Effect of diet composition and ration size on key enzyme activities of glycolysis-gluconeogenesis, pentose phosphate pathway and amino acid metabolism in liver of Sparus aurata

The effects of diet composition and ration size on the activities of key enzymes involved in intermediary metabolism were studied in the liver of gilthead sea bream (Sparus aurata). Highcarbohydrate, low-protein diets stimulated 6-phosphofructo 1-kinase (EC 2.7.1.11), pyruvate kinase (EC 2.7.1.40), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44) enzyme activities, while they decreased alanine aminotransferase (EC 2.6.1.2) activity. A high degree of correlation was found between food ration size and the activity of the enzymes 6-phosphofructo 1-kinase, pyruvate kinase, glucose-6-phosphate dehydrogenase (positive correlations) and fructose-1,6-bisphosphatase (EC 3.1.3.11) (negative correlation). These correlations matched well with the high correlation also found between ration size and growth rate in starved fish refed for 22 d. Limited feeding (5 g/kg body weight) for 22 d decreased the activities of the key enzymes for glycolysis and lipogenesis, and alanine aminotransferase activity. The findings presented here indicate a high level of metabolic adaptation to both diet type and ration size. In particular, adaptation of enzyme activities to the consumption of a diet with a high carbohydrate level suggests that a carnivorous fish like Sparus aurata can tolerate partial replacement of protein by carbohydrate in the commercial diets supplied in culture. The relationship between enzyme activities, ration size and fish growth indicates that the enzymes quickly respond to dietary manipulations of cultured fish.

Effect of diet composition and ration size on key enzyme activities of glycolysis-gluconeogenesis, pentose phosphate pathway and amino acid metabolism in liver of Sparus aurata

The effects of diet composition and ration size on the activities of key enzymes involved in intermediary metabolism were studied in the liver of gilthead sea bream (Sparus aurata). Highcarbohydrate, low-protein diets stimulated 6-phosphofructo 1-kinase (EC 2.7.1.11), pyruvate kinase (EC 2.7.1.40), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44) enzyme activities, while they decreased alanine aminotransferase (EC 2.6.1.2) activity. A high degree of correlation was found between food ration size and the activity of the enzymes 6-phosphofructo 1-kinase, pyruvate kinase, glucose-6-phosphate dehydrogenase (positive correlations) and fructose-1,6-bisphosphatase (EC 3.1.3.11) (negative correlation). These correlations matched well with the high correlation also found between ration size and growth rate in starved fish refed for 22 d. Limited feeding (5 g/kg body weight) for 22 d decreased the activities of the key enzymes for glycolysis and lipogenesis, and alanine aminotransferase activity. The findings presented here indicate a high level of metabolic adaptation to both diet type and ration size. In particular, adaptation of enzyme activities to the consumption of a diet with a high carbohydrate level suggests that a carnivorous fish like Sparus aurata can tolerate partial replacement of protein by carbohydrate in the commercial diets supplied in culture. The relationship between enzyme activities, ration size and fish growth indicates that the enzymes quickly respond to dietary manipulations of cultured fish.

Determinação de carbaril utilizando testes ELISA (Enzyme-linked immunosorbent assay) e CLAE com detecção por arranjo de diodos

ELISAs have been applied to pesticide residue analysis due to their high sensitivity and selectivity. However, some ELISAs performance may be affected by matrix components. In this work, ELISA for carbaryl in water samples was checked for interference by naturally occurring fulvic acids. The results suggested that the high fulvic acid concentration (ž³30 mg L-1) and acidic pH conditions (pH 4.0) interfere with the signal detection decreasing the method sensitivity. A dilution of the samples and adjust to pH 8.0 are appropriate to minimize the matrix interferences in the ELISA method. Good correlation between ELISA and HPLC-DAD results was observed.

