Endophytic fungi producing of esterases: evaluation in vitro of the enzymatic activity using pH indicator

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Phytase production by Rhizopus microsporus var. microsporus biofilm: characterization of enzymatic activity after spray drying in presence of carbohydrates and nonconventional adjuvants

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Enzymatic activity profile of a Brazilian culture collection of Candida albicans isolated from diabetics and non-diabetics with oral candidiasis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

In vitro evaluation of the enzymatic activity profile of non-albicans Candida species isolated from patients with oral candidiasis with or without diabetes

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Conformational changes in a hyperthermostable glycoside hydrolase: enzymatic activity is a consequence of the loop dynamics and protonation balance

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Diffusivity and enzymatic activity control the exchange of carbonyl sulfide (COS) between soils and the atmosphere

Carbonylsulfid (COS) ist eines der stabilsten reduzierten schwefelhaltigen Spurengase in der Atmosphäre. In der gut durchmischten Troposphäre bewegt sich seine Konzentration um 500 ppt. COS spielt eine wichtige Rolle in der Produktion von stratosphärischem Aerosol und im Ozon Zyklus. Dieses Spurengas hat eine Vielfalt an natürlichen und anthropogenen Quellen, denen gleichstarke Senken, darunter die dominanten wie Vegetation und Boden, gegenüber stehen. Die Stärke der Senken ist trotz langjähriger Forschungen immer noch Gegenstand der Diskussionen. Daher ist es wichtig die kontrollierenden Parameter zu charakterisieren. Alle Austauschmessungen vor 1990 vermuteten Böden als Quelle von COS, was aber durch Castro and Galloway (1991) klar widerlegt wurde. Heute werden Böden in Ergänzung zur Vegetation grundsätzlich als Senke betrachtet. Vor diesem Hintergrund wurden Bodenproben auf den Austausch von Carbonylsulfid mit der Atmosphäre unter verschiedenen Umgebungsbedingungen untersucht. Drei Ackerböden aus Deutschland, China und Finnland und zwei Waldböden aus Sibirien und Surinam konnten parametrisiert werden in Relation zur atmosphärischen Umgebungskonzentration, Temperatur und Bodenfeuchte (WC). Neben Umgebungskonzentration und Bodenfeuchte, scheinen Bodenstruktur und enzymatische Aktivität die Richtung und Größe des Austauschflusses zu kontrollieren. Die übereinstimmenden Optima für boreale Böden in Relation zum wassergefüllten Porenvolumen des Bodens (WFPS) und die Linearität zwischen Depositionsgeschwindigkeit (Vd) und Bulk density lassen auf eine Dominanz der Abhängigkeit der COS-Aufnahme von der durch WFPS bestimmten Diffusionsfähigkeit schließen. WFPS ist abhängig von WC, Bodenstruktur und Bodenporosität. In Ergänzung zu diesen eher physikalischen Parametern konnte die Carboanhydrase (CA) als kontrollierendes Enzym in Böden identifiziert werden. Erste Versuche zur direkten Bestimmung der CA in den untersuchten Böden erlaubten eine erste, aber noch sehr ungenaue Abschätzung der Enzymaktivität.

