Direct Evidence for Aligned Binding of Cellulase Enzymes to Cellulose Surfaces.

The conversion of biomass into green fuels and chemicals is of great societal interest. Engineers have been designing new cellulase enzymes for the breakdown of otherwise insoluble cellulose materials. A barrier to the rational design of new enzymes has been our lack of a molecular picture of how cellulase binding occurs. A critical factor is the attachment via the enzyme's carbohydrate binding module (CBM). To elucidate the structural and mechanistic details of cellulase adsorption, we have combined experimental data from sum frequency generation spectroscopy with molecular dynamics simulations to probe the equilibrium structure and surface alignment of a 14-residue peptide mimicking the CBM. The data show that binding is driven by hydrogen bonding and that tyrosine side chains within the CBM align the cellulase with the registry of the cellulose surface. Such an alignment is favorable for the translocation and effective cellulose breakdown and is therefore likely an important parameter for the design of novel enzymes.

Freezing from the inside: Ice nucleation in Escherichia coli and Escherichia coli ghosts by inner membrane bound ice nucleation protein InaZ.

Ice nucleation (IN) active bacteria such as Pseudomonas syringae promote the growth of ice crystals more effectively than any material known. Using the specialized ice nucleation protein (INP) InaZ, P. syringae-the well studied epiphytic plant pathogen-attacks plants by frost damage and, likewise fascinating, drives ice nucleation within clouds when airborne in the atmosphere by linkage to the Earth's water cycle. While ice nucleation proteins play a tremendous role for life on the planet, the molecular details of their activity on the bacterial membrane surface are largely unknown. Bacterial ghosts (BGs) derived from Escherichia coli can be used as simplified model systems to study the mode of action of InaZ. In this work, the authors used BGs to study the role of InaZ localization on the luminal side of the bacterial inner membrane. Naturally, P. syringae INPs are displayed on the surface of the outer membrane; so in contrast, the authors engineered an N-terminal truncated form of inaZ lacking the transport sequence for anchoring of InaZ on the outer membrane. This construct was fused to N- and C-terminal inner membrane anchors and expressed in Escherichia coli C41. The IN activity of the corresponding living recombinant E. coli catalyzing interfacial ice formation of supercooled water at high subzero temperatures was tested by a droplet-freezing assay and surface spectroscopy. The median freezing temperature (T50) of the parental living E. coli C41 cells without INP was detected at -20.1 °C and with inner membrane anchored INPs at a T50 value between -7 and -9 °C, demonstrating that the induction of IN from the inside of the bacterium by inner membrane anchored INPs facing the luminal inner membrane side is very similar to IN induced by bacterial INPs located at the outer membrane. Bacterial ghosts derived from these different constructs showed first droplet freezing values between -6 and -8 °C, whereas E. coli C41 BGs alone without carrying inner membrane anchored INPs exhibit a T50 of -18.9 °C. Sum frequency generation spectroscopy showed structural ordered water at the BG/water interface, which increased close to the water melting point. Together, this indicates that the more efficient IN of INP-BGs compared to their living parental strains can be explained by the free access of inner membrane anchored INP constructs to ultrapure water filling the inner space of the BGs.

Ice-nucleating bacteria control the order and dynamics of interfacial water.

Ice-nucleating organisms play important roles in the environment. With their ability to induce ice formation at temperatures just below the ice melting point, bacteria such as Pseudomonas syringae attack plants through frost damage using specialized ice-nucleating proteins. Besides the impact on agriculture and microbial ecology, airborne P. syringae can affect atmospheric glaciation processes, with consequences for cloud evolution, precipitation, and climate. Biogenic ice nucleation is also relevant for artificial snow production and for biomimetic materials for controlled interfacial freezing. We use interface-specific sum frequency generation (SFG) spectroscopy to show that hydrogen bonding at the water-bacteria contact imposes structural ordering on the adjacent water network. Experimental SFG data and molecular dynamics simulations demonstrate that ice-active sites within P. syringae feature unique hydrophilic-hydrophobic patterns to enhance ice nucleation. The freezing transition is further facilitated by the highly effective removal of latent heat from the nucleation site, as apparent from time-resolved SFG spectroscopy.

Biomimetic vaterite formation at surfaces structurally templated by oligo(glutamic acid) peptides.

Previous studies have reported that the metastable vaterite phase of calcium carbonate can be stabilized in solution by acidic additives. Here we demonstrate that vaterite can also be stabilized directly at surfaces by engineered peptides. Our data show that the mineralisation occurs in a 'self-templating' process where calcium ions restructure the peptide backbone, which in turn allows for effective vaterite precipitation.

Stabilization of Insulin by Adsorption on a Hydrophobic Silane Self-Assembled Monolayer.

