Determinação das propriedades catalíticas em meio aquoso e orgânico da lipase de Candida rugosa imobilizada em celulignina quimicamente modificada por carbonildiimidazol

Microbial lipase from Candida rugosa was immobilized by covalent binding on wood cellulignin (Eucaliptus grandis) chemically modified with carbonyldiimidazole. The immobilized system was fully evaluated in aqueous (olive oil hydrolysis) and organic (ester synthesis) media. A comparative study between free and immobilized lipase was carried out in terms of pH, temperature and thermal stability. A higher pH value (8.0) was found optimal for the immobilized lipase. The optimal reaction temperature shifted from 37 °C for the free lipase to 45 °C for the immobilized lipase. The pattern of heat stability indicated that the immobilization process tends to stabilize the enzyme. Kinetics tests at 37 °C following the hydrolysis of olive oil obeyed the Michaelis-Menten rate equation. Values for Km = 924.9 mM and Vmax = 198.3 U/mg were lower than for free lipase, suggesting that the affinity towards the substrate changed and the activity of the immobilized lipase decreased during the course of immobilization. The immobilized derivative was also tested in the ester synthesis from several alcohols and carboxylic acids.

Biossensor enzimático para detecção de fungicidas ditiocarbamatos: estudo cinético da enzima aldeído desidrogenase e otimização do biossensor

Initially, all major factors that affect the rate of the AldH-catalyzed reaction (enzyme concentration, substrate concentration, temperature and pH) were investigated. Optimal activity was observed between pH values of 7.5 and 9.5 in the temperature range of 25 to 50 ºC. Kinetic parameters, such as Km (2.92 µmol L-1) and Vmax (1.33 10-2 µmol min-1) demonstrate a strong enzyme-substrate affinity. The sensors were based on screen-printed electrodes modified with the Meldola Blue-Reinecke salt (MBRS) combination. Operational conditions (NAD+ and substrate contents, enzyme loading and response time) were optimized. Also, two enzyme immobilization procedures were tested: entrapment in poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ) and crosslinking with glutaraldehyde. Chronoamperometry was employed to observe the biosensor responses during enzymatic hydrolysis of propionaldehyde and also to construct inhibition curves with maneb and zineb fungicides. Best results were found with the following conditions: [NAD+] = 0.25 mmol L-1; [propionaldehyde] = 80 µmol L-1; enzyme loading = 0.8 U per electrode; response time = 10 min, and inhibition time = 10 min. Current intensities around 103 ± 13 nA with the sensors and good stability was obtained for both immobilization procedures. Detection limits, calculated using 10% inhibition were 31.5 µg L-1 and 35 µg L-1 for maneb and zineb, respectively. Results obtained with other MBRS-modified electrodes consisting of mono and bi-enzymic sensors were compared. The ability to catalyze NADH oxidation by MB was also highlighted.

Influência de desproteinizantes ácidos na quantificação da glutationa reduzida eritrocitária por CLAE-UV

Large differences in reduced glutathione (GSH) levels have been found in different investigations, also in healthy people. GSH oxidation in vitro has been associated with sample acidification in the presence of oxihemoglobin. In this work, the influence of different acids on GSH determination utilizing HPLC with UV detection was evaluated. The results showed that metaphosphoric acid and sulfosalicylic acid were inadequate for analysis, because metaphosphoric acid showed to be inefficient for deproteinization and with sulfosalicylic acid loss of GSH was observed. Trichloroacetic acid did not effect GSH quantification, since the deproteinized form was immediately derivatized with 5, 5´-dithio-bis (2-nitrobenzoic) acid. Methods with TCA deproteinization presented linear results from 0.5 to 3.0 mM. The correlation coefficient between aqueous curves and GSH spiked RBC exceeded 0.99. Precision calculations showed CV lower than 10% and bias within ± 10% for concentrations of 0.5; 1.5 and 3.0 mM GSH. The recovery was higher than 94%. Moreover, GSH blood concentrations were independent of hemoglobin concentrations.

