Partial purification, heat stability and kinetic characterization of the pectinmethylesterase from Brazilian guava, Paluma cultivars

Pectinmethylesterase (PME) was extracted from guava fruit (Psidium guajava L.), cultivar Paluma, by 70% ammonium sulphate saturation and partially purified by gel filtration on Sephadex G100. Gel filtration showed PME isoenzymes with different values of molecular mass. Two samples were examined: concPME (70% saturation by ammonium sulphate) and Iso4 PME (one of the isoforms from gel filtration with the greatest specific activity). Optimum pH of the enzyme (for both samples) was 8.5 and optimum temperature ranged from 75 and 85 degrees C. The optimum sodium chloride concentration was 0.15 M. The K-M and V-max ranged from 0.32 to 0.23 mg m1(-1) and 244 to 53.2 mu mol/min, respectively, for concPME and Iso4PME. The activation energies (E-a) were 64.5 and 103 kJ/mol, respectively, for concPME and Iso4PME. Guava PME, cv Paluma, is a very thermostable enzyme, showing great heat stability at all temperatures studied. (c) 2005 Elsevier Ltd. All rights reserved.

Peroxidase from peach fruit: Thermal stability

Peroxidase from peach fruit was purified 28.9-fold by DEAE-cellulose, Sephadex G-100 and hydroxylapatite chromatography. The purified enzyme showed only one peak of activity with an optimum pH of 5.0 and temperature of 40 degreesC. The calculated activation energy (Ea) for the reaction was 7.97 kcal/mol. The enzyme was heat-labile in the temperature range of 60 to 80 degreesC with a fast inactivation at 80 degreesC. PAGE of the inactivation course at 70 degreesC showed only one band of activity. Different sugars increased the heat stability of the activity in the following order: sucrose>lactose>glucose>fructose. Measurement of residual activity showed a stabilizing effect of sucrose at various temperature/sugar concentrations (10 to 40%, w/w) with the Ea for inactivation increasing with sucrose concentration from 0 to 20% (w/w). After inactivation at 70 degreesC and 75 degreesC the enzyme was able to be reactivated by up to 40% of the initial activity when stared at 30 degreesC.

Ionically bound peroxidase from peach fruit

Soluble, ionically bound peroxidase (POD) and polyphenoloxidase (PPO) were extracted from the pulp of peach fruit during ripening at 20degreesC Ionically bound form was purified 6.1 -fold by DEAE-cellulose and Sephadex G-100 chromatography. The purified enzyme showed only one peak of activity on Sephadex G-100 and PAGE revealed that the enzyme was purified by the procedures adopted. The purified enzyme showed a molecular weight of 29000 Da, maximum activity at pH 5.0 and at 40degreesC the calculated apparent activation energy (Ea) for the reaction was 10.04 kcal/mol. The enzyme was heat-labile in the temperature range of 60 to 75degreesC with a fast inactivation at 75degreesC Measurement of residual activity showed a stabilizing effect of sucrose at various temperature/sugar concentrations (0, 10, 20 %, w/w), with an activation energy (Ea) for inactivation increasing with sucrose concentration from 0 to 20% (w/w). The Km and V-max values were 9.35 and 15.38 mM for O-dianisidine and H2O2, respectively. The bound enzyme was inhibited competitively by (.)ferulic, caffeic and protocatechuic acids with different values of Ki,. L-cysteine, p-coumaric and indolacetic acid and Fe++ also inhibited the enzyme but at a lower grade. N-ethylmaleimide and p-CMB were not effective to inhibit the enzyme demonstrating the non-essentiality of SH groups.

Pectin methylesterase activity and ascorbic acid content from guava fruit, cv. Predilecta, in different phases of development

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

Partial purification and characterization of pectin methylesterase from acerola (Malpighia glabra L.)

