PGase Activity Research Articles

Exploring pectinolytic yeast diversity: toward effective polygalacturonase producers for applications in wine-making.

Pectinolytic enzymes secreted by yeasts have an untapped potential in industry, particularly in wine-making. This study addresses the limitations of the current screening methods in reliably predicting the capacity of pectinolytic yeast strains to secrete polygalacturonase (PGase) under industrial conditions, suggesting a novel screening approach. Using the context of wine-making as an example, a diverse collection of 512 yeast strains from 17 species was analyzed for PGase secretion, a key enzyme in pectinolysis. The traditional halo assay on solid YPD medium revealed 118 strains from nine genera being PGase positive. Screening these strains by incubating them at 20°C on a solid synthetic grape juice medium containing polygalacturonic acid (PG) significantly reduced the number of promising strains to 35. They belong to five genera: Kluyveromyces sp. Cryptococcus, Pichia, Torulaspora, and Rhodotorula. Afterward, a newly developed pectin-iodine assay was used to precisely quantify the PGase activity of the best-performing strains in a liquid medium. Strains from Kluyveromyces and Cryptococcus sp. stood out regarding high pectinolytic activity. Our methodological advancements tailored to identify highly promising pectinolytic yeasts for industrial use open new avenues for wine-making and other industrial processes encompassing media rich in pectin and sugars.

Statistical bioprocess optimization for enhanced production of a thermo alkalophilic polygalacturonase (PGase) from Pseudomonas sp. 13156349 using solid substrate fermentation (SSF)

In this study, a polygalacturonase (PGase) producing bacterial strain was isolated and identified as Pseudomonas sp. 13159349 from fruit market soils, and TLC analysis confirmed its pectinolytic activity. Additionally, SSF, Plackett-Burman design (PB), and response surface methodology (RSM) were used to optimize the production of this thermostable and alkalophilic PGase. Wheat bran demonstrated the highest activity (60.13 ± 3.39 U/gm) among the various agricultural wastes used as solid substrates. To further enhance the enzyme production, statistical optimization of media components was investigated using the PB design. Among the 11 variables tested, pH (p < 0.0001), inoculum size (p < 0.0001), incubation time (p < 0.0001), and temperature (p < 0.0041) were found to have a positive effect on the production. The interaction and concentration of the selected factors were examined by RSM, which demonstrated the optimal conditions for maximum production (315.65 U/gm) of the enzyme using wheat bran as the solid substrate were pH 10.5, 61–66 h of incubation, and 6–7.5% inoculum size. The model was highly significant, with a p-value of <0.0001, an F-value of 95.33, and a low CV of 2.31. The RSM model was validated by a laboratory-scale experiment showing 30600 ± 400.32 U/100 gm PGase activity. Thus, SSF and the statistical design of media components resulted in a significant 5.2-fold increase in PGase output solely by using agro waste and optimizing the physical parameters, making this a highly cost-effective bioprocess.

Production of microalgae-derived exo-polygalacturonase for fruit juice clarification

Due to the growing demand for commercial enzymes derived from renewable and sustainable resources with higher efficacy, algal biomass has gained more attention. Thus, the production of exo-polygalacturonase (PGase) from Bacillus subtilis under submerged fermentation (SmF) using algal feedstocks as a carbon source in fermentation medium and its potential use for the clarification of apple and carrot juices with the optimization of the enzyme loading, pH, and time using Box–Behnken response surface methodology (RSM) were prompted. The maximum PGase activity of 510.61 ± 7.86 U/L was achieved at pH 5.0 after 48 h at 40°C using 0.5% (w/v) pectin, 0.1% (w/v) yeast extract, 0.02% (w/v) MgSO47H2O, 0.02% (w/v) K2HPO4, 0.3% (w/v) glucose, and 0.583% (w/v) microalgae hydrolysate as a fermentation medium. PGase production without glucose was favored by SmF with a shake flask working volume of 100 ml (248.90 ± 4.58 U/L) as compared to a bioreactor with a working volume of 1 L (192.99 ± 0.84) for 48 h. Both carrot and apple juices were treated with the crude PGase (248.9 U/L) at different concentrations (0.1–0.5%), temperature (30–50°C), and time (30–120 min), and the maximal clarification conditions were obtained as 0.3% (w/v) enzyme concentration at 50°C for 30 min exposure time. This study revealed that microalgae-derived PGase in crude form could be effectively used for clarification of the juices.