Urinary 2,5-hexanedione in workers exposed to n-hexane: influence of the sample treatment

The aim of the present study was to determine 2,5-hexanedione (2,5-HD), a metabolite of n-hexane, by gas chromatography/flame ionization detection in 31 workers exposed to n-hexane after two types of sample pretreatment, i.e., with (total 2,5-HD) and without (free 2,5-HD) acid hydrolysis. The mean urinary 2,5-HD was 0.52 mg/L (free) and 2.88 mg/L (total), this difference being significant (Student t-test, p < 0.05). The differences in the results according to the sample treatment support the need to modify the current Brazilian legislation, which proposes the analysis of 2,5-HD without indicating whether it is the free or total metabolite.

Fenilsilicato dopado com Eu III obtido pelo método sol-gel

In this work, we report the synthesis and the photoluminescence features of a Eu(III)-doped modified silica matrix obtained by the sol-gel method. The matrix was prepared by reaction between tetraethylorthosilicate and phenyltriethoxysilane alkoxide. The hydrolysis occurred using basic catalysis. The solids were treated at 100, 200 and 300 ºC during 4 h and the structure was determined by thermogravimetric analysis (TG/DTG), nuclear magnetic resonance (NMR 29Si and 13C), infrared spectroscopy (IR) and photoluminescence (PL). The PL spectra display the Eu(III) lines characteristic of the ion, 5D0 -> 7F J (J=0, 1, 2, 3, 4), the blue emission as ascribed in the silica matrix. The NMR and TG showed the stability of hybrid silica.

Comparação do desempenho da lipase de candida rugosa imobilizada em suporte híbrido de polissiloxano-polivinilálcool empregando diferentes metodologias

The efficiency for immobilizing microbial Candida rugosa lipase on a hybrid matrix of polysiloxane polyvinyl alcohol, by adsorption, covalent coupling and encapsulation was compared. The activities of immobilized derivatives were evaluated using p-nitrophenylpalmitate (hydrolysis) and butyric acid and butanol (esterification) as substrates. Operational stability and storage tests were also performed. Among the procedures tested, the proposed matrix was efficient for immobilizing C. rugosa lipase by adsorption and covalent coupling techniques and unsuitable for encapsulation purposes. The results reveal that better catalytic properties in both aqueous and organic media were demonstrated by the covalent coupling POS-PVA immobilized lipase, including also satisfactory half-life and good storage stability.

Avaliação da estabilidade do marcador plasmático do estresse oxidativo: malondialdeído

Malondialdehyde (MDA) is one of the lipid peroxidation products widely used as indicator of cellular injury. However, the short-term and the long-term stability of this biomarker remain unclear. The objective of this work was to evaluate the stability of plasmatic MDA at -20 ºC, utilizing thiobarbituric acid (TBA) as derivative in spectrophotometric and chromatographic analysis. The results showed that MDA was stable for 24 h after blood collection, was not stable when stored after alkaline hydrolysis, remained stable for 30 days after TBA derivatization and was stable for 3 days when stored after n-butanol extraction, all at -20 ºC.

