RESUMO
Textiles represent promising support materials for enzymes. The goal of the present work was to investigate the immobilization of commercial peroxidase on a polyester needle felt and the repeated use in the gentle degradation of norbixin in whey from dairy cheese as a practical application. High enzyme loads were obtained by a 2-step immobilization procedure. First, the number of functional groups on the textile surface was increased by a modification with amino-functional polyvinylamine. Second, the enzyme was immobilized by using 2 types of crosslinking agents. Due to the iron content of peroxidase, inductively coupled plasma-optical emission spectrometry was used for the quantitative determination of the enzyme load on the textile. The enzyme activity was evaluated using common 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid) assay for peroxidases. By the variation of enzyme input and crosslinker concentration, a maximal enzyme load of 80 mg/g of textile was achieved, and a maximum specific activity of 57 U/g of textile. For the visualization of the enzyme on the fiber surface, fluorescence microscopy as well as scanning probe microscopy were used. The immobilized peroxidase showed significant activity, even after 50 reuse cycles. In addition, the potential of the new support and enzyme combination in commercial whey bleaching was demonstrated successfully on a 10-L scale.
Assuntos
Clareadores/química , Carotenoides/metabolismo , Queijo , Peroxidase/química , Soro do Leite/química , Clareadores/metabolismo , Cor , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Peroxidase/metabolismo , Poliésteres/química , TêxteisRESUMO
Colloids are responsible for undesirable haze formation in wine. Here, we characterized 20 colloid batches after isolation by ultrafiltration of musts and wines from five cultivars obtained from four consecutive vintages. Polysaccharide and protein concentrations of the colloids ranged from 0.10 to 0.65 and 0.03 to 0.40 mg/L, respectively. Protein profiling in must and wine colloids by fast protein liquid chromatography (FPLC) and liquid chromatography-high-resolution tandem mass spectrometry (LC-HR-MS/MS) analyses indicated a lower number of proteins in wine than in must colloids. Molar mass distribution analyses revealed all colloids to consist of two carbohydrate- (424-33,390 and 48-462 kg/mol) and one protein-rich (14-121 kg/mol) fractions. The observed barely negative ζ potentials (-3.1 to -1.1 mV) in unstable wines unraveled that colloid instability might be partly related to their poor electrostatic repulsion in the wine matrix. ζ potentials of the colloids from pH 1 to 10 are also presented. Our data support future developments to eliminate haze-forming colloids from wine.
Assuntos
Vinho , Vinho/análise , Espectrometria de Massas em Tandem , Cromatografia Líquida , Cromatografia Líquida de Alta Pressão , ColoidesRESUMO
The adaptive advantage of epibenthic articulate brachiopods over inarticulate forms resulted from a modification of the mechanics of shell opening from an indirect hydraulic system to a direct muscular one. As a consequence, the articulate brachiopods were able to reduce the complex muscular system of the ancestral inarticulates, freeing two-thirds of the space within the shell for enlargement of the feeding apparatus. The original hydraulic mechanism of the inarticulate brachiopods most likely evolved from the hydrostatic skeleton of metameric lower invertebrates, probably polychaete-like annelids, as shown by a biomechanical analysis. The transitional stages between such annelids and inarticulate brachiopods are presented and explained as adaptive improvements in body construction.
RESUMO
Proton spin-lattice relaxation times of pyridine and 1-methylimidazole complexed on Fe(III)protoporphyrin IX dimethyl ester, Fe(III)tetraphenylporphyrin in chloroform and on metmyoglobin in 2H2O have been measured. Caused by chemical exchange of the ligand molecules into the bulk solvent phase, the decay of their MZ-magnetization is given by a superposition of two exponentials from which the mean lifetime of the complexed species can be determined. This method offers the possibility to study exchange kinetics of ligand molecules from a well defined molecular configuration. The present data are compared with the results from linewidth measurements of the bulk phase molecules. From both sets of parameters a detailed picture of the ligand exchange can be gained, particularly if spin transition of the paramagnetic organic metal complex occurs, as is the case for some ferriporphyrins. For metmyoglobin, the NMR result is compared with relaxation times extracted from temperature jump experiments under similar conditions.
