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Human kallikrein 7 (KLK7) is a potential target for the treatment of skin inflammation and cancer. Despite its potential, few KLK7-specific small-molecule inhibitors have been reported in the literature. As an extension of our program to design serine protease inhibitors, here we describe the in vitro assays and the investigation of the binding mechanism by molecular dynamics simulation of a novel class of pseudo-peptide inhibitors derived from isomannide. Of the inhibitors tested, two inhibited KLK7 with K(i) values in the low micromolar range (9g=1.8µM; 9j=3.0µM). Eadie-Hofstee and Dixon plots were used to evaluate the competitive mechanism of inhibition for the molecules. Calculated binding free energies using molecular MM/PB(GB)SA approach are in good agreement with experimental results, suggesting that the inhibitors share the same binding mode, which is stabilized by hydrophobic interactions and by a conserved network of hydrogen bonds. The promising results obtained in this study make these compounds valid leads for further optimization studies aiming to improve the potency of this new class of kallikrein inhibitors.

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A quantitative structure-activity relationship model for Morita-Baylis-Hillman adducts with leishmanicidal activities was developed which correlates molecular orbital energy and dipole with percentage in the promastigote stage.

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The main objective of this work is to present the optimization of the biotransformation of R-(+)-limonene and (-)-ß-pinene aiming at the production of α-terpineol by strains of fungal and yeasts previously isolated by our research group using the methodology of experimental design. New optimized experimental data on α-terpineol production by the biotransformation of R-(+)-limonene and (-)-ß-pinene using newly isolated microorganisms are reported in this work. Conversion of about 1,700 mg/L was achieved when R-(+)-limonene was used as substrate and the newly isolated strain 05.01.35 as microorganism at the central point of the experimental design, corresponding to a substrate concentration of 1.75%, mass of inoculum of 2 g, and substrate to ethanol volume ratio of 1:1. The same experimental condition led to higher conversions when (-)-ß-pinene was used as substrates and the strains coded as 04.05.08 and 01.04.03 as microorganism. Here, conversions of about 770 mg/L were achieved.

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Verbenol is a bicyclicbicycle secondary allylic alcohol, with pronounced camphor and mint flavor notes, mainly used as food flavoring. This compound is also used to control harmful insects, and hence has potential for using in agriculture, and is an intermediate in the synthesis of valuable perfume and medicinal substances. This work is focused on the microbial oxidation of (-)-α-pinene to verbenol production. To carry out the present study, 405 microorganisms were tested for their ability to bioconvert the substrate. From the isolated microorganisms, 193 were selected in the pre-screening using mineral medium for limonene degradation. At the screening step, 31 strains were able to convert (-)-α-pinene in verbenol. The highest concentration in verbenol from (-)-α-pinene was about 125.6 mg/L for yeast isolated from orange juice industrial residue.

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This work reports the optimization of 2-ethylhexyl palmitate production by esterification reaction in a solvent-free system using a commercial lipase as catalyst. For this, a sequential strategy was performed applying three experimental designs. An empirical model was built so as to assess the effects of process variables on the reaction conversion. Afterward, the operating conditions that optimized 2-ethylhexyl palmitate production were determined to be acid to alcohol molar ratio of 1:5.5, 70 degrees C, 150 rpm and 10.5 wt% of enzyme, leading to a reaction conversion as high as 93%. From this point, a kinetic study was carried out evaluating the influence of acid to alcohol molar ratio, enzyme concentration and temperature on product yield. Results obtained in this step allow to conclude that an excess of alcohol (acid to alcohol molar ratio of 1:6), relatively low enzyme concentration (10 wt%) and temperature of 70 degrees C led to nearly complete reaction conversion.

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This work reports the application of a lipase in the 2-ethylhexyl palmitate esterification in a solvent-free system with an immobilized lipase (Lipozyme RM IM). A sequential strategy was used applying two experimental designs to optimize the 2-ethylhexyl palmitate production. An empirical model was then built so as to assess the effects of process variables on the reaction conversion. Afterwards, the operating conditions that optimized 2-ethylhexyl palmitate production were established as being acid/alcohol molar ratio 1:3, temperature of 70 degrees C, stirring rate of 150 rpm, 10 wt.% of enzyme, leading to a reaction conversion as high as 95%. From this point, a kinetic study was carried out evaluating the effect of acid:alcohol molar ratio, the enzyme concentration and the temperature on product conversion. The results obtained in this step permit to verify that an excess of alcohol (acid to alcohol molar ratio of 1:6), relatively low enzyme concentration (10 wt.%) and temperature of 70 degrees C, led to conversions next to 100%.

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This work is focused on the biotransformation of R-(+)-limonene and (-)-beta-pinene to bioflavor production. To carry out the present study, 405 microorganisms were tested for their ability to bioconvert the substrates. From the isolated microorganisms, 193 were selected in the prescreening using mineral medium for limonene degradation. At the screening step, eight strains were able to convert R-(+)-limonene and 15 to transform (-)-beta-pinene, both in alpha-terpineol. The highest concentration in alpha-terpineol from R-(+)-limonene was about 3,450 mg/L for Penicillium sp. isolated from eucalyptus steam. From (-)-beta-pinene, the highest product concentration of 675.5 mg/L was achieved using an Aspergillus sp. strain isolated from orange tree stem.

