Fast and economic immobilization methods described for non-commercial Pseudomonas lipases

There is an increasing interest to seek new enzyme preparations for the development of new products derived from bioprocesses to obtain alternative bio-based materials. In this context, four non-commercial lipases from Pseudomonas species were prepared, immobilized on different low-cost supports, and examined for potential biotechnological applications. Results: To reduce costs of eventual scaling-up, the new lipases were obtained directly from crude cell extracts or from growth culture supernatants, and immobilized by simple adsorption on Accurel EP100, Accurel MP1000 and Celite (R) 545. The enzymes evaluated were LipA and LipC from Pseudomonas sp. 42A2, a thermostable mutant of LipC, and LipI. 3 from Pseudomonas CR611, which were produced in either homologous or heterologous hosts. Best immobilization results were obtained on Accurel EP100 for LipA and on Accurel MP1000 for LipC and its thermostable variant. Lip I. 3, requiring a refolding step, was poorly immobilized on all supports tested ( best results for Accurel MP1000). To test the behavior of immobilized lipases, they were assayed in triolein transesterification, where the best results were observed for lipases immobilized on Accurel MP1000. Conclusions: The suggested protocol does not require protein purification and uses crude enzymes immobilized by a fast adsorption technique on low-cost supports, which makes the method suitable for an eventual scaling up aimed at biotechnological applications. Therefore, a fast, simple and economic method for lipase preparation and immobilization has been set up. The low price of the supports tested and the simplicity of the procedure, skipping the tedious and expensive purification steps, will contribute to cost reduction in biotechnological lipase-catalyzed processes.

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).

Desenvolvimento de procedimento de extração para análise de sedativos e B-bloqueadores em rim suíno

A procedure was developed for determination of 5 sedatives and 14 β-blockers in swine kidney and subsequent analysis by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Three different procedures for extraction were tested, evaluated through recovery studies. The procedure using acetonitrile for extraction and cleanup with freezing at low temperature and dispersive solid phase extraction using 500 mg celite® 545 before the concentration step presented the better results. The dried samples were redissolved with methanol and analyzed using a LC-MS/MS system with electrospray ionization (ESI) operating in positive MRM mode. The recovery values for this procedure were in the 75-88% range. The robustness of the method was tested against small variations. The method was used to analyze carazolol, azaperone and azaperol in collaborative assay, obtaining results close to designed value.

Immobilization of Burkholderia cepacia Lipase: Kinetic Resolution in Organic Solvents, Ionic Liquids and in Their Mixtures

Immobilization of Burkholderia cepacia Lipase: Kinetic Resolution in Organic Solvents, Ionic Liquids and in Their Mixtures Biocatalysis opens the door to green and sustainable processes in synthetic chemistry allowing the preparation of single enantiomers, since the enzymes are chiral and accordingly able to catalyze chemical reactions under mild conditions. Immobilization of enzymes enhances process robustness, often stabilizes and activates the enzyme, and enables reuse of the same enzyme preparation in multiple cycles. Although hundreds of variations of immobilization methods exist, there is no universal method to yield the highly active, selective and stable enzyme catalysts. Therefore, new methods need to be developed to obtain suitable catalysts for different substrates and reaction environments. Lipases are the most widely used enzymes in synthetic organic chemistry. The literature part together with the experimental part of this thesis discusses of the effects of immobilization methods mostly used to enhance lipase activity, stability and enantioselectivity. Moreover, the use of lipases in the kinetic resolution of secondary alcohols in organic solvents and in ionic liquids is discussed. The experimental work consists of the studies of immobilization of Burkholderia cepacia lipase (lipase PS) using three different methods: encapsulation in sol-gels, cross-linked enzyme aggregates (CLEAs) and supported ionic liquids enzyme catalysts (SILEs). In addition, adsorption of lipase PS on celite was studied to compare the results obtained with sol-gels, CLEAs and SILEs. The effects of immobilization on enzyme activity, enantioselectivity and hydrolysis side reactions were studied in kinetic resolution of three secondary alcohols in organic solvents, in ionic liquids (ILs), and in their mixtures. Lipase PS sol-gels were shown to be active and stable catalysts in organic solvents and solvent:IL mixtures. CLEAs and SILEs were highly active and enantioselective in organic solvents. Sol-gels and SILEs were reusable in several cycles. Hydrolysis side reaction was suppressed in the presence of sol-gels and CLEAs.

