Sodium Polypectate Research Articles

Purification of Geotrichum candidum endopolygalacturonase from culture and from host tissue by affinity chromatography on cross-linked polypectate

A homogeneous endopolygalacturonase preparation was obtained from culture filtrates of Geotrichum candidum by a single-step purification, using affinity chromatography on cross-linked polypectate. The enzyme has a molecular weight of 38 000, an isoelectric point at pH 7·8, and a K m of 1·33 mg ml −1 with sodium polypectate as the substrate. The purified enzyme, at pH 4·2, macerated the albedo of lemon peel and produced typical sour rot symptoms when injected into lemon peel. An endopolygalacturonase was purified from sour rot infected tissue which was identical with the enzyme obtained from culture filtrate as judged by isoelectric focusing, SDS polyacrylamide gel electrophoresis and antigenic properties.

Production of Pectin Lyase by Colletotrichum lindemuthianum in Culture and in Infected Bean (Phaseolus vulgaris) Tissue

SUMMARY: Colletotrichum lindemuthianum race γ secreted two forms of pectin lyase, having pI values of 8.2 and 9.7, when grown in culture with sodium polypectate or isolated Phaseolus vulgaris hypocotyl cell walls as the main source of carbon. A single form of polygalacturonase was also present in both media. A pectin lyase, with a pI value of 9.7 was detected in necrotic P. vulgaris tissue infected with race γ, but polygalacturonase activity was absent.

Host-Pathogen Interactions

Heat-labile elicitors of phytoalexin accumulation in soybeans (Glycine max L. Merr. cv Wayne) were detected in culture filtrates of Erwinia carotovora grown on a defined medium containing citrus pectin as the sole carbon source. The heat-labile elicitors were highly purified by cation-exchange chromatography on a CM-Sephadex (C-50) column, followed by agarose-affinity chromatography on a Bio-Gel A-0.5m gel filtration column. The heat-labile elicitor activity co-purified with two alpha-1,4-endopolygalacturonic acid lyases (EC 4.2.2.2). Endopolygalacturonic acid lyase activity appeared to be necessary for elicitor activity because heat-inactivated enzyme preparations did not elicit phytoalexins. The purified endopolygalacturonic acid lyases elicited pterocarpan phytoalexins at microbial-inhibitory concentrations in the soybean-cotyledon bioassay when applied at a concentration of 55 nanograms per milliliter (1 x 10(-9) molar). One of these lyases released heat-stable elicitors from soybean cell walls, citrus pectin, and sodium polypectate. The heat-stable elicitor-active material solubilized from soybean cell walls by the lyase was composed of at least 90% (w/v) uronosyl residues. These results demonstrate that endopolygalacturonic acid lyase elicits phytoalexin accumulation by releasing fragments from pectic polysaccharides in plant cell walls.

Isolation, purification and assay of the macerating enzyme produced by Penicillium oxalicum Curie and Thom.

Penicillium oxalicum produced two isozymes of polygalacturonase (PG) and a pectate lyase (PL). The enzymes were separated and purified following ammonium sulphate precipitation, ion exchange chromatography, ultrogel column chromatography and isoelectric focusing. The first isozyme of polygalacturonase (PGI) was rather unstable hence its properties could not be much assayed. PGII macerated and killed yam tissue in 4 hours but PL was unable to do so. Enzyme assay for the end-products of degradation of sodium polypectate and yam tissue showed that PGI was an exo-enzyme while PGII and PL were endo-enzymes. Endo-polygalacturonase (PGII) appears to play the major role (as the macerating enzyme) in the pathogenesis of yam tissue infected by P. oxalicum.

Effect of calcium-ion concentration on leaf maceration by Tetrachaetum elegans

Increasing Ca2+ concentration enhances the pectolytic activity and growth of T. elegans on a sodium polypectate substrate at pH 6.5 + A similar effect is obtained when the fungus is grown on alder-leaf strips as the sole carbon source with pectolytic activity measured as maceration of leaf tissue. Stimulation of pectic enzymes of aquatic hyphomycetes by Ca2+ may contribute to the increase in the rate of decay of plant material in streams of increasing Ca2+ concentration.

