29 research outputs found
Enzymatic treatment of acrylic and cellulose acetate fibres
Thesis for Doctoral (degree in Textile
Engineering Textile Chemistry)The background theme of the present thesis is the multidisciplinary area of textile
biotechnology, which is of major importance for the textile industry and its sustainable
development. The work here described was devoted to the treatment with enzymes of two manmade
fibres - acrylic and cellulose acetate. The thesis is divided in several chapters being the
first one a general introduction. Biocatalysis is addressed, especially in the context of textile
industry and surface modification of polymers, followed by a general description on the
properties and applications of both fibres. The enzymes used throughout the work – nitrilase
(EC 3.5.5.1) and cutinase (EC 3.1.1.74), are briefly mentioned as well as the major methods to
manipulate and improve enzymes. The general aim of the work is the formation of reactive
and/or hydrophilic groups at the surface of acrylic and cellulose acetate fibres by enzymatic
hydrolysis of their pendent groups. As follows, the purpose is to preserve the desirable bulk
properties of the fibres acting only at the surface by using eco-friendly catalysts.
In chapter 2, the modification of the surface of acrylic fabric with a commercial nitrilase is
reported. The enzymatic conversion of nitrile groups into the carboxylic groups, on the fibre
surface, was monitored for 36 hours by the release of ammonia to the media and by the
improvement in the affinity of the treated fabric for a basic dye. The steady release of ammonia
along the enzymatic treatment showed that the adsorption of nitrilase to the acrylic led to an
increase in its operational stability, resembling the immobilization procedures used to stabilize
proteins. A maximum affinity for the basic dye was observed for a treatment period of 8 hours,
which corresponded to a relative K/S of 135% when the colouration of acrylic was performed at
70 ºC. A surface erosion phenomenon took place causing the “oscillatory” behaviour of the
amount of dye uptake with the time of treatment. Polyacrylic acid was determined in solution as
a non desirable, secondary product of the modification of acrylic with nitrilase. These results
showed that the outcome of nitrilase application is closely dependent on reaction parameters
like time, enzyme activity and media formulation.
The chapter 3 describes the modification of the comonomer vinyl acetate of the acrylic
used with two enzymes: cutinase from the fungus Fusarium solani pisi and a commercial
esterase (Texazym PES). The effect of acrylic solvents and stabilizing polyalcohols on cutinase
operational stability in solution was studied. The influence of these additives and mechanical
agitation on the enzymatic modification of acrylic fabric was also investigated. The hydroxyl
groups produced on the fibre surface reacted with the dye Remazol Brilliant Blue R, C.I. 61200,
increasing the colour of treated fabric. The best colour level was obtained with a high level of
mechanical agitation and with the addition of 1% (v/v) N,N-dimethylacetamide. Under these
conditions, the increase in the acrylic fabric colour depth was around 30% for cutinase and 25%
for Texazym, comparing to the respective controls. The crystallinity degree, determined by wide
angle X-ray scattering, was not significantly changed between control samples and samples treated with cutinase. The results showed, once more, that the success of the application of
enzymes, in this case cutinase and a commercial esterase, depends closely on the conditions in
which the treatment takes place.
Cutinase was also chosen to modify the surface of cellulose diacetate and triacetate
fibres. This work is reported in chapter 4. The enzymatic hydrolysis of acetyl groups on the fibre
surface was evaluated by the release of acetic acid and by the specific chemical colouration of
the fabrics with Remazol Brilliant Blue R. The treatment for 8 hours, at 30 ºC and pH 8, resulted
in an acetyl esterase activity of 0.010 U and 0.0072 U on cellulose diacetate and triacetate,
respectively. The colour levels for samples treated with cutinase for 24 hours increased 25% for
cellulose diacetate and 317% for cellulose triacetate, comparing to the controls. Cross-sections
of both fibres were analysed by fluorescence microscopy and the superficial action of cutinase
was confirmed. Comparing to other enzymes described in literature, cutinase is a catalyst to
consider for the superficial regeneration of cellulose hydrophilicity and reactivity on highly
substituted acetates.
