One-Step Purification of Recombinant Cutinase from an E. coli Extract Using a Stabilizing Triazine-Scaffolded Synthetic Affinity Ligand.

Cutinase from Fusarium solani pisi is an enzyme that bridges functional properties between lipases and esterases, with applications in detergents, food processing, and the synthesis of fine chemicals. The purification procedure of recombinant cutinase from E. coil extracts is a well-established but time-consuming process, which involves a sequence of two anionic exchange chromatography steps followed by dialysis. Affinity chromatography is the most efficient method for protein purification, the major limitation of its use being often the availability of a ligand selective for a given target protein. Synthetic affinity ligands that specifically recognize certain sites on the surface of proteins are highly desirable for affinity processes due to their cost-effectiveness, durability, and reusability across multiple cycles. Additionally, these ligands establish moderate affinity interactions with the target protein, making it possible to purify proteins under gentle conditions while maintaining high levels of activity recovery. This study aimed to develop a new method for purifying cutinase, utilizing triazine-scaffolded biomimetic affinity ligands. These ligands were previously screened from a biased-combinatorial library to ensure their binding ability to cutinase without compromising its biological function. A lead ligand, designated as 11/3', [4-({4-chloro-6-[(2-methylbutyl)amino]-1,3,5-triazin-2-yl}amino)benzoic acid], was chosen and directly synthesized onto agarose. Experiments conducted at different scales demonstrated that this ligand (with an affinity constant Ka ≈ 104 M-1) exhibited selectivity towards cutinase, enabling the purification of the enzyme from an E. coli crude production medium in a single step. Under optimized conditions, the protein and activity yields reached 25% and 90%, respectively, with a resulting cutinase purity of 85%.

Design of Quercetin-Loaded Natural Oil-Based Nanostructured Lipid Carriers for the Treatment of Bacterial Skin Infections.

The biological activity of natural plant-oil-based nanostructured lipid carriers (NPO-NLCs) can be enhanced by the encapsulation of bioactive compounds, and they in turn can improve topical delivery of the drugs. Quercetin (QR), a vital plant flavonoid, expresses antibacterial properties, and we recently showed that empty NPO-NLCs also have antimicrobial activity. The main objective of this study was to evaluate the synergetic effect of loading natural plant-oil-based nanostructured lipid carriers with quercetin (QR-NPO-NLCs) as a topical delivery system for the treatment of bacterial skin infections. Five nanostructured lipid carrier systems containing different oils (sunflower, olive, corn, coconut, and castor) were engineered. The particles' stability, structural properties, bioavailability, and antimicrobial activity were studied. NLCs with an average size of <200 nm and Z-potential of −40 mV were developed. Stable QR-NPO-NLCs were obtained with high encapsulation efficiency (>99%). The encapsulation of QR decreased cytotoxicity and increased the antioxidant effect of nanocarriers. An increase in antibacterial activity of the systems containing QR was demonstrated against Staphylococcus aureus. QR-NPO-NLCs could transport QR to an intranuclear location within HaCaT cells, indicating that QR-NPO-NLCs are promising candidates for controlled topical drug delivery.

Green Extraction of Annatto Seed Oily Extract and Its Use as a Pharmaceutical Material for the Production of Lipid Nanoparticles.

This work developd nanomaterials formulated from annatto seed oily extract (ASE), myristic acid (tetradecanoic acid), and their fatty acid esters. The annatto seed oily extract was obtained using only soybean oil (ASE + SO) and Brazil nut oil (ASE + BNO). The UV/VIS analysis of the oily extracts showed three characteristic peaks of the bixin molecule at 430, 456 and 486 nm. The lipid nanoparticles obtained using myristic acid and ASE + BNO or only BNO showed better results than the oil soybean extract, i.e., the particle size was <200 nm, PDI value was in the range of 0.2−0.3, and had no visual physical instability as they kept stable for 28 days at 4 °C. Lipid nanoemulsions were also produced with esters of myristic acid and ASE + BNO. These fatty acid esters significantly influenced the particle size of nanoemulsions. For instance, methyl tetradecanoate led to the smallest particle size nanoemulsions (124 nm), homogeneous size distribution, and high physical stability under 4 and 32 °C for 28 days. This work demonstrates that the chemical composition of vegetable oils and myristic acid esters, the storage temperature, the chain length of fatty acid esters (FAE), and their use as co-lipids improve the physical stability of lipid nanoemulsions and nanoparticles from annatto seed oily extract.