In Vitro Secretion of Pectolytic Enzymes by Xanthomonas campestris

Summary Xanthomonas campestris (Pammel) Dowson produces pectic enzymes under the influence of different carbon sources. Polymethyl galacturonase (PMG) was a pectin-inducible enzyme in X. campestris , being best extruded with pectin as carbon source. Addition of glucose to pectin had practically no effect, to a smaller extent NaPP (sodium polypectate) effectuated PMG secretion. Addition of CMC to NaPP lowered the PMG secretion, glucose and maltose were found to be poor carbon sources for PMG induction. Polygalacturonase (PG), too, was adaptively produced in the culture filtrate of the pathogen, grown with NaPP as the carbon source. Addition of glucose to NaPP enhanced PG secretion and that of CMC retarded it. Glucose, pectin, and maltose individually caused PG liberation, but in a very minor way. 30 °C and 60 min of enzyme-substrate incubation were found to be optimum physical conditions for the secretion of PMG and PG. Polygalacturonase trans-eliminase (PGTE) was an exo-cellular enzyme, produced by X. campestris under adapted condition with NaPP as the best inducible carbon source. Separate addition of glucose and CMC to NaPP reduced PGTE extrusion. Pectin, glucose, and maltose separately effected PGTE liberation, but to a very limited extent, being much less than with NaPP. Addition of glucose to pectin suppressed whatever PGTE the pectin was alone able to produce.

Partial Purification and Characterization of an Endo-Polygalacturonase from Monilinia fructicola and its Implication in the Brown Rot Disease of Peaches)

A polygalacturonase (PG) from cultural media of Monilinia fructicola was purified 50-fold with 30% yield by ultrafiltration followed by successive chromatography on Sephadex G-75, Ecteola cellulose, and Biogel P-150 columns. The enzyme moved as a single band on disc gel electrophoresis and exhibited a single symmetrical peak on ultracentrifugation. Optimal PG 4 ) activity was at pH 5.2 and 50 ˚C with good enzyme stability between pH 4 and 6. The active form of the enzyme appeared to be a tetramer of molecular weight near 80,000. The enzyme was stable at acid pH but dissociated at pH > 9 to form an inactive monomer with a molecular weight of 20,000. Disc gel electrophoretic patterns supported this hypothesis. Kinetic studies indicated a V max of 2,700 µmoles/min/mg protein and a km of 1 x 10- 6 M with sodium polypectate as substrate. Although viscosity and uronic acid dehydrogenase measurements indicated the endo nature of this PG, hydrolysis of pectate yielded galacturonic acid as the major end product. Anthocyanins extracted from grapes, peaches, and cranberries caused varied inhibition while various polyphenol compounds and benlate, a fungicide, did not inhibit this PG at all. The properties of this enzyme in relation to host-pathogen interaction were discussed.

Polysaccharide-degrading enzymes of Botrytis allii and Sclerotium cepivorum. Enzyme production in culture and the effect of the enzyme on isolated onion cell walls

Botrytis allii and Sclerotium cepivorum were grown on liquid media with purified onion cell walls as the sole or major carbon source respectively. Polysaccharide-degrading activity determined at daily intervals for 10 days after inoculation increased over the course of the experiment. For both fungi, degrading activity against sodium polypectate was the highest at all times. Xylan, galactan and citrus pectin-degrading activities appeared at similar rates to each other with some variation in relative amounts from the two fungi. Carboxymethyl cellulase activity increased markedly after 6 days and activity against araban remained relatively low. Cell walls were incubated with enzyme preparations and the release of total carbohydrate and of individual sugars was measured. Cell wall carbohydrate was solubilized by enzyme preparations in amounts which increased with the age of the culture. The pattern of release of the individual sugars showed two groups, one containing uronic acid, galactose, rhamnose and arabinose and the second xylosem glucose, fucose and mannose. The release of the first group was related to polygalacturonase activity and the second to the cellulase and possibly xylanase activities. These findings emphasize the importance of polygalacturonase activity in the solubilization of onion cell walls by both fungi.

Multiple Forms of Polygalacturonase in Two Strains of Rhizoctonia solani

An unspecialized strain of Rhizoctonia solani (FC895) produced in vitro seven isoenzymes (four major and three minor peaks) which were separated by isoelectric focusing over a narrow pH range. The same isoenzyme pattern was detected when hypocotyls of cauliflower seedlings inoculated with the fungus were used as the enzyme source. A specialized strain (FC1241) produced two isoenzymes (one major and one minor peak). The four major isoenzymes of FC895 and the major isoenzyme of FC1241 had the same pH optimum and mode of action on sodium polypectate.

Laboratory-Scale Preparation of Sodium Polypectate for Use in Selective Media for PectolyticErwiniaspp.

Laboratory-Scale Preparation of Sodium Polypectate for Use in Selective Media for Pectolytic<i>Erwinia</i>spp.