Green polygalacturonase production by Aspergillus awamori NRC-F18 under solid-state fermentation

Background and objective Pectin-degrading enzymes applied in the food industries are preferred to be obtained from fungal origin because fungi are potent producers of pectic enzymes and the optimal pH of fungal enzymes is very close to the pH of many fruit juices, ranging from pH 3.0 to 5. Pectinolytic enzymes, have several industrial applications in different industries such as food industries to concentrate fruit juices. They increased the yield of fruit juice from the pulp and removal of haze from juices to get a clear product. Research to find microorganisms to produce high-quality polygalacturonase (PGase) and technical constraint include supply of cheap and pure raw materials needed to produce inexpensive enzymes. Utilization of agroindustrial residues offers potential benefits for solid-state fermentation, which is attractive for the implementation of sustainable bioprocesses.Materials and methods Fungal strain screening for PGase production was studied in 250 ml Erlenmeyer flasks containing 5 g of tested substrate moistened to 50% (v/w, ml/g) with distilled water. One milliliter of spore suspension (106 spores) from each fungus was used as inoculum. The cultures were incubated at 30°C for 4 days. At the end of the incubation period, 100 ml of distilled water was added to each flask, blended by shaking at 150 rpm for 30 min, and harvested by filtration. The filtrates were saved as sources of crude enzyme. The selected fungal strain and substrate were incubated for 120 h at 30°C and culture was taken at 24 h intervals to detect the optimum incubation period. Sugar beet pulp was moistened to different moisture levels, that is, 1 : 1, 1 : 2, 1 : 3, and 1 : 4 (v/w) under the optimum incubation period to determine the more suitable moisture content for enzyme production. Sodium phosphate buffer at 0.1 M was used for adjusting the initial pH of fermentation medium to different values from 3.5 to 7.5 to study the effect of pH on enzyme secretion. The fungus was incubated under different temperatures, that s, 20, 25, 30, 35, and 40°C to study the temperature effect on enzyme production. Inorganic nitrogen sources (at level 0.92 mg N/g solid substrate) were applied in the fermentation medium to study their effect on enzyme yield.Results and conclusion Aspergillus awamori NRC-F18 showed promising PGase’ production activity than other fungi screened. The study showed that the fungus gave promising results when the moisture content was adjusted to 70% (v/w), initial pH value 5.0, and incubation temperature at 35°C for 72 h. The enzyme activity was increased when urea was the sole nitrogen source at a level of 0.92 mg/g solid substrate supplemented to fermentation medium. Under the above conditions, 396.4 U/g original substrate was obtained. A study on the obtained PGase revealed that it has an optimum pH that ranged from 4.5 to 5.5, as well as it gave the highest activity when incubated at 50°C. Eighty percent ammonium sulfate (w/v) was applied to precipitate enzyme protein, as 24% of total protein involved 61% of total PGase activity obtained with a specific activity of 36.94 U/mg protein against 12.8 U/mg protein in the crude culture supernatant. After fermentation the mixture from unutilized raw material and fungal biomass after enzyme elution was 76% (w/w) from the original substrate dry weight involving 14.2% protein related to 9.2% before beet pulp fermentation which could be utilized as feedstuff component in rations for ruminant feed.

Production of an endopolygalacturonase from Wickerhanomyces anomalus with disintegration activity on plant tissues

Abstract The objective of the present study was to produce an endo-polyglacturonase by Wickerhanomyces anomalus, in a low cost fermentation medium, by batch and fed-batch cultures in order to be used in the enzyme. The effect of several nutrients on PGase production, were evaluate at shakes flasks, in a reference fermentation medium, using one factor at a time method, Plackett-Burman design and response surface methodology. The optimized fermentation medium was used to evaluate the growth and production of the enzyme by batch and fed-batch cultures, at a lab scale bioreactor. PGase activity obtained in the RF medium was ∼20 U/mL. The absence of trace element solution had a repressive effect on the enzyme synthesis. The addition of yeast extract, instead of vitamins and amino acids, in the culture medium, improved the production of the enzyme. Plackett-Burman design determined that only pectin and yeast extract had significant and positive effect on PGase production. The Doehlert design determined that maximum PGase synthesis was obtained with 6.0 and 0.8 g/L of pectin and yeast extract, respectively. The final optimized fermentation medium included glucose, pectin, urea, yeast extract and salts. In this medium, PGase synthesis reached ∼25 U/mL, and ∼49 U/mL, in batch and fed-batch cultures, respectively, at lab scale bioreactor. In this study, it was able to obtain enzymatic extract with high PGase activity, by the growth of W. anomalus in a low cost culture medium, by fed-batch system, for its future use in the enzymatic cassava starch extraction.