Orthologous and heterologous expression of a protein from Saccharomyces cerevisiae: Optimization for a biotechnological application

In this thesis (TFG) the results of the comparison between different methods to obtain a recombinant protein, by orthologous and heterologous expression, are exposed. This study will help us to identify the best way to express and purify a recombinant protein that will be used for biotechnology applications. In the first part of the project the goal was to find the best expression and purification system to obtain the recombinant protein of interest. To achieve this objective, a system expression in bacteria and in yeast was designed. The DNA was cloned into two different expression vectors to create a fusion protein with two different tags, and the expression of the protein was induced by IPTG or glucose. Additionally, in yeast, two promoters where used to express the protein, the one corresponding to the same protein (orthologous expression), and the ENO2 promoter (heterologous expression). The protein of interest is a NAD-dependent enzyme so, in a second time, its specific activity was evaluated by coenzyme conversion. The results of the TFG suggest that, comparing the model organisms, bacteria are more efficient than yeast because the quantity of protein obtained is higher and better purified. Regarding yeast, comparing the two expression mechanisms that were designed, heterologous expression works much better than the orthologous expression, so in case that we want to use yeast as expression model for the protein of interest, ENO2 will be the best option. Finally, the enzymatic assays, done to compare the effectiveness of the different expression mechanisms respect to the protein activity, revealed that the protein purified in yeast had more activity in converting the NAD coenzyme.

Interactions of bacteria and fungi on decomposing litter: differential extracellular enzyme activities

Fungi and bacteria are key agents in plant litter decomposition in freshwater ecosystems. However, the specific roles of these two groups and their interactions during the decomposition process are unclear. We compared the growth and patterns of degradativeenzymes expressed by communities of bacteria and fungi grown separately and in coexistence on Phragmites leaves. The two groups displayed both synergistic and antagonistic interactions. Bacteria grew better together with fungi than alone. In addition, there was a negative effect of bacteria on fungi, which appeared to be caused by suppression of fungal growth and biomass accrual rather than specifically affecting enzyme activity. Fungi growing alone had a high capacity for the decomposition of plant polymers such as lignin, cellulose, and hemicellulose. In contrast, enzyme activities were in general low when bacteria grew alone, and the activity of key enzymes in the degradation of lignin and cellulose (phenol oxidase and cellobiohydrolase) was undetectable in the bacteria-only treatment. Still, biomass-specific activities of most enzymes were higher in bacteria than in fungi. The low total activity and growth of bacteria in the absence of fungi in spite of apparent high enzymatic efficiency during the degradation of many substrates suggest that fungi provide the bacteria with resources that the bacteria were not able to acquire on their own, most probably intermediate decomposition products released by fungi that could be used by bacteria

Extração de esterase de fígado suíno (PLE)

A simple, fast and low-cost methodology was optimized, seeking preparation of a crude pig liver esterase (PLE) concentrate. Basically, the method consisted of the following steps: liver homogenization, acetone washing, enzyme extraction and purification/concentration. Starting from 1 kg of fresh liver more than 200 kU of PLE suspension were obtained after 8 hours, at an estimated cost of US$0.21/kU. The PLE concentrate thus obtained was stable, showing 96-100% of the initial activity after 7 months in refrigerator at 4°C.

Ultrasuodatuskalvojen valinta, likaantuminen ja puhdistus entsyymiliuosten suodatuksessa

