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In plants, polygalacturonase-inhibiting proteins (PGIPs) play critical roles for resistance to fungal disease by inhibiting the pectin-depolymerizing activity of endopolygalacturonases (PGs), one type of enzyme secreted by pathogens that compromises plant cell walls and leaves the plant susceptible to disease. Here, the interactions between PGIPs from Phaseolus vulgaris (PvPGIP1 and PvPGIP2) and PGs from Aspergillus niger (AnPG2), Botrytis cinerea (BcPG1 and BcPG2), and Fusarium moniliforme (FmPG3) were reconstituted through a yeast two hybrid (Y2H) system to investigate the inhibition efficiency of various PvPGIP1 and 2 truncations and mutants. We found that tPvPGIP2_5-8, which contains LRR5 to LRR8 and is only one-third the size of the full length peptide, exhibits the same level of interactions with AnPG and BcPGs as the full length PvPGIP2 via Y2H. The inhibitory activities of tPvPGIP2_5-8 on the growth of A. niger and B. cinerea were then examined and confirmed on pectin agar. On pectin assays, application of both full length PvPGIP2 and tPvPGIP2_5-8 clearly slows down the growth of A. niger and B. cinerea. Investigation on the sequence-function relationships of PGIP utilizing a combination of site directed mutagenesis and a variety of peptide truncations suggests that LRR5 could have the most essential structural feature for the inhibitory activities, and may be a possible target for the future engineering of PGIP with enhanced activity. This study highlights the potential of plant-derived PGIPs as a candidate for future in planta evaluation as a pest control agent.

During ripening, European pear (Pyrus communis L. cv. ‘La France’) fruit undergo dramatic softening in response to increased ethylene production, whereas Chinese pear (Pyrus bretschneideri Rehd. cv. ‘Yali’) fruit remain firm, despite producing large amounts of ethylene. The molecular basis of this differential softening behavior is not well understood. In this study, we combined a yeast-based signal sequence trap (YSST) and macroarray gene expression analysis to identify putative genes encoding secreted proteins that control pear fruit softening. We identified 22 cDNAs annotated as encoding proteins with diverse cell wall-associated functions that were up- or down-regulated during fruit ripening in ‘La France’. Gene expression analysis in fruit that were treated with the ethylene perception inhibitor 1-methylcyclopropene (1-MCP) at 4 d after the onset of ripening revealed that 16 of the targeted genes are ethylene-regulated, while the others appear to be ethylene independent. Comparative gene expression analyses of ‘La France’ and ‘Yali’ fruit during ripening suggested that four ethylene-regulated cDNAs encoding cell wall modifying proteins, contig 2 (polygalacturonase 3), contig 15 (expansin), contig 19 (expansin) and contig 55 (pectate lyase) contribute to the different softening behaviors of ‘La France’ and ‘Yali’ fruit. Additionally, one ethylene-independent cell wall related gene, contig 36 (expansin), and three genes encoding proteins of unknown function, contigs 1, 13 and contig 75 showed differential expression between ‘La France’ and ‘Yali’ fruit during ripening. The results presented herein represent promising candidates for future functional analysis and elucidation of softening mechanisms.

To disentangle the role of polygalacturonase (PG) genes in strawberry softening, the two PG genes most expressed in ripe receptacles, FaPG1 and FaPG2, were down-regulated. Transgenic ripe fruits were firmer than those of the wild type when PG genes were silenced individually. Simultaneous silencing of both PG genes by transgene stacking did not result in an additional increase in firmness. Cell walls from ripe fruits were characterized by a carbohydrate microarray. Higher signals of homogalacturonan and rhamnogalacturonan I pectin epitopes in polysaccharide fractions tightly bound to the cell wall were observed in the transgenic genotypes, suggesting a lower pectin solubilization. At the transcriptomic level, the suppression of FaPG1 or FaPG2 alone induced few transcriptomic changes in the ripe receptacle, but the amount of differentially expressed genes increased notably when both genes were silenced. Many genes encoding cell wall-modifying enzymes were down-regulated. The expression of a putative high affinity potassium transporter was induced in all transgenic genotypes, indicating that cell wall weakening and loss of cell turgor could be linked. These results suggest that, besides the disassembly of pectins tightly linked to the cell wall, PGs could play other roles in strawberry softening, such as the release of oligogalacturonides exerting a positive feedback in softening.

Tannins are present in grape skins and seeds from where they are transferred into the must-wine matrix during the maceration stages of winemaking. However, tannin transfer is often incomplete. This could be due, among other reasons, to tannins becoming bound to grape cell wall polysaccharides, including soluble polymers, which are released during vinification and are present in high concentrations in the must/wine. The use of cell wall deconstructing enzymes offers the possibility of reducing these interactions, releasing more tannins into the final wine. The main aim of this study was to evaluate the optimal addition (individually, in combination or sequentially) of hydrolytic enzymes that would prevent tight polysaccharide-tannin associations. The use of comprehensive microarray polymer profiling (CoMPP) methodology provided key insights into how the enzyme treatments impacted the grape cell wall matrix and tannin binding. The results demonstrated that polygalacturonase + pectin-lyase promoted the highest release of tannins into solution.

The objective of the present research was to evaluate the effect of the cultivation conditions on the production of a polygalacturonase (PGase) by Wickerhamomyces anomalus. Fermentations were carried out in agitated flasks, in a synthetic medium, varying culture conditions (temperature, pH and agitation). A 23 factorial design was initially applied and then response surface methodology. The macerating capacity of the enzymatic extract on a vegetal tissue was evaluated. PH and temperature significantly influenced the production of PGase enzyme, while the influence of the stirring rate was not significant. The highest titles were obtained at 30°C and pH of 5.1; with a maximum PGase activity of 18.84 EU / mL. The enzymatic extract was able to macerate bell pepper tissues. Enzymatic extracts with high PGase activity were obtained, under selected conditions, of interest in food technology., El objetivo del presente trabajo fue evaluar el efecto de las condiciones de cultivo sobre la producción de una Poligalacturonasa (PGasa) por Wickerhamomyces anomalus. Las fermentaciones se realizaron en frascos agitados, en un medio sintético, variando las condiciones de cultivo (temperatura, pH y velocidad de agitación). Se aplicó inicialmente un diseño factorial 23 y posteriormente la metodología de superficie de respuesta. Se evaluó la capacidad macerante del extracto enzimático sobre un tejido vegetal. El pH y la temperatura influyeron significativamente en la producción de la enzima PGasa, mientras que la influencia de la velocidad de agitación fue no significativa. Los mayores títulos se obtuvieron a 30° C y pH de 5,1; con un valor máximo de actividad PGasa de 18,84 UE/mL. El extracto enzimático fue capaz de macerar tejidos de pimiento morrón. Se obtuvieron extractos enzimáticos con elevada actividad PGasa, en las condiciones seleccionadas, de interés en tecnología de los alimentos.

El objetivo del presente trabajo fue evaluar el efecto de diferentes factores en la extracción de pectina a partir de albedo de limón, mediante el extracto enzimático con actividad poligalacturonasa de Wickerhamomyces anomalus (WA cepa 110), una levadura autóctona de la provincia de Misiones, Argentina. El proceso de extracción enzimática de pectina se realizó en frascos Erlenmeyers conteniendo albedo de limón, buffer y el extracto enzimático crudo de WA cepa 110, con agitación (pH 5,0). Los factores evaluados fueron: relación sólido/líquido, temperatura, tiempo y actividad enzimática. El material polimérico se precipitó con etanol y el peso seco del gel obtenido se denominó material insoluble en etanol (MIE). Se obtuvieron rendimientos de MIE de 35,93±0,67% (p/p), con una relación sólido/líquido de 1/50, 12,5 mL del EE (actividad PGasa en el medio de reacción: 4,28 UE/mL), 40º C, 4 h y pH 5,0. Este valor fue superior al obtenido utilizando el método químico, lo cual confirma la bondad del proceso enzimático utilizado., The objective of the present work was to evaluate the effect of different factors on the extraction of pectin from lemon albedo, using the enzymatic extract with polygalacturonase activity of Wickerhamomyces anomalus (WA strain 110), a native yeast from the Province of Misiones, Argentina. Pectin extraction process was carried out in Erlenmeyer flasks containing lemon albedo, buffer and the crude enzymatic extract of WA strain 110, with agitation (pH 5.0). Factors studied were: solid-liquid ratio, temperature, time and enzyme activity. The soluble polymeric material was precipitated with ethanol and the weight of the dried gel obtained was named as ethanol insoluble material (EIM). It was obtained a yield of MIE of 35.93± 0.67% (w/w), with a solid / liquid ratio of 1/50, 12.5 mL of EE (PGase activity in the reaction medium: 4.28 UE/mL), 40º C, 4 h, and pH 5.0. This value was higher than that obtained using the chemical method, which confirms the goodness of the enzymatic process used.

