The extraction and characterisation of a polysaccharide from Moniliophthora perniciosa CCMB 0257.

The alpha-D-glucans are worldwide acknowledged as powerful immune system stimulants found in several sources; however, the fungal-derived sources appear to respond with higher activity. The present study has investigated polysaccharide production in Moniliophthora perniciosa. The dry biomass was subjected to thermal treatment in alkaline solution after fermentation. The biopolymers dissolved in this solution were precipitated after three volumes of absolute ethanol were added to the supernatant. The pure polysaccharide MPS1 was obtained through molecular exclusion chromatography using the Sephacryl S-200 column. The HPLC-RI analysis showed that MPS1 was a glucose homopolysaccharide. Nuclear magnetic resonance (NMR) spectra indicated the α-form as the anomeric carbon configuration in glucose residue. The structure of the polysaccharide was further confirmed as (1→4)-α-D-glucan through the chemical shift of C4. The molecular weight of MPS1 was 31.0 kDa.

Extraction optimization and antinociceptive activity of (1→3)-ß-d-glucan from Rhodotorulamucilaginosa.

ß-d-glucans are polymers of d-glucose monomers found in the cell walls of many bacteria, plants, fungi and yeasts. A variety of ß-d-glucans differing in structures have been isolated from various sources and their biological activity to be regulated by various structural factors, such as the primary structure, molecular weight, solubility, and conformation. This study investigated the effect of extraction time and temperature on the yield of ß-d-glucan produced by Rhodotorulamucilaginosa. A statistical Doehlert design was applied to determine the important effects and interactions of these independent variables on the yield of ß-d-glucan, the dependent variable. Significant models were obtained. The best yield was of 25% obtained after 128min of extraction in a temperature of 72°C. The polysaccharides were characterized as (1⟶3)-ß-d-glucan by methods spectroscopic (FT-IR, (1)HNMR and (13)CNMR). In addition, the antinociceptive effect was evaluated using different experimental tests (acetic acid-induced writhing test, formalin test and tail immersion test). The (1⟶3)-ß-d-glucan showed a potent peripheral antinociceptive effect, possibly by the inhibition of inflammatory mediators.

Aqueous extraction of pectin from sisal waste.

In this work, sisal waste was used as a source of pectin. Sisal is known worldwide as a source of hard fibres, and Brazil is the largest producer of sisal, producing more than 246,000 tonnes. However, the process of removing the fibres of the sisal leaf generates 95% waste. This study investigated the effect of the liquid/solid ratio (%), time (min), and temperature (°C) on the yield of the pectin obtained from sisal waste by attractive environmentally friendly process. A statistical Box-Behnken design was applied to determine the important effects and interactions of these independent variables on the yield of pectin, the dependent variable. Significant models were obtained. The yield of the extracted pectin ranged from 4.61 to 19.2%. The conditions that produced the highest yield (19.2%) were a temperature of 85 °C, extraction time of 60 min and a liquid/solid ratio of 2%.

Purification, characterization and structural determination of chitinases produced by Moniliophthora perniciosa.

The enzyme chitinase from Moniliophthora perniciosa the causative agent of the witches' broom disease in Theobroma cacao, was partially purified with ammonium sulfate and filtration by Sephacryl S-200 using sodium phosphate as an extraction buffer. Response surface methodology (RSM) was used to determine the optimum pH and temperature conditions. Four different isoenzymes were obtained: ChitMp I, ChitMp II, ChitMp III and ChitMp IV. ChitMp I had an optimum temperature at 44-73ºC and an optimum pH at 7.0-8.4. ChitMp II had an optimum temperature at 45-73ºC and an optimum pH at 7.0-8.4. ChitMp III had an optimum temperature at 54-67ºC and an optimum pH at 7.3-8.8. ChitMp IV had an optimum temperature at 60ºC and an optimum pH at 7.0. For the computational biology, the primary sequence was determined in silico from the database of the Genome/Proteome Project of M. perniciosa, yielding a sequence with 564 bp and 188 amino acids that was used for the three-dimensional design in a comparative modeling methodology. The generated models were submitted to validation using Procheck 3.0 and ANOLEA. The model proposed for the chitinase was subjected to a dynamic analysis over a 1 ns interval, resulting in a model with 91.7% of the residues occupying favorable places on the Ramachandran plot and an RMS of 2.68.

Production, extraction and characterization of exopolysaccharides produced by the native Leuconostoc pseudomesenteroides R2 strain.

The genus Leuconostoc belongs to a group of lactic acid bacteria usually isolated from fermented vegetables, which includes species involved in the production of exopolysaccharides (EPS). These biopolymers possess considerable commercial potential. Because of the wide variety of industrial applications of EPS, this study aimed to produce and characterize the native exopolysaccharide strain Leuconostoc pseudomesenteroides R2, which was isolated from cabbage collected in a semi-arid region of Bahia. We employed the following conditions for the production of EPS: 10.7% sucrose, pH 8.2, without agitation and incubation at 28ºC for 30 hours. The fermentation broth was treated with ethanol and generated two types of polysaccharide substances (EPS I and EPS II). The identification of EPS I and EPS II was conducted using FT-IR, (1)H, (13)C and DEPT-135 NMR spectra. The two substances were identified as linear dextran α polysaccharides (1 → 6) which indicated different characteristics with respect to thermal analysis and density of free packaging, viscosity and time of solubilization. Both dextrans are of low density, possess high thermal stability and exhibited the behavior characteristic of pseudoplastic polymers.