O corrência natural de desoxinivalenol e toxina T-2 em milho pós-colheita

Foi verificada a incidência natural de desoxinivalenol (DON) e toxina T-2 em 115 amostras de milho em grão, pós-colheita, procedentes de diferentes localidades do Brasil, Argentina e Paraguai, safra 1994/1995. Os grãos de milho foram obtidos da Associação Brasileira de Milho. DON foi extraído do grão com acetonitrila-água (84+16) e o extrato foi submetido a uma purificação em coluna de carvão ativo, alumina e Celite. A detecção foi feita por cromatografia em camada delgada, impregnando a placa com solução de cloreto de alumínio, aquecida após desenvolvimento e visualização em luz ultravioleta (366 nm). Para a toxina T-2, um método direto e competitivo de ELISA foi utilizado, após extração da amostra com metanol a 70%. As recuperações médias para o DON e toxina T-2 foram superiores a 70%. O limite de detecção foi cerca de 90 ng/g e 50 ng/g, para DON e toxina T-2, respectivamente. DON foi detectado em 7 das amostras (102-542 ng/g) e a toxina T-2 em uma amostra (104 ng/g).

Determinação de tricotecenos em cerveja e avaliação de incidência no produto comercializado no Rio Grande do Sul

Este trabalho adaptou um método e avaliou os seus indicadores de mérito para determinação de tricotecenos, desoxinivalenol (DON) e toxina T-2, em cervejas comercializadas na região Sul do Rio Grande do Sul, empregando cromatografia gasosa. As adaptações foram realizadas na etapa de extração, purificação, derivação e condições cromatográficas usando amostras de cerveja artificialmente contaminadas, avaliando a adequação do procedimento pela recuperação obtida. Foram estudados os solventes acetonitrila, metanol e cloreto de metileno puro ou em misturas para a etapa de extração. A purificação do extrato foi realizada empregando ou não minicoluna de carvão:alumina:celite (7:5:3). Para a derivação testou-se basicidade do meio (bicarbonato de sódio, piridina), agente acilante (TFAA e HFBI), tempo (30 e 60 minutos) e temperatura (60 e 80ºC). Foram otimizadas as condições cromatográficas em cromatógrafo gasoso equipado com injetor split/splitless e detector de ionização de chama. A melhor recuperação foi obtida quando se empregou partição líquido-líquido com cloreto de metileno, seguida da limpeza em minicoluna e derivação com TFAA e piridina a 60°C durante 60 minutos. A recuperação média foi de 83 e 103% para DON e toxina T-2 respectivamente, para níveis de adição entre 50 e 200ng/mL. O coeficiente de variação assumiu valores de 2% e 4% e limite de detecção de 21 e 60 ng/mL. A aplicabilidade do método foi testada em um levantamento realizado em 72 amostras de cervejas. Destas 9,7% estavam contaminadas, sendo 5,3% com DON (50 a 336ng/mL) e 4,5% com toxina T-2 (114 a 249ng/mL).

Revestimento de sementes de milho superdoce (sh2)

A semente de milho doce é leve e rugosa. A rugosidade torna difícil a classificação das sementes quanto à forma e ao tamanho e isso dificulta a semeadura. Uma solução para esse problema seria a utilização da técnica de revestimento. Assim, este trabalho teve por objetivo avaliar diferentes materiais de enchimento, cimentantes e corantes na peletização de sementes de milho superdoce e verificar quais combinações de materiais seriam eficientes na manutenção da qualidade fisiológica das sementes após o armazenamento e que permitissem vazão e distribuição uniformes durante a semeadura. Foram então testados doze materiais de enchimento (calcários 1 e 2, caulim, carvão vegetal ativado, areia, vermiculita, fubá de milho, farinha de trigo, polvilho de mandioca, amido de milho, celite e terra de diatomáceas), dois cimentantes (goma arábica e cascorez extra) e seis corantes (tintas guache, acrílica, plástica e para tecido, corante para alimento e gelatina). As avaliações da qualidade física e fisiológica das sementes revestidas e nuas foram efetuadas por meio dos testes: teor de água, fragmentação, peso de mil sementes, volume aparente e plantabilidade, germinação, primeira contagem da germinação e emergência de plântulas em campo. O revestimento de sementes de milho superdoce proporciona homogeneidade de forma e tamanho às sementes, melhora a vazão e a distribuição dos péletes na semeadura e não compromete a emergência de plântulas em campo depois de quatro meses de armazenamento.