El objetivo del presente trabajo fue evaluar el efecto de diferentes factores en la extracción de pectina a partir de albedo de limón, mediante el extracto enzimático con actividad poligalacturonasa de Wickerhamomyces anomalus (WA cepa 110), una levadura autóctona de la provincia de Misiones, Argentina. El proceso de extracción enzimática de pectina se realizó en frascos Erlenmeyers conteniendo albedo de limón, buffer y el extracto enzimático crudo de WA cepa 110, con agitación (pH 5,0). Los factores evaluados fueron: relación sólido/líquido, temperatura, tiempo y actividad enzimática. El material polimérico se precipitó con etanol y el peso seco del gel obtenido se denominó material insoluble en etanol (MIE). Se obtuvieron rendimientos de MIE de 35,93±0,67% (p/p), con una relación sólido/líquido de 1/50, 12,5 mL del EE (actividad PGasa en el medio de reacción: 4,28 UE/mL), 40º C, 4 h y pH 5,0. Este valor fue superior al obtenido utilizando el método químico, lo cual confirma la bondad del proceso enzimático utilizado., The objective of the present work was to evaluate the effect of different factors on the extraction of pectin from lemon albedo, using the enzymatic extract with polygalacturonase activity of Wickerhamomyces anomalus (WA strain 110), a native yeast from the Province of Misiones, Argentina. Pectin extraction process was carried out in Erlenmeyer flasks containing lemon albedo, buffer and the crude enzymatic extract of WA strain 110, with agitation (pH 5.0). Factors studied were: solid-liquid ratio, temperature, time and enzyme activity. The soluble polymeric material was precipitated with ethanol and the weight of the dried gel obtained was named as ethanol insoluble material (EIM). It was obtained a yield of MIE of 35.93± 0.67% (w/w), with a solid / liquid ratio of 1/50, 12.5 mL of EE (PGase activity in the reaction medium: 4.28 UE/mL), 40º C, 4 h, and pH 5.0. This value was higher than that obtained using the chemical method, which confirms the goodness of the enzymatic process used.

The objective of the present research was to evaluate the effect of the cultivation conditions on the production of a polygalacturonase (PGase) by Wickerhamomyces anomalus. Fermentations were carried out in agitated flasks, in a synthetic medium, varying culture conditions (temperature, pH and agitation). A 23 factorial design was initially applied and then response surface methodology. The macerating capacity of the enzymatic extract on a vegetal tissue was evaluated. PH and temperature significantly influenced the production of PGase enzyme, while the influence of the stirring rate was not significant. The highest titles were obtained at 30°C and pH of 5.1; with a maximum PGase activity of 18.84 EU / mL. The enzymatic extract was able to macerate bell pepper tissues. Enzymatic extracts with high PGase activity were obtained, under selected conditions, of interest in food technology., El objetivo del presente trabajo fue evaluar el efecto de las condiciones de cultivo sobre la producción de una Poligalacturonasa (PGasa) por Wickerhamomyces anomalus. Las fermentaciones se realizaron en frascos agitados, en un medio sintético, variando las condiciones de cultivo (temperatura, pH y velocidad de agitación). Se aplicó inicialmente un diseño factorial 23 y posteriormente la metodología de superficie de respuesta. Se evaluó la capacidad macerante del extracto enzimático sobre un tejido vegetal. El pH y la temperatura influyeron significativamente en la producción de la enzima PGasa, mientras que la influencia de la velocidad de agitación fue no significativa. Los mayores títulos se obtuvieron a 30° C y pH de 5,1; con un valor máximo de actividad PGasa de 18,84 UE/mL. El extracto enzimático fue capaz de macerar tejidos de pimiento morrón. Se obtuvieron extractos enzimáticos con elevada actividad PGasa, en las condiciones seleccionadas, de interés en tecnología de los alimentos.

Chitosan (1.0% and 2.0%) and putrescine (1.0 and 2.0 mmol/L) treatments were used to investigate the effects of these compounds on the postharvest quality and shelf‐life of two banana cultivars, “Native” and “Cavendish.” Fruits were stored at 15 ± 2°C and a relative humidity of 85%–90% during a 20‐day period. In the controls, increases in weight loss, microbial population, total soluble solids, and ethylene production and decreases in firmness, ascorbic acid contents, and fruit lightness occurred gradually during storage. All these changes were delayed significantly after treatments with chitosan and putrescine. Application of putrescine and chitosan also caused small increases in phenolic compound contents and antioxidant activity at the end of the storage period. Results obtained suggest that a treatment with 1% chitosan is effective in improving the postharvest quality and shelf‐life of banana, and open the possibility that lower concentrations of chitosan may be also effective.

A highly prevalent IgE-binding protein band of 28 kDa is observed when Salsola kali pollen extract is incubated with individual sera from Amaranthaceae pollen sensitized patients. By an immunoproteomic analysis of S. kali pollen extract, we identified this protein band as an allergenic polygalacturonase enzyme. The allergen, named Sal k 6, exhibits a pI of 7.14 and a molecular mass of 39,554.2 Da. It presents similarities to Platanaceae, Poaceae, and Cupressaceae allergenic polygalacturonases. cDNA-encoding sequence was subcloned into the pET41b vector and produced in bacteria as a His-tag fusion recombinant protein. The far-UV CD spectrum determined that rSal k 6 was folded. Immunostaining of the S. kali pollen protein extract with a rSal k 6-specific pAb and LC-MS/MS proteomic analyses confirmed the co-existence of the 28 kDa band together with an allergenic band of about 47 kDa in the pollen extract. Therefore, the 28 kDa was assigned as a natural degradation product of the 47 kDa integral polygalacturonase. The IgE-binding inhibition to S. kali pollen extract using rSal k 6 as inhibitor showed that signals directed to both protein bands of 28 and 47 kDa were completely abrogated. The average prevalence of rSal k 6 among the three populations analyzed was 30%, with values correlating well with the levels of grains/m3 of Amaranthaceae pollen. Sal k 6 shares IgE epitopes with Oleaceae members (Fraxinus excelsior, Olea europaea and Syringa vulgaris), with IgE-inhibition values ranging from 20% to 60%, respectively. No IgE-inhibition was observed with plant-derived food extracts., Ministerio de Economía y Competitividad (MINECO), Instituto de Salud Carlos III, Sección Deptal. de Bioquímica y Biología Molecular (Biológicas), Fac. de Ciencias Biológicas, TRUE, pub

[EN] A highly prevalent IgE-binding protein band of 28 kDa is observed when Salsola kali pollen extract is incubated with individual sera from Amaranthaceae pollen sensitized patients. By an immunoproteomic analysis of S. kali pollen extract, we identified this protein band as an allergenic polygalacturonase enzyme. The allergen, named Sal k 6, exhibits a pI of 7.14 and a molecular mass of 39,554.2 Da. It presents similarities to Platanaceae, Poaceae, and Cupressaceae allergenic polygalacturonases. cDNA-encoding sequence was subcloned into the pET41b vector and produced in bacteria as a His-tag fusion recombinant protein. The far-UV CD spectrum determined that rSal k 6 was folded. Immunostaining of the S. kali pollen protein extract with a rSal k 6-specific pAb and LC-MS/MS proteomic analyses confirmed the co-existence of the 28 kDa band together with an allergenic band of about 47 kDa in the pollen extract. Therefore, the 28 kDa was assigned as a natural degradation product of the 47 kDa integral polygalacturonase. The IgE-binding inhibition to S. kali pollen extract using rSal k 6 as inhibitor showed that signals directed to both protein bands of 28 and 47 kDa were completely abrogated. The average prevalence of rSal k 6 among the three populations analyzed was 30%, with values correlating well with the levels of grains/m(3) of Amaranthaceae pollen. Sal k 6 shares IgE epitopes with Oleaceae members (Fraxinus excelsior, Olea europaea and Syringa vulgaris), with IgE-inhibition values ranging from 20% to 60%, respectively. No IgE-inhibition was observed with plant-derived food extracts.

[EN] A highly prevalent IgE-binding protein band of 28 kDa is observed when Salsola kali pollen extract is incubated with individual sera from Amaranthaceae pollen sensitized patients. By an immunoproteomic analysis of S. kali pollen extract, we identified this protein band as an allergenic polygalacturonase enzyme. The allergen, named Sal k 6, exhibits a pI of 7.14 and a molecular mass of 39,554.2 Da. It presents similarities to Platanaceae, Poaceae, and Cupressaceae allergenic polygalacturonases. cDNA-encoding sequence was subcloned into the pET41b vector and produced in bacteria as a His-tag fusion recombinant protein. The far-UV CD spectrum determined that rSal k 6 was folded. Immunostaining of the S. kali pollen protein extract with a rSal k 6-specific pAb and LC-MS/MS proteomic analyses confirmed the co-existence of the 28 kDa band together with an allergenic band of about 47 kDa in the pollen extract. Therefore, the 28 kDa was assigned as a natural degradation product of the 47 kDa integral polygalacturonase. The IgE-binding inhibition to S. kali pollen extract using rSal k 6 as inhibitor showed that signals directed to both protein bands of 28 and 47 kDa were completely abrogated. The average prevalence of rSal k 6 among the three populations analyzed was 30%, with values correlating well with the levels of grains/m(3) of Amaranthaceae pollen. Sal k 6 shares IgE epitopes with Oleaceae members (Fraxinus excelsior, Olea europaea and Syringa vulgaris), with IgE-inhibition values ranging from 20% to 60%, respectively. No IgE-inhibition was observed with plant-derived food extracts.