For further improvement of cutinase activity on cellulose modified fibres, chimeric
cutinases were produced, by recombinant DNA technologies, and used to treat cellulose
acetate fabrics, as described in chapter 5. Two distinct carbohydrate-binding modules were
fused independently to the C-terminal of cutinase: the carbohydrate-binding module of
cellobiohydrolase I, from the fungus Trichoderma reesei, and the carbohydrate-binding module
of endoglucanase C, from the bacterium Cellulomonas fimi. Both chimeric cutinases had a more
efficient performance than the wild type enzyme, but the interaction of these bifunctional
enzymes with cellulose acetate needs to be further characterized for a better assessment of the
nature and yield of the observed modifications.
The chapter 6 is dedicated to the general discussion, final remarks and future
perspectives. In this thesis, evidences are presented showing that enzymes, more specifically,
nitrilase and cutinase are important tools for the acrylic and cellulose acetate surface
functionalization. This work also evidenced that this is only the first step towards the efficient
utilization of these resources that Nature provide us.O tema de fundo deste trabalho é a área multidisciplinar da biotecnologia têxtil que tem
vindo a afirmar-se como uma área de grande importância para a indústria têxtil e para o seu
desenvolvimento sustentável. O trabalho aqui apresentado consistiu no tratamento com
enzimas de duas fibras, a acrílica e o acetato de celulose. A tese encontra-se dividida em
vários capítulos consistindo o primeiro numa introdução geral a diversos tópicos abordados
pelo trabalho. A biocatálise é referida, especialmente, no contexto da indústria têxtil e
modificação superficial de polímeros, seguida de uma descrição geral das propriedades e
aplicações das duas fibras. As enzimas utilizadas ao longo do trabalho – nitrilase (EC 3.5.5.1) e
cutinase (EC 3.1.1.74), são mencionadas de forma sucinta assim como os métodos principais
de manipulação da actividade enzimática. O objectivo geral do trabalho assentou no
desenvolvimento de metodologias não poluentes conducentes à formação de grupos
reactivos/hidrofílicos à superfície das fibras, via hidrólise dos grupos laterais dos respectivos
polímeros de forma a preservar as propriedades nucleares e desejáveis das fibras.
No capítulo 2 é reportada a modificação da superfície da fibra acrílica por uma nitrilase
comercial. A conversão enzimática dos grupos nitrilo em grupos carboxílicos à superfície da
fibra foi avaliada durante 36 horas através da libertação de amoníaco para a solução e através
do aumento da afinidade do tecido tratado para um corante básico. O aumento linear do
amoníaco, libertado durante o tratamento, mostrou que a adsorção da nitrilase à fibra acrílica
conduziu a um aumento da sua estabilidade operacional assemelhando-se aos procedimentos
de imobilização usados para estabilizar enzimas. Um valor máximo de K/S foi observado para
um período de incubação de 8 horas, o que corresponde a um K/S relativo de 135% quando se
fez uma coloração a 70 ºC. Teve lugar um fenómeno de erosão superficial que determinou o
comportamento oscilatório da quantidade de corante fixada com o tempo de tratamento. Foi
determinado ácido poliacrílico nas soluções de tratamento como produto secundário não
desejado da modificação da acrílica pela nitrilase. Estes resultados revelam que o efeito final
da aplicação da nitrilase no tratamento da acrílica está intimamente dependente dos
parâmetros da reacção como o tempo, a actividade da enzima e a composição do meio.
