Effect of air flow rate on scleroglucan synthesis by Sclerotium glucanicum in an airlift bioreactor with an internal loop

The effects of several plant cell wall polysaccharides degrading enzymes on sugar beet pulps pressing were studied. Study was carried out using three two level fractional factorial experiment designs. With only 36 experiments, the effects of the presence of pectin methylesterase, pectin lyase, polygalacturonase, cellulase, arabinase, xylanase and two rhamnogalacturonases on pressing were examined. Pectin lyase, pectin methylesterase and cellulase had a negative effect and caused the decrease of sugar beet pulp pressability. On the contrary, the presence of polygalacturonase, arabinase and xylanase increased pressing efficiency. When increasing enzymes concentrations, these effects varied and positive interactions between xylanase and polygalacturonase appeared. The presence of each of the two rhamnogalacturonases improved pressability despite their antagonistic effects. These enzymes had a complex effect and strongly interacted with polygalacturonase, arabinase and xylanase.     

Changes in cell-wall polysaccharides from the mesocarp of grape berries during veraison

Softening of grape berries ( Vitis vinifera L. × V. labruscana cv. Kyoho) was evaluated by studying changes in composition and degradation of cell-wall polysaccharides. The grape berry softens at the beginning of the second growth cycle many weeks before harvest. The softening stage is called 'veraison' by viticulturists. On day 50 after full bloom, green hard berries (before veraison [BV]), softening berries (veraison [V]) and partly peel colored berries (C) were selected from the same clusters. In addition, mature berries (M) were collected on day 78 after full bloom. Mesocarp tissues at each stage were fractionated into hot water-soluble (WS), hot EDTA-soluble (pectin), alkali-soluble (hemicellulose) and residual (cellulose) fractions. Neutral and acidic sugar contents of WS and pectin fractions decreased only after the V stage, while the neutral sugar content of the hemicellulose fraction decreased from the BV to V stages. Cellulose content constantly decreased as the berry ripened, but the large decrease was found from the BV to V stages. Molecular masses of pectic and hemicellulosic polysaccharides decreased from the BV to V stages. Hemicellulosic xyloglucan was markedly depolymerized from the BV to V stages. The neutral and acidic sugar composition of each fraction changed little during the berry ripening. These data indicated that softening of berry during veraison involved the depolymerization of pectin and xyloglucan molecules and decrease in the amounts of hemicellulose and cellulose.     

Changes in Cell Wall Composition during Ripening of Grape Berries

Cell walls were isolated from the mesocarp of grape (Vitis vinifera L.) berries at developmental stages from before veraison through to the final ripe berry. Fluorescence and light microscopy of intact berries revealed no measurable change in cell wall thickness as the mesocarp cells expanded in the ripening fruit. Isolated walls were analyzed for their protein contents and amino acid compositions, and for changes in the composition and solubility of constituent polysaccharides during development. Increases in protein content after veraison were accompanied by an approximate 3-fold increase in hydroxyproline content. The type I arabinogalactan content of the pectic polysaccharides decreased from approximately 20 mol % of total wall polysaccharides to about 4 mol % of wall polysaccharides during berry development. Galacturonan content increased from 26 to 41 mol % of wall polysaccharides, and the galacturonan appeared to become more soluble as ripening progressed. After an initial decrease in the degree of esterification of pectic polysaccharides, no further changes were observed nor were there large variations in cellulose (30–35 mol % of wall polysaccharides) or xyloglucan (approximately 10 mol % of wall polysaccharides) contents. Overall, the results indicate that no major changes in cell wall polysaccharide composition occurred during softening of ripening grape berries, but that significant modification of specific polysaccharide components were observed, together with large changes in protein composition.     