LipNanoCar Technology - A Versatile and Scalable Technology for the Production of Lipid Nanoparticles.

The extensive knowledge in the miniemulsion technique used in biocatalysis applications by the authors allowed the development of drug delivery systems that constitutes the LipNanoCar technology core for the production of lipid nanoemulsions and solid lipid nanoparticles. The LipNanoCar technology, together with adequate formulations of different oils, fatty acids, surfactants, and temperature, allows the entrapment of several bioactive and therapeutic compounds in lipid nanoparticles for cosmetic, nutrition, and pharmaceutical applications.The LIpNanoCar technology allowed lipid nanoparticles production with average sizes ranging from 100 to 300 nm and Zeta Potentials between -55 and -20 mV. Concomitantly, high entrapment or encapsulation efficiencies (%EE) were achieved, as illustrated in this work for ß-carotene and vitamins derivatives (>85%) for cosmetic application, and for antibiotics currently used in chemotherapy, like rifampicin (69-85%) and pyrazinamide (14-29%) against Mycobacterium tuberculosis (TB), and ciprofloxacin (>65%) and tobramycin (~100%) in Cystic Fibrosis (CF) respiratory infections therapy. Ciprofloxacin presented, for example, a quick-release from the lipid nanoparticles using a dialysis tubing (96% in the first 7 h), but slower than the free antibiotic (95% in the first 3 h). This result suggests that ciprofloxacin is loaded near the external surface of the lipid nanoparticles.The toxicity and validation of entrapment of antibiotics in lipid nanoparticles for Cystic Fibrosis therapy were assessed using Caenorhabditis elegans as an animal model of bacterial infection. Fluorescence microscopy of an entrapped fluorescent dye (DiOC) confirmed the uptake of the lipid nanoparticles by ingestion, and their efficacy was successfully tested in C. elegans. Burkholderia contaminans IST408 and Burkholderia cenocepacia K56-2 infections were tested as model bacterial pathogens difficult to eradicate in Cystic Fibrosis respiratory diseases.

Dermal Delivery of Lipid Nanoparticles: Effects on Skin and Assessment of Absorption and Safety.

During the recent decades, dermal delivery has achieved visible popularity mainly due to the increase of chronic skin diseases and the demand for targeted delivery and patient compliance. Dermal delivery provides an attractive alternative to oral drug delivery, promoting the drug application directly at the site of action, resulting in higher localized drug concentration with reduced systemic drug exposure. Among several types of drug delivery systems used in dermal delivery are the lipid nanoparticles, which include solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs). These lipid nanocarriers have attracted great interest and have been intensively studied for their use in dermal applications. Lipid nanoparticles increase the transport of active compounds through the skin by improving drug solubilization in the formulation, drug partitioning into the skin, and fluidizing skin lipids. Moreover, these nanocarriers are composed of biologically active and biodegradable lipids that show less toxicity and offer many favorable attributes such as adhesiveness, occlusion, skin hydration, lubrication, smoothness, skin penetration enhancement, modified release, improvement of formulation appearance providing a whitening effect, and offering protection of actives against degradation.This chapter focuses on the effects of lipid nanoparticles in dermal delivery, on the types of active compounds that are used in their formulation and application, some aspects related to their possible toxicity, and a description of the most commonly used techniques for the evaluation of drug absorption on the skin.

Bioelectricity generation using long-term operated biocathode: RFLP based microbial diversity analysis.