Ultrasuodatus on tehokas entsyymiliuosten konsentrointi- ja puhdistusmenetelmä. Prosessin ongelmana on kuitenkin kalvojen likaantumisesta johtuva suodatuksen aikainen vuon pienentyminen ja kalvojen hankala puhdistaminen. Oikeilla kalvovalinnoilla ja optimaalisilla pesustrategioilla voidaan suodatus- ja pesuaikoja lyhentää ja nain parantaa suodatusprosessin tehokkuutta ja tuotteen laatua. Työn kirjallisessa osassa on käsitelty entsyymiliuosten ja polymeerikalvojen suodatusprosessiin vaikuttavia ominaisuuksia. Työssä on esitelty myös pesuaineiden ja pesukemikaalien ominaisuuksia sekä pesuprosessiin vaikuttavia tekijöitä. Työn kokeellinen osa koostui kahdesta osasta. Työn ensimmäisessä osassa etsittiin entsyymiliuosten ultrasuodatusprosessiin sopivaa kalvoa vertailemalla kalvojen permeabiliteetteja, hydrofiilisyytta, varausta ja peseytyvyyttä. Suodatuksissa käytettiin kahta Roal Oy:n tuottamaa entsyymiliuosta ja suodatukset tehtiin DSS Labstak M20-laitteistolla. Työn toisen osan tarkoituksena oli etsiä entsyymiliuosten suodatuksessa käytettävien kalvojen likaantumisen syyt sekä tehokas puhdistusaine kalvojen pesuun. Tehokasta pesuainetta etsittiin liotuskokeilla ja laboratoriomittakaavan poikkivirtauslaitteistolla. Analysointeja tehtiin muun muassa FTIR- spektroskoopilla, vuomittauksilla, pyyhkäisyelektronimikroskoopilla ja kontaktikulmamittauksilla. Entsyymiliuosten suodatuksiin sopivan kalvon etsinnässä testattujen kalvojen välillä oli suuria eroja niin permeabiliteeteissa kuin likaantumisessa ja puhdistumisessa, mutta mikään kalvoista ei erottunut ylivoimaisesti parhaaksi. Pesuaineista kaupalliset membraanien puhdistukseen tarkoitetut emäksiset pesuaineet osoittautuivat tehokkaimmiksi lian poistajiksi.

Chelating Adsorbents in Purification of Hydrometallurgical Solutions

In this thesis, equilibrium and dynamic sorption properties of weakly basic chelating adsorbents were studied to explain removal of copper, nickel from a concentrated zinc sulfate solution in a hydrometallurgical process. Silica-supported chelating composites containing either branched poly(ethyleneimine) (BPEI) or 2-(aminomethyl)pyridine (AMP) as a functional group were used. The adsorbents are commercially available from Purity Systems Inc, USA as WP-1® and CuWRAM®, respectively. The fundamental interactions between the adsorbents, sulfuric acid and metal sulfates were studied in detail and the results were used to find the best conditions for removal of copper and nickel from an authentic ZnSO4 process solution. In particular, the effect of acid concentration and temperature on the separation efficiency was considered. Both experimental and modeling aspectswere covered in all cases. Metal sorption is considerably affected by the chemical properties of the studied adsorbents and by the separation conditions. In the case of WP-1, acid affinity is so high that column separation of copper, nickel and zinc has to be done using the adsorbent in base-form. On the other hand, the basicity of CuWRAM is significantly lower and protonated adsorbent can be used. Increasing temperature decreases the basicity and the metals affinity of both adsorbents, but the uptake capacities remain practically unchanged. Moreover, increasing temperature substantially enhances intra-particle mass transport and decreases viscosities thus allowing significantly higher feed flow rates in the fixed-bed separation. The copper selectivity of both adsorbents is very high even in the presence of a 250-fold excess of zinc. However, because of the basicity of WP-1, metal precipitation is a serious problem and therefore only CuWRAM is suitable for the practical industrial application. The optimum temperature for copper removal appears to be around 60 oC and an alternative solution purification method is proposed. The Ni/Zn selectivity of both WP-1 and CuWRAM is insufficient for removal of the very small amounts of nickel present in the concentrated ZnSO4 solution.

A convenient method for lecithin purification from fresh eggs

The increasing demand for fatty acid-free lecithin required modifications in existing purification methods. In this technical note we describe a purification procedure with the following steps: a) homogenization and extraction of yolks obtained from fresh eggs with acetone, b) solubilization with ethanol and solvent elimination and c) repeated solubilization/precipitation with petroleum ether/acetone. This crude extract was chromatographed on neutral alumina, which was exhaustively washed with chloroform before elution with chloroform:methanol, allowing the sequential separation of fatty acids and lecithin. Chromatographic behavior and mass spectra of the product are presented. This fast procedure yields fatty acid-free lecithin at a competitive cost.

Triterpenoids from Azorella trifurcata (Gaertn.) Pers and their effect against the enzyme acetylcholinesterase

The inhibition of the enzyme acetylcholinesterase is considered as a strategy for the treatment of Alzheimer's disease, senile dementia, ataxia, and myasthenia gravis. Three lanostane- and two cycloartane-type triterpenes, together with two mulinane-type diterpenes were isolated from petroleum ether extract of the whole shrub of Azorella trifurcata (Gaertn.) Pers. Their effect on the enzyme acetylcholinesterase was assessed as well. In addition, this is the first report of these triterpenes in the genus Azorella.

