Osvaldo Cascone

Effect of a high-temperature stress on endo-β-mannanase and α- and β-galactosidase activities during tomato fruit ripening

Abstract Tomato fruit show physiological disorders when exposed to temperatures at or above 30 °C. It has been suggested that the α- and β-galactosidases (α- and β-Gal), as well as the endo-β-mannanase could play roles during fruit ripening. Their pattern of activities was followed in detached tomato fruit, and the degree of inhibition of the activity at high temperatures was evaluated. The potential resumption of the enzyme activity when fruits were transferred to a room-temperature environment was also studied. Mature-green fruits were incubated at: (a) 36 °C for 2 days, (b) 40 °C for 2 days, or (c) 40 °C for 2 days +36 °C for another 2 days. After heat treatments, fruits were transferred to a 21 °C dark environment. Control fruit were stored at 21 °C. In the control fruit, activity was higher for α-Gal than for β-Gal and revealed slightly increasing patterns toward the red-ripe stage. Endo-β-mannanase activity was not detected in mature-green tomatoes but appeared at the breaker stage and rose rapidly, reaching constant values at the red-ripe stage. The firmness and enzymatic activities of fruits initially treated at 36 °C for 2 days were similar to those of the control group. Galactosidase activity decreased in tomatoes incubated at 40 °C for 2 days; and in those whose heat treatment was extended another 2 days at 36 °C the α-and β-Gal reached a minimum of 1 5 and 1 25 , respectively, of the initial activity on the fourth day. The activity of the endo-β-mannanase could be detected only 6 days after the fruit had been transferred to a 21 °C environment and increased slowly up to 24% of that attained by the control group. α-Gal showed a low rate of recovery but β-Gal activity recovered after 16 days at 21 °C. Results could be associated with an active role of these enzymes on the fruit softening process.

Horseradish peroxidase extraction and purification by aqueous two-phase partition

The effect of poly(ethyleneglycol) (PEG) molecular weight, system pH, and sodium chloride concentration on the partitioning behavior of horseradish peroxidase fromArmomcia rusticana root extract was investigated in poly(ethyleneglycol)/sodium phosphate systems.PEG molecular weight strongly affects the enzyme partition coefficient, whereas pH variation from 5.5 to 8.0 has little effect. The addition of sodium chloride (8% w/w) to a PEG 1540/phosphate system, pH 7.0, raises the peroxidase partition coefficient 13.5-fold without important changes in that of total horseradish root proteins. Moreover, these conditions allow direct homogenization of theA. rusticana roots with the selected aqueous two-phase system with the clear top phase containing over 90% of the enzyme and the purification factor being 4.8.

Acid protease recovery from a solid-state fermentation system

Investigations have been carried out on an acid protease produced by Mucor bacilliformis under solid-state fermentation conditions as a model of recovery and purification of a protein from a solid culture medium. The leaching efficiency of sodium chloride solutions to recover the enzyme from the fermented mass was higher than that of non-ionic detergents (Triton X-100 or Tween 80) or distilled water. Concentration and coarse fractionation of the crude extract could be achieved with a high yield of protease by precipitation with ethanol or acetone and by ultrafiltration and diafiltration in the presence of NaCl. Diafiltration was selected for the prechromatographic treatment of the crude extract as it enhances the maximum fraction of enzyme bound to an anion-exchanger up to 0.9. An overall purification factor of 15.5 was reached, with a total enzyme recovery of 46%, with the purification procedure developed by using this methodology. The final product obtained was a highly purified food-grade protease preparation.

Purification and characterization of a milk clotting protease from Mucor Bacilliformis

An acid protease having milk clotting activity has been isolated fromMucor bacilliformis cultures. The enzyme was basically purified by ionic exchange chromatography. An average yield of 29 mg purified product was obtained from 100 mL crude extract. As purity criteria, SDS-PAGE, reverse-phase HPLC, and N-terminal analysis were performed. The protease is a protein composed of a single polypeptide chain with glycine at the N-terminus. The mol wt is approx 32,000, and its amino acid composition is very similar to those of other fungal proteases. As expected, its clotting activity was drastically inhibited by pepstatin A action. On the other hand, its instability against heat treatment and its clotting/proteolytic activity ratio indicate that it may be considered as a potential substitute for bovine chymosin. Index Entries:Mucor bacilliformis protease; milk clotting enzyme; acid protease; fungal protease; aspartyl protease.