A low-temperature polygalacturonase from P. occitanis: characterization and application in juice clarification

An extracellular endo-polygalacturonase (PGase) was purified, after a single purification step, from the constitutive and hyperpectinolytic CT1 mutant of Penicillium occitanis. This enzyme named PG2 has a molecular weight of 42kDa. It was optimally active at 35°C and pH6 with more than 85% of activity at pH7 in contrast to the majority of fungal PGase, generally acting at 50°C and pH5. The specific activity obtained was among the highest ones, 31397.26U/mg. The PGase activity increased with the decrease of the degree of methylation (DM) of pectin, but it was also able to degrade the highly methyl-esterified substrates, 70% (DM) and 90% (DM), with almost 80% and 40% of residual activity respectively. Interestingly, PG2 is completely inhibited by DEPC, suggesting the implication of a Histidine residue in the active site. The sequencing of P. occitanis whole genome allowed us to identify the pga2 gene encoding PG2 and to localize the His residue, target of DEPC, while it was absent in the PG1 that resisted to DEPC. Besides that, the potentialities of PG2 have been put in use in juice clarification of pear, banana and citrus juice.

Screening of Yeast Strains for Pectinolytic Activity: Effects of Different Carbon and Nitrogen Sources in Submerged Fermentations

Objectives: In the industrial production of fruit juices it is necessary to eliminate the pectin released during fruit processing in order to reduce the time of filtration and enhance the production at the end of the process. The present investigation was to select yeast strains with the best pectinase production and characterize some of the properties of the enzymes produced by submerged fermentation (SmF) processes. Materials and Methods: For plate assay, yeast strains were inoculated in the solid medium including citrus or apple pectin (1.0%) and incubated for 72h at 30oC. After, iodine-potassium iodide solution or 6M HCI was added to detect clearance zones around the colonies. Strains presenting large clearing zones were used for enzyme production assays on liquid medium. SmF was carried out using 250 ml Erlenmeyer flasks with 40 ml of medium in a rotary shaker (120 rpm) at 30oC. After 96h the biomass was separated by centrifugation at 11000 rpm for 10 min and the supernatant was used to evaluate polygalacturonase (PGase) activity. Assay of PGase activity was determined by measuring the release of reducing groups using the 3,5-dinitrosalicylic acid (DNS) reagent. The reaction mixture containing 250 µl of 1% citric pectin in 250 µl citrate-phosphate, pH 6.0 buffer and 100 µl of culture supernatant, was incubated at 30oC for 5 min. Results: Yeast strains were tested for pectin hydrolysis by plate assay, and were classified as very good producers of pectin depolymerizing enzymes when presented clear halos around colonies of at least 1.5 cm (1), good producers when the halos were of at least 1 cm (4), weak producers when halos were at least 0.5 cm (4) and poor producers when no pectinolytic activity and no clear lysis zones were observed (11). Maximal PGase production activity was obtained for strains Kluyveromyces marxianus NRRL-Y-1195 and Pichia pastoris. Pectinolytic activity was the highest at pH 5.0 and 5.5 for these strains respectively, except for Candida rugosa NRRL-Y-95, which presented optimal pH between 6.0. Assay for determination of the optimal temperature for PGase activity indicated maximal activities between 40-50oC for high pectinolytic yeast strains. PGase secreted by these yeasts showed an activity between pH 3.5 and 6.0, which is typical of PGase secreted by yeasts. Conclusion: The pectinolytic activity of yeasts has specific properties which may offer advantages over currently available pectinase preparations. The enzyme solution can be applied directly to fruit juice industries without the need for pH modification. Furthermore, because of the temperature stability of the enzyme, it can be possibly used at processing temperature of 40-50oC, which is sufficient for industrial process.

