Enzymatic treatment of polyamide 6.6 fibres

Tese de Doutoramento em Engenharia Têxtil - Ramo de Conhecimento Química Têxtil.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.A hidrólise controlada da superfície das fibras de poliamida conduz à formação de grupos terminais amina e carboxílicos. A presença destes grupos melhora a hidrofilidade das fibras e cria possíveis locais para a ligação de certos compostos. A ligação de agentes retardadores da chama, de proteínas e outros compostos conduz à obtenção de fibras com maior valor acrescentado. No entanto, a hidrólise química tradicionalmente usada para modificar a superfície destas fibras, é um mecanismo “tudo-ou-nada” que provoca o amarelecimento e perda de resistência das fibras. Dado que as enzimas são proteínas globulares grandes, a sua acção catalítica reduz-se somente à superfície das fibras, preservando assim a sua estrutura. Esta dissertação apresenta com sucesso o uso de enzimas na funcionalização da superfície de fibras de poliamida 6.6. Foi testada a capacidade de uma cutinase, do fungo Fusarium solani pisi, e de uma protease, da bactéria Bacillus sp., para hidrolisar as fibras de poliamida 6.6. Foram desenvolvidas diferentes metodologias para monitorizar a formação dos produtos resultantes da hidrólise enzimática. Uma delas é baseada na reacção do composto 2,4,6 - ácido trinitrobenzenosulfónico (TNBS) com os grupos amina libertados para o banho de tratamento. A outra baseia-se na reacção de um corante reactivo específico para a lã com os grupos amina terminais que permanecem na superfície dos tecidos tratados após hidrólise. Neste trabalho, foi provado que a hidrólise enzimática com a cutinase ou com a protease, conduz apenas a uma modificação superficial, sendo a reacção selectiva. Foram efectuados estudos para reduzir o tempo de tratamento e aumentar a adsorção da enzima, aumentando assim a eficiência hidrolítica sobre as fibras de poliamida 6.6. Para isso, os tecidos de poliamide 6.6 foram incubados com cutinase na presença de solventes orgânicos. Os solventes orgânicos usados foram o álcool benzílico e a dimetilacetamida, os quais são geralmente usados no processamento da poliamida como auxiliares no seu tingimento. Foi também avaliada a estabilidade da cutinase na presença destes solventes. Constatou-se que a actividade da cutinase é preservada durante algumas horas na presença de baixas concentrações de solventes orgânicos (tempo de meia-vida de 26 horas para 1.5% álcool benzílico e tempo de meia-vida de 14 horas para 1.5% álcool benzílico + 10% dimetilacetamida). Na presença destes solventes, a estrutura da poliamida torna-se mais apta a ser modificada por acção enzimática. Os resultados confirmam um aumento de grupos terminais à superfície da fibra nos tecidos incubados com cutinase na presença dos referidos solventes. A cutinase foi assim apresentada como uma enzima versátil com invulgar actividade esteriolítica sobre substratos de poliamida, no entanto os níveis de modificação obtidos foram muito baixos. A análise da estrutura tridimensional da cutinase do fungo Fusarium solani pisi (PDB code 1CEX), mostrava que o centro activo externo, mas fechado, da enzima impedia o acesso ao substrato. De modo a ultrapassar esta dificuldade, foi efectuada a modificação genética da cutinase nativa. A técnica de mutagénese dirigida foi usada para efectuar modificações na enzima, substituindo aminoácidos específicos perto do centro activo por alanina, obtendo-se cinco mutações (L81A, N84A, L182A, V184A e L189A). Todas as mutações foram feitas de modo a criar mais espaço no centro activo onde o substrato polimérico pudesse encaixar. Os estudos de modelação molecular foram efectuados incorporando o substrato modelo de poliamida 6.6 no centro activo da enzima. Estes estudos mostraram que a mutação L182A apresentou os melhores resultados de estabilização do substrato modelo, os quais são suportados pelos resultados experimentais obtidos (+19% aminas na solução do banho de tratamento; 25% de adsorção de proteína) A cutinase e a protease não foram “desenhadas” pela natureza para interagir com substratos sintéticos como a poliamida 6.6, pelo que a sua acessibilidade à superfície das fibras assim como a sua adsorção pode ser restringida pela estrutura compacta das fibras de poliamida. Como referido por outros autores, o processo de adsorção enzimática é de extrema importância na hidrólise de fibras sintéticas. Diferentes estudos revelaram que a adsorção de proteínas segue diferentes passos e que a agitação mecânica tem um papel importante em todo o processo. De modo a estudar o sinergismo entre a agitação mecânica, adsorção de enzima e actividade enzimática, foram realizados estudos usando diferentes níveis de agitação mecânica. Os resultados revelaram que a funcionalização da superfície das fibras de poliamida com cutinases ou com a protease deve ser efectuada usando níveis baixos de agitação mecânica durante curtos períodos de tempo (4 horas usando agitação vertical – Rotawash MKIII, sem discos). Foi posteriormente encontrada uma aplicação prática para os tecidos previamente funcionalizados. As superfícies dos tecidos ricas em grupos amina, obtidas por acção enzimática de uma protease do Bacillus sp., foram usadas para imobilizar uma enzima (lacase de Trametes hirsuta). Foi desenvolvido um procedimento para a imobilização da lacase nos suportes de poliamida 6.6 usando o glutaraldeído como agente bifuncional, incluindo a 1,6-hexanodiamina em alguns casos, de forma a aumentar a distância entre a matriz e a enzima. Neste estudo foi usado o design factorial (24) para estudar a influência do pH, da 1,6-hexanodiamina, da concentração de enzima e de glutaraldeído, na eficiência da imobilização. A dosagem de enzima e a presença de 1,6-hexanodiamina tiveram um papel determinante no processo de imobilização. Em condições the trabalho optimizadas (29 UmL-1 de lacase; 10% de glutaraldeído, pH=5.5, na presença de 1,6-hexanodiamina), o tempo de meia-vida obtido foi de cerca de 78 h (18% mais elevado do que o obtido para a enzima livre), a retenção de proteína foi de cerca de 34% e o rendimento de imobilização foi de 2%. A imobilização da lacase em matrizes de poliamida 6.6 pode ser um sistema promissor na bioremediação de solos contaminados, no tratamento de águas residuais, na estabilização de vinhos e outra bebidas e até mesmo na aplicação de biosensores. Os resultados obtidos demonstram que a cutinase e a protease são enzimas capazes de modificar a superfície de fibras de poliamida 6.6 e podem ser usadas em diferentes etapas do processamento da fibra. Através da funcionalização da poliamida 6.6 com enzimas podem ser obtidos produtos de maior valor acrescentado, no entanto o processo de hidrólise enzimática necessita de ser mais detalhadamente caracterizado de forma a poder ser aplicado num processo industrial.Biosyntex European ProjectFundação para a Ciência e a Tecnologia (FCT