In the present work, power generation and substrate removal efficiencies of long-term operated microbial fuel cells, containing abiotic cathodes and biocathodes, were evaluated for 220 days. Among the two microbial fuel cell (MFC) types, the one containing biocathode showed higher power density (54 mW/m2), current density (122 mA/m2) coulombic efficiency (33%), and substrate removal efficiency (94%) than the abiotic cathode containing MFC. Voltammetric analysis also witnessed higher and sustainable electron discharge for the MFC with biocathode, when compared with the abiotic cathode MFC. Over the tested period, both MFC have shown a cell voltage drop, after 150 and 165, days, for the MFC with biocathode and abiotic cathodes, respectively. Polymerase chain reaction (PCR) based restriction fragment length polymorphism (RFLP) analysis identified 281 clones. Bacteria belonging to Acinetobacter, Acidovorax, Pseudomonas and Burkholderia were observed in the abiotic cathode MFC. Bacteria belonging to Geobacter, Cupriavidus and Acidobacteria were observed in the biocathode MFC. Almost similar types of archaea (Methanosarcinales, Methanolinea, Nitrososphaera and Methanomicrobiales) were observed in both MFCs.

Fast identification of off-target liabilities in early antibiotic discovery with Fourier-transform infrared spectroscopy.

Structural modifications of known antibiotic scaffolds have kept the upper hand on resistance, but we are on the verge of not having antibiotics for many common infections. Mechanism-based discovery assays reveal novelty, exclude off-target liabilities, and guide lead optimization. For that, we developed a fast and automatable protocol using high-throughput Fourier-transform infrared spectroscopy (FTIRS). Metabolic fingerprints of Staphylococcus aureus and Escherichia coli exposed to 35 compounds, dissolved in dimethyl sulfoxide (DMSO) or water, were acquired. Our data analysis pipeline identified biomarkers of off-target effects, optimized spectral preprocessing, and identified the top-performing machine learning algorithms for off-target liabilities and mechanism of action (MOA) identification. Spectral bands with known biochemical associations more often yielded more significant biomarkers of off-target liabilities when bacteria were exposed to compounds dissolved in water than DMSO. Highly discriminative models distinguished compounds with predominant off-target effects from antibiotics with well-defined MOA (AUROC > 0.87, AUPR > 0.79, F1 > 0.81), and from the latter predicted their MOA (AUROC > 0.88, AUPR > 0.70, F1 > 0.70). The compound solvent did not affect predictive models. FTIRS is fast, simple, inexpensive, automatable, and highly effective at predicting MOA and off-target liabilities. As such, FTIRS mechanism-based screening assays can be applied for hit discovery and to guide lead optimization during the early stages of antibiotic discovery.

Technologies for High-Throughput Identification of Antibiotic Mechanism of Action.

There are two main strategies for antibiotic discovery: target-based and phenotypic screening. The latter has been much more successful in delivering first-in-class antibiotics, despite the major bottleneck of delayed Mechanism-of-Action (MOA) identification. Although finding new antimicrobial compounds is a very challenging task, identifying their MOA has proven equally challenging. MOA identification is important because it is a great facilitator of lead optimization and improves the chances of commercialization. Moreover, the ability to rapidly detect MOA could enable a shift from an activity-based discovery paradigm towards a mechanism-based approach. This would allow to probe the grey chemical matter, an underexplored source of structural novelty. In this study we review techniques with throughput suitable to screen large libraries and sufficient sensitivity to distinguish MOA. In particular, the techniques used in chemical genetics (e.g., based on overexpression and knockout/knockdown collections), promoter-reporter libraries, transcriptomics (e.g., using microarrays and RNA sequencing), proteomics (e.g., either gel-based or gel-free techniques), metabolomics (e.g., resourcing to nuclear magnetic resonance or mass spectrometry techniques), bacterial cytological profiling, and vibrational spectroscopy (e.g., Fourier-transform infrared or Raman scattering spectroscopy) were discussed. Ultimately, new and reinvigorated phenotypic assays bring renewed hope in the discovery of a new generation of antibiotics.

Simultaneous elucidation of antibiotic mechanism of action and potency with high-throughput Fourier-transform infrared (FTIR) spectroscopy and machine learning.