The interaction between many proteins and hydrophobic functionalized surfaces is known to induce ß-sheet and amyloid fibril formation. In particular, insulin has served as a model peptide to understand such fibrillation, but the early stages of insulin misfolding and the influence of the surface have not been followed in detail under the acidic conditions relevant to the synthesis and purification of insulin. Here we compare the adsorption of human insulin on a hydrophobic (-CH3-terminated) silane self-assembled monolayer to a hydrophilic (-NH3(+)-terminated) layer. We monitor the secondary structure of insulin with Fourier transform infrared attenuated total reflection and side-chain orientation with sum frequency spectroscopy. Adsorbed insulin retains a close-to-native secondary structure on both hydrophobic and hydrophilic surfaces for extended periods at room temperature and converts to a ß-sheet-rich structure only at elevated temperature. We propose that the known acid stabilization of human insulin and the protection of the aggregation-prone hydrophobic domains on the insulin monomer by adsorption on the hydrophobic surface work together to inhibit fibril formation at room temperature.

Development and validation of analytical methodology with focus on the qualification of powder mixers / Misturadores de pós/validação analítica; Espectrofotometria; Óxido férrico/quantificação; Lactose monoidratada/análise quantitativa

This study aims at developing an analytical procedure capable of quantifying the ferric oxide present in the mixture of ferric oxide/lactose monohydrate (0.4% w/w). The analytical procedure was checked for specificity, linearity, precision (system repeatability, procedure repeatability and intermediate precision), accuracy, stability of solutions and robustness of the procedure. The concentration of Fe (III) was determined by spectrophotometry at 480 nm based on calibration curves. The specificity was verified. The linearity was obtained in the range of 11.2 to 16.8 µg of ferric oxide/mL. The relative standard deviation (RSD) of the system repeatability, procedure repeatability and intermediate precision, were not more than 2%. The RSD of the accuracy values were less than 0.75%. The stability of the samples was checked over a 24 hours assay. In the robustness evaluation, the wavelength and the concentration of hydrochloric acid varied. The maximum absorbance deviation due to wavelength variation was 0.14 percent, and the maximum deviation due to the hydrochloric acid concentration variation was 2.4%, indicating that the concentration of hydrochloric acid is critical to the analysis of ferric oxide. The procedure developed was validated and is suitable to the performance qualification of powder mixers..

O objetivo deste trabalho foi desenvolver um método analítico capaz de quantificar o óxido férrico presente na mistura óxido férrico/lactose monoidratada (0,4% w/w). Verificou-se a especificidade, linearidade, precisão (repetibilidade do sistema, repetibilidade do método e precisão intermediária), exatidão, estabilidade das soluções e robustez. A concentração de Fe(III) foi determinada por espectrofotometria em 480 nm com base em curvas de calibração. Verificou-se a especificidade verificada. A linearidade foi obtida na faixa de 11,2 a 16,8 µg de óxido férrico/mL. O desvio padrão relativo (DPR) da repetibilidade do sistema, método e precisão intermediária foram inferiores a 2%. Os valores de DPR da exatidão foram inferiores a 0,75%. A estabilidade das amostras foi verificada ao longo de 24 horas de ensaio. Na avaliação da robustez variou-se o comprimento de onda e a concentração de ácido clorídrico. O desvio máximo de absorbância ao se variar o comprimento de onda foi de 0,14%, enquanto que para a concentração de ácido clorídrico o desvio foi de 2,4% para a concentração de 0,8 M. Assim, a concentração de ácido clorídrico é crítica para a análise de óxido férrico. O método desenvolvido foi validado e é adequado à qualificação do desempenho de misturadores de pós e granulados...

Cytotoxicity of doxycycline effluent generated by the Fenton process.

This study aims at determining the Minimum Inhibitory Concentration with Escherichia coli ATCC 25922 and cytotoxicity to L929 cells (ATCC CCL-1) of the waste generated by doxycycline degradation by the Fenton process. This process has shown promise in this treatment thanks mainly to the fact that the waste did not show any relevant inhibitory effect on the test organism and no cytotoxicity to L-929 cells, thus demonstrating that the antibiotic properties were inactivated.

Tetracycline: production, waste treatment and environmental impact assessment

The frequent occurrence of pharmaceuticals in the aquatic environment requires an assessment of their environmental impact and their negative effects in humans. Among the drugs with high harmful potential to the environment are the antibiotics that reach the environment not only, as may be expected, through the effluents from chemical and pharmaceutical industries, but mainly through the sewage and livestock; because around 25 to 75% of the ingested drugs are excreted in unchanged form after the passage through the Gastro-Intestinal Tract. Tetracycline has high world consumption, representing a human consumption of about 23 kg/day in Brazil in 2007. At the moment, researches are being made to develop new tetracycline that incorporate heavy metals (Hg, Cd, Re, Pt, Pd) to their structures in order to increase their bactericidal effect. The conventional wastewater treatment plants are not able to degrade complex organic molecules to reduce their toxicity and improve their biodegradability. For this reason new technologies, i.e., the advanced oxidation processes, are being developed to handle this demand. The objectives of this study are to review the literature on the processes of obtaining tetracycline, presenting its waste treatment methods and evaluation of their environmental impact.