Tratamento alcalino e enzimático de fibras sintéticas

Dissertação de mestrado em Tecnologias de Fabricação.As enzimas são produtos naturais encontrados em abundância no corpo humano e na natureza, regulando um grande número de processos biológicos de forma específica e única. Por esta razão, as enzimas desempenham um papel importante na indústria têxtil, como substituição de produtos químicos que têm um impacto negativo sobre o meio ambiente. Uma das utilizações que tem sido investigada nos últimos anos, é o tratamento enzimático das fibras sintéticas, que normalmente era efectuado com produtos alcalinos, para melhorar o toque e a hidrofilidade das mesmas. É possível, através da utilização de nitrilases, proteases e esterases, a conversão de nitrilos em ácidos carboxílicos e a cisão hidrolítica de ligações amida e éster, respectivamente. Este trabalho consiste numa análise comparativa, entre o tratamento alcalino e o tratamento enzimático e de que forma estes dois tratamentos contribuem para o melhoramento das propriedades físicas e químicas das fibras sintéticas estudadas. Na execução deste estudo, foram aplicadas várias técnicas, como FTIR (espectroscopia por infravermelhos com transformadas de Fourrier), para a detecção dos grupos terminais formados, a técnica DSC (differential scanning calorimetry), usada para determinação da temperatura de transição vítrea das fibras estudadas, entre outras. O tratamento enzimático, mostra-se uma boa alternativa no melhoramento de algumas propriedades químicas e físicas, como a afinidade tintorial e a diminuição da rigidez à flexão, revelando-se assim, uma boa alternativa à caustificação das fibras sintéticas, poliéster, poliamida e acrílica.Enzymes are natural products founded in nature, wich catalyses a great variety of biologic processes. For this reason, enzymes have an important role on textile industry as substitutes of some chemical products, wich have a negative effect on the environment. In the past, the treatment of synthetic fibres used to be done with alkaline products, to improve their handle and hidrofilicity. Nowadays, the enzymatic treatment has been investigated as a replacement of the conventional process. With the enzymes Nitrilases, Proteases and Esterases it is possible to convert nitrils into carboxylic acids and promote the hydrolytic cleavage of amide and ester bonds, respectively. This reactions can be very usefull for the treatment of synthetic fibres, wich contain these groups. This study is based on a comparative analysis between the alkaline and enzymatic treatment, and in wich way this treatments influence the improvement of physical and chemical properties of the synthetic fibres studied. In this work, several techniques as FT . IR (infrared spectroscopy), for the determination of formed groups, DSC (differential scanning calorimetry), for the determination of the glass transition temperature of the fibres, and others, were used. The enzymatic treatment seams to be a good alternative for the improvement of some chemical and physical properties, such as dyeing affinity, and bending rigidity, and can be used as a substitute of the alkaline treatment on synthetic fibres

