LC-MS characterization of intermediates and products of acid orange dyes after laccase treatment

Poster apresentado em COST 847 & D32 actions joint meeting, na Povoa de Varzim, 200

Enzymatic degradation of azo dyes under long time oxidative conditions

Trametes villosa laccase was used for direct azo dye degradation for which the reaction products were analyzed over long periods of time. Laccases have been extensively studied for the degradation of azo dyes [1-3].These enzymes are multicopper phenol oxidases that decolorize azo dyes through a highly non-specific free radical mechanism forming phenolic type compounds, thereby avoiding the formation of toxic aromatic amines [4,5].In the literature, there are a large number of papers reporting on decolorization of azo dyes however the fate of the products of azo dye laccase reactions is ignored [6-8]. Therefore, the purpose of this work is the study of the azo dye degradation products in the presence of laccase. Direct azo dye laccase degradation and amino-phenols polymerization was performed for several days. The formed soluble products were studied by LC-MS while the polymerized insoluble products were studied by 13C -NMR. LC-MS analysis shows the formation of phenolic compounds in the dye oxidation process as well as a large amount of polymerized products that retain the azo group integrity. The amino-phenols reactions were also investigated by 13C-NMR and LC-MS analysis and the real polymerization character of laccase enzymes was shown. This study highlights the fact that laccases polymerize the reaction products obtained in long time batch decolorization processes of the azo dyes. These polymerized products provide unacceptable color levels in effluents limiting the application of laccases as bioremediation agents

Monitoring biotransformations in polyamide fibres

The enzymatic hydrolysis of polyamide fibres yields amino and carboxylic groups. These groups can be found in solution treatments as polyamide monomers and soluble oligomers. The amino groups can also be found at the surface of the fibres as end group chains. In this paper we report two methods to quantify the formation of these groups as a result of the enzymatic action. Soluble amino groups can be quantified with 2,4,6-trinitrobenzenesulfonic acid (TNBS), which yields a coloured complex which can be determined spectrophotometrically. The amino groups on the fibre surface can be quantified by reaction with a wool reactive dye and determination of colour intensities after a dyeing procedure below the glass transition temperature of polyamide

Development and characterization of PLA nanoparticles as carriers for topical delivery

Nanoparticles are seen today as one of the best approaches for the delivery of drugs into the skin. Poly (Lactic Acid) (PLA) is biocompatible and biodegradable and already approved for clinical use. Thus, this work aimed to study the effect of several parameters on the properties of PLA nanoparticles (PLA-NPs) intended for topical delivery. The yield of nanoparticles formation and entrapment efficiencies of lipophilic and hydrophilic model compounds in PLA-NPs were assessed. We evaluated the effects of mechanical stirring, solvent composition and presence of tri-bloc polymers on the protocol for the production of PLA-NPs. The best protocol provided a monodispersed population of non-cytotoxic spherical particles of !150 nm and a yield of nanoparticles formation of !90%. This formulation also proved to be efficient in the encapsulation of lipophilic and hydrophilic model compounds (>80%). The best protocol for the production of PLA-NPs includes a nanoprecipitation step, which is easily up scalable

Phosphorylated silk fibroin matrix for methotrexate release

Silk-based matrix was produced for delivery of a model anticancer drug, methotrexate (MTX). The calculation of net charge of silk fibroin and MTX was performed to better understand the electrostatic interactions during matrix formation upon casting. Silk fibroin films were cast at pH 7.2 and pH 3.5. Protein kinase A was used to prepare phosphorylated silk fibroin. The phosphorylation content of matrix was controlled by mixing at specific ratios the phosphorylated and unphosphorylated solutions. In vitro release profiling data suggest that the observed interactions are mainly structural and not electrostatical. The release of MTX is facilitated by use of proteolytic enzymes and higher pHs. The elevated -sheet content and crystallinity of the acidified-cast fibroin solution seem not to favor drug retention. All the acquired data underline the prevalence of structural interactions over electrostatical interactions between methotrexate and silk fibroin.The authors would like to acknowledge the support, granted by European NOVO Project, Contract No. FP7-HEALTH 2011-two-stage 278402. This work was partially supported by FEDER through POFC-COMPETE and by national funds from FCT through the projects PEst-C/BIA/UI4050/2011 (CBMA). V.V. also wants to thank Dr. Claudia Botelho for her helpful discussion and comments made during the critical reading of the manuscript

Folate-based nanobiodevices for integrated diagnosis/therapy targeting chronic inflammatory diseases