The low rate of discovery and rapid spread of resistant pathogens have made antibiotic discovery a worldwide priority. In cell-based screening, the mechanism of action (MOA) is identified after antimicrobial activity. This increases rediscovery, impairs low potency candidate detection, and does not guide lead optimization. In this study, high-throughput Fourier-transform infrared (FTIR) spectroscopy was used to discriminate the MOA of 14 antibiotics at pathway, class, and individual antibiotic level. For that, the optimal combinations and parametrizations of spectral preprocessing were selected with cross-validated partial least squares discriminant analysis, to which various machine learning algorithms were applied. This coherently resulted in very good accuracies, independently of the algorithms, and at all levels of MOA. Particularly, an ensemble of subspace discriminants predicted the known pathway (98.6%), antibiotic classes (100%), and individual antibiotics (97.8%) with exceptional accuracy, and similar results were obtained for simulated novel MOA. Even at very low concentrations (1 µg/mL) and growth inhibition (15%), over 70% pathway and class accuracy was achieved, suggesting FTIR spectroscopy can probe the grey chemical matter. Prediction of inhibitory effect was also examined, for which a squared exponential Gaussian process regression yielded a root mean square error of 0.33 and a R2 of 0.92, indicating that metabolic alterations leading to growth inhibition are intrinsically reflected on FTIR spectra beyond cell density. KEY POINTS: • Antibiotic MOA and potency estimated with high-throughput FTIR spectroscopy • Sub-inhibitory MOA identification suggests ability to explore grey chemical matter • Data analysis optimization improved MOA identification at antibiotic level by 38.

Metabolic Fingerprinting with Fourier-Transform Infrared (FTIR) Spectroscopy: Towards a High-Throughput Screening Assay for Antibiotic Discovery and Mechanism-of-Action Elucidation.

The discovery of antibiotics has been slowing to a halt. Phenotypic screening is once again at the forefront of antibiotic discovery, yet Mechanism-Of-Action (MOA) identification is still a major bottleneck. As such, methods capable of MOA elucidation coupled with the high-throughput screening of whole cells are required now more than ever, for which Fourier-Transform Infrared (FTIR) spectroscopy is a promising metabolic fingerprinting technique. A high-throughput whole-cell FTIR spectroscopy-based bioassay was developed to reveal the metabolic fingerprint induced by 15 antibiotics on the Escherichia coli metabolism. Cells were briefly exposed to four times the minimum inhibitory concentration and spectra were quickly acquired in the high-throughput mode. After preprocessing optimization, a partial least squares discriminant analysis and principal component analysis were conducted. The metabolic fingerprints obtained with FTIR spectroscopy were sufficiently specific to allow a clear distinction between different antibiotics, across three independent cultures, with either analysis algorithm. These fingerprints were coherent with the known MOA of all the antibiotics tested, which include examples that target the protein, DNA, RNA, and cell wall biosynthesis. Because FTIR spectroscopy acquires a holistic fingerprint of the effect of antibiotics on the cellular metabolism, it holds great potential to be used for high-throughput screening in antibiotic discovery and possibly towards a better understanding of the MOA of current antibiotics.

Miniemulsion in biocatalysis, a new approach employing a solid reagent and an easy protocol for product isolation applied to the aldol reaction by Rhizopus niveus lipase.

Miniemulsion systems presented a great potential for biocatalytic reactions. However, different limitations jeopardized the applications of this non-conventional medium. In this work, miniemulsion systems (emulsion reactors) were applied for the first time to the aldol reaction between cyclohexanone and 4-nitrobenzaldehyde by Rhizopus niveus lipase allowing a decrease in the catalyst concentration from 20 mg mL-1 to 6 mg mL-1 in comparison with organic solvents. Moreover, the yield increased from ~25% to ~65% for 48 h reactions and the enantiomeric excess increased from ~10% to ~30% for (R,S)-anti-aldol product, showing the potentiality of this non-conventional reaction medium. The advances reported in this work expands the possibilities of the miniemulsion reaction medium to a whole new level, increasing the scope to solid reagents and products, and also different reactions (biocatalytic or not) that requires pH control by buffers with a simple product isolation procedure, enabling future applications of this poorly studied reaction medium.

Antibiotic Discovery: Where Have We Come from, Where Do We Go?