Decolourization of paprika dye effluent with hydrogen peroxide produced by glucose oxidase

Hydrogen peroxide was produced from bran by a two-step process using cellulase/xylanase and glucose oxidase, sequentially. The decolourization efficiency of the produced reagent was tested using paprika oil dye (effluent from industrial source) and high levels of colour removal (96%) were achieved after saponification pre-treatment and hydrogen peroxide application. The method is economically and environmentally advantageous since lower energy and chemical input are needed and wastewater pollution is considerably reduced. At the same time, the utilization of waste materials was successfully achieved.The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article. Carla Silva would like to acknowledge the Portuguese Fundacao para a Ciencia e a Tecnologia (FCT) for funding under the scholarship SFRH/BPD/46515/2008

Interfacial stabilization of enzymes in microemulsions

One of the major constrains to the use of enzymes in industrial processes is their insufficient stability under processing conditions, namely high temperatures, presence of ultrasounds, among others. Herein, we investigated the use of oil-in-water proteinaceous (BSA) microemulsions as a novel methodology for the stabilization of laccase from ascomycete Micelliophthora thermophila. The immobilization of laccase onto the produced microemulsions benefitiated its stability under ultrasonic conditions. The half life time of immobilized laccase was 2.4-fold higher (from 23 to 56 minutes) than laccase in the free form. This technique show promising potentialities for the stabilization of enzymes used onto a variety of processes, namely textile bleaching, surface hydrolysis, among others

Laccase immobilization on enzymatically functionalized polyamide 6,6 fibres

Polyamide matrices, such as membranes, gels and non-wovens, have been applied as supports for enzyme immobilization, although in literature the enzyme immobilization on woven nylon matrices is rarely reported. In this work, a protocol for a Trametes hirsuta laccase immobilization using woven polyamide 6,6 (nylon) was developed. A 24 full factorial design was used to study the influence of pH, spacer (1,6-hexanediamine), enzyme and crosslinker concentration on the efficiency of immobilization. The factors enzyme dosage and spacer seem to have played a critical role in the immobilization of laccase onto nylon support. Under optimized working conditions (29 U mL−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 30% and the immobilization yield was 2%. The immobilized laccase has potential for application in the continuous decolourization of textile effluents, where it can be applied into a membrane reactor

Laccase/ultrasound system for cotton bleaching : an ultrasonic pilot-scale reactor

This work exploited the bleaching efficiency of cotton fabrics using a combined laccase-hydrogen peroxide system assisted by ultrasound. The main goal was to reach the whiteness levels obtained by conventional treatments, reducing the amount of chemical and energy consumption. Laccase promoted the oxidation of flavonoids responsible for the natural color of the fabric. In addition, ultrasound energy enhanced the mass transfer and speed-up of bleaching reactions. Laboratory experiments demonstrated that the biobleaching process allowed higher whitening levels than those obtained by standard methods. Thus, an adjustment of different operational parameters such as hydrogen peroxide concentration, temperature and incubation time was possible. As result, comparing with conventional processes, the amount of hydrogen peroxide was reduced 50% as well as the energy consumption in terms of temperature (reduction of 40 °C) and processing time (reduction of 90 minutes). Further, a pilot reactor for the explored technology was scaled-up by adapting an existing dyeing machine with piezoelectric ultrasonic devices. The developed ultrasonic pilot-scale reactor contributes for a sustainable bleaching process with reduced environmental impact as well as offers a better performance for the finishing operations

Influence of mechanical agitation on cutinases and protease activity towards polyamide substrates

Two polyamide 6,6 substrates with different constructions, namely a model substrate and a fabric, were hydrolyzed using native cutinase and L182A cutinase mutant (from Fusarium solani pisi) and a protease (subtilisin from Bacillus sp.). The catalytic efficiency of these enzymes, measured in terms of hydrolysis products release, was measured for both substrates and the protease released five times more amines to the bath treatment. The L182A cutinase mutant showed higher activity when compared with the native enzyme. All enzymes have shown activity additive effects with higher levels of mechanical agitation for polyamide fabrics. The results achieved are of paramount importance on the design of a process of enzymatic functionalization of polyamide.European Community - Biosyntex Project, G5RD-CT-2000-30110Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/22490/200