Synthesis of naringin 6"-ricinoleate using immobilized lipase.

BACKGROUND: Naringin is an important flavanone with several biological activities, including antioxidant action. However, this compound shows low solubility in lipophilic preparations, such as is used in the cosmetic and food industries. One way to solve this problem is to add fatty acids to the flavonoid sugar unit using immobilized lipase. However, there is limited research regarding hydroxylation of unsaturated fatty acids as an answer to the low solubility challenge. In this work, we describe the reaction of naringin with castor oil containing ricinoleic acid, castor oil's major fatty acid component, using immobilized lipase from Candida antarctica. Analysis of the 1H and 13 C NMR (1D and 2D) spectra and literature comparison were used to characterise the obtained acyl derivative. RESULTS: After allowing the reaction to continue for 120 hours (in acetone media, 50°C), the major product obtained was naringin 6″-ricinoleate. In this reaction, either castor oil or pure ricinoleic acid was used as the acylating agent, providing a 33% or 24% yield, respectively. The chemical structure of naringin 6″-ricinoleate was determined using NMR analysis, including bidimensional (2D) experiments. CONCLUSION: Using immobilized lipase from C. antarctica, the best conversion reaction was observed using castor oil containing ricinoleic acid as the acylating agent rather than an isolated fatty acid. GRAPHICAL

Production, characterization and application of inulinase from fungal endophyte CCMB 328.

Inulinase (ß-2,1-D- fructan fructanohydrolase), EC 3.2.1.7, targets the ß-2,1 linkage of inulin, a polyfructan consisting of linear ß-2,1 linked fructose, and hydrolyzes it into fructose. This use provides an alternative to produce fructose syrup through the hydrolysis of inulin. The objective of this work was to study the production, characterization and applications of inulinases from the fungal endophyte CCMB 328 isolated from the Brazilian semi-arid region. Response Surface Methodology (RSM) was employed to evaluate the effect of variables (concentration of glucose and yeast extract), on secreted inulinase activities detected in the culture medium and also in the inulin hydrolysis. The results showed that the best conditions for inulinase production by CCMB 328 are 9.89 g / L for glucose and 1.09 g / L for yeast extract. The concentration of 0.20 mol/L of NaCl and KCl increased the activity of inulinase from CCMB 328 by approximately 63% and 37%, respectively. The results also showed that the inulinase has potential for inulin hydrolysis, whose conversion yields roughly 72.48 % for an initial concentration of inulin at 1% (w/v).

Production, purification and characterization of a thermostable ß-1,3-glucanase produced by Moniliophthora perniciosa.

The enzyme glucanase from Moniliophthora perniciosa was produced in liquid medium and purified from the culture supernatant. A multivariate statistical approach (Response Surface Methodology - RSM) was employed to evaluate the effect of variables, including inducer (yeast extract) and fermentation time, on secreted glucanase activities M. perniciosa detected in the culture medium. The crude enzyme present in the supernatant was purified in two steps: precipitation with ammonium sulfate (70%) and gel filtration chromatography on Sephacryl S-200. The best inducer and fermentation time for glucanase activities were 5.9 g L(-1) and 13 days, respectively. The results revealed three different isoforms (GLUI, GLUII and GLUIII) with purification factors of 4.33, 1.86 and 3.03, respectively. The partially purified enzymatic extract showed an optimum pH of 5.0 and an optimum temperature of 40°C. The enzymatic activity increased in the presence of KCl at all concentrations studied. The glucanase activity was highest in the presence of 0.2 M NaCl. The enzyme showed high thermal stability, losing only 10.20% of its specific activity after 40 minutes of incubation at 90°C. A purified enzyme with relatively good thermostability that is stable at low pH might be used in future industrial applications.

Production, purification and characterization of a thermostable β-1,3-glucanase (laminarinase) produced by Moniliophthora perniciosa

The enzyme glucanase from Moniliophthora perniciosa was produced in liquid medium and purified from the culture supernatant. A multivariate statistical approach (Response Surface Methodology - RSM) was employed to evaluate the effect of variables, including inducer (yeast extract) and fermentation time, on secreted glucanase activities M. perniciosa detected in the culture medium. The crude enzyme present in the supernatant was purified in two steps: precipitation with ammonium sulfate (70 percent) and gel filtration chromatography on Sephacryl S-200. The best inducer and fermentation time for glucanase activities were 5.9 g L-1 and 13 days, respectively. The results revealed three different isoforms (GLUI, GLUII and GLUIII) with purification factors of 4.33, 1.86 and 3.03, respectively. The partially purified enzymatic extract showed an optimum pH of 5.0 and an optimum temperature of 40°C. The enzymatic activity increased in the presence of KCl at all concentrations studied. The glucanase activity was highest in the presence of 0.2 M NaCl. The enzyme showed high thermal stability, losing only 10.20 percent of its specific activity after 40 minutes of incubation at 90°C. A purified enzyme with relatively good thermostability that is stable at low pH might be used in future industrial applications.

