Assessment of the application of cathodic stripping voltammetry to the analysis of diazo reactive dyes and their hydrolysis products

Two reactive dyes, C.I. Reactive Red 120 (RR120) and C.I. Reactive Green 19 (RG19), each bearing two azo groups as the chromophoric moiety and two monochloro-s-triazine groups as reactive groups, can be detected at nanomolar levels using cathodic stripping voltammetry. Linear calibration graphs were obtained for both reactive dyes, from 0.015 to 0.14 mu mol l(-1) for RR120 in pH 4 buffer and from 0.012 to 0.26 mu mol l(-1) for RG19 in pH 3 buffer, using a preconcentration at 0 V during 180 and 240 s on the mercury electrode, respectively. (C) 2001 Elsevier B.V. Ltd. All rights reserved.

Assignment of the disulfide bridges in bothropstoxin-I, a myonecrotic Lys49 PLA(2) homolog from Bothrops jararacussu snake venom

Bothropstoxin-I (BthTX-1), a Lys49 phospholipase A(2) homolog with no apparent catalytic activity, was first isolated from Bothrops jararacussu snake venom and completely sequenced in this laboratory. It is a 121-amino-acid single polypeptide chain, highly myonecrotic, despite its inability to catalyze hydrolysis of egg yolk phospholipids, and has 14 half-cystine residues identified at positions 27, 29, 44, 45, 50, 51, 61, 84, 91, 96, 98, 105, 123, and 131 (numbering according to the conventional alignment including gaps, so that the last residue is Cys 131). In order to access its seven disulfide bridges, two strategies were followed: (1) Sequencing of isolated peptides from (tryptic + SV8) and chymotryptic digests by Edman-dansyl degradation; (2) crystallization of the protein and determination of the crystal structure so that at least two additional disulfide bridges could be identified in the final electron density map. Identification of the disulfide-containing peptides from the enzymatic digests was achieved following the disappearance of the original peptides from the HPLC profile after reduction and carboxymethylation of the digest. Following this procedure, four bridges were initially identified from the tryptic and SV8 digests: Cys50-Cys131, Cys51-Cys98, Cys61-Cys91, and Cys84-Cys96. From the chymotryptic digest other peptides were isolated either containing some of the above bridges, therefore confirming the results from the tryptic digest, or presenting a new bond between Cys27 and Cys123. The two remaining bridges were identified as Cys29-Cys45 and Cys44-Cys105 by determination of the crystal structure, showing that BthTX-1 disulfide bonds follow the normal pattern of group II PLA(2)s.

Controlled hydrolysis of cheese whey proteins using trypsin and alpha-chymotrypsin

This study examined the production of protein hydrolysates with controlled composition from cheese whey proteins. Cheese whey was characterized and several hydrolysis experiments were made using whey proteins and purified beta -lactoglobulin, as substrates, and trypsin and a-chymotrypsin, as catalysts, at two temperatures and several enzyme concentrations. Maximum degrees of hydrolysis obtained experimentally were compared to the theoretical values and peptide compositions were calculated. For trypsin, 100% of yield was achieved; for alpha -chymotrypsin, hydrolysis seemed to be dependent on the oligopeptide size. The results showed that the two proteases could hydrolyze beta -lactoglobulin. Trypsin and alpha -chymotrypsin were stable at 40 degreesC, but a sharp decrease in the protease activity was observed at 55 degreesC.

Enzymatic Logic Gates with Noise-Reducing Sigmoid Response

Biochemical computing is an emerging field of unconventional computing that attempts to process information with biomolecules and biological objects using digital logic. In this work we survey filtering in general, in biochemical computing, and summarize the experimental realization of an and logic gate with sigmoid response in one of the inputs. The logic gate is realized with electrode-immobilized glucose-6-phosphate dehydrogenase enzyme that catalyzes a reaction corresponding to the Boolean and functions. A kinetic model is also developed and used to evaluate the extent to which the performance of the experimentally realized logic gate is close to optimal.

Thermal behaviour of corn starch granules under action of fungal alpha-amylase

Corn starch, partially hydrolyzed by fungal alpha-amylase was investigated by using thermal analysis, microscopy and X-ray diffraction. After enzymatic treatment lower degradation onset temperatures were observed. DSC analysis showed almost similar range of gelatinization temperature, however, the enthalpies of gelatinization increased for the partially hydrolyzed starch granules. According to the X-ray diffraction analysis, stronger cereal pattern peaks were recognized after enzymatic digestion. The results suggested that the hydrolysis was more pronounced in the amorphous part of the starch granules.

Characterization of glycerol kinase from baker's yeast: Response surface modeling of the enzymatic reaction

The present study describes a methodology of dosage of glycerol kinase (GK) from baker's yeast. The standardization of the activity of the glycerol kinase from baker's yeast was accomplished using the diluted enzymatic preparation containing glycerol phosphate oxidase (GPO) and glycerol kinase. The mixture was incubated at 60 degrees C by 15 min and the reaction was stopped by the SDS solution addition. A first set of experiments was carried out in order to investigate the individual effect of temperature (7), pH and substrate concentration (S), on GK activity and stability. The pH and temperature stability tests showed that the enzyme presented a high stability to pH 6.0-8.0 and the thermal stability were completely maintained up to 50 degrees C during 1 h. The K(m) of the enzyme for glycerol was calculated to be 2 mM and V(max) to be 1.15 U/mL. In addition, modeling and optimization of reaction conditions was attempted by response surface methodology (RSM). Higher activity values will be attained at temperatures between 52 and 56 degrees C, pH around 10.2-10.5 and substrate concentrations from 150 to 170 mM.This low cost method for glycerol kinase dosage in a sequence of reactions is of great importance for many industries, like food, sugar and alcohol. RSM showed to be an adequate approach for modeling the reaction and optimization of reaction conditions to maximize glycerol kinase activity. (C) 2007 Elsevier B.V. All rights reserved.