Kapillaarielektroforeesin soveltaminen uuttofaasin koostumuksen analysointiin

Työn tarkoituksena oli löytää kapillaarielektroforeesimenetelmä (CE), joka soveltuisi metallien neste-nesteuutossa käytettävien orgaanisten uuttofaasien koostumuksen analysointiin. Kapillaarielektroforeesissa käytetyn elektrolyyttiliuoksen analyytti-kohtaista optimointia ei tässä työssä tehty, vaan liikkeelle lähdettiin fenoleille tarkoitetulla menetelmällä. Tarkasteltavia uuttoreagenssiryhmiä olivat hydroksi-oksiimit sekä fosfiinihappo- ja fosforihappopohjaiset reagenssit. Tutkittavia kaupallisia laimentimia olivat Orfom SX 11 ja Shellsol D70. Lisäksi tutkittiin kahta modifiointiainetta, TOPOa (tri-n-oktyylifosfiinioksidi) ja TXIB:tä (2,2,4-trimetyyli-1,3-pentaanidiolidi-isobutyraatti). Työssä tavoiteltiin kapillaarielektroforeesin hyötyjä erityisesti hydrometallurgisessa teollisuudessa. Suurimpana hyötynä ennakoitiin mahdollisuus analysoida suuria molekyylejä, kuten uuttoreagenssi-metallikomplekseja, joita ei pystytä analysoimaan kaasukromatografilla (GC). Näytteet voidaan myös analysoida ilman hidasta ja usein ei-kvantitatiivista derivatisointia. Kirjallisuudesta ei löytynyt aiempia artikkeleita CE:n soveltamisesta kyseisille aiheille. Kapillaarielektroforeesianalyyseissa pystyttiin esimerkiksi havaitsemaan hydroksi-oksiimin kuparikompleksi orgaanisessa faasissa. Seulonta-ajoissa yleisenä ongelmana oli kuitenkin tulosten heikko toistettavuus. Kapillaari-elektro-foreesi-menetelmä tarjoaa selvästi mahdollisuuksia tulevaisuudessa, mutta vielä sillä ei päästy luotettavaan toistoon sähkökentän häiriöiden ja elektrolyyttiliuoksen riittämättömän optimoinnin vuoksi. Lisäksi teollisissa olosuhteissa käytetyille autenttisille hydroksioksiimi- ja fosfiinihapponäytteille tehtiin perinteisiä kaasukromatografia-analyysejä, joiden perusteella voitiin nähdä uuttofaasin koostumuksen muuttuneen prosessissa. Hapettuminen sekä eri hydrolyysireaktiot ovat tärkeimmät syyt reagenssien ja laimentimien muuttumiselle. Näitä hajoamistuotteita ei tässä työssä onnistuttu analysoimaan kapillaarielektroforeesilla.

Purificação e caracterização de uma gentioexaose obtida de botriosferana por hidrólise ácida parcial

A hexa-oligosaccharide was obtained by partial acid hydrolysis from botryosphaeran, an exopolysaccharide (EPS) β(1→3; 1→6)-D-glucan type, produced by the ascomyceteous fungus Botryosphaeria rhodina. The oligosaccharide was purified by gel filtration and charcoal-Celite column chromatography and the analysis was followed by HPAEC/PAD. The structure was determined by NMR spectroscopy and mass spectrometry, which showed that the oligosaccharide consists of six β-D-glucopiranosyl units O-6 substituted (gentiohexaose).

Constituintes químicos dos frutos de Copaifera langsdorffii Desf.

Phytochemical investigation of the hexane extract of fruit shells of Copaifera langsdorffii Desf. (Caesalpinioideae) afforded ent-kaur-16-en-19-oic acid, polyalthic acid, nivenolide and the mixture of caryophyllene oxide and ent-kaur-16-en-19-oic acid. The chloroform extract of unripe seeds led to the isolation of coumarin and the GC/MS analysis of the extract allowed the identification of 81.8% of the fatty acid composition after hydrolysis followed by methylation. The main fatty acid identified was oleic acid (33.1%). The isolation of all secondary metabolites was accomplished by modern chromatographic methods and the structure determination was accomplished by spectrometric methods (IR, MS, NMR ¹H and 13C).

Produção de etanol a partir de torta de mamona (Ricinus communis L.) e avaliação da letalidade da torta hidrolisada para camundongos

The castor bean cake is rich in starch (48 ± 0.53%) and bears a problem linked to the occurrence of a toxic protein (ricin). The chemical hydrolysis (ratio solid:liquid = 1:6; H2SO4= 0.1 mol L-1; 120 °C; 40 min) generated a medium with 27 g L-1 of reducing sugars (hydrolysis efficiency= 32%). The hydrolyzed product was fermented and produced 11 g L-1 of ethanol (volumetric productivity=1.38 g L-1 h-1 and ethanol yield on substrate consumed=0.45 g g-1). In vivo experiments (DL50) revealed a reduction of roughly 240 times in the CBC toxicity (2.11 µg g-1).