Assuntos
Hemeproteínas , Imidazóis , Metamioglobina , Porfirinas , Piridinas , Calorimetria , Espectroscopia de Ressonância Magnética , Matemática , Ligação ProteicaRESUMO
BACKGROUND: Fungi of the phylum Basidiomycota are well-known to form a broad spectrum of biologically active secondary metabolites, especially low molecular weight compounds such as terpenoids. Hericium erinaceus produces various cyathane type diterpenoids including erinacines. However, no quantitative data and production kinetics have been reported on the biosynthesis of the erinacines C and P in submerged cultures. In the present study, the production of erinacine C was optimized, and the product formation kinetics as well as the antimicrobial activity were studied by high-performance liquid chromatography (HPLC), high-performance thin-layer chromatography (HPTLC) and direct bioautography. RESULTS: Oatmeal and Edamin® K were identified to be crucial media components for an efficient production of erinacine C. The highest concentrations of erinacine C were obtained in the optimized culture medium on the 9th culture day (approximately 260 mg L-1). The production of erinacine P was strongly time dependent. The maximum concentration of erinacine P of 184 mg L-1 was observed on the third culture day. Afterwards, the concentrations of erinacine P decreased while the concentrations of erinacine C steadily increased. Comparable results were obtained by HPTLC with UV detection and HPLC with diode-array detection (DAD) analyses. Direct bioautography allowed for an additional analysis of the antimicrobial activity of the secondary metabolites. CONCLUSIONS: The C and N sources oatmeal and Edamin® K induced the formation of erinacine C. Detailed product formation kinetics of the erinacines C and P have been reported for the first time. HPTLC combined with the Aliivibrio fischeri bioassay allowed for an instant detection of cyathane diterpenoids in crude extracts and for an evaluation of the antimicrobial activity of the secondary metabolites directly on the plate.
RESUMO
Lignosulfonates are abundantly available byproducts of the paper and pulping industry, and they therefore represent a promising feedstock for new sustainable processes. For industrial applications of lignosulfonates, their molecular weight distribution is a critical factor. In order to decrease the average molecular weight of lignosulfonates, Seventeen basidiomycetes were screened for their capability to depolymerize lignosulfonates from spent sulfite liquor (SSL) in surface and liquid cultures. Five basidiomycetes polymerized the lignosulfonates under the selected conditions. Only Irpex consors was found to efficiently degrade calcium lignosulfonates when SSL (0.5%, w/w) was used as the sole carbon and nitrogen source. The average molecular weight of the lignosulfonates was reduced from â¼26 to â¼4 kDa as determined by size exclusion chromatography (SEC) within two weeks. Various extracellular enzyme activities of I. consors were determined over the culture period. High peroxidase activities were correlating with a high degradation rate and the culture was harvested at the day of highest peroxidase activity. A putative versatile peroxidase was isolated by fast protein liquid chromatography (FPLC) and its encoding cDNA was cloned.
Assuntos
Proteínas Fúngicas/metabolismo , Lignina/análogos & derivados , Peroxidase/metabolismo , Polyporaceae/metabolismo , Sequência de Aminoácidos , Biodegradação Ambiental , Biotecnologia , Cromatografia em Gel , Clonagem Molecular , Proteínas Fúngicas/genética , Genes Fúngicos , Lignina/química , Lignina/metabolismo , Dados de Sequência Molecular , Peso Molecular , Peroxidase/genética , Filogenia , Polimerização , Polyporaceae/enzimologia , Polyporaceae/genética , Homologia de Sequência de AminoácidosRESUMO
When submerged cultured Pseudomonas fluorescens NCIMB 11761 was fed-batch supplemented with alpha-pinene oxide, a rapid formation of 2,6-dimethyl-5-methylene-hept-(2Z)-enal (I) (isonovalal) was observed. Biotransformation and isomerisation of (I) to the (2E)-isomer (II) (novalal) were enhanced by Lewatit OC 1064, a macroporous polystyrene adsorbent. Accelerated isomerisation in the presence of an amino donor (glycine) at pH 7.3 pointed to a merely chemical mechanism. A maximum yield of 48 g of aldehydesl(-1) was achieved, but quantitative analysis of the volatile fraction showed that the molar conversion of the pinene oxide substrate reached no more than 67%. To fill this gap of the mass balance, the acidic fraction was isolated. It contained several compounds which suggested a beta-oxidation-like catabolism starting from 2,6-dimethyl-5-methylene-hept-(2E)-enoic acid (III) (novalic acid). Using [2H7]-2,5,6-dimethyl-hept-(2E)-enoic acid as a conversion substrate and gas chromatography coupled to atomic emission detection and mass spectrometry a degradation pathway via labelled 3,4-dimethylpentenoic and methylpropanoic acids was evidenced. This pathway may play a predominant role in isoprenoid degradation by soil bacteria.
