Sodium Polypectate Research Articles

Effect of propagule size on the in vitro production of polygalacturonase and cellulase by Rhizoctonia solani

The physiological behavior of large (250–500 microns (μ)) infective and small (50–150 μ) non-infective mycelial fragments (propagules) of Rhizoctonia solani was compared. Liquid Czapek's medium containing sodium polypectate (NaPP) as the sole carbon source was inoculated with equal amounts (on a dry weight basis) of each kind of propagule. At NaPP concentrations of 0.005%–0.25% large propagules produced more polygalacturonase (PG) after a shorter period of incubation than small ones, whereas no differences in PG activity were observed at 1% NaPP. Differences in PG activity between large and small propagules were also observed in a medium consisting of 1:50 dilution of potato extract, but not in richer media such as potato dextrose broth and bean hypocotyl medium. These differences did not depend on pH of the medium or dry weight of the mycelium. Germination percentage of the large propagules and length of their emerging hyphae were greater than those of the small ones in media containing 0.005%–0.25% NaPP. Extracellular cellulase activity in Czapek's medium containing 0.25% carboxymethyl cellulose was also higher and appeared earlier in large propagules as compared with small ones. Viscometric and colorimetric methods for detecting PG and cellulase activities produced similar results. It is suggested that the capacity of the large propagules to produce hydrolytic enzymes at low nutrient levels may be important for their parasitic activity.

Polygalacturonate lyase production by Bacillus subtilis and Flavobacterium pectinovorum.

Nutritional factors relating to the production of polygalacturonate lyases by strains of Bacillus subtilis and Flavobacterium pectinovorum were examined. Studies were carried out in shake flask cultures. In the case of B. subtilis the enzyme was produced constitutively, whereas in the case of F. pectinovorum it was only produced in quantity in the presence of pectic substances. Glucose was the most suitable carbon source for production of the polygalacturonate lyase of B. subtilis; of the nitrogen sources examined, the highest activities per milliliter of supernatant and per milligram of cells were obtained with glutamine and ammonium sulfate, respectively. The pattern of enzyme production and growth was similar although enzyme production ceased at pH 5.3. Sodium polypectate was the best inducer of polygalacturonate lyase with F. pectinovorum. Highest activity per milliliter of cell-free supernatant was obtained with skin milk powder as nitrogen source, although ammonium sulfate gave highest enzyme production per unit of biomass. Growth of F. pectinovorum occurred between pH 5.7 and 7.2. Enzyme production occurred during active growth and was independent of the pH of the medium.

Studies on the α-L-Arabinofuranosidase Complex from Sclerotinia fructigena in Relation to Brown Rot of Apple

α-L-Arabinofuranosidase (AF) was detected in apple fruitlets experimentally infected by Sclerotinia fructigena. In extracts of such fruitlets, three AF isoenzymes were separated by preparative isoelectric focusing. When the fungus was grown in shake culture with different carbon sources, AF was detected in each culture filtrate and mycelial homogenate. Although fungus growth and total AF varied with the carbon source, the AF isoenzyme pattern was similar in each instance to that obtained when grown on sodium polypectate. Each of the partially purified AF isoenzymes behaved differently in substrate specificity and inhibitor studies; however, each showed a specificity for α-L-arabino-furanosides. The two extracellular AF isoenzymes released monomeric arabinose when incubated with araban or apple cell walls. External AF III (pI 6·5) was more active on a substrate of apple cell wall material than external AF I (pI 3·0). The latter form of the enzyme was less susceptible to inhibition by either oxidized or unoxidized apple juice. Two isolates of Sclerotinia fructigena with low growth rate in vivo secreted no AF III in vitro.

Polygalacturonate Lyase Production by Bacillus subtilis and Flavobacterium pectinovorum

Nutritional factors relating to the production of polygalacturonate lyases by strains of Bacillus subtilis and Flavobacterium pectinovorum were examined. Studies were carried out in shake flask cultures. In the case of B. subtilis the enzyme was produced constitutively, whereas in the case of F. pectinovorum it was only produced in quantity in the presence of pectic substances. Glucose was the most suitable carbon source for production of the polygalacturonate lyase of B. subtilis; of the nitrogen sources examined, the highest activities per milliliter of supernatant and per milligram of cells were obtained with glutamine and ammonium sulfate, respectively. The pattern of enzyme production and growth was similar although enzyme production ceased at pH 5.3. Sodium polypectate was the best inducer of polygalacturonate lyase with F. pectinovorum. Highest activity per milliliter of cell-free supernatant was obtained with skin milk powder as nitrogen source, although ammonium sulfate gave highest enzyme production per unit of biomass. Growth of F. pectinovorum occurred between pH 5.7 and 7.2. Enzyme production occurred during active growth and was independent of the pH of the medium.

