Tatiana Souza Porto

Kinetic and Thermodynamic Investigation on Ascorbate Oxidase Activity and Stability of a Cucurbita maxima Extract

The kinetic and thermodynamic properties of ascorbate oxidase (AO) activity and stability of a Cucurbita maxima extract were investigated. Activity tests performed at 25 °C using initial ascorbic acid concentration in the range 50–750 μM allowed estimating the Michaelis constant for this substrate (Km = 126 μM) and the maximum initial rate of ascorbic acid oxidation (A0,max = 1.57 mM min−1). The main thermodynamic parameters of the enzyme reaction (ΔH* = 10.3 kJ mol−1; ΔG* = 87.2 kJ mol−1; ΔS* = –258 J mol−1 K−1) were estimated through activity tests performed at 25–48 °C. Within such a temperature range, no decrease in the initial reaction rate was detected. The long‐term thermostability of the raw extract was then investigated by means of residual activity tests carried out at 10–70 °C, which allowed estimating the thermodynamic parameters of the irreversible enzyme inactivation as well (ΔH*D = 51.7 kJ mol−1; ΔG*D = 103 kJ mol−1; ΔS*D = –160 J mol−1 K−1). Taking into account the specific rate of AO inactivation determined at different temperatures, we also estimated the enzyme half‐life (1047 min at 10 °C and 21.2 min at 70 °C) and predicted the integral activity of a continuous system using this enzyme preparation. This work should be considered as a preliminary attempt to characterize the AO activity of a C. maxima extract before its concentration by liquid‐liquid extraction techniques.

Partition of proteases from Lentinus citrinus DPUA 1535 by the Peg/Phosphate Aqueous Two-Phase System

A full two-level factorial design was employed to study the influence of PEG molar mass (MMPEG), PEG concentration (CPEG) and phosphate concentration (CPHOSPH) on proteases partition by Lentinus citrinus DPUA 1535 in a PEG/phosphate aqueous two-phase system (ATPS). For all ATPS studied, proteases partitioned for the top phase and the best proteases extraction condition was obtained with MMPEG = 6000 g mol-1, CPEG = 17.5% (w/w) and CPHOSPH = 25% (w/w) with (1.1) purification factor and (151%) activity yield. Findings reported here demonstrate a practical strategy that serves as a first step for proteases purification from crude extract by L. citrinus.

Purification of a fibrinolytic protease from Mucor subtilissimus UCP 1262 by aqueous two-phase systems (PEG/sulfate)

A fibrinolytic protease from M. subtilissimus UCP 1262 was recovered and partially purified by polyethylene glycol (PEG)/sodium sulfate aqueous two-phase systems (ATPS). The simultaneous influence of PEG molar mass, PEG concentration and sulfate concentration on the enzyme recovery was first investigated using a 2(3) full factorial design, and the Response Surface Methodology used to identify the optimum conditions for enzyme extraction by ATPS. Once the best PEG molar mass for the process had been selected (6000g/mol), a two-factor central composite rotary design was applied to better evaluate the effects of the other two independent variables. The fibrinolytic enzyme was shown to preferentially partition to the bottom phase with a partition coefficient (K) ranging from 0.2 to 0.7. The best results in terms of enzyme purification were obtained with the system formed by 30.0% (w/w) PEG 6000g/mol and 13.2% (w/w) sodium sulfate, which ensured a purification factor of 10.0, K of 0.2 and activity yield of 102.0%. SDS-PAGE and fibrin zymography showed that the purified protease has a molecular mass of 97kDa and an apparent isoelectric point of 5.4. When submitted to assays with different substrates and inhibitors, it showed selectivity for succinyl-l-ala-ala-pro-l-phenylalanine-p-nitroanilide and was almost completely inhibited by phenylmethylsulfonyl fluoride, behaving as a chymotrypsin-like protease. At the optimum temperature of 37°C, the enzyme residual activity was 94 and 68% of the initial one after 120 and 150min of incubation, respectively. This study demonstrated that M. subtilissimus protease has potent fibrinolytic activity compared with similar enzymes produced by solid-state fermentation, therefore it may be used as an agent for the prevention and therapy of thrombosis. Furthermore, it appears to have the advantages of low cost production and simple purification.

Lichtheimia blakesleeana as a new potencial producer of phytase and xylanase.

