Specific cloning of human DNA as yeast artificial chromosomes by transformation-associated recombination.

DNA molecules undergoing transformation into yeast are highly recombinogenic, even when diverged. We reasoned that transformation-associated recombination (TAR) could be employed to clone large DNAs containing repeat sequences, thereby eliminating the need for in vitro enzymatic reactions such as restriction and ligation and reducing the amount of DNA handling. Gently isolated human DNA was transformed directly into yeast spheroplasts along with two genetically marked (M1 and M2) linearized vectors that contained a human Alu sequence at one end and a telomere sequence at the other end (Alu-CEN-M1-TEL and Alu-M2-TEL). Nearly all the M1-selected transformants had yeast artificial chromosomes (YACs) containing human DNA inserts that varied in size from 70 kb to > 600 kb. Approximately half of these had also acquired the unselected M2 marker. The mitotic segregational stability of YACs generated from one (M1) or two (M1 and M2) vector(s) was comparable, suggesting de novo generation of telomeric ends. Since no YACs were isolated when rodent DNAs or a vector lacking an Alu sequence was used, the YACs were most likely the consequence of TAR between the repeat elements on the vector(s) and the human DNA. Using the BLUR13 Alu-containing vector, we demonstrated that human DNA could be efficiently cloned from mouse cells that contained a single human chromosome 16. The distribution of cloned DNAs on chromosome 16 was determined by fluorescence in situ hybridization. We propose that TAR cloning can provide an efficient means for generating YACs from specific chromosomes and subchromosome fragments and that TAR cloning may be useful for isolating families of genes and specific genes from total genome DNA.

Regulation of bone biology by prostaglandin endoperoxide H synthases (PGHS): A rose by any other name...

It is well established that prostaglandins are essential mediators of bone resorption and formation. In the early 1990s, it was discovered that enzymatic reactions producing prostaglandins were regulated by two cyclooxygenase enzymes, one producing prostaglandins constitutively in tissues like the stomach, prostaglandin endoperoxide H synthase-1 (PGHS-1 or COX-1), and another induced by mitogens or inflammatory mediators (PGHS-2 or COX-2). This neat distinction has not been maintained because both enzymes act in different cell systems to provide physiological signaling, constitutively or by induction under certain conditions. For example, the regulation patterns of PGHS-1 and PGHS-2 are distinct, but the evidence shows that PGHS-2 functions constitutively in the skeleton. PGHS-2 hits quickly been established, therefore, as a key regulator of bone biology, capable of rapid and transient expression in bone cells, and mediating osteoclastogenesis, mechanotransduction, bone formation and fracture repair. The goal of this review is to Summarize the current state of our knowledge of PGHS regulation of bone metabolism and to identify some of the key unresolved challenges and questions that require further study. (c) 2006 Elsevier Ltd. All rights reserved.

Speciation, metabolism, toxicity, and protein-binding of different arsenic species in human cells