Given the increase in antibiotic-resistant bacteria, alongside the alarmingly low rate of newly approved antibiotics for clinical usage, we are on the verge of not having effective treatments for many common infectious diseases. Historically, antibiotic discovery has been crucial in outpacing resistance and success is closely related to systematic procedures-platforms-that have catalyzed the antibiotic golden age, namely the Waksman platform, followed by the platforms of semi-synthesis and fully synthetic antibiotics. Said platforms resulted in the major antibiotic classes: aminoglycosides, amphenicols, ansamycins, beta-lactams, lipopeptides, diaminopyrimidines, fosfomycins, imidazoles, macrolides, oxazolidinones, streptogramins, polymyxins, sulphonamides, glycopeptides, quinolones and tetracyclines. During the genomics era came the target-based platform, mostly considered a failure due to limitations in translating drugs to the clinic. Therefore, cell-based platforms were re-instituted, and are still of the utmost importance in the fight against infectious diseases. Although the antibiotic pipeline is still lackluster, especially of new classes and novel mechanisms of action, in the post-genomic era, there is an increasingly large set of information available on microbial metabolism. The translation of such knowledge into novel platforms will hopefully result in the discovery of new and better therapeutics, which can sway the war on infectious diseases back in our favor.

Fed-Batch Production of Saccharomyces cerevisiae L-Asparaginase II by Recombinant Pichia pastoris MUT s Strain.

L-Asparaginase (ASNase) is used in the treatment of acute lymphoblastic leukemia, being produced and commercialized only from bacterial sources. Alternative Saccharomyces cerevisiae ASNase II coded by the ASP3 gene was biosynthesized by recombinant Pichia pastoris MUT s under the control of the AOX1 promoter, using different cultivation strategies. In particular, we applied multistage fed-batch cultivation divided in four distinct phases to produce ASNase II and determine the fermentation parameters, namely specific growth rate, biomass yield, and enzyme activity. Cultivation of recombinant P. pastoris under favorable conditions in a modified defined medium ensured a dry biomass concentration of 31 gdcw.L-1 during glycerol batch phase, corresponding to a biomass yield of 0.77 gdcw.g glycerol - 1 and a specific growth rate of 0.21 h-1. After 12 h of glycerol feeding under limiting conditions, cell concentration achieved 65 gdcw.L-1 while ethanol concentration was very low. During the phase of methanol induction, biomass concentration achieved 91 gdcw.L-1, periplasmic specific enzyme activity 37.1 U.g dcw - 1 , volumetric enzyme activity 3,315 U.L-1, overall enzyme volumetric productivity 31 U.L-1.h-1, while the specific growth rate fell to 0.039 h-1. Our results showed that the best strategy employed for the ASNase II production was using glycerol fed-batch phase with pseudo exponential feeding plus induction with continuous methanol feeding.

Characterization of gastric cells infection by diverse Helicobacter pylori strains through Fourier-transform infrared spectroscopy.

The infection of Helicobacter pylori, covering 50% of the world-population, leads to diverse gastric diseases as ulcers and cancer along the life-time of the human host. To promote the discovery of biomarkers of bacterial infection, in the present work, Fourier-transform infrared spectra were acquired from adenocarcinoma gastric cells, incubated with H. pylori strains presenting different genotypes concerning the virulent factors cytotoxin associated gene A and vacuolating cytotoxin A. Defined absorbance ratios were evaluated by diverse methods of statistical inference, according to the fulfillment of the tests assumptions. It was possible to define from the gastric cells, diverse absorbance ratios enabling to discriminate: i) The infection; ii) the bacteria genotype; and iii) the gastric disease of the patients from which the bacteria were isolated. These biomarkers could fasten the knowledge of the complex infection process while promoting a platform for a new diagnostic method, rapid but also specific and sensitive towards the diagnosis of both infection and bacterial virulence.

Stability of lipases in miniemulsion systems: Correlation between secondary structure and activity.