Pectolytic enzymes in inoculated and uninoculated red clover seedlings

Pectolytic enzymes were studied in inoculated and uninoculated red clover (Trifolium pratense) seedlings grown aseptically in flasks containing distilled water or nitrogen-free salts media. Enzyme activity in root exudates and root extracts depended on the conditions of seedling growth. Tests with sodium polypectate and citrus pectin indicated the presence of two enzymes, specific for pectic acid and pectin respectively. Both enzymes were produced by uninoculated seedlings, and in seedlings inoculated with Rhizobium trifolii, R. leguminosarum or R. lupini, enzyme activity was not correlated with infectivity of the strains. re]19720814

Polygalacturonate Lyase of a Bacillus Species Associated with Increase in Permeability of Sitka Spruce (Picea sitchensis)

SUMMARY: The extracellular polygalacturonate lyase of a Bacillus species isolated from water-stored Sitka spruce (Picea sitchensis) was purified by ammonium sulphate fractionation and CM-cellulose chromatography. Its specific activity increased 59-fold and amylase, protease and xylanase activities were removed. Degradation of the substrate was accompanied by a large decrease in viscosity, suggesting random attack. While calcium and strontium ions activated the enzyme, most divalent cations caused inhibition. Addition of EDTA resulted in complete inactivation. Enzyme activity was higher with acid soluble pectic acid than with sodium polypectate. The purified enzyme was stable over a wide pH range and had considerable resistance to thermal inactivation. This stability explains the enzyme's prolonged activity in water-stored spruce sapwood.

Pectolytic enzyme production by Phoma betae

Phoma betae from decayed sugar beet storage root tissue grew most rapidly in culture at 15C but produced more polygalacturonase (PG) at 20C. When the fungus was supplied with six different nitrogen sources, it produced the most PG on (NH4)2SO4.Assays of dialyzed culture filtrates using sodium polypectate and pectin or cell wall material from storage roots as the carbon sources showed the production of exopolygalacturonase (exo-PG) and endopolygalacturonate trans-eliminase (endo-PGTE). No pectin methyl esterase was detected. Exo-PG and endo-PGTE also were present in decayed sugar beet tissue. Only endo-PGTE was detected within 3 mm of tissue surrounding the rotted area.In culture, cell wall material from the susceptible variety A58 induced more endo-PGTE formation than the resistant 2B. But 2B induced more exo-PG formation than A58. It is suggested that endo-PGTE plays a major role in cell wall degradation because pH 7.5 was optimum for tissue maceration and pH 8.5 for enzyme activity and the advancing margins of rotted tissue contained only endo-PGTE.

Pectinases from boll weevil larvae, Anthonomus grandis

Assays were conducted throughout a wide pH range to measure the ability of extracts from third instar boll weevil larvae to degrade pectic substances. Pectin was deesterified and was depolymerized by both hydrolytic and trans-eliminative mechanisms. Sodium polypectate (deesterified pectin, or pectic acid) was depolymerized solely by trans-elimination. Addition of Ca 2+ failed to stimulate the trans-eliminative cleavage of either pectin or polypectate. It is possible that one or more of the pectinases from boll weevil larvae contribute to the abscission of infested cotton flower buds.

Characterization of two polygalacturonases produced by Verticillium albo-atrum.

Two polygalacturonases (poly-α-1,4-galacturonide glycanohydrolase, EC 3.2.1.15) from the culture fluids of Verticillium albo-atrum were purified and characterized. Gel permeation and exchange chromatography followed by double isoelectric focusing have been used to separate and purify an exopolygalacturonase and an endopolygalacturonase. The exopolygalacturonase had a molecular weight of 44 000, pI of 6.5, and pH optima of 4.5 for pectin and 5.25 for sodium polypectate. The endopolygalacturonase had a molecular weight of 34 500, pI of 9.7, and pH optima of 5.0 for pectin and 6.0 for sodium polypectate. Both purified enzymes were electrophoretically homogeneous on polyacrylamide gels and exceptionally stable when stored in solution at 4°. The endo-enzyme was 20 times as effective in macerating plant tissue as the exo-enzyme.