O capítulo 3 descreve a modificação do comonómero acetato de vinilo da fibra acrílica
usada pela acção da cutinase do fungo Fusarium solani pisi e de uma esterase comercial
(Texazym PES). Foi estudado o efeito de solventes da acrílica e de poli-álcoois na estabilidade
operacional da cutinase em solução, bem como o impacto desses aditivos e da agitação
mecânica na modificação enzimática do tecido de acrílica. Os grupos hidroxilo produzidos na
superfície da fibra reagiram com o corante Remazol Brilliant Blue R, C.I. 61200, aumentando a
cor do tecido tratado. O melhor nível de coloração foi obtido com elevada agitação mecânica e
com a adição de 1% (v/v) de N,N-dimetilacetamida. Sob estas condições, o aumento da
intensidade de cor, em relação aos controlos, foi de 30% para o tratamento com a cutinas e 25% para a Texazym. O grau de cristalinidade determinado por difracção de raios-X não foi
alterado significativamente entre amostras controlo e amostras tratadas com cutinase. Uma vez
mais, os resultados mostraram que o sucesso da aplicação de enzimas, neste caso a cutinase
e uma esterase comercial, depende muito das condições em que se realiza o tratamento.
A cutinase foi escolhida para modificar também a superfície de fibras de diacetato e
triacetato de celulose, trabalho este que é abordado no capítulo 4. A hidrólise enzimática dos
grupos acetilo na superfície da fibra foi monitorizada pela libertação de ácido acético e pela
coloração específica dos tecidos com o corante Remazol Brilliant Blue R. O tratamento do
tecido durante 8 horas a 30 ºC e pH 8 resultou numa actividade acetil esterase de 0.010 U e
0.0072 U tendo como substrato a fibra de diacetato e triacetato de celulose, respectivamente.
Os níveis de cor das amostras tratadas com cutinase durante 24 horas aumentaram 25% para
o diacetato e 317% para o triacetato, comparando com os controlos. Foram analisadas secções
transversais de ambas as fibras, por microscopia de fluorescência, e confirmou-se a acção
superficial da cutinase. Por comparação com outras enzimas já descritas, a cutinase é um
catalizador a ter em consideração para a regeneração superficial da hidrofilicidade e
reactividade da celulose em acetatos com grau de substituição elevado.
Para melhorar a actividade da cutinase nas fibras modificadas de celulose foram
produzidas, através de tecnologias de ADN recombinante, cutinases quiméricas que foram,
posteriormente, usadas no tratamento dos tecidos de acetato de celulose descrito no capítulo
5. Dois módulos distintos de ligação a carbohidratos foram fundidos, independentemente, ao
terminal carboxílico da cutinase: o módulo da celobiohidrolase I, do fungo Trichoderma reesei e
o da endoglucanase C, da bactéria Cellulomonas fimi. Ambas cutinases quiméricas tiveram
uma performance mais eficiente que a cutinase nativa, mas a interacção destas enzimas
bifuncionais com os acetatos de celulose carece de mais estudos para melhor caracterizar a
natureza e extensão destas modificações.
O capítulo 6 é dedicado a uma discussão geral, observações finais e perspectivas
futuras. Nesta tese são apresentadas evidências que mostram que as enzimas, mais
concretamente, a nitrilase e a cutinase são ferramentas importantes para a funcionalização da
superfície das fibras acrílicas e de acetato de celulose. Com este trabalho também fica claro
que apenas se deu o primeiro passo num percurso que eventualmente nos conduzirá a um
aproveitamento eficiente destes recursos que a Natureza nos providencia.eFundação para a Ciência e Tecnologia (FCT)
by means of a PhD grant (SFRH / BD / 13423 / 2003BioSYNTEX (G5RD-CT-2001-00560) from the European Union 5th FrameworkProgram - GROWT
Cellulose biomodification with cutinase fusion proteins
The textile industry presents well succeed examples of
enzyme applications on the processing of natural fi
bres. The design of
hybrid enzymes mimics the strategies that Nature uses to evolve and it
is a powerful tool in biotechnology. The production and application of
the cutinase fused to carbohydrate
-
binding modules (CBMs) has
provided strong evidences of bei
ng an interesting strategy to pursuit.