The exploitation of an efficient enzymatic system to perform biopolymers synthesis, namely polyesters from dicarboxylic acids and dialcohols, requires the evaluation of the enzyme operational stability. This becomes particularly relevant when non-conventional media, such as miniemulsions, are used due to the inhomogeneity of the reaction media and presence of surfactants and high concentrations of organic compounds which might be deleterious to the structure-function of the enzyme. The stability of three lipases, Candida sp., Candida rugosa and Burkholderia cepacia, in miniemulsions during polyester synthesis, was accessed through the secondary structure integrities and activities in order to establish any putative correlations between secondary structure and activity. The effect of the individual components that constitute the emulsion system was also evaluated to identify those which are more disruptive to the secondary structure-function of the enzyme. Depending on the lipase and the presence of different reaction components, three scenarios were observed: a close correlation between secondary structural changes and activity, a drop in activity with no secondary structure alterations but unfolding of tertiary structure and disruption of secondary structure that allows regain of activity in the presence of substrate.

Synthesis and characterization of acetylated amylose and development of inclusion complexes with rifampicin.

Amylose (AM) tends to form single helical inclusion complexes with suitable agents. These complexes are considered promising biomaterial carrier since the guest molecules can be released later, leading to many applications, especially in the pharmaceutical industry. Rifampicin (RIF) has long been recognized as an active drug against Mycobacterium tuberculosis, however, the administration of RIF in high dosages can originate unwanted side-effects. Due to the fact that the use of native amylose (AM) in the formation of complexes is limited by their low water solubility, it was acetylated with a medium degree of substitution (DS), allowing solubilizing (0.5gL-1) acetylated amylose (AMA) in water at neutral pH, in opposition to that observed with native amylose (trace solubility). The resulting acetylated amylose was characterized by means of Fourier Transform Infrared (FT-IR) spectroscopy and Scanning Electron Microscopy (SEM). FT-IR results indicated that the acetylation of anhydroglucose units of amylose corresponds to a low DS, whereas SEM results suggested that the smooth surfaces of amylose granules were changed into rougher surfaces after acetylation. Ultraviolet absorption spectroscopy (UV-vis) analysis confirmed the formation and allowed the quantification of both native (AM-RIF) and acetylated (AMA-RIF) amylose inclusion complexes. Their characterization in solution was performed by dynamic light scattering (DLS) and zeta potential (ZP) measurements. The average size of inclusion complexes as determined by DLS, ranged between 70 and 100nm. Besides, ZP analysis showed that both complexes are more stable in the presence of RIF. This study may lead to the development of an effective method for the preparation of amylose inclusion complexes, which is beneficial to their further application in drug delivery systems.

In situ near-infrared (NIR) versus high-throughput mid-infrared (MIR) spectroscopy to monitor biopharmaceutical production.

The development of biopharmaceutical manufacturing processes presents critical constraints, with the major constraint being that living cells synthesize these molecules, presenting inherent behavior variability due to their high sensitivity to small fluctuations in the cultivation environment. To speed up the development process and to control this critical manufacturing step, it is relevant to develop high-throughput and in situ monitoring techniques, respectively. Here, high-throughput mid-infrared (MIR) spectral analysis of dehydrated cell pellets and in situ near-infrared (NIR) spectral analysis of the whole culture broth were compared to monitor plasmid production in recombinant Escherichia coli cultures. Good partial least squares (PLS) regression models were built, either based on MIR or NIR spectral data, yielding high coefficients of determination (R(2)) and low predictive errors (root mean square error, or RMSE) to estimate host cell growth, plasmid production, carbon source consumption (glucose and glycerol), and by-product acetate production and consumption. The predictive errors for biomass, plasmid, glucose, glycerol, and acetate based on MIR data were 0.7 g/L, 9 mg/L, 0.3 g/L, 0.4 g/L, and 0.4 g/L, respectively, whereas for NIR data the predictive errors obtained were 0.4 g/L, 8 mg/L, 0.3 g/L, 0.2 g/L, and 0.4 g/L, respectively. The models obtained are robust as they are valid for cultivations conducted with different media compositions and with different cultivation strategies (batch and fed-batch). Besides being conducted in situ with a sterilized fiber optic probe, NIR spectroscopy allows building PLS models for estimating plasmid, glucose, and acetate that are as accurate as those obtained from the high-throughput MIR setup, and better models for estimating biomass and glycerol, yielding a decrease in 57 and 50% of the RMSE, respectively, compared to the MIR setup. However, MIR spectroscopy could be a valid alternative in the case of optimization protocols, due to possible space constraints or high costs associated with the use of multi-fiber optic probes for multi-bioreactors. In this case, MIR could be conducted in a high-throughput manner, analyzing hundreds of culture samples in a rapid and automatic mode.