When nematodes invade and subsequently migrate within plant roots, they generate cell wall fragments (in the form of oligogalacturonides; OGs) that can act as damage-associated molecular patterns and activate host defence responses. However, the molecular mechanisms mediating damage responses in plant-nematode interactions remain unexplored. Here, we characterized the role of a group of cell wall receptor proteins in Arabidopsis, designated as polygalacturonase-inhibiting proteins (PGIPs), during infection with the cyst nematode Heterodera schachtii and the root-knot nematode Meloidogyne incognita. PGIPs are encoded by a family of two genes in Arabidopsis, and are involved in the formation of active OG elicitors. Our results show that PGIP gene expression is strongly induced in response to cyst nematode invasion of roots. Analyses of loss-of-function mutants and overexpression lines revealed that PGIP1 expression attenuates infection of host roots by cyst nematodes, but not root-knot nematodes. The PGIP1-mediated attenuation of cyst nematode infection involves the activation of plant camalexin and indole-glucosinolate pathways. These combined results provide new insights into the molecular mechanisms underlying plant damage perception and response pathways during infection by cyst and root-knot nematodes, and establishes the function of PGIP in plant resistance to cyst nematodes.

Polygalacturonase breaks down pectin chains generally found in the cell wall of plant. Primarily, enzymes have high tendency to be degraded by improper extraction method. Therefore, it is essential to use an economical, simple and efficient extraction method. In this study, polygalacturonase (PG) was extracted from durian seed with ultrasound assisted-extraction. The effects of extraction time, ultrasound temperature, pH of buffer and solvent-to-seed ratio for optimization of the extraction were determined. The optimum combination of extraction was achieved at 30 min extraction time, 50°C temperature and 5:1 ml/g of solvent-to-seed ratio at pH 5.5. Conventional purification processes are multistep, tedious and expensive; thus, it is vital to develop an economical, highly efficient and environmental friendly process for the purification of polygalacturonase with required properties. A novel aqueous two-phase system (ATPS) process composed of surfactant and acetonitrile was employed to purify polygalacturonase from Durio zibethinus seed at laboratory scale. In this study, the effect of Tie Line Length (TLL), crude loads and pH on purification of the enzyme were investigated. The results of the ATPS process indicated polygalacturonase was partitioned in the novel method of ATPS composed of 23% (w/w) Triton X-100 and 19% (w/w) acetonitrile, at 55.6% of TLL (tie line length) crude load of 25% (w/w) at pH 6.0. It was determined that the phase components, Tie Line Length (TLL), crude loads and pH effected the polygalacturonase partitioning. This study also showed that ATPS can be used as an economical and effective method for purification of the enzyme from a novel source with potential industrial application and alternative to the conventional ATPS. Characterization of the purified polygalacturonase was done to determine the polygalacturonase stability in vary conditions. In this study, it indicated that polygalacturonase extracted from durian seed was stable with the pre

The goals of the study were to evaluate yield of the blackberry juice preparation, and at same time of the extraction of sweetener glycosides from dehydrated stevia leaves (Stevia rebaudiana Bertoni), by using enzymatic preparations. Five enzyme preparations were evaluated during the preparation of the sweetener with these glycosides. Two levels of the variables studied were evaluated: enzyme concentration (100 and 200ppm), and incubation period (15 30min), and using two extraction solvents at two temperatures: water and blackberry juice (at 50 and 35 °C, respectively). Efficiency and yield was analyzed by determining suspended insoluble solids (SIS), soluble solids (SS), and quantitation by cromatografía líquida de alta eficacia o High Performance Liquid Chromatography (HPLC) of the main esteviolglucósidos (stevioside and rebaudioside A). Statistical analysis data shown that the incubation time and the enzyme concentration in the enzyme complex did not affect total soluble solids values, but it does in the concentration of SIS, when using the commercial preparation Pectinex Ultra AFPL, as compared with the rest of the tested samples. Similarly, it is shown statistically significant differences in assessing the enzyme-time binomial, in the steviolglycosides extraction in water and blackberry juice. A high concentration of stevioside and rebaudioside A were achieved when using Pectinex Clear, at a concentration of 200 ppm during 30 seconds. However, the obtained values are similar to concentrations quantified in the control sample showing a slight influence of enzymes in the extraction of both steviolglycosides. This result corroborates that the diffusion of these compounds from the leaves to the medium, occurs primarily by the solubility thereof, rather than by hydrolysis caused by specific enzymes used in the study., Como estrategia para alcanzar mayor rendimiento en el procesamiento de pulpa de mora y al mismo tiempo extraer los esteviolglucósidos presentes en hojas deshidratadas de estevia (Stevia rebaudiana Bertoni), fueron evaluados 5 preparados enzimáticos durante la elaboración de un jugo de mora. Se evaluaron dos niveles de las variables consideradas: concentración de enzima (100 y 200ppm) y tiempo de incubación (15 y 30min) en dos medios de extracción: agua y pulpa de mora (a 50 y 35°C, respectivamente). La eficiencia del tratamiento fue analizado a través de la determinación de sólidos insoluasbles en suspensión, sólidos solubles y cuantificación por HPLC de esteviósido y rebaudiósido A. En base al análisis estadístico aplicado se pudo afirmar que no existe ningún efecto, ni en el tiempo de incubación, ni en la concentración del complejo enzimático, sobre los valores resultantes de sólidos solubles totales, pero si en los SIS, del preparado comercial ®Pectinex Ultra AFPL. En relación a la extracción de esteviolglucósidos, tanto en los ensayos de extracción en jugo de mora como en agua, hay diferencias estadísticamente significativas al valorar el binomio enzima*tiempo. Las concentraciones más altas de esteviolglucósidos fueron alcanzadas empleando 200ppm de ®Pectinex Clear durante 30 segundos. A pesar de ello, los valores obtenidos son similares a las concentraciones cuantificadas en la muestra testigo lo que corrobora que la difusión de estos compuestos de las hojas al medio, ocurre principalmente por la solubilidad de los mismos, más que por la hidrólisis originada por las enzimas especificas empleadas en el estudio.

Polygalacturonase breaks down pectin chains generally found in the cell wall of plant. Primarily, enzymes have high tendency to be degraded by improper extraction method. Therefore, it is essential to use an economical, simple and efficient extraction method. In this study, polygalacturonase (PG) was extracted from durian seed with ultrasound assisted-extraction. The effects of extraction time, ultrasound temperature, pH of buffer and solvent-to-seed ratio for optimization of the extraction were determined. The optimum combination of extraction was achieved at 30 min extraction time, 50°C temperature and 5:1 ml/g of solvent-to-seed ratio at pH 5.5. Conventional purification processes are multistep, tedious and expensive; thus, it is vital to develop an economical, highly efficient and environmental friendly process for the purification of polygalacturonase with required properties. A novel aqueous two-phase system (ATPS) process composed of surfactant and acetonitrile was employed to purify polygalacturonase from Durio zibethinus seed at laboratory scale. In this study, the effect of Tie Line Length (TLL), crude loads and pH on purification of the enzyme were investigated. The results of the ATPS process indicated polygalacturonase was partitioned in the novel method of ATPS composed of 23% (w/w) Triton X-100 and 19% (w/w) acetonitrile, at 55.6% of TLL (tie line length) crude load of 25% (w/w) at pH 6.0. It was determined that the phase components, Tie Line Length (TLL), crude loads and pH effected the polygalacturonase partitioning. This study also showed that ATPS can be used as an economical and effective method for purification of the enzyme from a novel source with potential industrial application and alternative to the conventional ATPS. Characterization of the purified polygalacturonase was done to determine the polygalacturonase stability in vary conditions. In this study, it indicated that polygalacturonase extracted from durian seed was stable with the pre

Enzymatic processing contributes to a better touch and comfort of knitted fabrics. Therefore, in this paper, the influence of pretreatment time to process efficiency was researched. After pretreatment, few creations were designed from such knitted fabric as a material for lightweight jacket, coat or poncho. Models were created with an inspiration for everyday clothes, for different occasions and all generations. For the purpose of researching the influence of process parameters to the scouring efficiency, raw cotton knitted fabric was thoroughly washed, bio-scoured with neutral pectinase, alkali scoured, chemically bleached with hydrogen peroxide, and dyed with basic green dyestuff. The efficiency was monitored through the degree of polymerization, hydrophilicity, whiteness degree, fabric hand and depth in color. Enzimatske obrade pletiva doprinose boljem opipu i udobnosti, te je u ovom radu istražen utjecaj predobrade na ucinkovitost naknadnih procesa. Nakon predobrade osmišljeno je nekoliko kreacija iz predobradenog i oplemenjenog pletiva kao materijala prikladnog za izradu gornjih odjevnih predmeta. Modeli su nastali kao inspiracija za jednostavne odjevne predmete, koji su mogu koristiti za svaki dan, za razlicite prigode i za razlicite generacijske uzraste. U svrhu istraživanja utjecaja procesnih parametara u iskuhavanju, sirovo pamucno pletivo je temeljito oprano, bio-iskuhano neutralnom pektinazom, alkalno iskuhano, kemijski bijeljeno vodikovim peroksidom, te bojadisano baznim bojilom zelenog tona. Ucinci iskuhavanja su praceni kroz stupanj polimerizacije, hidrofilnost, stupanj bjeline, opip te dubinu obojenja, Postprint (published version)