Revestimento de sementes de milho superdoce (sh2)

A semente de milho doce é leve e rugosa. A rugosidade torna difícil a classificação das sementes quanto à forma e ao tamanho e isso dificulta a semeadura. Uma solução para esse problema seria a utilização da técnica de revestimento. Assim, este trabalho teve por objetivo avaliar diferentes materiais de enchimento, cimentantes e corantes na peletização de sementes de milho superdoce e verificar quais combinações de materiais seriam eficientes na manutenção da qualidade fisiológica das sementes após o armazenamento e que permitissem vazão e distribuição uniformes durante a semeadura. Foram então testados doze materiais de enchimento (calcários 1 e 2, caulim, carvão vegetal ativado, areia, vermiculita, fubá de milho, farinha de trigo, polvilho de mandioca, amido de milho, celite e terra de diatomáceas), dois cimentantes (goma arábica e cascorez extra) e seis corantes (tintas guache, acrílica, plástica e para tecido, corante para alimento e gelatina). As avaliações da qualidade física e fisiológica das sementes revestidas e nuas foram efetuadas por meio dos testes: teor de água, fragmentação, peso de mil sementes, volume aparente e plantabilidade, germinação, primeira contagem da germinação e emergência de plântulas em campo. O revestimento de sementes de milho superdoce proporciona homogeneidade de forma e tamanho às sementes, melhora a vazão e a distribuição dos péletes na semeadura e não compromete a emergência de plântulas em campo depois de quatro meses de armazenamento.

Poultry feed metabolizable energy determination using total or partial excreta collection methods

The aim of this work was to compare the efficiency of total (TC) or partial (PC) collection excreta methods to determine metabolizable energy in poultry feeds. A number of 180 12- to 21-day-old broilers were distributed into two treatments of six replicates of 10 birds each. A reference-diet was formulated to supply broiler requirements, and the test-diets consisted of 60% of reference diets and 40% of corn or soybean meal. Celite was added at 1% to the diets as a marker. Excreta and diet samples were analyzed for dry matter, energy, nitrogen, and acid-insoluble ash (AIA). AME of corn determined by partial collection (PC) was higher (3544 kcal/kg) as compared to total collection (TC) (3133 kcal/kg). However, no difference were observed for soybean meal (1797 vs. 1821 kcal/kg) between both methods. Marker recovery rates in the excreta were 101, 111, and 96% for the basal-diet, and the test-diets with corn or soybean meal, respectively. This result indicates the importance of marker recovery rate in the excreta to evaluate feed AME and digestibility.

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

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

Extraction and clean-up procedure for analysis of organochlorine pesticide residues in ethoxylated lanolin

In the present study an evaluation was made of a method for the determination of organochlorine pesticide residues in ethoxylated lanolin. Samples were homogenized with Celite, transferred to chromatographic columns, prepacked with silica gel deactivated to 10%. The pesticide elution was processed with n-hexane-dichloromethane and the concentrated eluate was analyzed using gas-liquid chromatography (GC) with electron capture detection (ECD). The composition of the elution solvent was a significant factor for the recovery of the pesticides. Mean recoveries obtained for fortified samples ranged from 87 to 94% for p,p'-DDE, dieldrin, endrin, p,p'-DDD and p,p'-DDT. Optimization of the experimental conditions resulted in a small-scale method that combines extraction and cleanup in a single step. (C) 2000 Elsevier B.V. S.A. All rights reserved.