The CBMs act synergistically with the catalytic domains by increasing
the effective enzyme concentration at the substrate surface and, for
some CBMs, by physical disruption of the solid substrate. Future work
is neede
d to improve the recombinant production of modular cutinases
and to study in detail their affinities toward the cellulose substrates
The effect of additives and mechanical agitation in surface modification of acrylic fibres by cutinase and esterase
The surface of an acrylic fibre containing about 7% of vinyl acetate was modified using Fusarium solani pisi cutinase and a commercial esterase, Texazym PES. The effect of acrylic solvents and stabilising polyols on cutinase operational stability was studied. The half-life time of cutinase increased by 3.5-fold with the addition of 15% N,N-dimethylacetamide (DMA) and by 3-fold with 1M glycerol. The impact of additives and mechanical agitation in the protein adsorption and in the hydrolysis of vinyl acetate from acrylic fabric was investigated. The hydroxyl groups produced on the surface of the fibre were able to react specifically with Remazol Brilliant Blue R (cotton reactive dye) and to increase the colour of the acrylic-treated fabric. The best staining level was obtained with a high level of mechanical agitation and with the addition of 1% DMA. Under these conditions, the raise in the acrylic fabric colour depth was 30% for cutinase and 25% for Texazym. The crystallinity degree, determined by X-ray diffraction, was not significantly changed between control samples and samples treated with cutinase. The results showed that the outcome of the application of these enzymes depends closely on the reaction media conditions.(undefined
A comprehensive review of mammalian pigmentation: paving the way for innovative hair colour-changing cosmetics
The natural colour of hair shafts is formed at the bulb of hair follicles, and it is coupled to the hair growth cycle. Three critical processes must happen for efficient pigmentation: (1) melanosome biogenesis in neural crest-derived melanocytes, (2) the biochemical synthesis of melanins (melanogenesis) inside melanosomes, and (3) the transfer of melanin granules to surrounding pre-cortical keratinocytes for their incorporation into nascent hair fibres. All these steps are under complex genetic control. The array of natural hair colour shades are ascribed to polymorphisms in several pigmentary genes. A myriad of factors acting via autocrine, paracrine, and endocrine mechanisms also contributes for hair colour diversity. Given the enormous social and cosmetic importance attributed to hair colour, hair dyeing is today a common practice. Nonetheless, the adverse effects of the long-term usage of such cosmetic procedures demand the development of new methods for colour change. In this context, case reports of hair lightening, darkening and repigmentation as a side-effect of the therapeutic usage of many drugs substantiate the possibility to tune hair colour by interfering with the biology of follicular pigmentary units. By scrutinizing mammalian pigmentation, this review pinpoints key targetable processes for the development of innovative cosmetics that can safely change the hair colour from the inside out.The authors thank the support of the Portuguese Foundation for Science and Technology (FCT), under the scope of the strategic funding of the UIDB/04469/2020 unit, and LABBELS—
Associate Laboratory in Biotechnology, Bioengineering and Microelectromechnaical Systems, LA/P/
0029/2020. The author Bruno Fernandes also acknowledges his PhD scholarship funded by FCT
(SFRH/BD/131824/2017).info:eu-repo/semantics/publishedVersio
Wound-healing evaluation of entrapped active agents into protein microspheres over cellulosic gauzes
The use of active ingredients in wound management have evolved alongside the pharmaceutical agents and dressings used to deliver them. However, the development of gauzes, dressings with specific properties, still remains a challenge for several medical applications. A new methodology for the controlled release of active components for the healing of burn wounds is proposed herein. Cotton and non-woven bandages have been cationised to promote the attachment of protein microspheres. The active agents, piroxicam and vegetable oil, were entrapped into the microspheres using ultrasound energy. Active agents were released from the microspheres by a change in pH. Wound healing was assessed through the use of standardised burn wounds induced