Improved production of alkaline polygalacturonate lyase by homologous overexpression pelA in Bacillus subtilis

A polygalacturonate lyase (PGL), PelA, was purified from the culture broth of Bacillus subtilis 7-3-3, with a molecular weight, optimal temperature, and pH of approximately 45kDa, 55°C, and 9.4, respectively. The PGL gene (pelA) was homologously overexpressed in B. subtilis 7-3-3 to increase the gene copies and enhance the PGL production. The resulting PGL activity was 2138UmL−1 at 44h, and the productivity reached 48.58U(mLh)−1 through the homologous overexpression of strain B-pN-pelA in a 7.5L fermentor, the highest PGL production compared to those reported in literature to the best of our knowledge. Crude enzyme has high PGL and PGase activity, which can remove 50.58% of pectin in unpretreatment ramie fibers at 50°C for 4h. Meanwhile, the enzyme system with a low level hemicellulase and almost no cellulase will further help in enhancing the efficiency of degumming besides maintaining tenacity of plant fiber. The B. subtilis B-pN-pelA shows high genetic stability and has great potential in the textile industry.

Optimization of Polygalacturonase Production from a Newly IsolatedThalassospira frigidphilosprofundusto Use in Pectin Hydrolysis: Statistical Approach

The present study deals with the production of cold active polygalacturonase (PGase) by submerged fermentation using Thalassospira frigidphilosprofundus, a novel species isolated from deep waters of Bay of Bengal. Nonlinear models were applied to optimize the medium components for enhanced production of PGase. Taguchi orthogonal array design was adopted to evaluate the factors influencing the yield of PGase, followed by the central composite design (CCD) of response surface methodology (RSM) to identify the optimum concentrations of the key factors responsible for PGase production. Data obtained from the above mentioned statistical experimental design was used for final optimization study by linking the artificial neural network and genetic algorithm (ANN-GA). Using ANN-GA hybrid model, the maximum PGase activity (32.54 U/mL) was achieved at the optimized concentrations of medium components. In a comparison between the optimal output of RSM and ANN-GA hybrid, the latter favored the production of PGase. In addition, the study also focused on the determination of factors responsible for pectin hydrolysis by crude pectinase extracted from T. frigidphilosprofundus through the central composite design. Results indicated 80% degradation of pectin in banana fiber at 20°C in 120 min, suggesting the scope of cold active PGase usage in the treatment of raw banana fibers.

Extracellular enzymes of Fusarium graminearum isolates

Fusarium graminearum isolates from three different agroecological regions in Argentina were examined according to the production of different extracellular enzyme activities of potential biotechnological interest: pectinases (PGase: polygalacturonase and PMGase: polymethylgalacturonase), cellulase (CMCase: carboxymethylcellulase) and hemicellulase (xylanase). The isolates were grown in minimum salt medium supplemented with 0.25% glucose, 0.125% citric pectin and 0.125% oat bran as carbon sources and/or enzyme inducers. PGase activity was detected early (after two days of incubation) in all the cultures; it was found to be the highest for all the isolates. PMGase was high only for those isolates of the II region. CMCase and endoxylanase activities were particularly found at late stages (after four and seven days of incubation, respectively) and the maximum values were lower than pectinase activities.

검류를 이용한 단세포 당근 제조 수율 향상 방법

In this study, the effects of gums (guar gum, xanthan gum, locust beam gum) on the activity of polygalacturonase( PGase) were examined. PGase activity was assayed by measuring the release of reducing groups from polygalacturonic acid. Guar gum, xanthan gum and locust bean gum were capable of increasing the catalytic activity of the PGase by 105%, 87% and 90%, respectively. Carrot was macerated by Macerozyme R-200 with gums and the yield of the maceration reaction for the production of carrot single cells was increased up to 13% in the presence of guar gum. This suggested that gums stated above can be used as good enhancers not only for the catalytic activity of the PGase but also for the production of carrot single cell.

Purification and Characterization of a Novel Protopectin-Solubilizing Enzyme from Aspergillus niger Z-25

A mutant, Aspergillus niger Z-25 derived from A. niger XZ-131 with N+ supplementation, produced a protopectin-solubilizing enzyme (protopectinase). The enzyme was purified to homogeneity by a procedure involving ammonium sulfate fractionation and gel chromatographies on DEAE-Sephadex A-50 and Sephadex G-100 columns. The molecular weight of the protopectinase was estimated at 68.4 KD by SDS-polyacrylamide gel electrophoresis. The enzyme was stable from pH 3.0 to 7.0 and up to 60°C. The optimum pH and temperature for enzyme activity was 4.0 and 50°C, respectively. The purified enzyme had 268-U/mL PPase (protopectinase) activity on citrus protopectin, and also showed 100-U/mL PGase (polygalacturonase) activity, which catalyzed the hydrolysis of polygalacturonic acid. The Km value for protopectin was 0.215 mg/mL, while the Km value for polygalacturonic acid was 39.09 mg/ml. The PPase/PGase q-value was 2.68, more than 0.5. Therefore the enzyme was considered a novel pectinase and classified as protopectinase.