A enzima glucanase de Moniliophthora perniciosa foi produzida em meio líquido e purificada a partir do sobrenadante da cultura. A metodologia de superfície de resposta (MSR) foi usada para avaliar os efeitos das variáveis, incluindo indutor (extrato de levedura) e tempo de fermentação, na atividade da glucanase de M. perniciosa detectada no meio de cultura. A enzima presente no sobrenadante foi purificada em duas etapas: precipitação com sulfato de amônio (70 por cento) e cromatografia de filtração em gel em Sephacryl S-200. A produção da enzima glucanase foi maior na concentração de 5,9 g L-1 de extrato de levedura e 13 dias de fermentação. Os resultados mostraram três diferentes isoformas (GLUI, GLUII e GLUIII) com fatores de purificação de 4,33, 1,86 e 3,03, respectivamente. O extrato enzimático parcialmente purificado mostrou um pH ótimo de 5,0 e uma temperatura ótima de 40°C. A atividade enzimática aumentou na presença de KCl em todas as concentrações estudadas. A atividade da glucanase foi maior na presença de NaCl 0,2 M. A enzima apresentou alta estabilidade térmica, perdendo apenas 10,20 por cento de sua atividade específica após 40 minutos de incubação a 90°C. Os resultados de termoestabilidade e a atividade em baixo pH mostraram que a enzima glucanase de M. perniciosa tem características promissoras para futuras aplicações industriais.

Polygalacturonase secreted by yeasts from Brazilian semi-arid environments.

Microbial pectinolytic enzymes are known to play a commercially important role in a number of industrial processes. The objective of this study was to investigate the extracellular polygalacturonases of yeasts isolated from Brazilian semi-arid environments. Among the 250 colonies tested, only 33 produced extracellular polygalacturonases: Aureobasidium pullulans (18 isolates), Candida boidinii (one isolate), Trichosporonoides sp. (three isolates), Kluyveromyces marxianus (one isolate), Cryptococcus liquefaciens (one isolate), Pseudozyma sp. (four isolates), and yeast-like related to fungal endophyte (five isolates). The highest activity of polygalacturonase was observed in Pseudozyma sp. CCMB 300 (14.17+/-0.08 micromol acid galacturonic released/min/mg protein). This study shows the potential of yeasts and yeast-like organisms isolated from Brazilian semi-arid environments to produce pectinolytic enzymes.

Thermostable inulinases secreted by yeast and yeast-like strains from the Brazilian semi-arid region.

The use of inulinases provides an alternative to the chemical process of inulin hydrolysis to obtain fructose syrup, and can reduce processing steps, time, and costs in the food industry. The objective of this work was to screen the thermostable inulinases produced by yeast and yeast-like strains isolated from the Brazilian semi-arid region. Thermostability was studied at different temperatures (60 degrees C, 70 degrees C, 80 degrees C and 90 degrees C) and for increasing periods of time (0-50 min). Thirty-three microorganisms were tested, and 27 showed inulinase activity with specific activities ranging from 0.98 to 73.79 micromol/mg protein/min. Three strains (CCMB 300, CCMB327 and CCMB328) showed the desired combination of high specific activity and a small reduction in residual activity when submitted to heat treatment (>or=60 degrees C). Our results indicate that the inulinases produced by these three yeast strains from the Brazilian semi-arid region have great potential to be used for inulin hydrolysis in the food industry.

Homology modelling of pyrophosphosrylase, enzyme involved in chitin pathway of Moniliophthora perniciosa.

Moniliophthora perniciosa (Sthael) (Singer) Phillips-Mora is the causal agent of witches' broom disease, which can infect Theobroma cacao decreasing the production of cocoa about 60%. M. perniciosa has a set of potential enzymes that can be useful targets for design of new inhibitors. After the release of the aminoacid sequence of pyrophosphorylase of M. perniciosa, a comparative modelling approach was carried out to obtain the 3D structure of this target. This model can be useful to develop new inhibitors against witches' broom disease.

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

The guava pectin methylesterase (PME) specific activity and vitamin C were assayed in samples from different phases of guava fruit development. The PME enzyme from guava was extracted with borate-acetate buffer, 50 mol/l, pH 8.0, in the presence of NaCl 0.3 mol/l. The results showed PME optimum activity at pH 9 and 95 degrees C, and it is a thermostable enzyme. Guava PME retained 96.8% of activity after 300 min in 90 degrees C. Electrophoresis showed that guava PME contained two isoforms, one with 57 kDa molecular mass. The analyses of the different phases of guava maturation showed that ascorbic acid decreases during the maturation process, but PME activity increases with maturation.