Fungal secretomes contain a wide range of hydrolytic and oxidative enzymes, including cellulases, hemicellulases, pectinases, and lignin-degrading accessory enzymes, that synergistically drive litter decomposition in the environment. While secretome studies of model organisms such as Phanerochaete chrysosporium and Aspergillus species have greatly expanded our knowledge of these enzymes, few have extended secretome characterization to environmental isolates or conducted side-by-side comparisons of diverse species. Thus, the mechanisms of carbon degradation by many ubiquitous soil fungi remain poorly understood. Here we use a combination of LC-MS/MS, genomic, and bioinformatic analyses to characterize and compare the protein composition of the secretomes of four recently isolated, cosmopolitan, Mn(II)-oxidizing Ascomycetes (Alternaria alternata SRC1lrK2f, Stagonospora sp. SRC1lsM3a, Pyrenochaeta sp. DS3sAY3a, and Paraconiothyrium sporulosum AP3s5-JAC2a). We demonstrate that the organisms produce a rich yet functionally similar suite of extracellular enzymes, with species-specific differences in secretome composition arising from unique amino acid sequences rather than overall protein function. Furthermore, we identify not only a wide range of carbohydrate-active enzymes that can directly oxidize recalcitrant carbon, but also an impressive suite of redox-active accessory enzymes that suggests a role for Fenton-based hydroxyl radical formation in indirect, non-specific lignocellulose attack. Our findings highlight the diverse oxidative capacity of these environmental isolates and enhance our understanding of the role of filamentous Ascomycetes in carbon turnover in the environment.

Fungal secretomes contain a wide range of hydrolytic and oxidative enzymes, including cellulases, hemicellulases, pectinases, and lignin-degrading accessory enzymes, that synergistically drive litter decomposition in the environment. While secretome studies of model organisms such as Phanerochaete chrysosporium and Aspergillus species have greatly expanded our knowledge of these enzymes, few have extended secretome characterization to environmental isolates or conducted side-by-side comparisons of diverse species. Thus, the mechanisms of carbon degradation by many ubiquitous soil fungi remain poorly understood. Here we use a combination of LC-MS/MS, genomic, and bioinformatic analyses to characterize and compare the protein composition of the secretomes of four recently isolated, cosmopolitan, Mn(II)-oxidizing Ascomycetes (Alternaria alternata SRC1lrK2f, Stagonospora sp. SRC1lsM3a, Pyrenochaeta sp. DS3sAY3a, and Paraconiothyrium sporulosum AP3s5-JAC2a). We demonstrate that the organisms produce a rich yet functionally similar suite of extracellular enzymes, with species-specific differences in secretome composition arising from unique amino acid sequences rather than overall protein function. Furthermore, we identify not only a wide range of carbohydrate-active enzymes that can directly oxidize recalcitrant carbon, but also an impressive suite of redox-active accessory enzymes that suggests a role for Fenton-based hydroxyl radical formation in indirect, non-specific lignocellulose attack. Our findings highlight the diverse oxidative capacity of these environmental isolates and enhance our understanding of the role of filamentous Ascomycetes in carbon turnover in the environment.

Kenaf fibres were produced by removing the pectin, a cementing material that binds the fibres together through the retting process. The traditional method of water retting requires long times and caused water pollution, while dew retting is weather and geographical dependence and produced fibres with low tensile strength. Retting using microbial pectinase is a promising approach as it reduces the times and produces fibres with high tensile strength, furthermore, it is environmental-friendly. The main objective of this study is to determine the efficacy of pectinase produced from a locally isolated fungus strain in kenaf bioretting. Pectinolytic fungi were isolated from the local sources and screened for their pectinase activity qualitatively and quantitatively. The selected pectinolytic fungus with the highest pectinase activity was identified by amplification of ITS region. The cultural conditions for maximum pectinase production from the selected strain were optimised using response surface methodology in solid state conditions,followed by purification using ammonium sulphate precipitation and gel chromatographic methods in order to characterise the pectinase produced. The pectinase produced was applied in kenaf bioretting to evaluate the possibility and efficiency of using pectinase in kenaf retting. A potential pectinolytic strain had been successfully isolated from the kenaf retting tank and was identified as Aspergillus fumigatus R6. Two types of pectinase were produced by A. fumigatus R6, which were polygalacturonase and pectin lyase using rice bran as the substrate in solid state conditions. The optimised cultural conditions for maximum production of polygalacturonase by A.fumigatus R6 was at an initial moisture level of 49.6%, 33°C, and 129 h of incubation time.A. fumigatus R6 polygalacturonase was purified using 60 – 80% ammonium sulphate precipitation and gel filtration chromatographic methods with a purification fold of 2.54 and polygalacturonase yield o

Kenaf fibres were produced by removing the pectin, a cementing material that binds the fibres together through the retting process. The traditional method of water retting requires long times and caused water pollution, while dew retting is weather and geographical dependence and produced fibres with low tensile strength. Retting using microbial pectinase is a promising approach as it reduces the times and produces fibres with high tensile strength, furthermore, it is environmental-friendly. The main objective of this study is to determine the efficacy of pectinase produced from a locally isolated fungus strain in kenaf bioretting. Pectinolytic fungi were isolated from the local sources and screened for their pectinase activity qualitatively and quantitatively. The selected pectinolytic fungus with the highest pectinase activity was identified by amplification of ITS region. The cultural conditions for maximum pectinase production from the selected strain were optimised using response surface methodology in solid state conditions,followed by purification using ammonium sulphate precipitation and gel chromatographic methods in order to characterise the pectinase produced. The pectinase produced was applied in kenaf bioretting to evaluate the possibility and efficiency of using pectinase in kenaf retting. A potential pectinolytic strain had been successfully isolated from the kenaf retting tank and was identified as Aspergillus fumigatus R6. Two types of pectinase were produced by A. fumigatus R6, which were polygalacturonase and pectin lyase using rice bran as the substrate in solid state conditions. The optimised cultural conditions for maximum production of polygalacturonase by A.fumigatus R6 was at an initial moisture level of 49.6%, 33°C, and 129 h of incubation time.A. fumigatus R6 polygalacturonase was purified using 60 – 80% ammonium sulphate precipitation and gel filtration chromatographic methods with a purification fold of 2.54 and polygalacturonase yield o

Prebiotics may be efficient for prevention of intestinal infections in humans and animals by increasing the levels of beneficial bacteria and thereby improving gut health. Using purified prebiotics may however not be cost-effective in the livestock production industry. Instead, prebiotic fibres may be released directly in the gastro-intestinal tract by feeding enzymes with a suitable substrate and allowing the prebiotics to be produced in situ. Using low doses, 0.03 % enzyme-to-substrate ratio, of the enzymes pectin lyase and polygalacturonase in combination with potato pulp, a low-value industrial by-product, we show that high molecular weight galacto-rhamnogalacturonan can be solubilized in the stomach of weaning piglets. The release of this fiber is in the order of 22–38 % of the theoretical amount, achieved within 20 min. The catalysis takes place mainly in the stomach of the animal and is then followed by distribution through the small intestines. To our knowledge, this is the first paper describing targeted production of prebiotics in an animal model.