Cell wall metabolism during maturation, ripening and senescence of peach fruit

Cell wall changes were examined in fruit of a melting flesh peach (Prunus persica L.) allowed to ripen on the tree. Three phases to softening were noted, the first of which began prior to the completion of flesh colour change and an increase in ethylene evolution. Softening in young mature fruit, prior to ripening, was associated with a depolymerization of matrix glycans both loosely and tightly attached to cellulose and a loss of Gal from all cell wall fractions. After the initiation of ripening, but before the melting stage, softening was associated with continuing, progressive depolymerization of matrix glycans. A massive loss of Ara from the loosely bound matrix glycan fraction was observed, probably from side chains of glucuronoarabinoxylan, pectin, or possibly arabinogalactan protein firmly bound into the wall and solubilized in this extract. An increase in the solubilization of polyuronides also occurred during this period, when softening was already well advanced. The extensive softening of the melting period was marked by substantial depolymerization of both loosely and tightly bound matrix glycans, including a loss of Ara from the latter, an increase in matrix glycan extractability, and a dramatic depolymerization of chelator-soluble polyuronides which continued during senescence. Depolymerization of chelator-soluble polyuronides thus occurred substantially after the increase in their solubilization. Ripening-related increases were observed in the activities of exo- and endo-polygalacturonase (EC 3.2.1.67; EC 3.2.1.15), pectin methylesterase (EC 3.1.1.11), endo-1,4-beta-glucanase (EC 3.2.1.4), endo-1,4-beta-mannanase (EC 3.2.1.78), alpha-arabinosidase (EC 3.2.1.55), and beta-galactosidase (EC 3.2.1.23), but the timing and extent of the increases differed between enzymes and was not necessarily related to ethylene evolution. Fruit softening in peach is a continuous process and correlated closely with the depolymerization of matrix glycans, which proceeded throughout development. However, numerous other cell wall changes also took place, such as the deglycosylation of particular polymers and the solubilization and depolymerization of chelator-soluble polyuronides, but these were transient and occurred only at specific phases of the softening process. Fruit softening and other textural changes in peach appear to have a number of stages, each involving a different set of cell wall modifications.     

Papaya Fruit Softening: Role of Hydrolases

Papaya (Carica papaya L.) cultivars show a wide variation in fruit softening rates, a character that determines fruit quality and shelf life, and thought to be the result of cell wall degradation. The activity of pectin methylesterase, β-galactosidase, endoglucanase, endoxylanase and xylosidase were correlated with normal softening, though no relationship was found between polygalacturonase activity and softening. When softening was modified by 1-MCP treatment, a delay occurred before the normal increase in activities of all cell wall activities except endoxylanase which was completely suppressed. Significant cell wall mass loss occurred in the mesocarp tissue during normal softening, but did not occur to the same extent following 1-MCP treatment. During normal softening, pectin polysaccharides and loosely bound matrix polysaccharides were solubilized and the release of xylosyl and galactosyl residues occurred. Cell wall changes in galactosyl residues after 1-MCP treatment were comparable to those of untreated fruit but 1-MCP treated fruit did not soften completely. The changes in the cell wall fractions containing xylosyl residues in 1-MCP treated fruit showed less solubilization and a higher association of xylosyl residues with the pectic polysaccharides. The results indicated that normal modification of cell wall xylosyl components during ripening did not occur following 1-MCP treatment at the color-break stage, this was associated with the failure of these fruit to fully soften and a selective suppression of endoxylanase activity. The results support a role for endoxylanase in normal papaya fruit softening and its suppression by 1-MCP lead to a failure to fully soften. Normal papaya ripening related softening was dependent upon the expression and activity of endoglucanase, β-galactosidase and endoxylanase.     

Temporal Sequence of Cell Wall Disassembly in Rapidly Ripening Melon Fruit

The Charentais variety of melon (Cucumis melo cv Reticulatus F1 Alpha) was observed to undergo very rapid ripening, with the transition from the preripe to overripe stage occurring within 24 to 48 h. During this time, the flesh first softened and then exhibited substantial disintegration, suggesting that Charentais may represent a useful model system to examine the temporal sequence of changes in cell wall composition that typically take place in softening fruit. The total amount of pectin in the cell wall showed little reduction during ripening but its solubility changed substantially. Initial changes in pectin solubility coincided with a loss of galactose from tightly bound pectins, but preceded the expression of polygalacturonase (PG) mRNAs, suggesting early, PG-independent modification of pectin structure. Depolymerization of polyuronides occurred predominantly in the later ripening stages, and after the appearance of PG mRNAs, suggesting the existence of PG-dependent pectin degradation in later stages. Depolymerization of hemicelluloses was observed throughout ripening, and degradation of a tightly bound xyloglucan fraction was detected at the early onset of softening. Thus, metabolism of xyloglucan that may be closely associated with cellulose microfibrils may contribute to the initial stages of fruit softening. A model is presented of the temporal sequence of cell wall changes during cell wall disassembly in ripening Charentais melon.     