Despite of its known toxicity and potential to cause cancer, arsenic has been proven to be a very important tool for the treatment of various refractory neoplasms. One of the promising arsenic-containing chemotherapeutic agents in clinical trials is Darinaparsin (dimethylarsinous glutathione, DMA III(GS)). In order to understand its toxicity and therapeutic efficacy, the metabolism of Darinaparsin in human cancer cells was evaluated. With the aim of detecting all potential intermediates and final products of the biotransformation of Darinaparsin and other arsenicals, an analytical method employing high performance liquid chromatography inductively coupled mass spectrometry (HPLC-ICP-MS) was developed. This method was shown to be capable of separating and detecting fourteen human arsenic metabolites in one chromatographic run. The developed analytical technique was used to evaluate the metabolism of Darinaparsin in human cancer cells. The major metabolites of Darinaparsin were identified as dimethylarsinic acid (DMAV), DMA III(GS), and dimethylarsinothioyl glutathione (DMMTAV(GS)). Moreover, the method was employed to study the conditions and mechanisms of formation of thiol-containing arsenic metabolites from DMAIII(GS) and DMAV as the mechanisms of formation of these important As species were unknown. The arsenic sulfur compounds studied included but were not limited to the newly discovered human arsenic metabolite DMMTA V(GS) and the unusually highly toxic dimethylmonothioarsinic acid (DMMTAV). It was found that these species may form from hydrogen sulfide produced in enzymatic reactions or by utilizing the sulfur present in protein persulfides. Possible pathways of thiolated arsenical formation were proposed and supporting data for their existence provided. In addition to known mechanism of arsenic toxicity such as protein-binding and reactive oxygen formation, it was proposed that the utilization of thiols from protein persulfides during the formation of thiolated arsenicals may be an additional mechanism of toxicity. The toxicities of DMAV(GS), DMMTA V, and DMMTAV(GS) were evaluated in cancer cells, and the ability of these cells to take the compounds up were compared. When assessing the toxicity by exposing multiple myeloma cells to arsenicals externally, DMMTAV(GS) was much less toxic than DMAIII(GS) and DMMTAV, probably as a result of its very limited uptake (less than 10% and 16% of DMAIII(GS) and DMMTAV respectively).^

Microbial diversity and metabolic potential in caves

Caves are dark and oligotrophic habitats where chemotrophic microbial communities interact with the inorganic mineral rocks and cooperate organizing themselves in complex biological formations, which are visible in caves as biofilms, biodeposits or biospeleothems. In these environments, microorganisms contribute to the turnover of the matter and activate peculiar enzymatic reactions leading to the modification of the mineral rocks and to the production of metabolites with possible industrial and pharmaceutical interest. In this PhD thesis, various molecular and geomicrobiological approaches were used to investigate the microbial diversity and potential activities in different cave systems, i.e. the orthoquartzite cave Imawarì Yeuta, the sufidic cave Fetida and the ice cave Cenote Abyss. This is aimed at gathering indications on the possible interactions that support microbial growth and its impact in cave environments. As a result, microbial taxa and functions associated to light-independent chemolithotroph and heterotrophic activities were identified in the three caves, indicating the involvement of microorganisms in i) silica mobilization and amorphization processes and the formation of a novel type of silica-based stromatolite in Imawarì Yeuta Cave, ii) the formation of three types of biofilm/biodeposit involved in sulphur cycle and in the speleogenesis of Fetida Cave, iii) the development of biofilms and their maintenance under psychrophilic conditions in samples collected from ice in Cenote Abyss. Additionally, the metabolic potentials of around one hundred isolates derived from these cave systems were evaluated in terms on anti-microbial activity. The results pointed out that unexplored and oligotrophic caves are promising environments for novel bioactive molecules discovery.

Enzymatic kinetic resolution of (RS)-1-(Phenyl)ethanols by Burkholderia cepacia lipase immobilized on magnetic nanoparticles

Lipase from Burkholderia cepacia immobilized on superparamagnetic nanoparticles using adsorption and chemisorption methodologies was efficiently applied as recyclable biocatalyst in the enzymatic kinetic resolution of (RS)-1-(phenyl)ethanols via transesterification reactions. (R)-Esters and the remaining (S)-alcohols were obtained with excellent enantiomeric excess (> 99%), which corresponds to a perfect process of enzymatic kinetic resolution (conversion 50%, E > 200). The transesterification reactions catalysed with B. cepacia lipase immobilized by the glutaraldehyde method showed the best results in terms of reusability, preserving the enzyme activity (conversion 50%, E > 200) for at least 8 successive cycles.