Brazil is known for its great potential for production of renewable resources such as agro-industrial residues. These residues can be used as alternative sources of new products. Meanwhile, solid-state fermentation, with its advantages of energy conservation and pollution reduction, has been identified as a process of great potential for the production of bioactive compounds, especially enzymes. In the present work, a 23 factorial design was used to evaluate the effects of pH, temperature and moisture on the production of phytase and xylanase by Lichtheimia blakesleeana URM 5604 through the fermentation of citrus pulp. Statistical analyses of the results showed that the only the pH influenced the production of these enzymes, with the best phytase production (264.68 U/g) ocurring at pH 6.0, 34 °C, initial moisture 50%, after 48 hours of culture. The best conditions for xylanase production (397.82 U/g) were fermentation for 120 hours at pH 4.0, 26 °C and initial moisture of 70%. The best parameters for the simultaneous production of phytase (226.92 U/g) and xylanase (215.59 U/g) were determined to be initial moisture of 50%, pH 6.0, 26 °C, and 48 hours of fermentation.

Large scale purification of Clostridium perfringens toxins: a review

Clostridium perfringens, a Gram-positive anaerobic bacterium, is widespread in the environment and commonly found in the intestines of animals, including humans. C. perfringens strains are classified into five toxinotypes (A, B, C, D and E) based on the production of four major toxins (±, ², µ, ¹). However the toxins (theta, delta, lambda and enterotoxin) are also synthesized by C. perfringens strain. Many attempts to purify the toxins produced by C. perfringens have been proposed. In this review we discuss the purification methods used to isolate toxins from C. perfringens reported in last four decades.

Biotechnological richness of the northeastern semi-arid region: antioxidant activity of casein hydrolysates from Moxotó goat milk (Capra hircus Linnaeus, 1758) obtained by papain action

This study aimed to identify antioxidant peptides from caprine casein hydrolysates by papain application using MALDI-TOF mass spectrometer, and a 2 2 full factorial design, with 4 axial points, in order to evaluate kinetic parameters (time and pH) effects on the degree of hydrolysis as well as the antioxidant activity of Moxoto goat milk casein peptides. Degree of hydrolysis was determined by total and soluble protein ratio in casein. Antioxidant activity was measured by ABTS method with 2, 2-cation-azinobis (3-ethylbenzothiazoline-6-sulfonic acid). TROLOX was used as standard. Peptide pattern and sequence of antioxidant amino acids were obtained using MALDI-TOF/MS. The highest degree of hydrolysis (28.5%) and antioxidant activity (2329.6 mmol.L TROLOX. mg –1 peptide) were observed in the permeate. NENLL, NPWDQVK and LLYQEPVLGPV peptides, detected in the permeate, were pointed as the responsible for antioxidant activity, suggesting their potential application as food supplement and pharmaceutical products.

Thermodynamic investigation of an alkaline protease from Aspergillus tamarii URM4634: A comparative approach between crude extract and purified enzyme

The thermostable crude proteolytic extract and purified protease produced by Aspergillus tamarii URM4634 were investigated at different temperatures. The activity results were used to estimate the activation energy of the hydrolysis reaction catalyzed by crude extract and purified protease (E*=34.2 and 16.2kJ/mol) as well as the respective standard enthalpy variations of reversible enzyme unfolding (ΔH°u=31.9 and 13.9kJ/mol). When temperature was raised from 50 to 80°C in residual activity tests, the specific rate constant of crude proteolytic extract thermoinactivation increased from 0.0072 to 0.0378min-1, while that of purified protease from 0.0099 to 0.0235min-1. These values, corresponding to half-life decreases from 96.3 to 18.3min and from 70.0 to 29.5min, respectively, enabled us to estimate the activation energy (E*d=49.7 and 28.8kJ/mol), enthalpy (ΔH*d=47.0 and 26.1kJ/mol), entropy (ΔS*d=-141.3 and -203.1J/molK) and Gibbs free energy (92.6≤ΔG*d≤96.6kJ/mol and 91.8≤ΔG*d≤98.0kJ/mol) of thermoinactivation. Such values suggest that this protease, which proved to be highly thermostable in both forms, could be profitably exploited in industrial applications. To the best of our knowledge, this is the first comparative study on thermodynamic parameters of a serine protease produced by Aspergillus tamarii URM4634.