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This work aimed to evaluate the effects of particle size, solid/solvent ratio (w/v), and reaction time on hydrolytic and esterification activities of a lipase extract from wheat seeds. The higher hydrolytic activity was 5.9 U/g with a particle size of 425 microm, solid/solvent ratio of 30:60 (w/v), and reaction time of 15 h, with maximum hydrolytic activity of 14.47 U/g after 24 h of precipitation. For esterification activity the best result was 57.88 U/g with a particle size of 425 microm and solid/solvent ratio of 30:120 (w/v) for 5 h, with a maximum value after 10 h of precipitation reaching 208.20 U/g. The partial characterization showed that the optimal pH and temperature were found to be 5.5 and 32-37 degrees C, respectively. The extract stability at low temperatures was kept after 48 h of storage in terms of esterification activity. The hydrolytic activity was kept constant at -10 degrees C during 72 h and diminished considerably after 24 h at 4 degrees C.

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Biginelli reactions have been monitored by direct infusion electrospray ionization mass spectrometry (ESI-MS) and key cationic intermediates involved in this three-component reaction have been intercepted and further characterized by tandem MS experiments (ESI-MS/MS). Density functional theory calculations were also used to investigate the feasibility of the major competing mechanisms proposed for the Biginelli reaction. The experimental and theoretical results were found to corroborate the iminium mechanism proposed by Folkers and Johnson, whereas no intermediates directly associated with either the more energy demanding Knoevenagel or enamine mechanisms could be intercepted.

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The lipase-catalyzed kinetic resolution of rac-1-phenylethanol with vinyl acetate as acyl donor and cyclohexane as solvent has been investigated applying both microwave dielectric heating and conventional thermal heating in order to probe the existence of nonthermal microwave effects. All transformations were conducted at 40 degrees C in a dedicated reactor setup that allowed accurate internal reaction temperature measurements with use of fiber-optic probes. Quartz reaction vessels that allow higher levels of microwave power to be administered to the reaction mixture were used for all experiments. For all five studied immobilized lipases, the observed reactivities and enantioselectivities in microwave and oil bath experiments were identical and thus not related to the presence of the microwave field. The effect of magnetic stirring proved critical as too rapid stirring in some instances destroyed the enzyme support structure and led to altered reactivities and selectivities.

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In this work, nine tetrasubstituted derivatives [NH(2), OCH(3), Li, Na, Si(CH(3))(3)/SiH(2)CH(3,) P(CH(3))(2), Cl, F, and CN] of the spiropentadiene dication were analyzed within the framework of QTAIM. In the studied series, the electron-withdrawing substituents destabilize the ptC-containing spiropentadiene dication. On the other hand, stabilization of this dication is possible for electron-donating substituents only through sigma bonds, such as Li and Na. In all studied systems, according to QTAIM, the pi-electron system does not participate in the stabilization of the ptC atom in the spiropentadiene dication. sigma-electron-donating groups stabilize the spiropentadiene dication system by increasing the charge density of C(ext)-ptC bonds, whereas electron-withdrawing groups remove the charge density from C(ext)-ptC bonds.

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Mate (Ilex paraguariensis) is an important natural product in the economic and cultural context of Brazil. Peroxidase and polyphenol oxidase have been responsible for quality deterioration and browning in mate. The objective of this work was to investigate a methodology of extraction and enzymatic activity determination of oxidases present in mate tea leaves and to evaluate the oxidases stability. The effects of raw-material mass, buffer molar concentration, Triton X-100 addition, extraction pH, pH activity measurement, polyvinylpyrrolidone K90 addition, and centrifugation time were evaluated by the experimental planning methodology. The storage of the oxidases along 150 days at low temperatures showed that no significant difference was found at -4 and -80 degrees C but significant difference was observed when compared to 4 degrees C. The results showed that higher activities of oxidases are obtained at similar conditions. The exposition to high-temperatures and the variation of the time of exposition affected the enzymatic activity significantly.

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Raman spectra of pure liquid dimethylsulfoxide (DMSO) and of binary mixtures of formamide (FA) and DMSO in different compositions were obtained. The vibrations involving the SO functional group in the band envelope at ca. 1050 cm(-1) of pure liquid DMSO are assigned to monomers, dimers and higher aggregates of DMSO. The appearance of a new band at 1024 cm(-1), whose intensity shows large dependence on the FA concentration, is assigned to a FA-DMSO adduct. This has been possible due to the two H-bond donor sites of FA and the strong donor character of DMSO that become the environment propitious for the donor-acceptor reaction. Quantitative analysis performed in the SO stretching region in the binary mixtures gives a 1:1 stoichiometry in this adduct in the limit of infinite dilution. This proportion is in full agreement with our previous determination for the FA-ACN adduct. The experimental evidence of the 1:1 FA-DMSO adduct is presented for the first time using Raman spectroscopy. The results described here open new possibilities to study the acid-base reactions nature of FA adducts.