The enzyme pectin methylesterase (PME) is present in acerola fruit and was partially purified by gel filtration on Sephadex G-100. The results of gel filtration showed different PME isoforms. The total PME (precipitated by 70% salt saturation) and one of these isoforms (fraction from Sephadex G-100 elution) that showed a molecular mass of 15.5 +/- 1.0 kDa were studied. The optimum pH values of both forms were 9.0. The total and the partially purified PME showed that PME specific activity increases with temperature, the total acerola PME retained 13.5% of its specific activity after 90 min of incubation at 98 degreesC. The partially purified acerola (PME isoform) showed 125.5% of its specific activity after 90 min of incubation at 98 degreesC. The K-m values of the total PME and the partially purified PME isoform were 0.081 and 0.12 mg/mL, respectively. The V-max values of the total PME and the partially purified PME were 2.92 and 6.21 mumol/min/mL/mg of protein, respectively.

Kinetic characterization of hypophosphatasia mutations with physiological substrates

We have analyzed 16 missense mutations of the tissue-nonspecific AP (TNAP) gene found in patients with hypophosphatasia. These mutations span the phenotypic spectrum of the disease, from the lethal perinatal/infantile forms to the less severe adult and odontohypophosphatasia. Site-directed mutagenesis was used to introduce a sequence tag into the TNAP cDNA and eliminate the glycosylphosphatidylinositol (GPI)-anchor recognition sequence to produce a secreted epitope-tagged TNAP (setTNAP). The properties of GPI-anchored TNAP (gpiTNAP) and setTNAP were found comparable. After introducing each single hypophosphatasia mutation, the setTNAP and mutant TNAP cDNAs were expressed in COS-1 cells and the recombinant flagged enzymes were affinity purified. We characterized the kinetic behavior, inhibition, and heat stability properties of each mutant using the artificial substrate p-nitrophenylphosphate (pNPP) at pH 9.8. We also determined the ability of the mutants to metabolize two natural substrates of TNAP, that is, pyridoxal-5'-phosphate (PLP) and inorganic pyrophosphate (PPi), at physiological pH. Six of the mutant enzymes were completely devoid of catalytic activity (R54C, R54P, A94T, R206W, G317D, and V365I), and 10 others (A16V, A115V, A160T, A162T, E174K, E174G, D277A, E281K, D361V, and G439R) showed various levels of residual activity. The A160T substitution was found to decrease the catalytic efficiency of the mutant enzyme toward pNPP to retain normal activity toward PPi and to display increased activity toward PLP. The A162T substitution caused a considerable reduction in the pNPPase, PPiase, and PLPase activities of the mutant enzyme. The D277A mutant was found to maintain high catalytic efficiency toward pNPP as substrate but not against PLP or PPi. Three mutations ( E174G, E174K, and E281K) were found to retain normal or slightly subnormal catalytic efficiency toward pNPP and PPi but not against PLP. Because abnormalities in PLP metabolism have been shown to cause epileptic seizures in mice null for the TNAP gene, these kinetic data help explain the variable expressivity of epileptic seizures in hypophosphatasia patients.

Purification and characterization of pectin methylesterase from acerola (Malpighia glabra L.)

The enzyme pectinmethylesterase (PME) from acerola was extracted and purified by gel anion-exchange chromatography (Q Sepharose) and filtration on Sephadex G-100. The results showed two different PME isoforms (PME1 and PME2), with molecular masses of 25.10 and 5.20 kDa, respectively. PMEI specific activity increased by 9.63% after 60 min incubation at 98 degrees C, while PME2 retained 66% of its specific activity under the same conditions. The K-m values of PMEI, PME2 and concentrated PME were 0.94, 0.08 and 0.08mg mL(-1), respectively. The V-max value of PMEI, PME2 and concentrated were 204.08, 2, 158.73 and 2.92 mu mol min(-1) mg(-1) protein, respectively. (c) 2007 Society of Chemical Industry.

Thermal structure, heat content and stability of two lakes in The National Park of Rio Doce Valley (Minas Gerais, Brazil)

The thermal structure, heat content and stability were studied in Lakes Dom Helvécio and Carioca during an annual cycle. It was found that the maximum heat content, stability and work of the wind in Lake Dom Helvécio correspond to two, four and four times, respectively, the values for the Lake Carioca. These difference can be attributed to morphometric differences in the lakes. A long-term record of heat content and stability for lake Carioca is also presented. Diel variations were studied in summer and winter. The tropicality of the lakes is discussed and compared with other lacustrine systems. © 1989 Kluwer Academic Publishers.