Influence of Media Composition on the Production of alpha‐Amylase and Amyloglucosidase by a Strain of Aspergillus niger

AbstractThe nature of thermophilic amylolytic enzymes produced by a strain of Aspergillus niger Van Tieghem was influenced by media composition. While glucose, maltose, dextrin and soluble starch induced the production of amyloglucosidase, pectin and sodium polypectate almost exclusively induced the formation of α‐amylase. Alkali ions suppressed the formation of amyloglucosidase. NH4NO3, (NH4)2HPO4 and (NH4)2C4H4O6 served as good inorganic nitrogen sources, while casein, groundnut protein and corn steep liquor served to a similar capacity as nitrogen sources from organic material. Deletion of the growth factors pyridoxine‐hydrochloride and inositol suppressed enzyme production, while the absence of riboflavin had the opposite effect.

Modellversuche zur Isolierung bakterieller Naßfäuleerreger mittels pektinhaltiger nährböden

1. Under aerobic conditions bacterial soft rots are caused in the most cases by pectolytic Erwinia, Pseudomonas, and Bacillus spp. 2. Using 7 soft rotting strains several selective media were tested for their ability to sure a quick and exact isolation and differentiation of these pathogens. By bile salt-lactose-medium all Erwinia spp. under test could be isolated. For isolation of pectinolytic Pseudomonas strains the D4 medium of Kado and Heskett was suitable as the best one. 3. By the use of substrats containing pectic substances the results of isolation could be essentially improved: So, a sure differentiation of soft rotting bacteria and saprophytic organisms already upon the substrate and in addition, the isolation of pectinolytic Bacillus strains also became possible. The pectinolytic activity of the test strains on pectin-double layer-media was dependent upon the composition of the basal medium. 4. On the base of the results we obtained an isolation scheme is proposed, that allowed to indicate the pectolytic active bacilli (after heating to 80 degrees C for 10 min) on thioglycollate medium covered by a pectin layer, the Erwinia spp. on Stewart-Medium covered by pectin layer and the pectolytic pseudomonads on FPA-medium described by Sands, Hankin and Zucker (containing citrus pectin) to the equal time. 5. The trials also demonstrated, that the preparing of double layer-media instead of sodium polypectate mostly being recommended in literature lower estered pectic compounds can be used successfully.

Production and properties of polygalacturonate lyase by an alkalophilic microorganism Bacillus sp. RK9.

Bacillus sp. RK9 was isolated from soil and produced a constitutive polygalacturonate lyase. Production of the enzyme required the presence of complex nitrogen (peptone and yeast extract). Highest activity was obtained with an initial pH of 9.7. The organism was alkalophilic. No growth occurred below pH 7.5. The enzyme was purified by salt precipitation and diethylaminoethyl (DEAE) cellulose ion-exchange chromatography. The pH optimum for activity was 10.0 in 0.01 M glycine-NaOH buffer. Calcium alone, of divalent cations, activated the enzyme by 2.9-fold. Complete inhibition of enzyme activity was achieved by 1 mM ethylenediaminetetraacetic acid (EDTA). Hydrolysis of substrate occurred in a random fashion and the enzyme was 50% more active towards acid soluble pectic acid (ASPA) than towards sodium polypectate.

Isolation and partial characterization of pectic enzymes produced by Blastocladia ramosa

Two pectic enzymes from culture filtrates of the obligately fermentative fungus Blastocladia ramosa were isolated and partially characterized in order to better understand the ability of this fungus to utilize plant substrates. Separation of the two activities was achieved by batch adsorption to carboxymethyl Sephadex, followed by carboxymethyl Sephadex chromatography and preparative isoelectric focusing. One of the enzymes was shown to be an endo-polygalacturonase (EC 3.2.1.15) with a molecular weight of 3.0 × 10 4 , a pH optimum of 6.0, a p I of 10.6, and an apparent K m for sodium polypectate of 0.025% (w/v). This enzyme was purified 1778-fold. The other enzyme was shown to be an endo-polymethylgalacturonate lyase (EC 4.2.2.3) with a molecular weight of 4.0 × 10 4 , a pH optimum of 8.6 (with no calcium requirement), a p I of 8.8, and an apparent K m for pectin of 0.020% (w/v). This enzyme was purified 78-fold.