Production, characterization and application of a thermostable polygalacturonase of a thermophilic mould Sporotrichum thermophile Apinis

The production of polygalacturonase (PGase) by Sporotrichum thermophile Apinis in stirred submerged fermentation (SmF) was high in comparison with that in static conditions. Yeast extract (0.25%) and citrus pectin (2%) at pH 7.0 and 45 °C supported a high enzyme production in flasks agitated at 200 rpm. An overall 1.75-fold enhancement in PGase production was achieved as a result of optimization. The enzyme was optimally active at pH 7.0 and 55 °C, and exhibited t 1/2 of 4 h at 65 °C. The K m and V max values of the enzyme (for pectin) were 0.416 mg ml −1 and 0.52 μmol mg −1 min −1, respectively. The PGase activity was stimulated by Mn 2+ and Fe 2+, but inhibited strongly by Mg 2+, and slightly by Tween 80 and Triton X-100. Among the additives tested, β-mercaptoethanol exerted a strong inhibitory effect, suggesting a critical role of disulphide linkages in maintaining a suitable conformation of the enzyme. An increase in the yield of banana, grape and apple juices was recorded due to the treatment of fruit pulps with the mixture of enzymes (pectinase, xylanases and cellulase) of S. thermophile as compared to that with only pectinase. The yield of fruit juices did not increase with enhanced titre of cellulase in the enzyme mixture.

Flax-retting by polygalacturonase-containing enzyme mixtures and effects on fiber properties

Enzyme-retting of flax was accomplished via individual treatment with four polygalacturonase (PGase) containing solutions of various fungal sources and the resulting fibers were characterized. The retting solutions were equilibrated to contain 2.19 U of PGase activity as determined via a dinitrosalicylic acid (DNS) reducing sugar assay. As compared with the buffer control, treatment with the various enzyme solutions increased the yield of fine fibers. Treatment with Aspergillus niger PGase resulted in a 62% increase in fine fiber yield as compared with the buffer control and fiber strength did not statistically differ ( P≤0.05) between these treatments. Retting via PGases of Rhizopus origin produced the weakest fibers. These results illustrate that the crude PGases differ in their ability to ret flax and that under the defined experimental conditions the A. niger PGase is a better retting agent. Light microscopy demonstrated the ability of all enzymes to separate fiber from shive and epidermal tissues. Enzyme profiles of the solutions were determined via viscometric assays. Pectinolytic activity was the predominant activity of all enzymes tested. Activity against carboxymethyl cellulose (CMC) was a minor component of all solutions except A. niger PGase for which no activity was detected.

Inhibition of polygalacturonase from Verticillium dahliae by a polygalacturonase inhibiting protein from cotton

An extracellular endo-polygalacturonase (PGase) [E.C. 3.2.1.15] was isolated from 18-day-old culture filtrates of Verticillium dahliae and partially purified using gel permeation chromatography. The band responsible for PGase activity was electrophoretically characterized as having a molecular mass of approximately 29 500 and an isoelectric point of 5.4. Kinetic studies indicate a Km of 3.3 mg ml−1 and Vmax of 0.85 μmol reducing units min−1 ml−1 with polygalacturonic acid as substrate. Polygalacturonase inhibitor protein (PGIP) in cotton seedlings was induced by 5 mM salicylic acid and immunochemical analysis indicated high levels in the hypocotyl tissues. PGIP was purified from roots and stems using affinity chromatography with endo-PGase from Aspergillus niger as an immobilised ligand. The purified PGIP contained monomeric and dimeric molecules with molecular masses of 34 and 66 kDa respectively. Purified cotton PGIP inhibited endo-polygalacturonase from A. niger in a non-competitive or mixed manner with an inhibition constant, KI of 15 nM. The isolated V. dahliae PGase was, however, inhibited in a positive cooperative manner, indicative of allosteric interactions between the enzyme and the inhibitor protein. In addition to reducing the reaction rate, decreased substrate affinity may contribute to the accumulation of elicitor-active oligouronides.