Assuntos
Aldeídos/metabolismo , Ácidos Graxos Monoinsaturados/síntese química , Ácidos Graxos Monoinsaturados/metabolismo , Ácidos Pentanoicos/síntese química , Ácidos Pentanoicos/metabolismo , Pseudomonas fluorescens/metabolismo , Terpenos/metabolismo , Monoterpenos Bicíclicos , Cromatografia Líquida de Alta Pressão , Isomerismo , Estrutura Molecular , Monoterpenos , Oxirredução , Ácidos Pentanoicos/análiseRESUMO
Volatile acyloins (alpha-hydroxy ketones) were obtained by condensing either aldehydes with pyruvate or 2-keto acids with acetaldehyde in a reaction catalyzed by yeast pyruvate decarboxylases (EC 4.1.1.1). Odor qualities and threshold values of 34 acyloins were evaluated, and 23 of them possessed distinct flavor properties. Sherry and soy sauce flavors were analyzed: 2-hydroxy-3-pentanone and 3-hydroxy-2-pentanone were identified in soy sauce for the first time; these and 2-hydroxy-5-methyl-3-hexanone and 3-hydroxy-1-phenyl-2-butanone were isolated from sherry for the first time. The biocatalytic efficiencies of crude pyruvate decarboxylase preparations from Zygosaccharomyces bisporus, Saccharomyces cerevisiae, Kluyveromyces lactis, and Kluyveromyces marxianus were compared. Product yields comparable to those of conversions with purified pyruvate decarboxylase demonstrated the suitability of crude enzyme extracts as cost-effective biocatalysts in acyloin formation. Conversion rates of >50% showed that the potential of this type of enzyme to catalyze the formation of aliphatic acyloins has been underestimated before.
Assuntos
Álcoois Graxos/metabolismo , Piruvato Descarboxilase/metabolismo , Zygosaccharomyces/enzimologia , Bebidas Alcoólicas/análise , Odorantes , VolatilizaçãoRESUMO
Although beta-boldenone (bBol) used to be a marker of illegal steroid administration in calves, its endogenous formation has recently been demonstrated in these vertebrates. However, research on the pathway leading to bBol remains scarce. This study shows the usefulness of in vivo invertebrate models as alternatives to vertebrate animal experiments, using Neomysis integer and Lucilia sericata. In accordance with vertebrates, androstenedione (AED) was the main metabolite of beta-testosterone (bT) produced by these invertebrates, and bBol was also frequently detected. Moreover, in vitro experiments using feed-borne fungi and microsomes were useful to perform the pathway from bT to bBol. Even the conversion of phytosterols into steroids was shown in vitro. Both in vivo and in vitro, the conversion of bT into bBol could be demonstrated in this study. Metabolism of phytosterols by feed-borne fungi may be of particular importance to explain the endogenous bBol-formation by cattle. To the best of our knowledge, it is the first time the latter pathway is described in literature.