Endopolygalacturonase from Rhizoctonia fragariae Purification and characterization of two isoenzymes

An electrophoretically homogeneous preparation of endo-polygalacturonase (poly(1,4-α- d-galacturonide)glycanohydrolase, EC 3.2.1.15) from culture filtrates of Rhizoctonia fragariae, a pathogenic agent in strawberry plants, was resolved into two isoenzymes when subjected to isoelectrofocusing in a narrow pH range. The isoelectric points of the two isoenzymes were 6.76 ± 0.03 and 7.08 ± 0.05. The two polygalacturonases exhibited similar substrate specificity, pH optimum and pattern of degradation of sodium polypectate. The two enzymes consisted of a sigle polypeptide chain which had an apparent molecular weight of 36 000 as determined by gel filtration on Sephadex G-100.

Pectin-polygalacturonase in Citrus

Extracts from citrus [Citrus sinensis (L.) Osbeck] pedicles rated as containing polygalacturonase and cellulase gave activities both as measured with carboxymethylcellulose and with sodium polypectate. Gel electrophrosis of the extract demonstrated two region areas that contained cellulytic activity.

Activity of faecal fluid of a leaf-cutting ant toward plant cell wall polysaccharides

The faecal fluid of the leaf-cutting ant, Atta colombica tonsipes, has been shown to contain enzymes active in the degradation of pectin, sodium polypectate, xylan, and carboxymethylcellulose. In addition, glycosidase activity has been detected in the faecal fluid using various naturally occurring disaccharides and synthetic p-nitrophenyl glycosides as substrates. The importance of these enzymes in the symbiosis between A. c. tonsipes and its food fungus is discussed, with particular emphasis on the rôle of the pectin-degrading enzymes.

Sequential production of polygalacturonase, cellulase, and pectin lyase by Rhizoctonia solani

The sequence of appearance of cell wall degrading enzymes of Rhizoctonia solani propagules was followed. Polygalacturonase (PG; EC 3.2.1.15) was induced earlier by sodium polypectate (NaPP) as compared with the induction of cellulase (Cx; EC 3.2.1.4) by carboxymethyl cellulose (CMC), cellobiose, or fibrous cellulose powder. Increasing CMC concentration to 0.5% shortened the time of Cx appearance. In Czapek medium containing citrus pectin, pectin lyase (PL; EC 4.2.2.10) was produced faster and at higher amounts than in a medium containing NaPP as the sole carbon source. PG appearance also preceded that of PL in media simultaneously supplemented with their respective inducers. NaPP, which induced production of PG, repressed Cx production. Among the Cx inducers, only CMC and cellobiose repressed PG production to any extent. At pH 6.0, either in a synthetic medium or on autoclaved bean hypocotyl segments, a delay in PG production as compared with Cx and Pl production was observed. Optimal pH levels for enzyme production and activity were 4.0 and 5.0 for PG, and 5.5 for Cx, and 8.0 and 7.5 for PL. PG was less repressed than Cx by glucose, cellobiose, and monogalacturonic acid, while PL was not affected.

Release of cell-bound polygalacturonase and cellulase from mycelium of Rhizoctonia solani.

Propagules of Rhizoctonia solani grown in modified Czapek's medium containing sodium polypectate or carboxymethyl cellulose as a sole carbon source produced both extracellular and cell-bound polygalacturonase (PG), and cellulase (Cx), respectively. The cell-bound enzymes can be released to various extents by shaking the germinating propagules in solutions of NaCl, KCl, phosphate buffer, Na2EDTA (ethylenediaminetetraacetate), detergents such as Triton X-100 (octyl phenoxypolyethoxyethanol), Tween 80 (polyoxyethylene sorbitan monooleate), Celmusol, and distilled water. Sodium dodecyl sulfate (SDS) inactivated both PG and Cx but did no affect Cx activity in phosphate buffer solution. PG was more easily released by salts from the mycelium of R. solani than Cx. The release of both enzymes was a passive process and was not due to an osmotic effect. The amount of the cell-bound fraction was correlated with the total amount of the extracellular fraction rather than with the mycelial growth. At least one-third of the cell-bound fractions of both enzymes was found to be associated with the cell wall fraction of the mycelium.

Polygalacturonase of Botrytis cinerea E-200 Pers

A polygalacturonase (poly(1,4-α- d-galacturonide)glycanohydrolase, EC 3.2.1.15) was purified from the culture fluid of Botrytis cinerea. The polygalacturonase preparation, homogeneous on the basis of disc-gel electrophoresis also showed pectinesterase activity. Some properties of the purified polygalacturonase were studied. It had a molecular weight about 69 000. It was inactivated by p-chloromercuribenzoate, tetranitromethane and urea. A 50% loss in viscosity of sodium polypectate solution occurred when 4.6% of the glycosidic bonds were hydrolyzed. The only end product of sodium polypectate and oligogalacturonides hydrolysis was monogalacturonic acid.