A ocorrência frequente de fármacos no meio aquático exige a avaliação do seu impacto ambiental e seus efeitos negativos em seres humanos. Dentre os fármacos com maior potencial de impacto ambiental estão os antibióticos, que chegam ao meio ambiente através dos efluentes de indústrias químico-farmacêuticas e, principalmente, através de esgotos domésticos e criação de animais, visto que 25% a 75% dos fármacos são excretados em forma inalterada após passagem pelo Trato Gastrointestinal. Parcela significativa do consumo mundial dos antibióticos corresponde à classe das tetraciclinas, representando consumo humano de 23 kg/dia no Brasil, em 2007. Atualmente, há pesquisas de novas tetraciclinas que incorporam metais pesados (Hg, Cd, Re, Pt, Pd) às suas estruturas com o intuito de aumentar suas atividades bactericidas. As estações de tratamento de águas residuais convencionais não são capazes de degradar moléculas orgânicas complexas, diminuir a sua toxicidade e melhorar a sua biodegradabilidade. Por esta razão, as novas tecnologias, como, por exemplo, os processos oxidativos avançados, estão sendo desenvolvidos para lidar com esta demanda. Os objetivos deste trabalho são fazer uma revisão da literatura sobre os processos de obtenção de tetraciclinas, apresentar métodos de tratamento de seus resíduos e avaliar o seu impacto ambiental.

Environmental contamination by fluoroquinolones

Over the past few decades, a high number of pharmaceuticals have been detected in surface, ground and drinking waters. This contamination comes from domestic sewage, livestock, hospitals and chemical-pharmaceutical industries. Typical examples of these pollutants are the fluoroquinolones - powerful antibiotics used in human and veterinary medicine. The presence of fluoroquinolones in the environment can pose a serious threat to the ecosystem and to human health due to their high consumption globally: in 1998, around 120 tons were produced. Even at low environmental concentrations, antibiotics stimulate bacterial resistance. The consequences of the presence of fluoroquinolones in the environment are not fully understood, but are known to be toxic to plants and aquatic organisms. Approximately 85% of the fluoroquinolones present in influents can be removed by conventional wastewater treatment plants, but the removed fraction is frequently accumulated in the sludge, which is sometimes used as fertilizer, representing an additional input route into the environment. The removal of fluoroquinolones by biological treatment is ineffective, and it is believed that only advanced oxidation technologies are able to destroy these emerging pollutants.

Nas últimas décadas, um grande número de fármacos tem sido identificado em águas superficiais, subterrâneas e potáveis. Tal contaminação advém do esgoto doméstico, hospitais, criação de animais e das indústrias químico-farmacêuticas. Exemplos típicos desses poluentes são as fluoroquinolonas – potentes antibióticos empregados na medicina humana e veterinária. A presença de fluoroquinolonas no meio ambiente pode representar uma séria ameaça para o ecossistema e para a saúde humana devido ao alto consumo mundial: em 1998 foram produzidas, aproximadamente, 120 toneladas. Mesmo em baixas concentrações, antibióticos podem estimular a resistência bacteriana. As consequências da presença de fluoroquinolonas no ambiente não são completamente compreendidas, mas sabe-se que são tóxicas para plantas e organismos aquáticos. Aproximadamente 85% das fluoroquinolonas presentes em efluentes podem ser removidos em estações de tratamento de efluentes convencionais, porém a fração removida é frequentemente acumulada no lodo, muitas vezes usado como fertilizante, o que representa uma rota adicional de entrada desses compostos no ambiente. A remoção de fluoroquinolonas por meio de tratamento biológico não é eficiente, e acredita-se que somente as tecnologias de oxidação avançada sejam capazes de degradar esses poluentes emergentes.

A new enzymatic process for the treatment of phenolic pollutants

This work aimed to develop a new economic enzymatic process to treat the phenolic pollutants using pure tyrosinase in stirred vessel and adopting temperature (T), pH, rotational speed (N), initial phenol (C P,o) and enzyme (C T) concentrations as independent variables. Experimental data of the residual phenol concentration (C P) were used to calculate the oxidation efficiency (η), initial oxidation rate (-r o) and time required to reach the end of reaction (t) that were selected as the responses. Under the optimal conditions (T = 45°C, pH 6.6, N = 400 rpm, C P,o = 100 ppm and C T = 50 U/mL), η was 88.1%, -r o = 10.2 mg L-1 min-1, t = 40 min. These results suggested that tyrosinase-rich crude extracts from vegetable byproducts could be quite promising.

Phenol oxidation by mushroom waste extracts: a kinetic and thermodynamic study.

Tyrosinase activity of mushroom extracts was checked for their ability to degrade phenol. Phenol oxidation kinetics was investigated varying temperature from 10 to 60 °C and the initial values of pH, enzyme activity and phenol concentration in the ranges 4.5-8.5, 1.43-9.54 U/mL and 50-600 mg/L, respectively. Thermodynamic parameters of phenol oxidation and tyrosinase reversible inactivation were estimated. Tyrosinase thermostability was also investigated through residual activity tests after extracts exposition at 20-50 °C, whose results allowed exploring the thermodynamics of enzyme irreversible thermoinactivation. This study is the first attempt to separate the effects of reversible unfolding and irreversible denaturation of tyrosinase on its activity. Extracts were finally tested on a real oil mill wastewater.