A novel use for sugarcane bagasse hemicellulosic fraction: Xylitol enzymatic production

The excess of sugarcane bagasse (SCB) from the sugar-alcohol industry is considered a by-product with great potential for many bioproducts production. This work had as objective to verify the performance of sugarcane bagasse hemicellulosic hydrolysate (SCBHH) as source of sugars for enzymatic or in vitro xylitol production. For this purpose, xylitol enzymatic production was evaluated using different concentrations of treated SCBHH in the commercial reaction media. The weak acid hydrolysis of SCB provided a hydrolysate with 18 g L(-1) and 6 g L(-1) of xylose and glucose, respectively. Considering the reactions, changes at xylose xylitol conversion efficiency and volumetric productivity in xylitol were not observed for the control experiment and using 20 and 40% v.v (1) of SCBHH in the reaction media. The conversion efficiency achieved 100% in all the experiments tested. The results showed that treated SCBHH is suitable as xylose and glucose source for the enzymatic xylitol production and that this process has potential as an alternative for traditional xylitol production ways. (C) 2011 Published by Elsevier Ltd.

Compositional and textural properties of milkfat-soybean oil enzymatic interesterification

Milkfat-soybean oil blends were enzymatically interesterified (EIE) by Aspergillus niger lipase immobilized on SiO(2)-PVA hybrid composite in a solvent free system. An experimental mixture design was used to study the effects of binary blends of milkfat-soybean oil (MF:SBO) at different proportions (0:100; 25:75; 33:67; 50:50; 67:33; 75:25; 100:0) on the compositional and textural properties of the EIE products, considering, as response variables, the interesterification yield (IY), consistency and hardness. Lipase-catalysed interesterification reactions increased the relative proportion of TAGs` C(46)-C(52) and decreased the TAGs` C(40)-C(42) and C(54) concentrations. The highest IY was attained (10.8%) for EIE blend of MF:SBO 67:33 resulting in a more spreadable material at refrigerator temperature in comparison with butter, milkfat or non-interesterified (NIE) blend. In this case, consistency and hardness values were at least 32% lower than values measured for butter. Thus, using A. niger lipase immobilized on SiO(2)-PVA improves the textural properties of milkfat and has potential for development of a product incorporating unsaturated and essential fatty acids from soybean oil. (C) 2010 Elsevier Ltd. All rights reserved.

An integrated approach to produce biodiesel and monoglycerides by enzymatic interestification of babassu oil (Orbinya sp)

Two screenings of commercial lipases were performed to find a lipase with superior performance for the integrated production of biodiesel and monoglycerides. The first screening was carried out under alcoholysis conditions using ethanol as acyl acceptor to convert triglycerides to their corresponding ethyl esters (biodiesel). The second screening was performed under glycerolysis conditions to yield monoglycerides (MG). All lipases were immobilized on silica-PVA composite by covalent immobilization. The assays were performed using babassu oil and alcohols (ethanol or glycerol) in solvent free systems. For both substrates, lipase from Burkholderia cepacia (lipase PS) was found to be the most suitable enzyme to attain satisfactory yields. To further improve the process, the Response Surface Methodology (RSM) was used to determine the optima operating conditions for each biotransformation. For biodiesel production, the highest transesterification yield (>98%) was achieved within 48 h reaction at 39 degrees C using an oil-to-ethanol molar ratio of 1:7. For MG production, optima conditions corresponded to oil-to-glycerol molar ratio of 1: 15 at 55 degrees C, yielding 25 wt.% MG in 6 h reaction. These results show the potential of B. cepacia lipase to catalyze both reactions and the feasibility to consider an integrated approach for biodiesel and MG production. (C) 2009 Elsevier Ltd. All rights reserved.

Synthesis of unnatural cyclitols via a combined enzymatic-palladium catalysis approach

The Suzuki-Miyaura cross-coupling reaction of a hydroxylated vinyl bromide obtained by a chemoenzymatic approach with a diverse range of potassium organotrifluoroborates has been accomplished catalyzed by Pd(PPh(3))(4) in satisfactory yields. A variety of functional groups are tolerated in the nucleophilic partner. (C) 2008 Elsevier B.V. All rights reserved.