Evaluation of 316L Stainless Steel Corrosion Resistance in Solution Simulating the Acid Hydrolysis of Biomass

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

Effects of HCl on the ultrasound catalyzed TEOS hydrolysis as determined by a calorimetric study

The acid catalyzed and ultrasound stimulated hydrolysis of solventless tetraethoxysilane-water mixtures was studied at 39°C as a function of HCl added to the mixtures (log[HCl]-1 ranged from 0.8 to 2.0), The reaction was carried out in a specially designed device, in which a steady state heat flow is maintained, while sonication is taking place, if no reaction is expected to occur. The exothermal hydrolysis reaction causes an increasing temperature (ΔTt) as a function of the reaction time, t. The isothermal hydrolysis rate constant, k, has been evaluated from the experimental ΔTt versus t data, after corrections for the increasing temperature effects, by using a method resulting from our theoretical modeling based on a dissolution and reaction mechanism. The hydrolysis rate constant fits closely a k α [H+] law as expected for this kind of hydrogen-ion catalyzed reaction.

Comparative time-course study of aminoacyl- and dipeptidyl-resin hydrolysis

The classic hydrolysis procedure for quantification of resin-bound aminoacyl and peptidyl groups with 12 N HCl: propionic acid was recvaluated by studying the influence of the nature of the resin and the resin-bound group. Their stability during acid hydrolysis was dependent on the C-terminal amino acid, and the order of acid stability was Phe > Val > Gly. Otherwise, the dipeptides Ala-Gly, Ala-Val, and Ala-Phe displayed enhanced rates of hydrolysis of the resin if compared with their parent aminoacyl groups. Amongthe resins assayed, the order of acid stability was: benzhydrylamine-resin > p-methylbenzhydrylamine-resin ≅4-(oxymethyl)-phenylacetamidomethyl-resin > chloromethyl-copolymer of styrene-1%-divinylbenzene. Important for peptide synthesis method, the findings demonstrate that longer hydrolysis times than previously recommended in the literature (1 h at 130°C and 15 min at 160°C for peptides attached to the chloromethyl-copolymer of styrene-1%-divinylbenzene) are necessary for the quantitative acid-catalyzed cleavage of some resin-bound groups. The observed broad range of hydrolysis time varied from less than 1 h to about 100 h.

Effect of micelles, microemulsions and other supramolecular aggregates on the hydrolysis of β-lactam antibiotics

A critical revision of the literature was made regarding the stability of β-lactam antibiotics in the presence of surfactants. The factors involved in the drug decomposition were analyzed in the development of the discussion. The analysis has indicated that some organized systems obtained from surfactants can be used to control rates and mechanisms of antibiotic decomposition. These organized systems can also be used to obtain specific information about the drug reactivity in a microenvironment similar to the site of pharmacological effect.

Formation of colloidal particles of hydrous iron oxide by forced hydrolysis

The mechanism of formation and growth of hydrous iron oxide (FeOOH) during the initial stages of forced hydrolyses of ferric chloride aqueous solution was studied by small angle X-ray scattering (SAXS). The effect of the hydrolysis temperature (60°C, 70°C and 80°C) and of the addition of urea on the formation of colloidal particles under isothermal conditions were investigated. Based on the experimental scattering functions in the Guinier range, we suggest the presence of elongated colloidal particles. The particle diameter and length, and their variation with time, were determined by fitting the form factor of prolate ellipsoids to the experimental scattering functions. We have assumed that our solutions are in a dilute state and that all colloidal particles are approximately of the same size. The colloidal particles have geometrical shapes similar to those of the subcrystals that build up the superstructure of β-FeOOH crystals, indicating that the formation of this hydrous iron oxide is governed by an aggregation process. Otherwise, the addition of urea hinders the growth and yields smaller particles, with a reduction in size greater than 50%. © 2000 Elsevier Science B.V. All rights reserved.

Assignment of the bisulfide bridges in bothropstoxin-I, a Myonecrotic Lys49 PLA2 homolog from bothrops jararacussu snake venom

Bothropstoxin-I (BthTX-I), a Lys49 phospholipase A2 homolog with no apparent catalytic activity, was first isolated from Bothrops jararacussu snake venom and completely sequenced in this laboratory. It is a 121-amino-acid single polypeptide chain, highly myonecrotic, despite its inability to catalyze hydrolysis of egg yolk phospholipids, and has 14 half-cystine residues identified at positions 27, 29, 44, 45, 50, 51, 61, 84, 91, 96, 98, 105, 123, and 131 (numbering according to the conventional alignment including gaps, so that the last residue is Cys 131). In order to access its seven disulfide bridges, two strategies were followed: (1) Sequencing of isolated peptides from (tryptic + SV8) and chymotryptic digests by Edman-dansyl degradation; (2) crystallization of the protein and determination of the crystal structure so that at least two additional disulfide bridges could be identified in the final electron density map. Identification of the disulfide-containing peptides from the enzymatic digests was achieved following the disappearance of the original peptides from the HPLC profile after reduction and carboxymethylation of the digest. Following this procedure, four bridges were initially identified from the tryptic and SV8 digests: Cys50-Cysl31, Cys51-Cys98, Cys61-Cys91, and Cys84-Cys96. From the chymotryptic digest other peptides were isolated either containing some of the above bridges, therefore confirming the results from the tryptic digest, or presenting a new bond between Cys27 and Cys 123. The two remaining bridges were identified as Cys29-Cys45 and Cys44-Cysl05 by determination of the crystal structure, showing that BthTX-I disulfide bonds follow the normal pattern of group II PLA2s. © 2001 Plenum Publishing Corporation.