Assuntos
Anabolizantes/metabolismo , Ração Animal/microbiologia , Alternativas ao Uso de Animais/métodos , Fungos/metabolismo , Testosterona/análogos & derivados , Androstenodiona/metabolismo , Animais , Vias Biossintéticas , Bovinos , Cromatografia Líquida de Alta Pressão , Crustáceos/metabolismo , Dípteros/metabolismo , Larva/metabolismo , Microssomos/metabolismo , Fitosteróis/metabolismo , Pleurotus/metabolismo , Detecção do Abuso de Substâncias/veterinária , Espectrometria de Massas em Tandem , Testosterona/metabolismoAssuntos
Ferro , Porfirinas , Protoporfirinas , Piridinas , Sítios de Ligação , Calorimetria , Clorofórmio , Cinética , Matemática , Oxirredução , Temperatura , TermodinâmicaAssuntos
Eletromiografia , Músculos/fisiologia , Potenciais de Ação , Humanos , Contração Muscular , Sistemas On-LineRESUMO
The present paper deals with a method permitting the isolation of haptoglobin 2-2 from human serum or plasma. The haptoglobin is adsorbed to DEAE-cellulose at pH 5.1 by batching. The loaded cellulose is given into a column and the haptoglobin eluted by a 0.1 M to 0.15 M acetate buffer. The last step is a gel-chromatography on Sephadex G-200. Disc- and immunoelectrophoresis were used to test the purity. As by-product acid alpha1-glycoprotein can be obtained.
Assuntos
Haptoglobinas/isolamento & purificação , Proteínas Sanguíneas/isolamento & purificação , Cromatografia DEAE-Celulose/métodos , Cromatografia em Gel/métodos , Eletroforese Descontínua , Humanos , Concentração de Íons de Hidrogênio , ImunoeletroforeseRESUMO
Proton nuclear magnetic resonance shifts, spin-lattice and spin-spin relaxation times have been measured of low-spin bis-pyridine ferriprotoporphyrin(IX) dimethyl ester in chloroform. From the relaxation behavior the hyperfine coupling constant has been obtained and the contact term of the chemical shift was calculated. Deviations between measured and calculated chemical shifts may be attributed to second-order Zeeman interactions. The geometry of pyridine coordinated to the fifth and sixth position of ferriprotoporphyrin(IX) dimethyl ester was estimated from measured relaxation rates. From the non-exponential decay of the Mz magnetization a mean lifetime of taub = 50 ms for pyridine attached to low-spin ferriprotoporphyrin(IX) dimethyl ester was found at 253 K.
Assuntos
Ferro/análise , Porfirinas , Protoporfirinas , Espectroscopia de Ressonância de Spin Eletrônica , Ésteres , Análise de Fourier , Espectroscopia de Ressonância Magnética , Matemática , Conformação MolecularRESUMO
The question of dose-effect relations of berylliosis was examined by measuring the Be-concentration in blood and urine, as well as the immunological behaviour of T-lymphocytes for a group of occupationally exposed men (smaller than 8 ng Be/m3, 4-6 h daily) and a non-exposed control group. Normal values 1.0 +/- 0.4 ng Be/g(n=10) in blood and 0.9 +/- 0.5 ng Be/g(n=10) in urine were found by an optimized flameless atomic absorption spectrometry method. The corresponding values for the exposed group (n=8) showed a mean increase of a factor of 4. The preferential enrichment of Be in the prealbumin and in the nu-globulin fractions was determined by a preparative isotachophoretic column with physiological pH conditions, especially developed for this application. The Be-specific stimulation of the T-lymphocytes of the exposed group was increased significantly. In blood and urine samples of exposed (2-40 mg Be/m3 as Be(NO3)2) Wistar-rats and guinea pigs Be values up to 36 ng Be/g in serum and up to 300 ng Be/g in urine were determined. After Be exposure, guinea pig serum showed a significant increase in the nu-globulins after the isotachophoretical separation of the serum proteins, which could not be correlated with increased Be-levels in this fraction. 70% of the total Be(10-100 ng/g serum) were detected in the prealbumin, only about 1% in the nu-globulin fraction. The same results were found in serum samples with in vitro addition of Be. The lymphocyte transformation rates for the exposed animals were increased. Skin tests were found to be positive in 70% for exposed rats and in 50-70% for guinea pigs.
Assuntos
Beriliose/imunologia , Berílio/sangue , Animais , Berílio/urina , Cobaias , Humanos , Pulmão/imunologia , Pré-Albumina/imunologia , Ratos , Linfócitos T/imunologia , Fatores de Tempo , gama-Globulinas/imunologiaRESUMO
Submerged cells of the basidiomycete Nidula niveo-tomentosa, a microbial producer of 4-(4-hydroxyphenyl)-butan-2-one, were supplemented with (13)C-labeled L-phenylalanines and with [1-(13)C]glucose. Labeled transformation products were detected by a novel method of analyzing stable isotope-labeled metabolites, gas chromatography (GC) coupled to an atomic emission detector, and by GC-mass spectrometry. A benzoate moiety was side chain elongated according to the poly-beta-keto scheme. The presence of an acetyl coenzyme A-carboxylase inhibitor shifted the spectrum of products to benzyl compounds. Hence, the fungal pathway differs from the one established for plant tissues.