Pectic substances,pectic enzymes and haze formation in fruit juices

The pectic substances, located primarily in the middle lamella between cells in higher plant tissues, are complex polysaccharides. They include the negatively charged rhamnogalacturonans, and the neutral arabinogalactans I and II and l-arabinans. These polysaccharides add viscosity to juices but may also form hazes and precipitates and retard maximum recovery of juices from the fruit. The rhamnogalacturonans are degraded by the enzymes pectin methylesterase and polygalacturonase normally present in plant tissues and by these enzymes and pectate lyase in microbially derived commercial pectic enzymes added during processing. The presence of a?abinofuranosidase, which degrades l-arabinans, in commercial pectic enzyme preparations, can cause haze formation in juices such as apple and pear.     

Production of polygalacturonase byByssochlamys fulva

Summary Byssochlamys fulva was grown in two fermentation media using shake flasks, stirred fermentor and disc fermentor under conditions to give maximum production of pectolytic enzymes. Only polygalacturonase activity was detected in the culture filtrates during all fermentations. In all production conditions studied, no evidence of pectin methylesterase, pectin lyase, cellulase or proteinase activities were found. The maximum polygalacturonase activity (4.5 units/ml) was achieved when the microorganism was grown on medium II in shake flasks at pH 4.0–4.5 and 30°C after 12 days of fermentation.     

Induction of lytic enzymes by the interaction of Ustilago maydis with Zea mays tissues

The nonpathogenic (FB-2) and pathogenic (FB-D12) strains of Ustilago maydis were grown in medium supplemented with different carbon sources including monosaccharides, polysaccharides, and plant tissues. Both strains were able to grow on all substrates, with doubling times varying from 2 to 25 h depending on the carbon source. Plant tissues supplied as carbon source induced lytic enzymes differentially; pectate lyase and cellulase activities were induced preferentially by apical stem meristem in strain FB-D12, whereas leaves preferentially induced xylanase and cellulase activities in strain FB2. Stems induced polygalacturonase activity in both strains. All enzyme activities, except cellulase in the FB-D12 strain, were detected at a low level when U. maydis was grown on glucose. In planta, chlorosis and production of teliospores were paralleled by an increase in pectate lyase activity. Anthocyanin production and formation of galls and teliospores correlated with polygalacturonase expression whereas cellulase activity increased only during the stage of anthocyanin production and gall formation. Expression of xylanase activity coincided with the last stage of teliospore formation.     

Three-dimensional structure of Erwinia chrysanthemi pectin methylesterase reveals a novel esterase active site

Most structures of neutral lipases and esterases have been found to adopt the common alpha/beta hydrolase fold and contain a catalytic Ser-His-Asp triad. Some variation occurs in both the overall protein fold and in the location of the catalytic triad, and in some enzymes the role of the aspartate residue is replaced by a main-chain carbonyl oxygen atom. Here, we report the crystal structure of pectin methylesterase that has neither the common alpha/beta hydrolase fold nor the common catalytic triad. The structure of the Erwinia chrysanthemi enzyme was solved by multiple isomorphous replacement and refined at 2.4 A to a conventional crystallographic R-factor of 17.9 % (R(free) 21.1 %). This is the first structure of a pectin methylesterase and reveals the enzyme to comprise a right-handed parallel beta-helix as seen in the pectinolytic enzymes pectate lyase, pectin lyase, polygalacturonase and rhamnogalacturonase, and unlike the alpha/beta hydrolase fold of rhamnogalacturonan acetylesterase with which it shares esterase activity. Pectin methylesterase has no significant sequence similarity with any protein of known structure. Sequence conservation among the pectin methylesterases has been mapped onto the structure and reveals that the active site comprises two aspartate residues and an arginine residue. These proposed catalytic residues, located on the solvent-accessible surface of the parallel beta-helix and in a cleft formed by external loops, are at a location similar to that of the active site and substrate-binding cleft of pectate lyase. The structure of pectin methylesterase is an example of a new family of esterases.     