From inulin to fructose syrups using sol-gel immobilized inulinase.

The present work aims to provide the basic characterization of sol-gel immobilized inulinase, a biocatalyst configuration yet unexploited, using as model system the hydrolysis of inulin to fructose. Porous xerogel particles with dimensions in slight excess of 10 µm were obtained, yielding an immobilization efficiency of roughly 80%. The temperature- and pH-activity profiles displayed a broader bell-shaped pattern as a result of immobilization. In the latter case, a shift of the optimal pH of 0.5 pH units was observed towards a less acidic environment. The kinetic parameters estimated from the typical Michaelis-Menten kinetics suggest that immobilization in sol-gel did not tamper with the native enzyme conformation, but on the other hand, entrapment brought along mass transfer limitations. The sol-gel biocatalyst displayed a promising operational stability, since it was used in more than 20 consecutive 24-hour batch runs without noticeable decay in product yield. The performance of sol-gel biocatalyst particles doped with magnetite roughly matched the performance of simple sol-gel particles in a single batch run. However, the operational stability of the former proved poorer, since activity decay was evident after four consecutive 24-hour batch runs.

Comparative study between probe focussed sonication and conventional stirring in the evaluation of cadmium and copper in plants.

Ultrasound (US)-assisted extraction has been widely used for metal ion extraction in plants due to its unique properties of decreased extraction time, minimal contamination, low reagent consumption and low cost. However, very few papers present a sound comparison between probe-focussed sonication and conventional stirring in the evaluation of metal ion extraction in plants. In this study, ultrasonic-assisted digestion has been evaluated and compared to magnetic stirring for total copper and cadmium determination by atomic absorption spectrometry in biological samples (plants, plankton and mussels). The same experimental conditions of sample amount and particle size, extractant solution and extraction time were applied for both ultrasound and magnetic stirring-assisted extraction methods in order to truly compare their effect on metal ion solubilisation. To gain further insight in this issue, dried and fresh plants were tested. The results obtained indicated that osmotic tension in cell walls, produced when dried and powdered samples were immersed in the extractant solution, had an important contribution to metal ion solubilisation, the enhancement due to US for the same purpose being negligible.

Improved specific productivity in cephalexin synthesis by immobilized PGA in silica magnetic micro-particles.

There is a marked trend in pharmaceutical industry towards the replacement of classical organic methods by "green" alternatives that minimize or eliminate the generation of waste and avoid, where possible, the use of toxic and/or hazardous reagents and solvents. In this work the kinetically controlled synthesis of cephalexin by soluble and penicillin G acylase immobilized in sol-gel micro-particles with magnetic properties was performed in aqueous media with PGME and 7-ADCA as substrates, at different concentrations of substrate, temperature, pH, enzyme to substrate ratio and acyl donor to nucleophile ratio. Excess acyl donor had a strong effect on cephalexin productivity. A PGME/7-ADCA ratio of 3 was considered optimum. A maximum specific productivity of 5.9 mmol h(-1), gbiocatalyst(-1) at 160 mM 7-ADCA, 480 mM PGME and low enzyme to substrate ratio at 32.5 U mmol(-1) 7-ADCA was obtained with immobilized PGA in full aqueous medium, suggesting that diffusional limitations were minimized when compared with other commercial biocatalysts. A half-life of 133 h for the immobilized biocatalyst was estimated during cephalexin synthesis in the presence of 100 mM 7-ADCA and 300 mM PGME, in 50 mM Tris/HCl at pH 7.2 and 14°C. These results compare quite favorably with those previously reported for the kinetically controlled synthesis of cephalexin.