Enzymatic Activity of Soybean Lipoxygenase-1 on Linoleoyl-Derivatized Agarose

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of linoleic acid to form 13(S) and 9(R) hydroperoxides. The manner in which substrates bind to the lipoxygenase family of enzymes is not known. It is believed fatty acid substrates may bind either with the aliphatic end first or with the carboxylate group facing the interior of the protein. This thesis tested a potential methyl-end first substrate binding mechanism by studying the activity of SBLO-1 to oxygenate immobilized linoleoyl residues attached to an insoluble polymer. Linoleic acid was attached to aminohexyl agarose in the presence of N-(3- dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) and Nhydroxysuccinimide (NHS). The concentration of the covalently attached residues was facilitated by enriching linoleic acid with a small amount of the radioactive 14C-isotope. Functionalization yields of 3% available primary amines on the resin were obtained. Enzymatic oxygenation of the linoleoyl-residues was verified using the ferrous oxidation in xylenol orange (FOX) assay. Approximately 30% of the attached linoleoyl moieties were converted to hydroperoxides in the presence of SBLO-1. A disulfide-containing cleavable linker, cystamine, was used as part of an improved method to isolate the product in a facile manner. Cystamine was attached to NHS-activated agarose with approximately 5% overall functionalization yield of available functional groups. 14C-linoleic acid was successfully covalently linked to the cystamine moieties in the presence of EDC and NHS. The FOX assay verified the enzymatic oxygenation of the linoleoyl residues attached to cystamine-derivatized agarose. The isolation of the peroxide product was attempted in a series of extractions in organic solvents. The product was analyzed using GC/MS which did not show a new peak indicative of product. Further work is needed to successfully analyze the stereoand regiochemistry of the oxygenated product. The presence of the peroxides in this study indicated the linoleoyl residues behave as substrates of SBLO-1. It is unknown how bulky substrates bind to the active site; however, it is difficult to rationalize a carboxylate group-first binding mode. Discovery of the 13(S)-hydroperoxide product on the linoleoyl-agarose would support the claim of a potential methyl-end first binding mechanism.

Cytochrome P-450 reductase FAD binding domain: Cloning, enzymatic activity and flavin binding characterization

The Mixed Function Oxidase System metabolizes a wide range of biochemicals including drugs, pesticides and steroids. Cytochrome P450 reductase is a key enzymatic component of this system, supplying reducing equivalents from NADPH to cytochrome P450. The electrons are shuttled through reductase via two flavin moieties: FAD and FMN. Although the exact mechanism of flavins action is not known, the enzymatic features of reductase greatly depleted of either FMN of FAD have been characterized. Additionally, flavin location within reductase has been proposed by homology and chemical modification studies. This study seeks to extend the flavin depletion analysis in a more controlled system by eliminating the proposed FMN binding domain with recombinant DNA techniques and biochemical analysis. Two P450 reductase cDNA clones containing only the FMN and NADPH binding domain were isolated, expressed and the protein products purified and analysed. This study confirms the proposed FAD binding site, role of FAD in electron shuttling pathway and provides new methods to study the FAD binding domain. ^

Distinct tyrosine phosphorylation sites in JAK3 kinase domain positively and negatively regulate its enzymatic activity

Cytokines are critically important for the growth and development of a variety of cells. Janus kinases (JAKs) associate with cytokine receptors and are essential for transmitting downstream cytokine signals. However, the regulation of the enzymatic activity of the JAKs is not well understood. Here, we investigated the role of tyrosine phosphorylation of JAK3 in regulating its kinase activity by analyzing mutations of tyrosine residues within the putative activation loop of the kinase domain. Specifically, tyrosine residues 980 and 981 of JAK3 were mutated to phenylalanine individually or doubly. We found that JAK3 is autophosphorylated on multiple sites including Y980 and Y981. Compared with the activity of wild-type (WT) JAK3, mutant Y980F demonstrated markedly decreased kinase activity, and optimal phosphorylation of JAK3 on other sites was dependent on Y980 phosphorylation. The mutant Y980F also exhibited reduced phosphorylation of its substrates, γc and STAT5A. In contrast, mutant Y981F had greatly increased kinase activity, whereas the double mutant, YY980/981FF, had intermediate activity. These results indicate that Y980 positively regulates JAK3 kinase activity whereas Y981 negatively regulates JAK3 kinase activity. These observations in JAK3 are similar to the findings in the kinase that is closely related to the JAK family, ZAP-70; mutations of tyrosine residues within the putative activation loop of ZAP-70 also have opposing actions. Thus, it will be important to determine whether this feature of regulation is unique to JAK3 or if it is also a feature of other JAKs. Given the importance of JAKs and particularly JAK3, it will be critical to fully dissect the positive and negative regulatory function of these and other tyrosine residues in the control of kinase activity and hence cytokine signaling.