Functionalization of gauzes with liposomes entrapping an anti-inflammatory drug: a strategy to improve wound healing

From ancientness, suitable materials have been developed to cover the wounds in order to prevent infections and promote proper wound healing. In this study, the successful development of functionalized nonwoven gauzes with liposomes entrapping anti-inflammatory piroxicam is reported. Piroxicam is a non-steroidal anti-inflammatory drug (NSAID) that can suppress a persistent inflammatory response, leading to improved wound healing. The results demonstrated that the highest NSAID concentration released is achieved when gauzes were previously cationized with poly(diallyldimethylammonium chloride) (PDDA) and high concentration of phospholipid (≈3000 μM) and multilamellar liposomes (MLVs) were used. MLVs were also the best vehicle considering their biocompatibility with skin human fibroblasts, where no toxicity was observed for neither of the tested conditions. The developed functionalized gauzes can be, therefore, a good strategy to treat chronic wounds.Helena Ferreira, Teresa Matama and Carla Silva thank POPH/FSE for co-financing and FCT for fellowships SFRH/BPD/38939/2007, SFRH/BPD/47555/2008, SFRH/BPD/46515/2008, respectively. This work was supported by FEDER through POFC - COMPETE and by national funds from FCT through the project PEst-C/BIA/UI4050/2011

Coloured and low conductive fabrics by in situ laccase-catalysed polymerization

Coloured and conductive fabrics were obtained through in situ laccase polymerization of catechol and p-phenylenediamine under high-pressure homogenization. Both monomers, catechol and p-phenylenediamine, were polymerized by different laccase forms, namely native, PEGylated and Epoxy-PEGylated. All the catalysts were placed inside a textile fabric bag which served simultaneously as enzyme support and as substrate for coating with the newly produced polymers. The PEGylated laccase forms gave rise to higher amount of oligomers/polymers and higher colouration level of polyethylene terephthalate (PET), cotton and wool fabrics compared to native laccase. Both functional polymers were able to confer conductivity to the substrates however in a different extent. Fabrics coated with poly(p-phenylenediamine) present higher conductivity, rather due to its polymerized structure than to the amount of polymer produced by enzyme catalysis. Herein a green approach was presented to produce polyphenols with increased fixation onto different textile substrates. These substrates reach high levels of colouration and good fastness behaviour after washing.Chinese Government Scholarship under China Scholarship Council [grant No. 201606790036]; and by the Chinese Foundation Key projects of governmental cooperation in international scientific and technological innovation [grant No. 2016 YFE0115700]; and by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. This work was also supported by the European Union through the European Regional Development Fund (ERDF) under the Competitiveness Operational Program [grant COP-A1.1.4-E-2015nr.30/01.09.2016]; and by the Project “Search-ON2: Revitalization of HPC infrastructure of UMinho” [grant NORTE-07-0162-FEDER-000086), co-funded by the North Portugal Regional Operational Programme (ON.2 – O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF). The work was also supported by the FCT- Fundação para a Ciência e a Tecnologia [grant SFRH/BD/121673/2016 and IF/00186/2015]info:eu-repo/semantics/publishedVersio

Polyoxometalate/laccase-mediated oxidative polymerization of catechol for textile dyeing

The synergistic effect between polyoxometalates (POMs), namely K5[SiW11VVO40]·11H2O and H5[PMo10 VV2O40]·13H2O and laccase from ascomycete Myceliophthora thermophila has been employed for the first time in oxidative polymerization of catechol. Such a laccase-mediator system allowed the formation of a relatively high molecular weight polycatechol as confirmed by size exclusion chromatography and electrospray ionization mass spectrometry (ESI-MS) (3990 Da when using K5[SiW11VVO40]·11H2O and 3600 Da with H5[PMo10VV 2O40]·13H2O). The synthesized polymers were applied as dyes for the dyeing of flax fabrics. The color intensity of flax fabrics colored with polymer solutions was evaluated by diffuse reflectance spectrophotometry via k/s measurements (+10% of fixation ratio). A new synthetic process allowed a dyeing polymer, provided upon flax coloration, better color fixation and color resistance when compared to that obtained by conventional synthesis with laccase solely or with addition of organic mediator (1-hydroxybenzotriazole)