Assuntos
Basidiomycota/metabolismo , Butanonas/metabolismo , Isótopos de Carbono/metabolismo , Cromatografia Gasosa , Cromatografia Gasosa-Espectrometria de Massas , Glucose/metabolismo , Fenilalanina/metabolismoRESUMO
Cells of the wild-type yeast strain Zygosaccharomyces bisporus CBS 702 form alpha-hydroxy ketones from aromatic amino acid precursors during fermentation. Pyruvate decarboxylase (PDC, E.C. 4.1.1.1), the key enzyme of this biotransformation catalysing the non-oxidative decarboxylation of pyruvate and other 2-oxo-acids, was purified and characterised. The active enzyme is homotetrameric (alpha4) with a molecular mass of about 244 kDa. Activation of PDC by its substrate pyruvate results in a sigmoidal dependence of the reaction rate from substrate concentration (apparent Km value 1.73 mM; Hill coefficient 2.10). A cDNA library was screened using a PCR-based procedure, and a 1856 bp cDNA of PDC was identified and sequenced. The cDNA encodes a polypeptide of 563 amino acid residues (monomeric unit). Sequence alignments demonstrate high homologies (> 80%) to PDC genes from Saccharomyces cerevisiae, Kluyveromyces lactis and Kluyveromyces marxianus.
Assuntos
Piruvato Descarboxilase/genética , Piruvato Descarboxilase/isolamento & purificação , Análise de Sequência de DNA , Zygosaccharomyces/enzimologia , DNA Complementar/genética , Álcoois Graxos/metabolismo , Biblioteca Gênica , Cetonas/metabolismo , Cinética , Dados de Sequência Molecular , Peptídeos/genética , Reação em Cadeia da Polimerase , Piruvato Descarboxilase/química , Alinhamento de Sequência , Leveduras/enzimologia , Leveduras/metabolismoRESUMO
More than 50 filamentous fungi and yeasts, known for de novo synthesis or biotransformation of mono-, sesqui-, tri-, or tetraterpenes, were screened for their ability to cleave beta,beta-carotene to flavor compounds. Ten strains discolored a beta,beta-carotene-containing growth agar, indicating efficient degradation of beta,beta-carotene. Dihydroactinidiolide was formed as the sole conversion product of beta,beta-carotene in submerged cultures of Ganoderma applanatum, Hypomyces odoratus, Kuehneromyces mutabilis, and Trametes suaveolens. When mycelium-free culture supernatants from five species were applied for the conversions, nearly complete degradation of beta,beta-carotene was observed after 12 h. Carotenoid-derived volatile products were detected in the media of Ischnoderma benzoinum, Marasmius scorodonius, and Trametes versicolor. beta-Ionone proved to be the main metabolite in each case, whereas beta-cyclocitral, dihydroactinidiolide, and 2-hydroxy-2,6,6-trimethylcyclohexanone were formed in minor quantities. Using a photometric bleaching test, the beta,beta-carotene cleaving enzyme activities of M. scorodonius were partially characterized.
Assuntos
Fungos/metabolismo , beta Caroteno/metabolismo , Ágar , Basidiomycota/enzimologia , Biodegradação Ambiental , Meios de Cultura , Aromatizantes/metabolismo , Fungos/classificação , Fungos/enzimologia , Fungos/crescimento & desenvolvimento , Norisoprenoides/metabolismo , Oxirredução , VolatilizaçãoRESUMO
Controversy exists as to whether chin tap force and bite force affect the duration of the electromyographic silent period following the jaw jerk reflex during isometric contraction of the elevator muscles of the mandible. This study demonstrates that statistical trends exist showing increased silent period duration for increased tap forces and decreased silent period duration with increased bite forces.