Wickerhamomyces anomalus, a yeast isolated from citrus fruit peels in the province of Misiones, Argentina, produces a polygalacturonase (endo-PG) with maceration activity of vegetable tissues. The objective of the present work was to determine kinetic and stoichiometric parameters of W. anomalus growth and polygalacturonase production in a synthetic culture medium, operating in a batch-type bioreactor at laboratory scale. Cultures were performed in a bioreactor of 4 l, containing 3 l of a synthetic medium composed of glucose, citrus pectin, vitamins, amino acids, ammonium sulfate and salts, and were incubated with agitation (450 rpm) and aeration at 30 °C, during 12 h. The course of the fermentation process was followed by measuring biomass, residual glucose, polygalacturonase activity and O2 and CO2 content of outlet gases from the reactor. The maximum specific growth rate (Um) of W. anomalus was 0.337 h-1 and the biomass yield (Yx/s) was 0.40 gx/gs. At the end of the culture, PG activity in the supernatant was ~84 UE/ml. The specific activity and the productivity obtained were ~1.91 104 UE/gx and ~9,301 UE/l.h, respectively. Respiratory quotient was approximately 1.0 throughout the fermentation process. No other product different from biomass and CO2 was detected. Batch culture could be an adequate alternative for the production of polygalacturonase from W. anomalus and an extract with high enzymatic activity using a synthetic and economic culture medium could be obtained., Wickerhamomyces anomalus, una levadura aislada de frutas cítricas en la provincia de Misiones, Argentina, produce una poligalacturonasa (endo-PG) con capacidad macerante de tejidos vegetales. El objetivo del presente trabajo fue determinai los parámetros cinéticos y estequiométricos del crecimiento de W. anomalus y la producción de la enzima poligalacturonasa en un medio de cultivo sintético, operado en sistema por lote, en un biorreactor a escala laboratorio. Los cultivos se realizaron en un biorreactor de 4 l que contenía 3 l de un medio sintético compuesto por glucosa, pectina de citrus, vitaminas, aminoácidos, sulfato de amonio y sales, y se incubaron con agitación y aireación, a 30 °C durante 12 h. El transcurso de proceso fermentativo se siguió por medidas de biomasa, glucosa residual, actividad poligalacturonasa y contenido de O2 y CO2 de los gases a la salida del reactor. La velocidad específica de crecimiento máxima (|im) de W. anomalus fue de 0,337 h-1 y el rendimiento de biomasa producida (Yx/s) de 0,401 gx/gs. Al finalizar el cultivo, la actividad PG en el sobrenadante fue de PG de ~ 83,7 UE/ml. La actividad específica y la productividad obtenidas fueron de ~ 1,91. 104UE/gx y ~ 9.301 UE/l.h, respectivamente. El cociente respiratorio fue cercano a 1 durante el proceso fermentativo. No se formó ningún otro producto, además de biomasa y CO 2 . El cultivo por lote resultó ser una buena alternativa para la producción de PG a partir de W. anomalus, obteniéndose un extracto con elevada actividad enzimática, en un medio de cultivo sintético y de bajo costo.

To reveal the ester distribution patterns in acetylated pectins, an enzymatic fingerprinting method usinga combined endo-polygalacturonase (PG) and pectin lyase (PL) treatment followed by hydrophilicinteraction liquid chromatography coupled to electrospray ionization ion trap mass spectrometry with evaporative light scattering detection was developed. This methodpaved the way for the development of the new quantitative parameters degree of hydrolysis by PG (DHPG) and degree of hydrolysis by PL (DHPL). These parameters distinguished the methylester and acetyl group distribution patterns within different sugar beet pectins (SBPs). In the case of pectin having a degree of methylesterification (DM) of >50 and acetylation of ~20, the above approach was insufficient. Hence, a seconddigestion was introduced using a fungal pectin methylesterase and a PG.More than 60% of the total GalA residues present in three SBPs were recovered as monomer and oligomers after the two digestions. The first digestion of the acid extracted commercial SBP revealed the presence of small blocks of nonesterified GalA residues and segments containing large blocks of PL degradable methylesterified and /or acetylated GalA residues. Blocks of partly methylesterified, non-acetylated GalA residues were recognized after the second digestion. These results show that the acetylation pattern is non-random. A pectin acetylesterase (BliPAE) and a pectin methylesterase (BliPME) from Bacillus licheniformis DSM13 were produced, purified and biochemically characterized. The mode of action of BliPAE and BliPME towards acetylated pectins was revealed using the newly developed enzymatic fingerprinting method. BliPAE specifically deacetylates the O-3 linked acetyl groups of nonmethylesterified galacturonic acid residues in the homogalacturan of pectin. BliPME efficiently de-methylesterifies lemon pectins (DM34-76 ®DM 0) and SBPs (DM 30-73 ®DM 14) in a blockwise manner. BliPME is quite tolerant towards the acetyl gro

The possible biotechnological application of a recombinant endopolygalacturonase of Kluyveromyces marxianus (KMPG) for the aroma enhancement of Albariño wine was studied. The addition of this enzyme to the must gives rise to a significant increase of the total compounds responsible for the aroma as opposed to the effect when using a commercial pectic enzyme. This increase also results in a significant rise of the odoriferous aglycones which are direct determinants of the aroma. Wines made by using the KMPG enzyme are characterised by a greater richness and diversity with regard to the number of aromatic compounds present, clearly differing from those obtained with a commercial pectic preparation. Based on compounds with odour activity values (OAV) > 1, the wines obtained with the enzyme KMPG are richer in citric, balsamic, spicy and above all floral (violet and rose) aromas than untreated wines or wines supplemented with a commercial enzyme. © 2013 Elsevier Ltd. All rights reserved.

© 2014 Elsevier Ltd. Despite high-pressure (HP) processed avocado products are nowadays commercialized, there is no information about the effect of pressure on avocado polygalacturonase (PG) yet. In the present study, PG inactivation kinetics in crude avocado extract was investigated under isobaric conditions at 20 °C. Moreover, PG inactivation kinetics under isothermal conditions at atmospheric pressure was also included to make comparisons with the processing technique usually employed in the industry. Both temperature and pressure inactivation of avocado PG, either at 60-70 °C/0.1 MPa or at 20 °C/350-500 MPa, could be mathematically described by first-order inactivation models. No thermal or pressure resistant fractions were detected. Thus, avocado PG appeared a rather labile enzyme which could be completely inactivated after relatively mild thermal or pressure treatments (10 min/70 °C/0.1 MPa or 15 min/20 °C/450 MPa). PG inactivation models developed for PG in crude avocado extract could predict PG inactivation in avocado purée relatively well. Industrial relevance High-pressure processed avocado products are, nowadays, commercially available, but no data exist about the effect of high-pressure on pectinases yet. This paper provides, for the first time in the literature, scientific data about kinetics of thermal and high-pressure inactivation of avocado polygalacturonase. Moreover, first-order inactivation models are issued to mathematically describe inactivation. All these data should be of high interest for the food industry since polygalacturonase can affect the texture of avocado halves and chunks and the rheological properties of guacamole, avocado purée and sauces during the storage.

Studies in zucchini (Cucurbita pepo L. spp. pepo) pollen have been limited to the viability and morphology of the mature pollen grain. The enzyme polygalacturonase (PG) is involved in pollen development and pollination in many species. In this work, we study anther and pollen development of C. pepo and present the cloning and characterisation of a putative PG CpPG1 (Accession no. HQ232488) from pollen cDNA in C. pepo. The predicted protein for CpPG1 has 416 amino acids, with a high homology to other pollen PGs, such as P22 from Oenothera organensis (76%) and PGA3 from Arabidopsis thaliana (73%). CpPG1 belongs to clade C, which comprises PGs expressed in pollen, and presents a 34 amino acid signal peptide for secretion towards the cell wall. DNA-blot analysis revealed that there are at least another two genes that code for PGs in C. pepo. The spatial and temporal accumulation of CpPG1 was studied by semi-quantitative- and qRT-PCR. In addition, mRNA was detected only in anthers, pollen and the rudimentary anthers of bisexual flowers (only present in some zucchini cultivars under certain environmental conditions that trigger anther development in the third whorl of female flowers). However, no expression was detected in cotyledons, stem or fruit. Furthermore, CpPG1 mRNA was accumulated throughout anther development, with the highest expression found in mature pollen. Similarly, exo-PG activity increased from immature anther stages to mature anthers and mature pollen. Overall, these data support the pollen specificity of this gene and suggest an involvement of CpPG1 in pollen development in C. pepo. © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands.

Lignocellulose-based polymers are presently being investigated for potential as raw materials for polymer industry. However, covalent bonds between carbohydrates and lignin, so called lignin carbohydrate complexes (LCCs) present one of the factors impeding clean fractionation of the polymers. Understanding the chemical structure of LCCs in relation to the cell wall ultra-structure therefore provides relevant insight for technical fractionation. In the present work, new mild analytical protocols for LCCs were developed and fractions thereof subjected to detailed structural/ultra-structural characterization. When combined with size exclusion chromatography, the application of mono-component ploysaccharidases proved to be crucial for the identification of LCC moieties and provided indirect evidence for existence of lignin-carbohydrate bonds. The carbohydrate composition of the LCC moieties was essential to determination of the ultra-structural origin of the LCC types. Analytical 31P NMR of the LCCs and of their thioacidolysis products was useful in unveiling lignin structure in LCCs. A complete structure/ultra-structure relationship of LCCs in softwood was determined., QC 20140120

The necrotrophic fungal plant pathogenBotrytis cinerea is able to infect over 200 host plants and cause severe damage to crops, both pre- and post-harvest. B. cinerea often penetrates host leaf tissue at the anticlinal cell wall and subsequently grows into and through the middle lamella, which consists mostly of low-methylesterified pectin. Effective pectin degradation thus is important for virulence of B. cinerea. Chapter 1 describes the chemical structures of plant cell wall polysaccharides, the cell wall-associated mechanisms that confer resistance against pathogens, and the microbial enzymes involved in cell wall decomposition. It then discusses the plant cell wall degrading enzymes of pathogenic fungi and illustrates with case studies the process of pectin decomposition by B. cinerea. Chapter 2describes the molecular identification and functional characterization of a novel MAMP receptor RBPG1, a Leucine-Rich Repeat Receptor-Like Protein (LRR-RLP), that recognizes fungal endo-polygalacturonases (endo-PGs), in particular the B. cinerea protein BcPG3. Infiltration of the BcPG3 protein into Arabidopsis thaliana accession Col-0 induced a necrotic response. Heat-inactivated protein and a catalytically inactive mutant protein retained the ability to induce necrosis. An 11-amino acid peptide stretch was identified that is conserved among many fungal but not plant endo-PGs. A synthetic peptide comprising this sequence was unable to induce necrosis. A map-based cloning strategy, combined with comparative and functional genomics, led to the identification of the RBPG1 gene, which is required for responsiveness of A. thaliana to the BcPG3 protein. Co-immunoprecipitation experiments demonstrated that RBPG1 and BcPG3 form a complex inNicotiana benthamiana, which also involves the A. thaliana LRR-RLK SOBIR1. The sobir1 mutant plants no longer respond to BcPG3. Furthermore, overexpression of RBPG1 in the BcPG3-non-responsive accession Br-0 did not enhance resistance to a numb

