Gestão da Água na Suinocultura

Embrap

Enzymatic surface hydrolysis of PET : effect of structural diversity on kinetic properties of cutinases from thermobifida

In this study cutinases from Thermobifida cellulosilytica DSM44535 (Thc_Cut1 and Thc_Cut2) and Thermobifida fusca DSM44342 (Thf42_Cut1) hydrolyzing poly(ethylene terephthalate) (PET) were successfully cloned and expressed in E.coli BL21-Gold(DE3). Their ability to hydrolyze PET was compared with other enzymes hydrolyzing natural polyesters, including the PHA depolymerase (ePhaZmcl) from Pseudomonas fluorescens and two cutinases from T. fusca KW3. The three isolated Thermobifida cutinases are very similar (only a maximum of 18 amino acid differences) but yet had different kinetic parameters on soluble substrates. Their kcat and Km values on pNP–acetate were in the ranges 2.4–211.9 s–1 and 127–200 μM while on pNP–butyrate they showed kcat and Km values between 5.3 and 195.1 s–1 and between 1483 and 2133 μM. Thc_Cut1 released highest amounts of MHET and terephthalic acid from PET and bis(benzoyloxyethyl) terephthalate (3PET) with the highest concomitant increase in PET hydrophilicity as indicated by water contact angle (WCA) decreases. FTIR-ATR analysis revealed an increase in the crystallinity index A1340/A1410 upon enzyme treatment and an increase of the amount of carboxylic and hydroxylic was measured using derivatization with 2-(bromomethyl)naphthalene. Modeling the covalently bound tetrahedral intermediate consisting of cutinase and 3PET indicated that the active site His-209 is in the proximity of the O of the substrate thus allowing hydrolysis. On the other hand, the models indicated that regions of Thc_Cut1 and Thc_Cut2 which differed in electrostatic and in hydrophobic surface properties were able to reach/interact with PET which may explain their different hydrolysis efficiencies.This study was performed within the Austrian Centre of Industrial Biotechnology ACIB, the MacroFun project and COST Action 868. This work has been supported by the Federal Ministry of Economy, Family and Youth (BMWFJ), the Federal Ministry of Traffic, Innovation and Technology (bmvit), the Styrian Business Promotion Agency SFG, the Standortagentur Tirol and ZIT - Technology Agency of the City of Vienna through the COMET-Funding Program managed by the Austrian Research Promotion Agency FFG. Financial support was also given from Sachsisches Staatsministerium fur Umwelt und Landwirtschaft, Germany. PET was kindly provided by Dr. Vincent Nierstrasz from Ghent University

Enzymatic reduction of azo and indigoid compounds

A customer- and environment-friendly method for the decolorization azo dyes was developed. Azoreductases could be used both to bleach hair dyed with azo dyes and to reduce dyes in vat dyeing of textiles. A new reduced nicotinamide adenine dinucleotide-dependent azoreductase of Bacillus cereus, which showed high potential for reduction of these dyes, was purified using a combination of ammonium sulfate precipitation and chromatography and had a molecular mass of 21.5 kDa. The optimum pH of the azoreductase depended on the substrate and was within the range of pH 6 to 7, while the maximum temperature was reached at 40°C. Oxygen was shown to be an alternative electron acceptor to azo compounds and must therefore be excluded during enzymatic dye reduction. Biotransformation of the azo dyes Flame Orange and Ruby Red was studied in more detail using UV-visible spectroscopy, high-performance liquid chromatography, and mass spectrometry (MS). Reduction of the azo bonds leads to cleavage of the dyes resulting in the cleavage product 2-amino-1,3 dimethylimidazolium and N∼1∼,N∼1∼-dimethyl-1,4-benzenediamine for Ruby Red, while only the first was detected for Flame Orange because of MS instability of the expected 1,4-benzenediamine. The azoreductase was also found to reduce vat dyes like Indigo Carmine (C.I. Acid Blue 74). Hydrogen peroxide (H2O2) as an oxidizing agent was used to reoxidize the dye into the initial form. The reduction and oxidation mechanism of Indigo Carmine was studied using UV-visible spectroscopy

Bone turnover markers for early detection of fracture healing disturbances: A review of the scientific literature