Pectic enzymes produced by Erwinia chrysanthemi and their effects on plant tissue

Erwinia chrysanthemi produced extracellular enzymes which degrade pectic acid in a trans-eliminative manner. Isolate 307, from carnation, produced at least two, and isolate EC 23, from Philodendron, produced at least four polygalacturonic acid trans-eliminases. The enzymes produced by each isolate were separated from each other and purified by ammonium sulfate fractionation, diethylaminoethylcellulose column chromatography, and electrofocusing. Molecular weights for the six enzymes ranged between 30,000 and 36,000 as determined by gel filtration in Sephadex G-75 and sucrose density gradient centrifugation. The isoelectric points of two enzyme fractions from isolate 307 were pH 9·1 and 7·9 and those for the four enzyme fractions from isolate EC 23 were pH 9·4, 8·4, 7·9 and 4·6. Random cleavage of pectic acid was greatest with the enzyme fractions having isoelectric points at pH 9·4 and 9·1, intermediate for the fraction with an isoelectric point at pH 4·6, and least with the fractions with isoelectric points at pH 8·4 and 7·9. All of the trans-eliminases exhibited a Ca 2+ requirement and had pH optima between pH 8·2 and 9·8. All but one of the purified polygalacturonic acid trans-eliminase fractions from both E. chrysanthemi isolates induced electrolyte loss, maceration and cell death of potato tissue. The enzyme fraction with an isoelectric point at pH 4·6 from isolate EC 23 did not macerate potato, carrot or cucumber tissue even though it readily degraded pectic acid prepared from onion, carrot and cucumber as well as commercial sources of sodium polypectate, polygalacturonic acid and pectin N.F.

Purification and characterization of endopolygalacturonase from Verticillium albo-atrum

An endopolygalacturonase (poly,α-1,4-galacturonide glycanohydrolase, EC 3.2.1.15) was purified 60-fold from the culture fluids of Verticillium albo-atrum by chromatography on columns of carboxymethyl cellulose and hydroxylapatite, gel filtration, and preparative electrophoresis. Final preparations were homogeneous on the basis of disc-gel electrophoresis, ion-exchange and gel-filtration chromatography, and sedimentation in the ultracentrifuge. The purified endopolygalacturonase had a molecular weight of about 30,000, was rich in basic amino acids, and contained 1.2% carbohydrate. The enzyme catalyzed the random hydrolysis of sodium polypectate to short-chain oligouronides and exhibited macerating activity on cucumber pericarp tissues. The endopolygalacturonase had a Michaelis constant and maximum velocity on sodium polypectate of 0.15% and 2150 μmoles galacturonate equivalents liberated/ min/mg protein, respectively, at 35 ° and at the optimum pH of 6.5. The enzyme was inhibited by heavy metals but not by classical sulfhydryl or serine antagonists.

Pectic enzyme production by two strains of pseudomonas fluorescens associated with the pinkeye disease of potato tubers

A bacterium identified asPseudomonas fluorescens was isolated from pinkeye lesions of infected Kennebec potato tubers. Two isolates which rapidly macerated tuber slices were selected for further study. Supernatants from macerated potato tuber slices inoculated previously with either isolate 13 or 31 ofP. fluorescens contained pectic enzyme activity. Trace amounts of pectin methylesterase activity were observed in the supernatants. Optimum viscosity reduction of sodium polypectate (NaPP) or pectin substrates by the enzyme complexes occurred at pH 8.6 and 7.2 respectively.

Pectic and cellulolytic enzymes of Stemphylium radicinum.

Two isolates of Stemphylium radicinum grown in liquid culture produced polygalacturonase (PG), and measurable amounts of pectin methylesterase (PME) and cellulase (Cx). PG was produced inductively. Sodium polypectate was hydrolyzed more rapidly by the PG than was pectin.

A Comparison of the Polygalacturonases Produced in vivo and in vitro by Penicillium expansum Thom.

Summary: Polygalacturonase (PG) was produced by Penicillium expansum in rotted apples and in liquid culture medium containing pectin; acetone precipitates of the enzyme were prepared from both sources. The PG extracted from apples (in vivo PG) behaved as an endo-PG and degraded sodium polypectate to tetragalacturonic acid. PG from culture filtrates (in vitro PG) before purification had quite different properties, the final hydrolysis product being mono-galacturonic acid. PG from both sources was purified by ion-exchange chromatography. The properties of some purified PG preparations were quite different from those of the crude material. None of the purified in vitro PG released monogalacturonic acid from sodium polypectate, whilst some preparations from both sources had identical endo-PG properties after purification. Changes in the properties of endo-PG produced in vivo and in vitro were apparent during purification and a series of distinct endo-PG’s were obtained which yielded unresolved oligomers, tri- or tetragalacturonic acid as final hydrolysis products. It is suggested that the changes in behaviour of the various PG preparations during purification were brought about by changes in the molecular configuration of the enzyme, and that such changes could account for some of the variability between enzymes produced in vivo and in vitro.