Acidic condition-inducible polygalacturonase of Aspergillus kawachii

Aspergillus kawachii IFO 4308, which can grow in an extremely acidic condition (pH 2), produced some extracellular polygalacturonases (PGase). However, pH 2 and pH 5 culture filtrates showed different pH PGase activity profiles. Anion exchange chromatographies revealed that the PGase compositions of the two culture filtrates were different, and dominant enzymes (PGase-A1 and -A2 in the pH 2 culture and PGase-B in the pH 5 culture) were purified and characterized. The optimal pH was pH 4 for A1, pH 3 for A2, and pH 5 for B. PGase-A1 and -A2 were more stable at low pH than PGase-B. Molecular masses of PGase-A1, -A2, and -B were 43, 83, and 71 kDa, respectively. The N-terminal amino acid sequence of PGase-B was similar to those of other fungal PGases, but distinctly different from those of PGase-A1 and -A2. These results suggest that PGase-A1 and -A2 may be acidic condition-inducible enzymes and that a pH-regulated expression system is involved in the PGase production of A. kawachii.

Quantification of protopectinase SE, an endopolygalacturonase with pectin-releasing activity from Geotrichum klebahnii

Pectin releasing activity of protopectinase SE (PPase-SE) from Geotrichum klebahnii (= G. penicillatum = Trichosporon penicillatum) ATCC 42397 was determined using different batches of lemon protopectin as substrate. Results obtained showed a high degree of variability depending on the batch of protopectin used. As PPase-SE also shows polygalacturonase (PGase) activity, a method for the assay of this activity was optimised. The best assay conditions were: substrate (polygalacturonic acid) concentration of 2.0 g 1−1, reaction time of 10 min and up to 0.17 PGase units per test tube.

Inhibitors of polygalacturonase in calli of Orobanche aegyptiaca

The presence of at least three distinct polygalacturonases (PGase) in callus of Orobanche was demonstrated. The PGase activity is labile and at pH 4.5 does not require activation by cations. It can be partially purified on Biogel P 100 columns and can be resolved by PAGE into several bands. Broomrape callus tissue also contains inhibitors of PGase activity. One of these is a low M r compound, stable to boiling and removable by dialysis. An additional inhibitor precipitable by ammonium sulphate is also present. © 1997 Elsevier Science Ltd. All rights reserved

Involvement of Suppressor‐Glucans and Plant Epidermal Cells in Host‐Selective Pathogenesis of Phytophthora capsici

Water‐soluble glucans (WSG) from a virulent isolate of Phytophthora capsici (PCAP‐3) which were released during germination of cystospore markedly suppressed the elicitor‐induced death of suspension‐cultured cells of susceptible sweet pepper and tomato but not that of resistant pepper and tobacco. PCAP‐3, its polygalacturonase (PGase)‐deficient mutant (PCAP3‐M16), and galacturonic acid non‐utilizable mutant carrying the PGase (PCAP‐1) activity could penetrate in epidermal cells of host leaves, but similarly caused a hypersensitive response (HR) on non‐injured leaves of resistant host (sweet pepper). In the case of inoculation on press‐injured leaves, however, both of the resistant and nonhost plant leaves became quite susceptible to PCAP‐3similar to susceptible hosts, but not to PCAP3‐M16 and PCAP‐1. The results suggested that host‐selectivity of P. capsici may be determined in the leaf epidermal cells where the suppressor glucans released during infection effectively suppressed the occurrence of hypersensitive reaction. Furthermore, during growth of the fungus in intercellular spaces of leaf tissues, PGase may contribute not only to the virulence experession but also the supply of initial nutrition for fungal growth in the intercellular space of host tissues.

Purification and characterization of a pectin-releasing enzyme produced by Kluyveromyces wickerhamii

A pectin-releasing enzyme produced by Kluyveromyces wickerhamii IFO 1675 (PPase-W) was purified to homogeneity from a culture filtrate by cation-exchange and size-exclusion chromatographies. This enzyme had a molecular weight of 35,000 determined by both size exclusion chromatography and ultracentrifugal analysis, and of 40,000 by SDS-PAGE. It contained 2.4% sugar, and its isoelectric point was at pH 5.2. PPase-W catalyzed the release of highly polymerized pectin from various protopectins, and also showed endopolygalacturonase (endo-PGase) activity. The purified enzyme had optimum PGase activity at about pH 5.2 and 50°C and was stable in the range of pH from 4.0 to 7.0 and up to 50°C. The properties of PPase-W were compared with those of PPase-F from Kluyveromyces fragilis IFO 0288, and some differences were found. Also, some preliminary data dealing with the relationship between enzyme activities (PPase and endo-PGase) and protein structure are discussed.