Development of a Validated Stability-Indicating LC Assay and Stress Degradation Studies of Linezolid in Tablets

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

Effect of heat treatment on stainless steel orthodontic wires

This study evaluated the effect of heat treatment on CrNi stainless steel orthodontic archwires. Half of forty archwires of each thickness - 0.014 (0.35 mm), 0.016 (0.40 mm), 0.018 (0.45 mm) and 0.020 (0.50 mm) (totalling 160 archwires) - were subjected to heat treatment while the remainder were not. All of the archwires had their individual thickness measured in the anterior and posterior regions using AutoCad 2000 software before and after compressive and tensile strength testing. The data was statistically analysed utilising multivariance ANOVA at a 5% significance level. All archwires without heat treatment that were subjected to tensile strength testing presented with anterior opening, which was more accentuated in the 0.020 archwires. In the posterior region, the opening produced by the tensile force was more accentuated in the archwires without heat treatment. There was greater stability in the thermally treated archwires, especially those subjected to tensile strength testing, which indicates that the heat treatment of orthodontic archwires establishes a favourable and indispensable condition to preserve the intercanine width.

Influence of microwave disinfection on the dimensional stability of intact and relined acrylic resin denture bases

Statement of problem. Microwave irradiation has been suggested as a method to disinfect denture bases. However, the effect of microwave heating on the dimensional stability of the relined denture bases is unknown.Purpose. The purpose of this study was to evaluate the dimensional stability of intact and relined acrylic resin denture bases after microwave disinfection.Material and methods. A standard brass cast simulating an edentulous maxillary arch was machined and used to fabricate 2- and 4-mm-thick denture bases (n=200), which were processed with heat-polymerized acrylic resin (Lucitone 550). The 2-mm thick-specimens (n=160) were relined with 2 mm of autopolymerizing resin (Tokuso Rebase Fast, Ufi Gel Hard, Kooliner, or New Truliner). Distances between 5 removable pins on the standard brass cast were measured with a Nikon optical comparator, and the area (mm(2)) formed by the distance between 5 pins was calculated and served as baseline. During fabrication, the pins were transferred to the intaglio surface of the specimens. Differences between the baseline area and those subsequently determined for the specimens were used to calculate the percent dimensional changes. The intact and relined denture bases were divided into 4 groups (n=10) and evaluated after: polymerization (control group P); 1 cycle of microwave disinfection (MW); daily microwave disinfection for 7 days (control group MW7); water storage for 7 days (WS7). Microwave irradiation was performed for 6 minutes at 650 W. Data were analyzed using 2-way ANOVA followed by Tukey's test (alpha=.05).Results. Intact specimens and those relined with Kooliner and New Truliner showed increased shrinkage after 1 (P=.05, .018, and .001, respectively) and 7 (P <.001, .003, and <.001, respectively) cycles of microwave disinfection. With the exception of specimens relined with Kooliner, intact specimens showed greater shrinkage than the relined specimens after 7 cycles of microwave disinfection.Conclusions. Microwave disinfection produced increased shrinkage of intact specimens and those relined with New Truliner and Kooliner.

Influence of Microwave Disinfection on the Dimensional Stability of Denture Reline Polymers

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

LOSS of EXPONENTIAL STABILITY FOR A THERMOELASTIC SYSTEM WITH MEMORY

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

THERMAL REGIME AND STABILITY OF JURUMIRIM RESERVOIR (PARANAPANEMA RIVER, SAO-PAULO, BRAZIL)

Thermal regime and stability in Jurumirim Reservoir (São Paulo, Brazil) were studied for a year. Isothermy and thermal stratification were observed from June to September and from October to March, respectively. The annual heat budget was 14.0 Kcal . cm-2 and average heat gain per day was 67.2 cal . cm-2 . day-1. Maximum stability and wind work reached 324.2 and 3,037.5 g . cm . cm-2, respectively. According to thermal structure and heat content, Jurumirim Reservoir was classified as a warm tropical monomictic lake.

Quantitative Phase Analyses Through The Rietveld Method with X-Ray Powder Diffraction Data of Heat-Treated Carbamazepine Form III

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