Immobilized metal ion affinity hollow-fibre membranes obtained by the direct grafting technique

Abstract An immobilized metal ion affinity hollow fibre was prepared by radiation-induced direct grafting of glycidylmethacrylate (GMA) on hydrophilized polyethylene membranes. The epoxy group was converted into an iminodiacetate by iminodiacetic acid treatment. The effect of the radiation dose, salt inhibitors, methanol and GMA concentration, on the grafting degree was studied. The degree of grafting was closely related to the GMA concentration. Salt inhibitors failed in the production of a differential effect on the grafting and homopolymerization processes. Between 30 and 35% of methanol, there is a maximum yield, and no grafting was obtained with a methanol concentration above 50%. Water flux dropped exponentially with the increase in the grafting degree. Scanning-electron microscopy showed that the graft branches are formed on the pores. The pectinesterase adsorption capacity of the membranes was of the same order as of the commercial chelating soft gels.

Screening of One-Bead-One-Peptide Combinatorial Library Using Red Fluorescent Dyes. Presence of Positive and False Positive Beads

To screen one-bead-one-compound (OBOC) combinatorial libraries, tens of thousands to millions of compound beads are first mixed with a target molecule. The beads that interact with this molecule are then identified and isolated for compound structure determination. Here we describe an OBOC peptide library screening using streptavidin (SA) as probe protein, labeled with a red fluorescent dye and using the COPAS BIO-BEAD flow sorting equipment to separate fluorescent from nonfluorescent beads. The red dyes used were ATTO 590 and Texas Red. After incubating the library with the SA-red fluorescent dye conjugate, we isolated positive beads caused by peptide-SA interaction and false positive beads produced by peptide fluorescent dye interaction. These false positives were a drawback when sorting beads by COPAS. However,an in depth analysis of both kinds of beads allowed the differentiation of positives from false positives. The false positive beads showed bright homogeneous fluorescence, while positive beads had a heterogeneous fluorescence, exhibiting a characteristic halo appearance, with fluorescence intensity greatest at the bead surface and lowest in the core. The difference was more evident when using Texas Red instead of ATTO 590. Thus, positive beads could be manually separated from false positive ones. The beads were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Most of the sequences obtained from positive beads had the His-Pro-Gln motif. Peptides from false positive beads were rich in Leu/Ileu, His, Phe, and Tyr.

Partition behaviour and purification of a Mucor bacilliformis acid protease in aqueous two-phase systems

Abstract The partitioning of a Mucor bacilliformis acids protease, a potential substitute for bovine chymosin in cheese manufacture, was accomplished in various aqueous two-phase systems in order to investigate how changes in factors such as PEG (poly(ethylene glycol) molecular weight, pH and sodium chloride concentration, can modify the partition coefficient value. PEG/Reppal and PEG-phosphate systems were evaluated, in the presence of contaminating material from the solid substrate fermentation of the microorganism. When PEG-phosphate systems were analysed, it was found that K AP depended strongly on the PEG molecular weight (at pH 5·0, an increase in PEG molecular weight from 600 to 20 000 leads to a decrease in the K value from > 50 to 0·1). A dependence between K AP and system pH was also noticed, this effect being important at lower/intermediate PEG molecular weight. When PEG 1540 was used, a V-shaped distribution of K AP values was obtained, with a minimum at pH 5·0 (K = 1·40) and maxima at pH values of 3·0 (K > 40) and 5·8 (K = 14). Furthermore, the addition of NaCl led to an increase in K AP (for PEG 3350/phosphate at pH 5·0, K increased from 1·1 to > 35 when 1·0 mol kg −1 NaCl was added). Suitable conditions for enzyme purification were found in PEG 3350-phosphate systems at pH 3·0 and NaCl 1·0 mol kg −1 (K AP > 35, K CP = 0·10) and PEG-Reppal at pH 3·0, NaCl 1·5 mol kg −1 (K AP = 13, K CP = 0·32). In these systems, proteinaceous and particulate contaminating materials precipitated and adsorbed at the interphase, thus yielding a clear upper phase containing the purified enzyme. Furthermore, direct extraction of the fermented was performed using a PEG 20 000-Reppal-NaCl system (K AP = 14, K CP = 0·19, PF (purification factor) = 5·9). The enzyme can be recovered in the PEG 20 000-rich phase and back-extracted by adding salt (K AP = 0·25, K CP = 1·10, PF = 1·7). This method provides a simpler process for leaching and purification of an enzyme produced by solid-state fermentation.