Germination of Botrytis cinerea conidia in the presence of quintozene, tecnazene and dichloran

Various substances alter the activity of quintozene, tecnazene and dichloran towards Botrytis cinerea . The toxicity of tecnazene and dichloran is increased in agar gels, and in 0·1 % solutions of agar, sodium carboxymethylcellulose, sodium polypectate and gelatine; there are no similar effects with quintozene. A glucose/casein hydrolysate solution enhances the activity of dichloran, but not that of the other two fungicides. Less striking, but still pronounced effects are caused by tecnazene and dichloran in the presence of these high molecular weight substances, and nutrients.

Polygalacturonase in the salivary apparatus of Lygus hesperus (Hemiptera)

A polygalacturonase was found in the posterior lobe of the principal salivary gland of Lygus hesperus Knight, which degraded the α-1,4 glycosidic bonds of sodium polypectate. The enzyme(s), which was heat stable in dry air and resistant to ethyl alcohol, had an activity of 800 based on viscosimetric measurements. Air-dried salivary glands, when mixed with buffer and potato tissue contained macerating enzymes which digested 11 mg of potato tissue/hr at an estimated dilution of 5 × 10 5. Optimum activity occurred at pH 7·0.

Pectin Methyl-trans-eliminase as the Maceration Factor of Sclerotinia fructigena and Its Significance in Brown Rot of Apple

SUMMARY: Good correlation between pectin methyl-trans-eliminase (PTE) activity and the ability to macerate plant tissue slices was found in fractions obtained from culture filtrates of the fungus Sclerotinia fructigena by gel filtration on dextran gel, or by ion-exchange chromatography. The presence of two isoenzymes was indicated, one of which was markedly activated by sodium polypectate or potato extract. Endo-polygalacturonase and α-L-arabinofuranosidase activities were not correlated with macerating ability. Negligible amounts of PTE were, however, detected in extracts of apple fruitlets rotted by S. fructigena; the pH optima of the enzyme components in the culture filtrates differed greatly from the pH of apple tissue. For this and other reasons PTE would appear to have little, and at most very localized, significance in the infection of apple fruits by S. fructigena.

EFFECT OF BUFFERS ON THE PECTOLYTIC ACTIVITY OF CULTURE FILTRATES OF FUSARIUM OXYSPORUM.

PECTOLYTIC enzymes have been implicated in the disintegration of plant tissue by a number of phytopathogenic bacteria and fungi1. One procedure to assay culture filtrates for pectolytic activity requires a demonstration of a loss of viscosity for a solution of sodium polypectate or pectin in buffer, using a viscosimeter2. This communication presents results of the effect of 4 buffers at different pH values on the pectolytic activity of culture nitrates from 7 formae speciales of Fusarium oxysporum Schl. emend. Snyder and Hansen.

A Selective Medium for the Isolation of Soft Rot Coliforms from Soil

BACTERIAL species which cause soft rot of plant tissue secrete pectic enzymes which destroy the middle lamella of the cell wall and, in vitro, liquefy pectate gel. This property has been utilized by Wieringa1 and Stewart2 in the preparation of selective media for isolating soft rotting Erwinia spp., and by Paton3 for the isolation of pectin-decomposing Pseudomonas spp. The preparation of these media involves a double-layer technique in which a calcium agar medium is overlaid with a sodium polypectate gel in which the formation of shallow pits serves as a diagnostic characteristic.

Infection of bean plants (Vicia faba L.) with Botrytis cinerea and B. fabae

SUMMARYThe infection of bean plants (Vicia faba L.) by Botrytis fabae has been studied by inoculating detached leaves with standard volumes of spore suspensions of known concentrations, or with single spores.Increasing the number of spores in the inoculation drops increased the number of lesions, and lesions were more readily produced on older than on younger leaves.The addition of glucose, a number of other carbohydrates, and also sodium polypectate and pectin, increased the number and rate of the spread of lesions. Xylose caused a decrease, and a number of nitrogen compounds had little effect.The number and rate of spread of lesions was increased if the surface of leaves were gently rubbed with a mild abrasive before inoculation. Calcium‐deficient plants were more susceptible than plants which had received normal nutrition. Deficiencies of other major elements had much less effect.A larger proportion of the spores in inoculation drops germinated when these drops were on older leaves, when the drops contained glucose, and on leaf surfaces that had been gently abraded. Addition of xylose reduced the amount of germination and the fungus grew very poorly in liquid culture when this was the only carbon source. The addition of small quantities of glucose allowed the fungus to use xylose as efficiently as glucose.When single spores were used to inoculate leaves, very different results were obtained with leaves taken from different plants, but under some conditions single spores often induced lesion formation. B. fabae caused lesions more readily than did B. cinerea, in spite of the fact that a greater proportion of B. cinerea spores in inoculation drops germinated.