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

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

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

New enzyme based process direction to prevent wool shrinking without substantial tensile strength loss

In this paper a new enzymatic process direction is described for obtaining machine washable wool with acceptable quality. In general, application of protease enzyme technology in wool processing results in considerable loss of tensile strength by diffusion of the enzyme into the interior of wool fibers. To overcome this disadvantage enzymatic activity has been more targeted to the outer surface of the scales by improving the susceptibility of the outer surface scale protein for proteolytic degradation. This has been realized by a pretreatment of wool with hydrogen peroxide at alkaline pH in the presence of high concentrations of salt

Azoreductase activity in intact cells of a non-conventional yeast strain

Poster apresentado no Annual Workshop COST 847 Textile Quality and Biotechnology, 2, Villa Olmo, Como, Itália, 10-11 Outubro 2002.Several non-conventional ascomycete yeast strains, isolated from dye-contaminated environments, display azo-reductive capabilities. Those strains can therefore decolorize azo dyes, which are widely used in the textile industries. The azoreductase activity has been described and investigated in a wide variety of anaerobic bacterial species, particularly from the intestinal microflora, and also, although more rarely, in aerobic bacteria. The present work describes some characteristics of the azoreductase present in one of the isolated yeast strains. This enzyme activity seems to be constitutive in the microorganism, as the decolourization process is not affected by pre-adaptation of the cells to the tested dyes. The azo reduction activity, measured at constant OD (optical density), has a maximum at the end of the exponential growth phase. For cells collected in this phase, the decolourization profile for the two dyes derived from N,N-dimethylaniline (I and II) presents a plateau between pH 3 and 5, while the analogue dyes derived from beta-naphtol (III and IV) have a maximum at pH 3. The azoreductase activity in resting cells shows a typical saturation kinetics in relation to the concentration of the dye. For dye II, at pH 4.0, a Km of 0,42 mM and a vmáx of 3,4 mmol*h-1*gDW-1 were obtained. Despite the low specificity of this yeast azoreductase, both the initial decolourization rate and the extent of the process are dependent on the dye structure.BIOEFTEX Project

Spectroscopic on-line monitoring and stopped-flow kinetic analysis of dye degradation by laccase/mediator systems

The laccase catalyzed transformation of the acid dye Indigo Carmine (CI Acid Blue 74) was studied using various redox mediators: violuric acid (VIO), 2,2,6,6-tetramethyl-1-piperidinyloxyl (TEMPO), 1-hydroxybenzotriazole (HOBT), and 2,2-azinobis-(3-ethylbenzothiazoline-6-disulfonic acid diammonium salt (ABTS). Inline UV/Vis and IR spectroscopy was employed to monitor the decolorization in real-time during batch decolorization. ABTS was the most effective mediator follwed by TEMPO. Stopped flow kinetics was employed to study the initial phase of dye degradation in more detail. While the batch decolorization experiments suggested zero-order rate laws for dye transformation at an early stage, the more accurate stopped-flow kinetic experiments revealed that the rate laws for the initial phase were actually more complicated. Different pH optima for dye decolorization were found for the laccase catalyzed reaction (pH 3.5) and for the oxidation brought about by the isolated ABTS radical cation (pH 6.7)

Making sense of light: the use of optical spectroscopy techniques in plant sciences and agriculture

As a result of the development of non-invasive optical spectroscopy, the number of prospective technologies of plant monitoring is growing. Being implemented in devices with different functions and hardware, these technologies are increasingly using the most advanced data processing algorithms, including machine learning and more available computing power each time. Optical spectroscopy is widely used to evaluate plant tissues, diagnose crops, and study the response of plants to biotic and abiotic stress. Spectral methods can also assist in remote and non-invasive assessment of the physiology of photosynthetic biofilms and the impact of plant species on biodiversity and ecosystem stability. The emergence of high-throughput technologies for plant phenotyping and the accompanying need for methods for rapid and non-contact assessment of plant productivity has generated renewed interest in the application of optical spectroscopy in fundamental plant sciences and agriculture. In this perspective paper, starting with a brief overview of the scientific and technological backgrounds of optical spectroscopy and current mainstream techniques and applications, we foresee the future development of this family of optical spectroscopic methodologies.info:eu-repo/semantics/publishedVersio

Characterization of the azo reductase activity in a novel ascomycete yeast strain

Poster apresentado no XIII Congresso Nacional de Bioquímica, Lisboa, 5-7 Dezembro 2002.An ascomycete yeast strain, UM41 , was isolated from a textile wastewater treatment facility, and selected on the basis of its azo dye decolourising activity. Growing yeast cultures in liquid media containing glucose and 0.2mM dye removed more than 80% of the colour in 15 h, and total decolourisation of the tested dyes occured in 24 h. Colour loss is due to a constitutive azoreductase activity, expressed in intact cells, but not detectable in supernatant samples or cell-free extracts. This activity suffers thermal inactivation at temperatures higher than 50ºC and its dependence on dye concentration is satisfactorily described by the Michaelis-Menten model. Specific decolourising activities of resting cells have a maximum during the late exponential growth phase and are enhanced by up to 3-fold by the redox mediator AQS. The effect of pH on the decolourisation rates is structure-dependent. UM41 growing on glucose-containing medium initially exhibits a predominantly fermentative metabolism and produces ethanol, while oxygen is quicky depleted from the culture medium. However the same batch of cells retained decolourising activity for up to five successive cycles of dye removal, without any further nutrient addition. The microorganism growth and dye decolourisation observed after glucose exhaustion probably occur at the expense of ethanol, suggesting that decoulorisation is also compatible with a respiratory metabolism.BIOEFTEX

Enzymatic phosphorylation of silk fibroins : a platform for the production of biocompatible, cell-static, materials

Silks are natural protein polymers produced by insects. Silk heavy chain of B.mori is primarily composed of hydrophobic, –(–Ala–Gly–)n– -sheet crystalline domains. Based on silk biocompatibility, biodegradability and strength, different materials were developed. Silk offers a stabilizing environment for incorporated proteins and molecules. Silk properties can be controlled via structure manipulation, by coupling molecules of biological significance; its Tyr and Ser residues can be modified. Once incorporated into a protein, the phosphate group establishes hydrogen bonds that affect intra- and inter-molecular interactions16. Phosphorylation is stable under physiological conditions, thus directing the formation and reorganization of protein networks. Curiously, using phosphorylation for protein functionalization is largely unexplored. Significant research is devoted to bio-inspired materials with various cell-differentiating and cell-supporting features. However, little attention is paid to develop cell-static bio-materials. Such materials do not promote cell growth. That can be achieved by lowering the probability of cell attachment to the material, via creation of negatively charged material surface. The goal of this study was to produce bio-compatible materials with the cell-static properties by phosphorylation. Silk solutions were made to cast films of variable pH and phosphorylated content. Obtained materials were tested and a dependency between amount of phosphorylation and bio-chemical properties confirmed