Imaging techniques are the standard method for assessment of fracture healing processes. However, these methods are perhaps not entirely reliable for early detection of complications, the most frequent of these being delayed union and non-union. A prompt diagnosis of such disorders could prevent prolonged patient distress and disability. Efforts should be directed towards the development of new technologies for improving accuracy in diagnosing complications following bone fractures. The variation in the levels of bone turnover markers (BTMs) have been assessed with regard to there ability to predict impaired fracture healing at an early stage, nevertheless the conclusions of some studies are not consensual. In this article the authors have revised the potential of BTMs as early predictors of prognosis in adult patients presenting traumatic bone fractures but who did not suffer from osteopenia or postmenopausal osteoporosis. The available information from the different studies performed in this field was systematized in order to highlight the most promising BTMs for the assessment of fracture healing outcome.As técnicas imagiológicas são o método convencional para a avaliação dos processos de cicatrização das fraturas. No entanto, estes métodos não são talvez totalmente confiáveis para a deteção precoce de complicações, as mais frequentes destas sendo o atraso da união e a não-união. Um diagnóstico eficaz destas desordens poderia prevenir a dor e a incapacidade prolongada do paciente. Esforços devem ser dirigidos no sentido do desenvolvimento de novas tecnologias para melhorar a exatidão no diagnóstico de complicações após fraturas ósseas. A variação nos níveis dos marcadores do turnover ósseo (BTMs) têm sido avaliados com vista à sua capacidade para prever o comprometimento da cicatrização das fraturas numa fase inicial, no entanto, as conclusões de alguns estudos não são consensuais. Neste artigo os autores fizeram uma revisão do potencial dos BTMs como fatores de previsibilidade precoce do prognóstico em doentes adultos que apresentavam fraturas ósseas traumáticas mas que não sofriam de osteopenia ou osteoporose pós-menopausa. A informação disponível nos diferentes estudos realizados neste campo foi sistematizada com vista a evidenciar-se os BTMs mais promissores para a avaliação da evolução da cicatrização das fraturas.SFRH/BD/45018/200

Influence of Ecto-Nucleoside Triphosphate Diphosphohydrolase Activity on Trypanosoma cruzi Infectivity and Virulence

The protozoan Trypanosoma cruzi is the causative agent of Chagas disease, an endemic zoonosis present in some countries of South and Central Americas. The World Health Organization estimates that 100 million people are at risk of acquiring this disease. The infection affects mainly muscle tissues in the heart and digestive tract. There are no vaccines or effective treatment, especially in the chronic phase when most patients are diagnosed, which makes a strong case for the development of new drugs to treat the disease. In this work we evaluate a family of proteins called Ecto-Nucleoside-Triphosphate-Diphosphohydrolase (Ecto-NTPDase) as new chemotherapy target to block T. cruzi infection in mammalian cells and in mice. We have used inhibitors and antibodies against this protein and demonstrated that T. cruzi Ecto-NTPDases act as facilitators of infection in mammalian cells and virulence factors in mice model. Two of the drugs used in this study (Suramin and Gadolinium) are currently used for other diseases in humans, supporting the possibility of their use in the treatment of Chagas disease

An Interspecific Nicotiana Hybrid as a Useful and Cost-Effective Platform for Production of Animal Vaccines

The use of transgenic plants to produce novel products has great biotechnological potential as the relatively inexpensive inputs of light, water, and nutrients are utilised in return for potentially valuable bioactive metabolites, diagnostic proteins and vaccines. Extensive research is ongoing in this area internationally with the aim of producing plant-made vaccines of importance for both animals and humans. Vaccine purification is generally regarded as being integral to the preparation of safe and effective vaccines for use in humans. However, the use of crude plant extracts for animal immunisation may enable plant-made vaccines to become a cost-effective and efficacious approach to safely immunise large numbers of farm animals against diseases such as avian influenza. Since the technology associated with genetic transformation and large-scale propagation is very well established in Nicotiana, the genus has attributes well-suited for the production of plant-made vaccines. However the presence of potentially toxic alkaloids in Nicotiana extracts impedes their use as crude vaccine preparations. In the current study we describe a Nicotiana tabacum and N. glauca hybrid that expresses the HA glycoprotein of influenza A in its leaves but does not synthesize alkaloids. We demonstrate that injection with crude leaf extracts from these interspecific hybrid plants is a safe and effective approach for immunising mice. Moreover, this antigen-producing alkaloid-free, transgenic interspecific hybrid is vigorous, with a high capacity for vegetative shoot regeneration after harvesting. These plants are easily propagated by vegetative cuttings and have the added benefit of not producing viable pollen, thus reducing potential problems associated with bio-containment. Hence, these Nicotiana hybrids provide an advantageous production platform for partially purified, plant-made vaccines which may be particularly well suited for use in veterinary immunization programs