Protein Adsorption onto Tentacle Cation-Exchange Hollow-Fiber Membranes

A sulfonic group (up to 200 μmol/mL) membrane was incorporated to epoxy‐activated microporous hollow fibers to obtain high‐capacity convective ion exchangers. The pure water flux through the membrane decreased exponentially with sulfonic group density and protein binding capacity increased accordingly. At sulfonic group density of 70 μmol/mL, the membrane lysozyme maximum binding capacity was 84 ± 9 mg/mL in comparison with its theoretical monolayer maximum binding capacity of 20 mg/mL, thus evidencing tentacle formation. After a cycle of adsorption in a 30 mM sodium phosphate buffer, pH 7.0, adsorbed lysozyme could be quantitatively recovered following elution with 0.5 M NaCl in the same buffer. Dynamic capacity for lysozyme was 67% of maximum binding, and this value did not change at space velocities ranging from 10 to 40 min−1 as shown by the superimposition of the corresponding breakthrough curves. A cartridge assembled with 21 fibers showed a dynamic‐to‐static capacity ratio for lysozyme of 0.60 with 1 mg/mL pure lysozyme solution, and 0.42 with a particulate feed composed of 1 mg/mL lysozyme and 0.1 mg/mL yeast.

Immobilization of soybean seed coat peroxidase on polyaniline: Synthesis optimization and catalytic properties

Soybean seed coat peroxidase (SBP) was immobilized on various polyaniline-based polymers (PANI), activated with glutaraldehyde. The most reduced polymer (PANIG2) showed the highest immobilization capacity (8.2 mg SBP g−1 PANIG2). The optimum pH for immobilization was 6.0 and the maximum retention was achieved after a 6-h reaction period. The efficiency of enzyme activity retention was 82%. When stored at 4°C, the immobilized enzyme retained 80% of its activity for 15 weeks as evidenced by tests performed at 2-week intervals. The immobilized SBP showed the same pH-activity profile as that of the free SBP for pyrogallol oxidation but the optimum temperature (55°C) was 10°C below that of the free enzyme. Kinetic analysis show that the Km was conserved while the specific Vmax dropped from 14.6 to 11.4 µmol min−1 µg−1, in agreement with the immobilization efficiency. Substrate specificity was practically the same for both enzymes. Immobilized SBP showed a greatly improved tolerance to different organic solvents; while free SBP lost around 90% of its activity at a 50% organic solvent concentration, immobilized SBP underwent only 30% inactivation at a concentration of 70% acetonitrile. Taking into account that immobilized HRP loses more than 40% of its activity at a 20% organic solvent concentration, immobilized SBP performed much better than its widely used counterpart HRP.

Antiproliferative effect of 1-deamino-8-D-arginine vasopressin analogs on human breast cancer cells

BACKGROUND Desmopressin (dDAVP), a synthetic nonapeptide derivative of arginine vasopressin, is a safe antidiuretic and hemostatic compound that acts as a selective agonist for the vasopressin V2 membrane receptor (V2R). It is known that dDAVP can inhibit progression of residual metastatic cells in preclinical models. Among other mechanisms, the compound induces an agonist effect on V2R present in tumor cells. RESULTS/DISCUSSION Looking for novel analogs with improved anti-tumor activity, positions 4 and 5, at the conformational peptide loop, were substituted. The analog [V(4)Q(5)]dDAVP ([4-valine 5-glutamine] desmopressin) exhibited a significantly higher antiproliferative effect than dDAVP in cultures of MCF-7, a V2R-expressing human breast carcinoma cell line. The chiral isomer of this analog and tetrapeptide fragments corresponding to the loop region were also assessed. CONCLUSION Preclinical evaluation of the anti-tumor activity of [V(4)Q(5)]dDAVP in animal models is warranted.