved for several hours in the presence of low amounts of organic solvents (half-life time of 26 hours for 1.5% of benzyl alcohol and half-life time of 14 hours for 1.5% benzyl alcohol+10% dimethylacetamide). In the presence of these organic solvents, the polyamide structure became more suitable to be modified by enzymatic action. The results obtained confirmed an increase of terminal amino groups at the surface of the fabrics incubated with cutinase in the presence of the referred organic solvents. Cutinase was presented as a versatile enzyme with unusual stereolytic activity towards polyamide substrates however turnover rates were very low. The analysis of the 3D structure of cutinase from Fusarium solani pisi, (PDB code 1CEX), showed that the external, but closed active site, was hindering the access to the fibre substrate. In order to overcome this difficulty, the genetic modification of native cutinase was performed. The site-directed mutagenesis was performed by changing specific amino acid residues around the active site by alanine (L81A, N84A, L182A, V184A and L189A) and five mutations were obtained. All mutations were done to create more space in order to fit the large inaccessible polymer in the active site of the cutinase. Molecular modelling studies were performed by docking the synthetic model substrate of polyamide 6.6 at the cutinase active site. These studies predicted that L182A mutation provided the best stabilization of polyamide model substrate which support the experimental results obtained (+19% of amines in the bath solution treatment; 25% of protein adsorption). Cutinase and protease were not designed by nature to interact with synthetic substrates like polyamide 6.6, therefore the accessibility of these enzymes to the fibre surface as well as their adsorption can be restricted by the compacted structure of polyamide fibres. As other authors have reported, the process of enzyme adsorption is of major importance to the enzymatic hydrolysis of synthetic fibres. Different studies have revealed that the adsorption of proteins follows different steps and that mechanical agitation plays an important role in all of them. In order to study the synergism among mechanical agitation, enzyme adsorption and enzyme activity, studies were performed using different levels of mechanical agitation. The results obtained revealed that the surface functionalisation of polyamide fibres with cutinases or protease should be performed using low levels of mechanical agitation for short periods of time (4 hours using a vertical agitation – Rotawash MKIII machine, in absence of discs). A practical application was found for the functionalised surfaces obtained. The amine-enriched surfaces obtained by enzymatic action of a protease from Bacillus sp. were used to immobilise an enzyme (a laccase from Trametes hirsuta). An immobilisation procedure was developed to immobilise laccase onto woven polyamide 6.6 supports using glutaraldeyde as the crosslinking agent with the inclusion of a spacer (1,6-hexanediamine) in some cases. A 24 full factorial design was applied to study the influence of pH, spacer, enzyme and crosslinker concentration on the efficiency of immobilisation. The factors enzyme dosage and spacer have played a critical role in the immobilisation process. Under optimised working conditions (29 UmL-1 of laccase, 10% of glutaraldehyde, pH=5.5, with the presence of the spacer), the half-life time attained was about 78 h (18% higher than that of free enzyme), the protein retention was about 34% and the immobilisation yield was 2%. Laccase immobilisation onto polyamide 6.6 matrices can be a promising system for bioremediation of contaminated soils, wastewater treatment, wine and other beverage stabilisation, and even biosensor applications. The results obtained reveal that cutinase and protease are able to modify the surface of polyamide 6.6 fibres and that these enzymes can be used in different steps of fibre processing. Higher added value products can be obtained by polyamide 6.6 functionalisation with enzymes, however the process of enzymatic hydrolysis needs to be characterized in more detail in order to be applied into an industrial process.

Name thesis:degree_name

Doutoramento em Engenharia Têxtil (especialidade em Química Têxtil)

Year dc:date.issued

2008

Author dc:creator

• Silva, Carla Manuela Pereira Marinho da

Advisor dc:contributor.advisor

• Paulo, Artur Cavaco

Statement dc:rights

• openAccess

Language dc:language.iso

eng

Handle dc:identifier.uri

https://hdl.handle.net/1822/8242

OAI record oai:identifier

http://repositorium.sdum.uminho.pt/oai/request?verb=GetRecord&metadataPrefix=dim&identifier=oai%3Arepositorium.uminho.pt%3A1822%2F8242