Preparative separation and purification of sesquiterpenoids from tussilago farfara l. by high-speed counter-current chromatography

Preparative high-speed counter-current chromatography (HSCCC) was successfully applied for separation and purification of sesquiterpenoids from an extract of Tussilago farfara L. with a two-phase solvent system composed of n-hexane-ethyl acetate- methanol-water (1:0.5:1.1:0.3, v/v/v/v). The separation produced a total of 32 mg of tussilagone, 18 mg of 14-acetoxy-7β-(3'-ethyl cis-crotonoyloxy)-lα-(2'-methyl butyryloxy)-notonipetranone and 21 mg of 7β-(3'-ethyl cis-crotonoyloxy)-lα-(2'- methyl butyryloxy)-3,14-dehydro-Z-notonipetranone from 500 mg of the crude extract in one step separation with the purity of 99.5, 99.4 and 99.1%, respectively, as determined by HPLC. The structures of these compounds were identified by ESI-MS, ¹H-NMR and 13C-NMR.

Treatment for purification water of biodiesel using electrofloculation

Biodiesel was created as a solution for a great economic and enviromental problem of petroleum, a resource with sustainable characteristics. But its production still needs optimization, because it uses a lot of water and generates a large volume of this residue, which appears improper to disposal without correct treatment. This work propose an economic, viable and efficient way to treat the Biodiesel purification water, not only aiming at a proper disposal, but the reuse as input in the process, generating a large industrial economy, and greater environmental progress. For both treatment uses the electrofloculation technique.

Enzyme loading dependence of cellulose hydrolysis of sugarcane bagasse

The enzymatic hydrolysis of steam-pretreated sugarcane bagasse, either delignified or non-delignified, was studied as a function of enzyme loading. Hydrolysis experiments were carried out using five enzyme loadings (2.5 to 20 FPU/g cellulose) and the concentration of solids was 2% for both materials. Alkaline delignification improved cellulose hydrolysis by increasing surface area. For both materials, glucose concentrations increased with enzyme loading. On the other hand, enzyme loadings higher than 15 FPU/g did not result in any increase in the initial rate, since the excess of enzyme adsorbed onto the substrate restricted the diffusion process through the structure.

Preparative separation and purification of bufadienolides from ChanSu by high-speed counter-current chromatography combined with preparative HPLC

Eight bufadienolides were successfully isolated and purified from ChanSu by high-speed counter-current chromatography (HSCCC) combined with preparative HPLC (prep-HPLC). First, a stepwise elution mode of HSCCC with the solvent system composed of petroleum ether - ethyl acetate - methanol - water (4:6:4:6, 4:6:5:5, v/v) was employed and four bufadienolides, two partially purified fractions were obtained from 200 mg of crude extract. The partially purified fractions III and VI were then further separated by prep-HPLC, respectively, and another four bufadienolides were recovered. Their structures were confirmed by ESI-MS and ¹H-NMR spectra.

Separation and purification of three stilbenes from the radix of Polygonum cillinerve (Nakai) Ohwl by macroporous resin column chromatography combined with high-speed counter-current chromatography

An effective method for the rapid separation and purification of three stilbenes from the radix of Polygonum cillinerve (Nakai) Ohwl by macroporous resin column chromatography combined with high-speed counter-current chromatography (HSCCC) was successfully established. In the present study, a two-phase solvent system composed of chloroform-n-butanol-methanol-water (4:1:4:2, v/v/v/v) was used for HSCCC separation. A one-step separation in 4 h from 150 mg of crude extract produced 26.3 mg of trans-resveratrol-3-O-glucoside, 42.0 mg of pieceid-2"-O-gallate, and 17.9 mg of trans-resveratrol with purities of 99.1%, 97.8%, and 99.4%, respectively, as determined by high-performance liquid chromatography (HPLC). The chemical structures of these compounds were identified by nuclear magnetic resonance (NMR) spectroscopy.