It is estimated that inflammatory diseases affect more than 80 million people worldwide and these numbers are expected to increase in the next 20 years. Disorders such as rheumatoid arthritis (RA) can shorten life span by 10 years and its treatment remains a challenge for the medical and scientific community. More efficient strategies are required in order to improve clinical benefit. Nano-enabled drug delivery systems aim to improve therapy of chronic inflammatory disorders by creating a new, highly specific and efficient strategy, with reduced treatment costs. The consortium of NANOFOL FP7 project produced FBN (liposomal, protein-based and PLA (poly (L-lactic acid)) nanoparticles) with encapsulated anti-inflammatory drugs that were shown to be biologically active, non cytotoxic and capable of specifically targeting folate receptor (FR)-positive cells, in particular activated macrophages, mediators of chronic inflammation in RA. The NANOFOL nanobiodevices targeting activated macrophages may be an interesting theranostics solution, i.e., simultaneous diagnosis and treatment of the site of inflammation in RA patients. The production of a validated, stable, specific FBN with incorporated imaging agent and therapeutic agent (drugs or siRNAs) by the NANOFOL consortium will have many applications in all inflammatory diseases but may also extend to cancer treatment

Formulações micelares proteicas e respectivo método de produção

The present invention describes micellar protein formulations for the controlled release of active ingredients, and method for preparing the same. The invention describes a new micelle composition for use in pharmaceuticals, cosmetics and detergents. In particular, it describes micelle formation formulations that comprise: an aqueous phase containing a protein or a natural or synthetic peptide; a lipophilic phase containing a hydrophobic compound; an adjuvant dissolved in the aqueous phase to regulate the size and stability of the micelles; the size of the micelles varying from 30 to 5000 nm, preferably from 30 to 100 nm, wherein the micelles can be obtained by two different methods, namely using ultrasound or a high-pressure homogeniser. The preparation method involves two distinct phases: an aqueous phase and a lipophilic phase. The aqueous phase can be water or any buffer that is best suitable for a given use, such as an aqueous solution of bovine serum albumen (BSA); human serum albumen (HSA); silk fibroin or a polypeptide fibroin.A presente invenção descreve em formulações micelares proteicas para libertação controlada de agentes e respetivo método de produção. A invenção descreve numa nova composição de micelas para aplicações farmacêuticas, cosméticas e 0 o0 detergência. Nomeadamente, formulações para a formação de micelas que compreendem: · uma fase aquosa contendo uma proteína ou um péptido natural ou sintético; · uma fase lipofílica que compreende um composto hidrofóbico; · um agente adjuvante dissolvido na fase aquosa que regula o tamanho e estabilidade das micelas; em que os tamanhos das referidas micelas varia entre 30 a 5000 nm, de preferência de 30-100 nm, as referidas micelas podem ser obtidas a partir de duas metodologias diferentes, nomeadamente ultra-sons ou homogeneizador de alta pressão. O método de preparação envolve duas fases distintas: fase aquosa e fase lipofílica. A fase aquosa pode ser água ou qualquer tampão que mais se adeque para uma determinada aplicação, como por exemplo uma solução aquosa de albumina sérica bovina (BSA); albumina sérica humana (HSA); fibroína da seda ou de um polipéptido.Universidade do Minh

Biotransformation of phenolics with laccase containing bacterial spores

Treatment of effluents containing phenols such as textile dyes with fungal laccases is usually limited to the acid to neutral pH range and moderate temperatures. Here we demonstrate for the first time that spore-bound laccases which are stable at high temperatures and pH values can be used for phenolic dye decolorisation. Laccase containing spores from Bacillus SF were immobilized on alumina pellets. Both immobilized and free spores were able to completely decolorize the common textile dyes Mordant Black 9, Mordant Brown 96/Mordant Brown 15, and Acid Blue 74 within 90 min of incubation time and decolorized solutions were successfully used in re-dyeing. spores from Bacillus SF were immobilized on alumina pellets. Both immobilized and free spores were able to completely decolorize the common textile dyes Mordant Black 9, Mordant Brown 96/Mordant Brown 15, and Acid Blue 74 within 90 min of incubation time and decolorized solutions were successfully used in re-dyeing.European Project BIOEFFTEXCompetence Centre Applied Biocatalysi

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

Protein matrices for improved wound healing : elastase inhibition by a synthetic peptide model

The unique properties of silk fibroin were combined with keratin to develop new wound-dressing materials. Silk fibroin/keratin (SF/K) films were prepared to reduce high levels of elastase found on chronic wounds. This improved biological function was achieved by the incorporation of a small peptide synthesized based on the reactive-site loop of the Bowman−Birk Inhibitor (BBI) protein. In vitro degradation and release were evaluated using porcine pancreatic elastase (PPE) solution as a model of wound exudate. It was found that biological degradation and release rate are highly dependent on film composition. Furthermore, the level of PPE activity can be tuned by changing the film composition, thus showing an innovative way of controlling the elastase−antielastase imbalance found on chronic wounds.We would like to acknowledge FCT - Portuguese Foundation for Science and Technology for the scholarship concession; European project Lidwine, contract no. NMP2-CT-2006-026741, and Silvia Cappellozza from "Sezione Specializzata per la Bachicoltura" for the supply of silk cocoons