Background: Cell separation that occurs during fleshy fruit abscission and dry fruit dehiscence facilitates seed dispersal, the final stage of plant reproductive development. While our understanding of the evolutionary context of cell separation is limited mainly to the eudicot model systems tomato and Arabidopsis, less is known about the mechanisms underlying fruit abscission in crop species, monocots in particular. The polygalacturonase (PG) multigene family encodes enzymes involved in the depolymerisation of pectin homogalacturonan within the primary cell wall and middle lamella. PG activity is commonly found in the separation layers during organ abscission and dehiscence, however, little is known about how this gene family has diverged since the separation of monocot and eudicots and the consequence of this divergence on the abscission process. Results: The objective of the current study was to identify PGs responsible for the high activity previously observed in the abscission zone (AZ) during fruit shedding of the tropical monocot oil palm, and to analyze PG gene expression during oil palm fruit ripening and abscission. We identified 14 transcripts that encode PGs, all of which are expressed in the base of the oil palm fruit. The accumulation of five PG transcripts increase, four decrease and five do not change during ethylene treatments that induce cell separation. One PG transcript (EgPG4) is the most highly induced in the fruit base, with a 700-5000 fold increase during the ethylene treatment. In situ hybridization experiments indicate that the EgPG4 transcript increases preferentially in the AZ cell layers in the base of the fruit in response to ethylene prior to cell separation. Conclusions: The expression pattern of EgPG4 is consistent with the temporal and spatial requirements for cell separation to occur during oil palm fruit shedding. The sequence diversity of PGs and the complexity of their expression in the oil palm fruit tissues contrast with data

Flocculation is a very useful phenotype for industrial yeast strains, since it facilitates cell harvest and represents an easy way of cell immobilization in continuous fermentation processes. The present work represents the first time that an inducible flocculation phenotype has been generated in a non flocculent strain of Kluyveromyces marxianus. This was accomplished by expressing Saccharomyces cerevisiae FLO5 gene in K. marxianus CECT 11769 strain. The FLO 5 gene was placed under the control of an EPG promoter, not repressed by glucose and induced by anoxia. Our experimental approach successfully generated two novel K. marxianus flocculent phenotypes: one inducible and one constitutive. The constitutive phenotype originated from deletions in the FLO5 promoter region, indicating the existence of putative upstream repressor site involved in oxygen regulation of the EPG1 promoter. The novel strains here generated had a unique set of characteristics that provided an advantage, over the wild-type strain, for the industrial co-production of ethanol and polygalacturonase.

Background: Cell separation that occurs during fleshy fruit abscission and dry fruit dehiscence facilitates seed dispersal, the final stage of plant reproductive development. While our understanding of the evolutionary context of cell separation is limited mainly to the eudicot model systems tomato and Arabidopsis, less is known about the mechanisms underlying fruit abscission in crop species, monocots in particular. The polygalacturonase (PG) multigene family encodes enzymes involved in the depolymerisation of pectin homogalacturonan within the primary cell wall and middle lamella. PG activity is commonly found in the separation layers during organ abscission and dehiscence, however, little is known about how this gene family has diverged since the separation of monocot and eudicots and the consequence of this divergence on the abscission process. Results: The objective of the current study was to identify PGs responsible for the high activity previously observed in the abscission zone (AZ) during fruit shedding of the tropical monocot oil palm, and to analyze PG gene expression during oil palm fruit ripening and abscission. We identified 14 transcripts that encode PGs, all of which are expressed in the base of the oil palm fruit. The accumulation of five PG transcripts increase, four decrease and five do not change during ethylene treatments that induce cell separation. One PG transcript (EgPG4) is the most highly induced in the fruit base, with a 700-5000 fold increase during the ethylene treatment. In situ hybridization experiments indicate that the EgPG4 transcript increases preferentially in the AZ cell layers in the base of the fruit in response to ethylene prior to cell separation. Conclusions: The expression pattern of EgPG4 is consistent with the temporal and spatial requirements for cell separation to occur during oil palm fruit shedding. The sequence diversity of PGs and the complexity of their expression in the oil palm fruit tissues contrast with data

Potato pulp is a poorly utilized, high-volume co-processing product resulting from industrial potato starch manufacturing. Potato pulp mainly consists of the tuber plant cell wall material and is particularly rich in pectin, notably galactan branched rhamnogalacturonan I type pectin which has previously been shown to exhibit promising properties as dietary fiber. The objective of this study was to solubilize dietary fibers from potato pulp by a one-step minimal treatment procedure and evaluate the prebiotic potential of the fibers. Statistically designed experiments were conducted to investigate the influence of enzyme type, dosage, substrate level, incubation time, and temperature on the enzyme catalyzed solubilization to define the optimal minimal enzyme treatment for maximal fiber solubilization. The result was a method that within 1 min released 75% [weight/weight (w/w)] dry matter from 1% (w/w) potato pulp treated with 1.0% (w/w) [enzyme/substrate (E/S)] pectin lyase from Aspergillus nidulans and 1.0% (w/w) E/S polygalacturonase from Aspergillus aculeatus at pH 6.0 and 60 °C. Molecular size fractionation of the solubilized fibers revealed two major fractions: one fraction rich in galacturonic acid of 10–100 kDa indicating mainly homogalacturonan, and a fraction >100 kDa rich in galactose, presumably mainly made up of β-1,4-galactan chains of rhamnogalacturonan I. When fermented in vitro by microbial communities derived from fecal samples from three healthy human volunteers, both of the solubilized fiber fractions were more bifidogenic than fructo-oligosaccharides (FOS). Notably the fibers having molecular masses of >100 kDa selectively increased the densities of Bifidobacterium spp. and Lactobacillus spp. 2–3 times more than FOS.

Les travaux présentés dans ce mémoire apportent un éclairage nouveau sur les mécanismes moléculaires et cellulaires impliqués dans l'abscission du fruit chez le palmier à huile (/Elaeis guineensis/ Jacq.). Des approches moléculaires et cellulaires ont été utilisées pour examiner les événements qui accompagnent la mise en place et le fonctionnement de la zone d'abscission (AZ). Tout d'abord, un protocole expérimental au champ a été mis en place pour définir avec précision, la cinétique d'abscission des fruits et examiner la réponse à l'éthylène en fonction de leur stade de développement. Les résultats indiquent que la réponse à l'éthylène est régulée au cours du développement et que l'abscission des fruits matures commencent après 9 h de traitement. La recherche des gènes codants des polygalacturonases (PG), exprimés à la base de fruit contenant l'AZ a permis l'identification de quatorze transcrits qui codent des PGs. L'un de ces transcrits EgPG4 augmente de 700 à 5000 fois au cours du temps, après traitement à d'éthylène. Sa localisation par hybridation /in situ/ a permis de montrer qu'il s'accumule préférentiellement dans les assises cellulaires qui composent la zone d'abscission, avant même que la séparation des cellules qui conduira à l'abscision du fruit soit initiée. L'analyse histologique de la base de fruits de palmier à huile montre la présence de mitoses essentiellement périclines. Il en résulte une organisation de la zone d'abscission en assises cellulaires (10 à 12 assises) dont les noyaux en position centrale sont alignés. Les cellules de l'AZ accumulent au cours du développement de grandes quantités de composés pectiques. Ces composés pectiques ne sont plus détectés dans les cellules de l'AZ lorsqu'elles se sont séparées, ce qui suggère une relation possible avec le fonctionnement de cette zone conduisant à la chute des fruits. L'analyse ultrastructurale par microscopie électronique à transmission a mis en évidence au cours du développement, une accumul

As pectinases são um grupo de enzimas que são encontradas na parede celular de vegetais e de alguns microrganismos fitopatogênicos, sendo capazes de degradar a pectina que é um polissacarídeo também encontrado na parede celular de vegetais. Essas enzimas, principalmente as poligalacturonases e a pectina metil esterase, atuam como fator de virulência em microrganismos, além disso possuem grande importância para utilização nas indústrias têxtil e alimentícia. O estudo objetivou caracterizar in silico genes de pectinases de Moniliophthora perniciosa, fungo causador da vassoura-de-bruxa, superexpressar uma pectinase em sistema heterólogo e detectar a atividade dessas enzimas nas diferentes isoformas do fungo em diferentes meios de cultura. Foram encontrados no banco de dados do M. perniciosa genes de poligalacturonase correspondentes aos do Genblank, sendo estas sequências de genes utilizadas para a confecção de primers específicos, possibilitando a síntese de cDNAs, a posteriores análises de expressão gênica e expressão heteróloga. Em paralelo, testes de atividade foram realizados para a pectina metil esterase; pouca atividade foi detectada para essa enzima, sendo necessária a utilização de novos protocolos e estudos na tentativa de se obter melhores resultados. A metodologia empregada está sendo discutida neste trabalho.