by a cautery in human full-thickness skin equivalents (EpidermFT). The best re-epithelialisation and fastest wound closure was observed in wounds treated with proteinaceous microspheres attached to gauzes, after six days of healing, in comparison with commercial collagen dressing and other controls. Furthermore, the ability of these materials to reduce the inflammation process, together with healing improvement, makes these biomaterials suitable for wound-dressing applications.We would like to acknowledge financial support from the European project Lidwine (contract no. NMP2-CT-2006-026741), and POPH/FSE for co-financing and Fundacao para a Ciencia e a Tecnologia (FCT) for fellowship SFRH/BPD/38939/2007 awarded to Helena Ferreira. The work at CBMA was supported by FEDER through POFC-COMPETE and by national funds from FCT through the project PEst-C/BIA/UI4050/2011
Functionalization of cellulose acetate fibers with engineered cutinases
In the present work, we describe for the first time the specific role of cutinase on surface modification of cellulose acetate fibers. Cutinase exhibits acetyl esterase activity on diacetate and triacetate of 0.010 U and 0.007 U, respectively. An increase on the hydroxyl groups at the fiber surface of 25% for diacetate and 317% for triacetate, after a 24 h treatment, is estimated by an indirect assay. Aiming at further improvement of cutinase affinity toward cellulose acetate, chimeric cutinases are genetically engineered by fusing the 3′-end coding sequence with a bacterial or a fungal carbohydrate-binding module and varying the linker DNA sequence. A comparative analysis of these genetic constructions is presented showing that, the superficial regeneration of cellulose hydrophilicity and reactivity on highly substituted cellulose acetates is achieved by chimeric cutinases.This work was supported by the Biosyntex Project, no. G5RD 2000-30110, from the European Community under the "Competitive and Sustainable Growth" Program, and by the PhD grant SFRH/BD/13423/2003, from Fundacao para a Ciencia e a Tecnologia
The structural properties of odorants modulate their association to human odorant binding protein
The following are available online at https://www.mdpi.com/2218-273X/11/2/145/s1. Odorant molecules CAS identification are listed in Table S1. Statistical analysis on the comparison of odorant properties (described in Table 1 and Table 2) in Table S2. The Vina grid box in Figure S1, the X-ray and Uniprot hOBP sequences alignment are presented in Figure S2 and the RMSD and cartoon structural superposition in Figures S3 and S4, respectively. Standard scatter plots/linear correlation regression are presented as SM, in Figures S5–S9, evaluating the relation of ΔGbinding to the other properties separately. The docking binding energies against the 4RUN X-ray structure are shown in Table S3.The binding of known odorant molecules to the human odorant-binding protein (hOBP) was evaluated in silico. Docking experiments elucidate the preferable binding site and binding affinity of odorant molecules to hOBP. The physicochemical properties molecular weight (MW), vapor pressure (Vp), hydrophobicity level (logP), number of double bonds (NºDB), degree of unsaturation (DoU) and the chemical classification, were selected for the study of odorant modulation. Here, these properties were analyzed concerning 30 pleasant and 30 unpleasant odorants, chosen to represent a wide variety of compounds and to determine their influence on the binding energy to hOBP. Our findings indicate that MW, logP and Vp are the most important odorant variables, directly correlated to odorant-binding energies (ΔGbinding) towards hOBP. Understanding how the odorants behave when complexed with the OBP in human olfaction opens new possibilities for the development of future biotechnological applications, including sensory devices, medical diagnosis, among others.The following funding is acknowledged: the authors thank the support received from the Portuguese Foundation for Science and Technology (FCT) through the strategic funding of UID/BIO/04469/2019 unit and BioTecNorte Operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte
Production of heterologous cutinases by E. coli and improved enzyme formulation for application on plastic degradation