Screening of supports for the immobilization of pectinmethylesterase from acerola (Malpighia glabra L)

A partially purified extract of pectinmethylesterase (PME) from acerola fruit was immobilized on various supports: glass, celite, chrysotile, agarose, concanavalin A Sepharose 4B, egg shell, polyacrylamide and gelatin. In addition, reticulation with glutaraldehyde was assessed, as well as the use of gelatin in the presence of celite, glass and silica. The highest immobilization yields were obtained when the pectinmethylesterase was immobilized in concanavalin A Sepharose 4B (81.7%) and in gelatin-water (78.0%). (C) 2004 Society of Chemical Industry.

Effect of a pilot washing system on dicofol levels in orange matrix

An efficient analytical method is described for the analysis of dicofol residues in pulp and orange peel. Samples are mixed with Celite and transferred to chromatographic columns prepacked with silica gel. Dicofol is eluted with ethyl acetate, and the extracts are analyzed by gas chromatography with electron capture detection. Mean recoveries for dicofol at levels of 0.5, 2.0, 5.0, and 10 mg/kg ranged from 87 to 95% with relative standard deviation values between 2.6 and 9.0%. To investigate the effect of a pilot washing system on dicofol residues in oranges, the analytical procedure was applied to samples submitted to different treatments with commercial formulations under field and laboratory conditions. The orange samples with and without washing were analyzed in duplicate, and the results indicated that washing under the described conditions did not allow a complete removal of dicofol residues from orange peel.

Comparison of different clean-up procedures for the determination of N-methylcarbamate insecticides in vegetable matrices by high-performance liquid chromatography with UV detection

Several clean-up procedures which included the use of glass chromatography columns (silica gel, alumina, Florisil, silanized Celite-charcoal), Sep-Pak cartridges and standard solutions were compared for the determination of the following N-methylcarbamate (NMC) insecticides: aldicarb, carbaryl, carbofuran, methomyl and propoxur. According to recovery results of the compounds after elution in a glass column, the most efficient systems employed 4.6% deactivated alumina and a silanized Celite-charcoal (4:1) as adsorbents, using dichloromethane-methanol (99:1) and toluene-acetonitrile (75:25) mixtures, respectively, as binary eluents. The recoveries of the compounds studied varied from 84 to 120%. Comparable recoveries (75-100%) for Sep-Pak cartridges in normal phase (NH2, CN) and reversed phase (C-8) were observed. Different temperatures were tested during the concentration step in a rotary evaporator, and we verified a strong influence of this parameter on the stability of some compounds, such as carbofuran and carbaryl. Recovery studies employing the best clean up procedures were performed at the Brazilian agricultural level in potato and carrot samples; Validation methodology of the US Food and Drug Administration was adapted for the N-methylcarbamate analysis. Their recoveries ranged between 79 and 93% with coefficients of variation of 2.3-8%. (C) 1998 Elsevier B.V. B.V.

Geophagy in the golden-faced saki monkey (Pithecia pithecia chrysocephala) in the Central Amazon

The golden-faced saki monkey Pithecia pithecia chrysocephala (Cebidae, Primates) was observed eating soil from termite nests during a long-term study of a family group in a Central Amazonian forest fragment. In this paper we describe the behaviour involved in the geophagy in these monkeys, and the results of geochemical and physical analyses of the termite nest material, as well as root mat and topsoil samples below the trees, in order to clarify the possible reasons for it. The sakis ate soil from nine arboreal termite nests on 26 soil feeding-bouts (in 853 observation hours); 25 soil feeding-bouts occurred in March 1987 (rainy season), during 19 days or 132 observation hours, and occupied 0.7% of the feeding time. Geophagy frequencies did not differ between sexes (17 feeding-bouts of four females and 8 for two males). Mineral composition was higher in arboreal termitaria than in the topsoil. Kaolinite was the major clay component. Tannin adsorptive capacity, tested through a modified radial diffusion method of Hagerman, was around 10-20%, similar to a control with kaolin (10-20%), but lower than bentonite or celite (30-45%). The observations reported here, although inconclusive as to the function of geophagy in this species, indicate that it is not a mineral supplement during times of scarcity or high consumption of leaves, as has been reported for other primates, nor that it is related to fruit consumption (redressing possible mineral imbalance), as has been suggested for some other frugivorous mammals. Our results do not rule out tannin adsorptive hypothesis for the ingestion of clays, but, being an irregular habit, we argue that it is most likely related to rare and occasional dietary components.