Cell wall metabolism during the development of chilling injury in cold-stored peach fruit: association of mealiness with arrested disassembly of cell wall pectins

Partially tree-ripened ripe fruit of peach (Prunus persica L.) were stored for 1-4 weeks at 5 degrees C and then ripened at 20 degrees C for 3 d to induce chilling injury. With increasing cold storage the incidence and severity of mealiness symptoms increased progressively, manifested as reduced quantities of free juice and internal flesh browning. Relative to juicy fruit, tissue of mealy fruit showed altered intercellular adhesion when examined by microscopy and, upon crushing, a higher proportion of cells remained intact and did not release cellular contents. Substantial alterations in the metabolism of cell wall polysaccharides were observed. Chelator-soluble polyuronides from mealy fruit were partially depolymerized during cold storage in a manner dissimilar to that in unripe or ripe juicy fruit, and were not depolymerized further during the ripening period. The solubility of these high molecular weight pectins remained low, and did not show the increase characteristic of juicy fruit. Furthermore, in mealy fruit the dramatic decline in the polymeric Ara content of base-soluble, matrix glycan-enriched fractions occurring during normal ripening was absent, indicating diminished disassembly of an arabinan-rich polysaccharide firmly attached to cellulose. A corresponding rise in the polymeric Ara content of the most soluble pectin fraction was also absent, as was a decline in the Gal content of this extract. The depolymerization of matrix glycans showed only minor differences between juicy and mealy fruit. After cold storage and ripening, the activities of endo-1,4-beta-glucanase (EC 3.2.1.4), endo-1,4-beta-mannanase (EC 3.2.1.78), beta-galactosidase (EC 3.2.1.23), alpha-arabinosidase (EC 3.2.1.55), and particularly endo-polygalacturonase (EC 3.2.1.15) were lower in mealy fruit than in juicy fruit, whereas pectin methylesterase activity (EC 3.1.1.11) was lower in slightly mealy and higher in very mealy fruit. The data suggest that cold storage affects the activities of numerous cell wall-modifying enzymes, with important consequences for pectin metabolism. These changes alter the properties of the primary wall and middle lamella, resulting in tissue breakage along enlarged air spaces, rather than across cells, which reduces the amount and availability of free juice upon tissue fragmentation.     

Cellular expansion and gene expression in the developing grape (Vitis vinifera L.)

Summary. Expression profiles of genes involved in cell wall metabolism and water transport were compared with changes in grape (Vitis vinifera L.) berry growth, basic chemical composition, and the shape, size, and wall thickness of cells within tissues of the berry pericarp. Expression of cell wall-modifying and aquaporin genes in berry pericarp tissues generally followed a bimodal expression profile with high levels of expression coinciding with the two periods of rapid berry growth, stages I and III, and low levels of expression corresponding to the slow-growth period, stage II. Cellular expansion was observed throughout all tissues during stage I, and only mesocarp cellular expansion was observed during stage III. Expansion of only exocarp cells was evident during transition between stages II and III. Cell wall-modifying and aquaporin gene expression profiles followed similar trends in exocarp and mesocarp tissues throughout berry development, with the exception of the up-regulation of pectin methylesterase, pectate lyase, two aquaporin genes (AQ1 and AQ2), and two expansin genes (EXP3 and EXPL) during stage II, which was delayed in the exocarp tissue compared with mesocarp tissue. Exocarp endo-(1→3)-β-glucanase and expansin-like gene expression was concurrent with increases in epidermal and hypodermal cell wall thickness. These results indicate a potential role of the grape berry skin in modulating grape berry growth. Correspondence: P. Bowen, Pacific Agri-Food Research Centre, 4200 Highway 97, Summerland, BC V0H 1Z0, Canada     

Molecular cloning of Erwinia chrysanthemi pectinase and cellulase structural genes

Erwinia chrysanthemi 3937 secretes four major pectate lyase isoenzymes (PL, EC 4.2.2.2) and one endocellulase (Cx, EC 3.2.1.4). A genomic library of this strain was constructed in the Lambda L47-1 vector, and screened for the presence of PL and Cx on pectate and caboxymethylcellulose agar. Among the seven Cx-positive phage clones, three were shown to encode an enzyme of the same mol. wt. as the one found in the culture supernatant of strain 3937. The 34 PL-positive phage clones were analyzed by electrofocusing and could, according to the PL they produced, be arranged in five classes. Phages from three classes produced three different single PL, named PLb, c and d. No common fragment was evidenced between the inserts of the phages of these three classes. This demonstrated that, in strain 3937, PLb, C, and d were encoded by three different genes called pelB, C, and D. Furthermore, our results suggest the existence of two additional genes encoding PLa and e. In addition, a pectin methylesterase gene was found closely linked to pelD.