Background: The hydrolytic action of cutinases has been applied to the degradation of plastics. Polyethylene terephthalate (PET) have long half-life which constitutes a major problem for their treatment as urban solid residues. The aim of this work was to characterize and to improve stable the enzyme to optimize the process of degradation using enzymatic hydrolysis of PET by recombinant cutinases. Results: The wild type form of cutinase from Fusarium solani pisi and its C-terminal fusion to cellulose binding domain N1 from Cellulomonas fimi were produced by genetically modified Escherichia coli. The maximum activity of cutinases produced in Lactose Broth in the presence of ampicillin and isopropyl β- D-1-thiogalactopyranoside (IPTG) was 1.4 IU/mL. Both cutinases had an optimum pH around 7.0 and they were stable between 30 and 50ºC during 90 min. The addition of glycerol, PEG-200 and (NH4)2SO4 to the metabolic liquid, concentrated by ultra filtration, stabilized the activity during 60 days at 28ºC. The treatment of PET with cutinases during 48 hrs led to maxima weight loss of 0.90%. Conclusions: Recombinant microbial cutinases may present advantages in the treatment of poly(ethylene terephthalate) PET through enzymatic treatments.This research study was financially supported by CAPES (Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior), FACEPE (Fundacao de Amparo a Ciencia e Tecnologia do Estado de Pernambuco) and CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico)
Using a nitrilase for the surface modification of acrylic fibres
The surface of an acrylic fibre was modified with a commercial nitrilase (EC 3.5.5.1). The effect of fibre solvents and polyols on nitrilase catalysis efficiency and stability was investigated. The nitrilase action on the acrylic fabric was improved by the combined addition of 1 M sorbitol and 4% N, N-dimethylacetamide. The colour levels for samples treated with nitrilase increased 156% comparing to the control samples. When the additives were introduced in the treatment media, the colour levels increased 199%. The enzymatic conversion of nitrile groups into the corresponding carboxylic groups, on the fibre surface, was followed by the release of ammonia and polyacrylic acid. A surface erosion phenomenon took place and determined the “oscillatory” behaviour of the amount of dye uptake with time of treatment. These results showed that the outcome of the application of the nitrilase for the acrylic treatment is intimately dependent on reaction media parameters, such as time, enzyme activity and formulation.(undefined
Chitosan–lignosulfonates sono-chemically prepared nanoparticles : characterisation and potential applications
Due to their recognised properties of biocompatibility, biodegradability and sustainability, chitosan nanocarriers have been successfully used as new delivery systems. In this work, nanoparticles combining chitosan and lignosulfonates were developed for the first time for cosmetic and biomedical applications. The ability of lignosulfonates to act as a counter polyion for stabilisation of chitosan particles, generated using high intensity ultrasound, was investigated. Several conditions for particles preparation were tested and optimised and the resulting nanoparticles were comprehensively characterised by measuring particle size, zeta potential and polydispersity index. The pH of chitosan solution, sonication time and the presence of an adequate surfactant, poloxamer 407, were determinant factors on the development of smaller particles with low polydispersity index (an average particle size of 230 nm was obtained at pH 5 after 8 min of sonication). The beneficial effects of lignosulfonates complex on chitosan nanoparticles were further characterised. Greater stability to lysozyme degradation, biocompatibility with human cells and antimicrobial activity was found upon lignosulfonates incorporation into chitosan nanoparticles. Furthermore, these particles were able to incorporate a hydrophilic model protein – RNase A. A burst release was observed when nanoparticles were loaded with low amount of protein while with high protein content, a sustained release was found, suggesting that the protein cargo maybe loaded both at the surface as in the bulk of the particle, depending on the concentration of drug incorporated.This work was financed by FP6 European project BioRenew (contract no. NMP2-CT-2006-026456), and by FEDER through POFC-COMPETE and by national funds from FCT through the project PEst-C/BIA/UI4050/2011. FCT - Portuguese Foundation for Science and Technology awarded scholarships to M.M. Fernandes (SFRH/BD/38363/2007), T. Matama (SFRH/BPD/47555/2008) and A. Loureiro (SFRH/BD/81479/2011)