Bahia grass, Paspalum notatum, is an important pollen allergen source with a long season of pollination and wide distribution in subtropical and temperate regions. We aimed to characterize the 55. kDa allergen of Bahia grass pollen (BaGP) and ascertain its clinical importance. BaGP extract was separated by 2D-PAGE and immunoblotted with serum IgE of a grass pollen-allergic patient. The amino-terminal protein sequence of the predominant allergen isoform at 55. kDa had similarity with the group 13 allergens of Timothy grass and maize pollen, Phl p 13 and Zea m 13. Four sequences obtained by rapid amplification of the allergen cDNA ends represented multiple isoforms of Pas n 13. The predicted full length cDNA for Pas n 13 encoded a 423 amino acid glycoprotein including a signal peptide of 28 residues and with a predicted pI of 7.0. Tandem mass spectrometry of tryptic peptides of 2D gel spots identified peptides specific to the deduced amino acid sequence for each of the four Pas n 13 cDNA, representing 47% of the predicted mature protein sequence of Pas n 13. There was 80.6% and 72.6% amino acid identity with Zea m 13 and Phl p 13, respectively. Reactivity with a Phl p 13-specific monoclonal antibody AF6 supported designation of this allergen as Pas n 13. The allergen was purified from BaGP extract by ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography. Purified Pas n 13 reacted with serum IgE of 34 of 71 (48%) grass pollen-allergic patients and specifically inhibited IgE reactivity with the 55. kDa band of BaGP for two grass pollen-allergic donors. Four isoforms of Pas n 13 from pI 6.3-7.8 had IgE-reactivity with grass pollen allergic sera. The allergenic activity of purified Pas n 13 was demonstrated by activation of basophils from whole blood of three grass pollen-allergic donors tested but not control donors. Pas n 13 is thus a clinically relevant pollen allergen of the subtropical Bahia grass likely to be important in eliciti

In the ripening process of the lulo (Solanum quitoense Lam.) physicochemical changes are produced by pectics enzymes as Polygalacturonase (PG), Pectinesterase (PE) and Pectateliase (PL) that acting on pectics substrates of plant tissue, being responsible of the physiological alteration of cells and tissues that results in the fruit softening and the beginning of the premature senescence period. This research explores the foundations of the softening enzymes behavior of lulo epicarp for the activity measurement of PL, PG and PE of fruit´s epicarp and determining their relationship with the softening process during the ripening and senescence process of fruits through follow up of the enzyme expression, the ripening index and instrumental hardness during the lulo fruit ripening under three storage treatments: 1) Control (18° C, 57 days), 2) Refrigeration (18° C, 1 day; 4° C, 14 days; 18° C, 42 days) and 3) Pre-cooling heat shock (27° C, 1 day; 4° C, 14 days; 18° C, 42 days) found that the enzymes expression and softening is reduced by heat treatment, compared with the control group; however, the cold storage inhibit the fruit softening process but chilling injuries was produced, while heat shock, in addition to inhibiting the enzymes expression, inhibited the fruit softening process and protect against chilling injuries prolonging the shelf life in 10 days, showing that it´s the best post-harvest treatment for this type of fruit.

Les travaux présentés dans ce mémoire apportent un éclairage nouveau sur les mécanismes moléculaires et cellulaires impliqués dans l'abscission du fruit chez le palmier à huile (/Elaeis guineensis/ Jacq.). Des approches moléculaires et cellulaires ont été utilisées pour examiner les événements qui accompagnent la mise en place et le fonctionnement de la zone d'abscission (AZ). Tout d'abord, un protocole expérimental au champ a été mis en place pour définir avec précision, la cinétique d'abscission des fruits et examiner la réponse à l'éthylène en fonction de leur stade de développement. Les résultats indiquent que la réponse à l'éthylène est régulée au cours du développement et que l'abscission des fruits matures commencent après 9 h de traitement. La recherche des gènes codants des polygalacturonases (PG), exprimés à la base de fruit contenant l'AZ a permis l'identification de quatorze transcrits qui codent des PGs. L'un de ces transcrits EgPG4 augmente de 700 à 5000 fois au cours du temps, après traitement à d'éthylène. Sa localisation par hybridation /in situ/ a permis de montrer qu'il s'accumule préférentiellement dans les assises cellulaires qui composent la zone d'abscission, avant même que la séparation des cellules qui conduira à l'abscision du fruit soit initiée. L'analyse histologique de la base de fruits de palmier à huile montre la présence de mitoses essentiellement périclines. Il en résulte une organisation de la zone d'abscission en assises cellulaires (10 à 12 assises) dont les noyaux en position centrale sont alignés. Les cellules de l'AZ accumulent au cours du développement de grandes quantités de composés pectiques. Ces composés pectiques ne sont plus détectés dans les cellules de l'AZ lorsqu'elles se sont séparées, ce qui suggère une relation possible avec le fonctionnement de cette zone conduisant à la chute des fruits. L'analyse ultrastructurale par microscopie électronique à transmission a mis en évidence au cours du développement, une accumul

As pectinases são um grupo de enzimas que são encontradas na parede celular de vegetais e de alguns microrganismos fitopatogênicos, sendo capazes de degradar a pectina que é um polissacarídeo também encontrado na parede celular de vegetais. Essas enzimas, principalmente as poligalacturonases e a pectina metil esterase, atuam como fator de virulência em microrganismos, além disso possuem grande importância para utilização nas indústrias têxtil e alimentícia. O estudo objetivou caracterizar in silico genes de pectinases de Moniliophthora perniciosa, fungo causador da vassoura-de-bruxa, superexpressar uma pectinase em sistema heterólogo e detectar a atividade dessas enzimas nas diferentes isoformas do fungo em diferentes meios de cultura. Foram encontrados no banco de dados do M. perniciosa genes de poligalacturonase correspondentes aos do Genblank, sendo estas sequências de genes utilizadas para a confecção de primers específicos, possibilitando a síntese de cDNAs, a posteriores análises de expressão gênica e expressão heteróloga. Em paralelo, testes de atividade foram realizados para a pectina metil esterase; pouca atividade foi detectada para essa enzima, sendo necessária a utilização de novos protocolos e estudos na tentativa de se obter melhores resultados. A metodologia empregada está sendo discutida neste trabalho.

Ethanol and endopolygalacturonase (endoPG) are simultaneously produced by the yeast Kluyveromyces marxianusCCEBI 2011. The aim of this study was to determine the optimal combination of seven environmental and nutritional variables, as well as the influence of each one, with respect to the fermentation process in yeast cultures in which sugarcanejuice was the substrate. Simplex sequential optimization showed that after 15 runs the optimal conditions were: pH, 4.6; temperature, 31ºC; total reducing sugars (TRS), 125 g/l; (NH4)2SO4, 2.48 g/l; (NH4)2HPO4, 2.73 g/l; CaCl2, 0.33 g/l andMgSO4·7H2O, 0.54 g/l. Under these conditions, the ethanol concentration was 47.6 g/l and endoPG concentration was 9.8 U/ml, which represented increases of 22% and 10%, respectively, over the concentrations obtained under suboptimal conditions. Temperature and (NH4)2SO4 supplementation were the most significant factors influencing the co-production process. [Int Microbiol 2011; 14(1):41-49]

Effects of different concentrations of calcium (Ca) from calcium chloride (CaCl2) on activities of two cell wall degrading enzymes; polygalacturonase (PG) and pectin methylesterase (PME) and fruit quality of dragon fruit (Hylocereus polyrhizus) were evaluated. Dragon fruit in this study were obtained from a commercial farm in Batang Benar in Nilai, Negeri Sembilan. Before any Ca treatments were given, it was important to establish the optimum temperature and pH for PG and PME enzymes as these were the two very important physical parameters influencing activity of an enzyme. PG and PME were assayed with different pH range of buffer solutions (pH 3.0 – 8.0) and different temperatures (30 – 70 °C) and results obtained were used in further experimentations. The activities of PG and PME were measured on dragon fruit of different maturity indices; index 3, 4 and 5. This study was carried out using a completely randomized design (CRD) with three replications. To determine the effect of Ca on PG and PME activities, different Ca concentrations (0, 2.5, 5 and 7.5 g/L Ca from CaCl2) were then treated to dragon fruit of two maturity indices (index 3 and 5) and stored for seven days at ± 20 °C. The experiment was conducted in a randomized completely block design (RCBD) with three replications. Effect of varying concentrations of Ca from CaCl2 (0, 2.5 and 7.5 g/L Ca from CaCl2) and dipping duration (0, 4, 8 and 12 mins) on PG and PME activities and fruit quality parameters (firmness, color, pH, calcium content, ascorbic acid content, titratable acidity and soluble solids concentration) of fresh-cut dragon fruit after five days of storage at 12 ± 1 oC were also examined. The experiment was carried out using RCBD with three replications. Results showed that the activity of PG was highest at 41 ºC (6.233 nkat/g) and at pH 6.0 (4.818 nkat/g) while PME activity reaches its maximum level at 47.8 ºC (60.864 neqv g-1 s-1) and at pH 5.8 (72.782 neqv g-1 s-1). Fruits of varying maturity in