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The use of microorganisms for biotransformations of monoterpenes has stimulated the biotechnological market. Aiming at the highest efficiency in the process of strains screening, the application of molecular biology techniques have been proposed. Based on these aspects, the objective of this work was to select different strains able to convert limonene using fermentative process and random amplified polymorphic DNA (RAPD) markers. The results obtained in the fermentative screening, from 17 strains tested, pointed out that four microorganisms were able to convert limonene into oxygenated derivatives. The RAPD study showed a polymorphism of 96.02% and a similarity from 16.02 to 51.51%. Based on this it was possible to observe a high genetic diversity, even among strains of same species, concluding that the RAPD was not able to correlate the genetic characteristics of the microorganism with the results obtained from the biotransformation process.

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Thrombovascular diseases result from imbalanced haemostasis and comprise important health problems in the aging population worldwide. The activity of enzymes pertaining to the coagulation cascade of mammalians exhibit several control mechanisms in order to maintain a proper balance between bleeding and thrombosis. For instance, human coagulation serine proteases carrying a F225 or Y225 are allosteric modulated by the binding of Na+ in a water-filled channel connected to the primary specificity pocket (S1 subsite) of these enzymes. We have characterized the structure, topography and lipophilicity of this channel in the ligand-free fast (sodium-bound) and slow (sodium-free) forms of thrombin, in the sole available structure of activated protein C and in several structures of the coagulation factors VIIa, IXa and Xa, differing in the nature of the bound inhibitor and in the occupancy of exosite-I as well as the Ca2+ and Na+ binding sites. Opposite to thrombin, the aqueous channels in all other coagulation enzymes sheltering a Na+ binding site do not have an aperture on the enzyme surface opposite to the S1 subsite entrance. In these enzymes, the lack of the three-residue insertion in loop 1 (183-189) as found in thrombin allied to compensatory mutations in the positions 187-185 and 222 effects a constriction in the water-filled channel that ends up by segregating the ion binding site from the S1 subsite. We also disclosed major topographical changes on the thrombin's surface upon sodium release and transition to the slow form that culminate in the narrowing of the S1 subsite entrance and, strikingly, in the loss of communication between the primary specificity pocket and the exosite-I. Such observation is in accordance with existing experimental data demonstrating thermodynamic linkage between these distant regions on the thrombin surface. Conformational changes in F34, L40, R73 and T74 were the main responsible for this effect. A path by which these changes in the vicinity of exosite-I could be transmitted to the S1 subsite and, consequently, to the sodium binding site is proposed.

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The main objective of this work was to investigate the biotransformations of (-)alpha-pinene, (-)beta-pinene, and (+) limonene by Aspergillus niger ATCC 9642. The culture conditions involved--concentration of cosolvent (EtOH), substrate applied, and sequential addition of substrates were--investigated. Adaptation of the precultures with small amounts of substrate was also studied. The experiments were performed in conical flasks with liquid cultures. This strain of A. niger was able to convert only (-)beta-pinene into alpha-terpineol. An optimum conversion of (-)beta-pinene into alpha-terpineol of about 4% was obtained when the substrate was applied as a diluted solution in EtOH and sequential addition of substrate was used.

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BACKGROUND: This work evaluates the role of subtype F human immunodeficiency virus type 1 (HIV-1) protease (PR) substitutions L89M and L90M in viral replication and resistance to PR inhibitors (PIs). METHODS: Subtype B and F PR genes were subjected to site-directed mutagenesis, to create and reverse the methionine at positions 89 and 90. Viruses were re-created in cell culture, and their replicative capacity was assessed by fitness assay. Generated viruses were also phenotyped for PI resistance. RESULTS: The subtype F clone (89M90L) showed a replicative capacity comparable to that of the PI-susceptible subtype B clone (89L90L) and was more fit than the L89M mutated subtype B clone (89M90L). Both 89M90M subtype B and F clones presented the lowest fitness s values. The L89M mutation impacted phenotypic resistance to all PIs in half of the subtype F isolates but not in the subtype B isolates. Subtype F isolates presented a phenotypic profile similar to that of subtype B isolates when the M89L mutation was introduced. CONCLUSION: The L89M mutation in subtype F viruses is a high genetic barrier to the accumulation of the L90M resistance mutation and can function as a resistance mutation, depending on the presence of other polymorphisms in the subtype F PR backbone.

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The biotransformation of isosafrole by Cladosporium sphaerospermum yielded piperonal, which is a compound of great commercial importance in the flavor and fragrance industries. The experiments were performed in 500-mL conical flasks containing 100 mL of Czapek-modified medium in an orbital shaker with controlled agitation and temperature. Spores of C. sphaerospermum were used as inocula, and after 96 h of incubation the substrate was added to the culture. Samples of 2 mL were withdrawn at 24-h intervals and analyzed by gas chromatography, (GC) and/or GC/MS spectroscopy.

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