Aqueous two-phase extraction for partial purification of Schizophyllum commune phytase produced under solid-state fermentation

Abstract This study describes a simple strategy for partial protein purification using an aqueous two- phase system (ATPS) of polyethyleneglycol (PEG)/sodium citrate. The protein studied was phytase produced by Schizophyllum commune under solid-state fermentation (SSF). A 24 experimental design (20 runs and 4 central points) was carried out with four factors (PEG molar mass, citrate concentration, PEG concentration, and pH) to evaluate the enzyme extraction. The responses’ partition coefficient (K), yield (Y), and the purification factor (PF) were analysed. The best system obtained was with 14% (w/w) sodium citrate, 22% (w/w) PEG with a molar mass of 1500 (g/mol), and pH of 7. The maximum partition coefficient (K) was 2.63 and also the citrate concentration had a positive effect. Under these conditions, in the top phase the highest phytase yield achieved was 367%, and the purification factor (PEG) was 5.43. Liquid–liquid extraction can therefore be used as a first step in the purification processes of phytase from S. commune.

Hydrophobicity-dependent effects of polymers on different protein conformations

We have previously shown that increasing the hydrophobicity of PEG by adding a methyl group to every other monomer unit allowed the resulting polymer to alter protein folding and inhibit protein aggregation to amyloid fibrils. As a continuation of this work, we analyzed here the effects of this substitution on the structural properties of proteins capable of adopting multiple conformations (folded, and different partially folded states, e.g. a molten globule-like intermediate) at mild denaturing conditions. To this end, we have selected several proteins (α-lactalbumin, apomyoglobin, carbonic anhydrase, staphylococcal nuclease, and cytochrome c) and examined them at different conditions where they exist in different partially folded conformations (pH, temperature, salt concentrations, presence of cofactors). We were especially interested in the relative sensitivity of partially folded (e.g. molten globule) conformations of these proteins to the presence of polymers as these conformations are often the most sensitive to the environment. We used far-UV CD to test the changes in the protein secondary structure, near-UV CD to monitor changes in the tertiary structure, and quenching of intrinsic protein fluorescence by acrylamide to evaluate changes in the solvent accessibility of aromatic residues. We found that the complexity of the effect of polymers on protein structure cannot be ascribed solely to macromolecular crowding since the behavior of proteins in solutions containing polymers is dependent on protein and polymer structure. We also cannot exclude the possibility that the structures of both proteins and polymers determine the balance between attractive and repulsive forces that drive protein–polymer interactions.

Optimization of production, biochemical characterization and in vitro evaluation of the therapeutic potential of fibrinolytic enzymes from a new Bacillus amyloliquefaciens

The capacity of fibrinolytic enzymes to degrade blood clots makes them of high relevance in medicine and in the pharmaceutical industry. In this work, forty-three microorganisms of the genus Bacillus were evaluated for their potential to produce fibrinolytic proteases. Thirty bacteria were confirmed as producers of fibrinolytic enzymes, the best results obtained for the strain Bacillus amyloliquefaciens UFPEDA 485. The optimization of the enzyme production conditions was done by a central composite design (CCD) star 23 that allowed to define the optimal conditions for soybean flour and glucose concentrations and agitation rate. The highest fibrinolytic activity (FA) of 813 U mL–1 and a degradation of blood clot in vitro of 62% were obtained in a medium with 2% (w/v) of soybean flour and 1% (w/v) glucose at 200 rpm after 48 h of cultivation, at pH 7.2 and 37 °C. The obtained fibrinolytic enzyme was characterized biochemically. Fibrinolytic activity was inhibited by PMSF (fluoride methylphenylsulfonyl - C7H7FO2S) 91.52% and EDTA (ethylenediaminetetraacetic acid - C10H16N2O8) 89.4%, confirming to be a serine-metallo protease. The optimum pH and temperature were 7.0 and 37 oC, respectively, and the enzyme was stable for 12 h. The fibrinolytic activity at physiological conditions of this enzyme produced by Bacillus amyloliquefaciens UFPEDA 485, as well as its long term stability, demonstrate that it has suitable characteristics for human and veterinary applications, and promises to be a powerful drug for the treatment of vascular diseases.