Effects of different concentrations of calcium (Ca) from calcium chloride (CaCl2) on activities of two cell wall degrading enzymes; polygalacturonase (PG) and pectin methylesterase (PME) and fruit quality of dragon fruit (Hylocereus polyrhizus) were evaluated. Dragon fruit in this study were obtained from a commercial farm in Batang Benar in Nilai, Negeri Sembilan. Before any Ca treatments were given, it was important to establish the optimum temperature and pH for PG and PME enzymes as these were the two very important physical parameters influencing activity of an enzyme. PG and PME were assayed with different pH range of buffer solutions (pH 3.0 – 8.0) and different temperatures (30 – 70 °C) and results obtained were used in further experimentations. The activities of PG and PME were measured on dragon fruit of different maturity indices; index 3, 4 and 5. This study was carried out using a completely randomized design (CRD) with three replications. To determine the effect of Ca on PG and PME activities, different Ca concentrations (0, 2.5, 5 and 7.5 g/L Ca from CaCl2) were then treated to dragon fruit of two maturity indices (index 3 and 5) and stored for seven days at ± 20 °C. The experiment was conducted in a randomized completely block design (RCBD) with three replications. Effect of varying concentrations of Ca from CaCl2 (0, 2.5 and 7.5 g/L Ca from CaCl2) and dipping duration (0, 4, 8 and 12 mins) on PG and PME activities and fruit quality parameters (firmness, color, pH, calcium content, ascorbic acid content, titratable acidity and soluble solids concentration) of fresh-cut dragon fruit after five days of storage at 12 ± 1 oC were also examined. The experiment was carried out using RCBD with three replications. Results showed that the activity of PG was highest at 41 ºC (6.233 nkat/g) and at pH 6.0 (4.818 nkat/g) while PME activity reaches its maximum level at 47.8 ºC (60.864 neqv g-1 s-1) and at pH 5.8 (72.782 neqv g-1 s-1). Fruits of varying maturity in

Ethanol and endopolygalacturonase (endoPG) are simultaneously produced by the yeast Kluyveromyces marxianusCCEBI 2011. The aim of this study was to determine the optimal combination of seven environmental and nutritional variables, as well as the influence of each one, with respect to the fermentation process in yeast cultures in which sugarcanejuice was the substrate. Simplex sequential optimization showed that after 15 runs the optimal conditions were: pH, 4.6; temperature, 31ºC; total reducing sugars (TRS), 125 g/l; (NH4)2SO4, 2.48 g/l; (NH4)2HPO4, 2.73 g/l; CaCl2, 0.33 g/l andMgSO4·7H2O, 0.54 g/l. Under these conditions, the ethanol concentration was 47.6 g/l and endoPG concentration was 9.8 U/ml, which represented increases of 22% and 10%, respectively, over the concentrations obtained under suboptimal conditions. Temperature and (NH4)2SO4 supplementation were the most significant factors influencing the co-production process. [Int Microbiol 2011; 14(1):41-49]

Potato pulp is a high-volume co-processing product resulting from industrial potato starch manufacturing. Potato pulp is particularly rich in pectin, notably galactan branched rhamnogalacturonan I polysaccharides, which are highly bifidogenic when solubilized. The objective of the present study was to characterize and compare four homogalacturonan degrading enzymes capable of catalyzing the required solubilization of these pectinaceous polysaccharides from potato pulp in a 1min reaction. An additional purpose was to assess the influence of the pH and the potential buffer chelating effects on the release of these polysaccharides from the potato pulp. The pH and temperature optima of two selected pectin lyases from Emericella nidulans (formerly known as Aspergillus nidulans) and Aspergillus niger were determined to 8.6 and 4.0, respectively, at ≥100°C within 1min of reaction. The optima for the two selected polygalacturonases from E. nidulans and Aspergillus aculeatus were determined to pH 4.4 and 46°C, and pH 3.7 and ≥80°C, respectively. The polygalacturonase from A. aculeatus was 4–42 times more heat-resistant at 50°C than the other enzymes. The difference in pH optima of the pectin lyases and the exceptional thermal stabilities of some of the enzymes are proposed to be related to specific amino acid substitutions, stabilizing hydrogen bonding and structural traits of the enzymes. The KM and Vmax values ranged from 0.3–0.6g/L and 0.5–250.5U/mg protein, respectively. Phosphate buffer induced release of a higher amount of dry matter than Tris–acetate buffer at pH 6, indicating a chelating effect of the phosphate. Moreover, the phosphate had a higher chelating effect at pH 6 than at pH 4. The optimal conditions for a high yield of polysaccharides from potato pulp were therefore: 1% (w/w) potato pulp treated with 1% (w/w) enzyme/substrate (E/S) pectin lyase from E. nidulans and 1% (w/w) E/S polygalacturonase from A. aculeatus at pH 6.0 and 60°C for 1min.

Leaf-cutting (attine) ants use their own fecal material to manure fungus gardens, which consist of leaf material overgrown by hyphal threads of the basidiomycete fungus Leucocoprinus gongylophorus that lives in symbiosis with the ants. Previous studies have suggested that the fecal droplets contain proteins that are produced by the fungal symbiont to pass unharmed through the digestive system of the ants, so they can enhance new fungus garden growth.

Als een spore is geland op het plantoppervlak, scheidt Botrytis cinerea enzymen en metabolieten uit waaronder pectine-afbrekende enzymen zoals de zes endopolygalacturonases (BcPGs). Elk van deze BcPGs breken pectine uit de plantencelwanden en middenlamel op hun eigen wijze af. Pectine zit verweven in de plantencelwanden en zorgt voor flexibiliteit van de cellen en de stabiliteit van de structuur van een weefsel. Op het moment dat pectine wordt afgebroken, wordt de verbinding tussen plantencellen verbroken, verliezen de celwanden hun flexibiliteit en het weefsel daarmee haar stabiliteit. Door middel van de BcPGs is Botrytis cinerea dus in staat het plantoppervlak van een gezonde plant binnen te dringen, door de middenlamel te groeien en plantenweefsel om te zetten in schimmelbiomassa en zodoende rot te veroorzaken.

In order to determine whether changes in pectin fractions facilitate or not the traditional drying of redpepperfruits (Capsicum annuum L.) and the possible consequences of these changes on the dehydration conditions for paprikaprocessing, that have an impact on the evolution of carotenoid content (responsible for colour), this evolution and other parameters were monitored during a traditional drying process in correlation with the temperature–time combinations used. Evolution of microbial flora was followed to analyse their possible contribution, as an exogenous source, to enzymatic polygalacturonase (PG) activity. Our results indicated that the mild temperature–time regime mediated the selection and proliferation of a microbial flora that contributed to enzymatic PG activity modifying the pectic fraction. The enzymatic activity generated rises in the calcium pectate (CaP) fraction, which favoured the drying of fruit with an initial low content of soluble pectins (SP) and CaP. Thus, the changes in texture helped during the transfer of moisture, facilitating the dehydration process, and therefore, a milder temperature–time regime was required. Consequently, bioactive compounds of the fruit, such as capsorubin, capsanthin and provitamin A carotenoids, remained almost unaltered. On the other hand, when the SP fraction increased during dehydration, the process was delayed, and this was also correlated with a higher content in SP and CaP in fresh fruit, indicating that the fruits were harvested at a later stage of ripeness. In this case a more intense temperature–time regime was needed, negatively affecting the carotenoid content by decreasing it significantly.

Leaf-cutting (attine) ants use their own fecal material to manure fungus gardens, which consist of leaf material overgrown by hyphal threads of the basidiomycete fungus Leucocoprinus gongylophorus that lives in symbiosis with the ants. Previous studies have suggested that the fecal droplets contain proteins that are produced by the fungal symbiont to pass unharmed through the digestive system of the ants, so they can enhance new fungus garden growth.

Leaf-cutting (attine) ants use their own fecal material to manure fungus gardens, which consist of leaf material overgrown by hyphal threads of the basidiomycete fungus Leucocoprinus gongylophorus that lives in symbiosis with the ants. Previous studies have suggested that the fecal droplets contain proteins that are produced by the fungal symbiont to pass unharmed through the digestive system of the ants, so they can enhance new fungus garden growth.

Leaf-cutting (attine) ants use their own fecal material to manure fungus gardens, which consist of leaf material overgrown by hyphal threads of the basidiomycete fungus Leucocoprinus gongylophorus that lives in symbiosis with the ants. Previous studies have suggested that the fecal droplets contain proteins that are produced by the fungal symbiont to pass unharmed through the digestive system of the ants, so they can enhance new fungus garden growth.