Oxygen isotope ratios of PO4: An inorganic indicator of enzymatic activity and P metabolism and a new biomarker in the search for life

The distinctive relations between biological activity and isotopic effect recorded in biomarkers (e.g., carbon and sulfur isotope ratios) have allowed scientists to suggest that life originated on this planet nearly 3.8 billion years ago. The existence of life on other planets may be similarly identified by geochemical biomarkers, including the oxygen isotope ratio of phosphate (δ18Op) presented here. At low near-surface temperatures, the exchange of oxygen isotopes between phosphate and water requires enzymatic catalysis. Because enzymes are indicative of cellular activity, the demonstration of enzyme-catalyzed PO4–H2O exchange is indicative of the presence of life. Results of laboratory experiments are presented that clearly show that δ18OP values of inorganic phosphate can be used to detect enzymatic activity and microbial metabolism of phosphate. Applications of δ18Op as a biomarker are presented for two Earth environments relevant to the search for extraterrestrial life: a shallow groundwater reservoir and a marine hydrothermal vent system. With the development of in situ analytical techniques and future planned sample return strategies, δ18Op may provide an important biosignature of the presence of life in extraterrestrial systems such as that on Mars.

The C-terminal region of human angiogenin has a dual role in enzymatic activity.

The ribonucleolytic activity of angiogenin (Ang) is essential to Ang's capacity to induce blood vessel formation. Previous x-ray diffraction and mutagenesis results have shown that the active site of the human protein is obstructed by Gln-117 and imply that the C-terminal region of Ang must undergo a conformational rearrangement to allow substrate binding and catalysis. As a first step toward structural characterization of this conformational change, additional site-directed mutagenesis and kinetic analysis have been used to examine the intramolecular interactions that stabilize the inactive conformation of the protein. Two residues of this region, Ile-119 and Phe-120, are found to make hydrophobic interactions with the remainder of the protein and thereby help to keep Gln-117 in its obstructive position. Furthermore, the suppression of activity by the intramolecular interactions of Ile-119 and Phe-120 is counterbalanced by an effect of the adjacent residues, Arg-121, Arg-122, and Pro-123 which do not appear to form contacts with the rest of the protein structure. They contribute to enzymatic activity, probably by constituting a peripheral subsite for binding polymeric substrates. The results reveal the nature of the conformational change in human Ang and assign a key role to the C-terminal region both in this process and, presumably, in the regulation of human Ang function.

Inhibition of transglutaminase 2 enzymatic activity ameliorates the anti-angiogenic effects of coeliac disease autoantibodies

Objective. Earlier work has demonstrated that serum autoantibodies from coeliac patients targeted against transglutaminase 2 (TG2) inhibit in vitro angiogenesis. The aim of this study was to establish whether coeliac patient-derived monoclonal TG2-targeted antibodies produced by recombination technology exert similar anti-angiogenic effects to serum-derived coeliac autoantibodies. In addition, we studied whether the monoclonal patient autoantibodies modulate endothelial cell TG2 activity and whether such modulation is related to the anti-angiogenic effects. Material and methods. The influence of coeliac patient-derived monoclonal TG2-targeted antibodies on endothelial cell tubule formation was studied using a three-dimensional angiogenic cell culture model. Endothelial cell TG2 enzymatic activity was determined by means of a live-cell enzyme-linked immunosorbent assay. Results. Coeliac patient-derived monoclonal TG2-targeted antibodies produced by recombination technology inhibited endothelial tubule formation and enhanced the crosslinking activity of TG2. When this enzymatic activity was inhibited using site-directed irreversible TG2 inhibitors in the presence of autoantibodies, in vitro angiogenesis reverted to the control level. Conclusions. Since we found a significant negative correlation between endothelial cell angiogenesis and TG2 activity, we suggest that the anti-angiogenic effects of coeliac patient-derived TG2-targeted autoantibodies are exerted by enhanced enzymatic activity of TG2.

Effects of Low Molecular Weight Sulfated Galactan Fragments From Botryocladia Occidentalis on the Pharmacological and Enzymatic Activity of Spla2 From Crotalus Durissus Cascavella

Low molecular weight fragments of sulfated galactans (Boc-5 and Boc-10) from the red algae Botryocladia occidentalis significantly inhibited Crotalus durissus cascavella sPLA2 enzymatic activity. Equimolar ratios of sPLA2 to Boc-5 or Boc-10 resulted in allosteric inhibition of sPLA2. Under the conditions tested, we observed that both Boc-5 and Boc-10 strongly decreased edema, myonecrosis, and neurotoxicity induced by native sPLA2.