An enzymatic route to a benzocarbazole framework using bacterial CotA laccase

The CotA laccase-catalysed oxidation of the meta, para-disubstituted arylamine 2,4-diaminophenyldiamine delivers, under mild reaction conditions, a benzocarbazole derivative (1) (74% yield), a key structural motif of a diverse range of applications. This work extends the scope of aromatic frameworks obtained using these enzymes and represents a new efficient and clean method to construct in one step C-C and C-N bonds.

Enzymatic activity mastered by altering metal coordination spheres

J Biol Inorg Chem (2008) 13:1185–1195 DOI 10.1007/s00775-008-0414-3

Synthesis of polyhydroxylated compounds from Derythrose: enzymatic inhibition studies

Dissertação de mestrado em Química Medicinal

Enzymatic hydrophobic modification of jute fibers via grafting to reinforce composites

Horseradish peroxidase (HRP)/H2O2 system catalyzes the free-radical polymerization of aromatic compounds such as lignins and gallate esters. In this work, dodecyl gallate (DG) was grafted onto the surfaces of lignin-rich jute fabrics by HRP-mediated oxidative polymerization with an aim to enhance the hydrophobicity of the fibers. The DG-grafted jute fibers and reaction products of their model compounds were characterized by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC). The results clearly indicated the grafting of DG to the jute fiber by HRP. Furthermore, the hydrophobicity of jute fabrics was determined by measuring the wetting time and static contact angle. Compared to the control sample, the wetting time and static contact angle of the grated fabrics changed from ~1 s to 1 h and from ~0° to 123.68°, respectively. This clearly proved that the hydrophobicity of jute fabrics improved considerably. Conditions of the HRP-catalyzed DG-grafting reactions were optimized in terms of the DG content of modified jute fabrics. Moreover, the results of breaking strength and elongation of DG-grafted jute/ polypropylene (PP) composites demonstrated improved reinforcement of the composite due to enzymatic hydrophobic modification of jute fibers.

Identification of new transformation products during enzymatic treatment of tetracycline and erythromycin antibiotics at laboratory scale by an on-line turbulent flow liquid-chromatography coupled to a high resolution mass spectrometer LTQ-Orbitrap

This work describes the formation of transformation products (TPs) by the enzymatic degradation at laboratory scale of two highly consumed antibiotics: tetracycline (Tc) and erythromycin (ERY). The analysis of the samples was carried out by a fast and simple method based on the novel configuration of the on-line turbulent flow system coupled to a hybrid linear ion trap – high resolution mass spectrometer. The method was optimized and validated for the complete analysis of ERY, Tc and their transformation products within 10 min without any other sample manipulation. Furthermore, the applicability of the on-line procedure was evaluated for 25 additional antibiotics, covering a wide range of chemical classes in different environmental waters with satisfactory quality parameters. Degradation rates obtained for Tc by laccase enzyme and ERY by EreB esterase enzyme without the presence of mediators were ∼78% and ∼50%, respectively. Concerning the identification of TPs, three suspected compounds for Tc and five of ERY have been proposed. In the case of Tc, the tentative molecular formulas with errors mass within 2 ppm have been based on the hypothesis of dehydroxylation, (bi)demethylation and oxidation of the rings A and C as major reactions. In contrast, the major TP detected for ERY has been identified as the “dehydration ERY-A”, with the same molecular formula of its parent compound. In addition, the evaluation of the antibiotic activity of the samples along the enzymatic treatments showed a decrease around 100% in both cases

Lipase-Mediated Enzymatic Catalysis For The Synthesis of New Chiral Polymers

Immobilized lipase B from Candida antarctica (N435) was investigated as a potential biocatalyst to generate silicone-based chiral polymers from monomers derived from the enzymatic dihydroxylation of bromobenzene. Several conditions and parameters have been investigated for this purpose and lipase transesterification preference to each of the free secondary alcohols in the chiral monomers was documented. The N435 was challenged with a series of substrates where the free alcohol moieties were systematically protected in order to study the substrate preference(s) for the transesterification reactions.