Biosynthesis of β-glucans by cell-free extracts from saccharomyces cerevisiae

Cell-free extracts from Saccharomyces cerevisiae catalyzed the incorporation of glucosyl residues from UDP-[U-¹⁴C]glucose into β-1, 3-glucans which contained a significant proportion of β-1, 6-glycosidic linkages. When GDP-[U-¹⁴C]-glucose was used as substrate only trace amounts of glucose were incorporated. Activity of β-glucan synthetase was distributed among membrane and cell wall fractions, specific activity being higher in this latter. β-Glucan synthesized by membrane and cell wall fractions contained 0.6% and 2.5% of β-1, 6-glycosidic linkages respectively. A marked decrease in the activity of β-glucan synthetase occurred as the cells aged. Significant activity of glycogen synthetase was detected only in cells which had reached the stationary phase of growth.     

beta-1,3-Glucan in Developing Cotton Fibers: Structure, Localization, and Relationship of Synthesis to That of Secondary Wall Cellulose

Evidence is presented for the existence of a noncellulosic β-1,3-glucan in cotton fibers. The glucan can be isolated as distinct fractions of varying solubility. When fibers are homogenized rigorously in aqueous buffer, part of the total β-1,3-glucan is found as a soluble polymer in homogenates freed of cell walls. The proportion of total β-1,3-glucan which is found as the soluble polymer varies somewhat as a function of fiber age. The insoluble fraction of the β-1,3-glucan remains associated with the cell wall fraction. Of this cell wall β-1,3-glucan, a variable portion can be solubilized by treatment of walls with hot water, a further portion can be solubilized by alkaline extraction of the walls, and 17 to 29% of the glucan remains associated with cellulose even after alkaline extraction. A portion of this glucan can also be removed from the cell walls of intact cotton fibers by digestion with an endo-β-1,3-glucanase. The glucan fraction which can be isolated as a soluble polymer in homogenates freed of cell walls is not associated with membranous material, and we propose that it represents glucan which is also extracellular but not tightly associated with the cell wall. Enzyme digestion studies indicate that all of the cotton fiber glucan is β-linked, and methylation analyses and enzyme studies both show that the predominant linkage in the glucan is 1 → 3. The possibility of some minor branching at C-6 can also be deduced from the methylation analyses. The timing of deposition of the β-1,3-glucan during fiber development coincides closely with the onset of secondary wall cellulose synthesis. Kinetic studies performed with ovules and fibers cultured in vitro show that incorporation of radioactivity from [¹⁴C]glucose into β-1,3-glucan is linear with respect to time almost from the start of the labeling period; however, a lag is observed before incorporation into cellulose becomes linear with time, suggesting that these two different glucans are not polymerized directly from the same substrate pool. Pulse-chase experiments indicate that neither the β-1,3-glucan nor cellulose exhibits significant turnover after synthesis.     

Estimation of soluble β-glucan content of yeast cell wall by the sensitivity to Glucanex 200G treatment

The soluble β-glucan contents in the cell wall of yeasts were estimated by treating cells with Glucanex^® 200G that contained mainly β1,3-glucanase and some β1,6-glucanase. The sensitivity of cell walls of 11 yeasts to various concentrations of β-glucanase was compared. The yeasts that are resistant to β-glucanase treatment are expected to contain higher β-glucan content and those that are sensitive to the β-glucanase treatment are expected to contain lower β-glucan content. Two yeast strains were selected for further study by comparing the sensitivity of cell wall to β-glucanase; Candida bombicola and Candida albicans. Candida bombicola was more resistant and C. albicans was more sensitive to the Glucanex^® 200G treatment. The results of enzyme sensitivity tests were verified by quantification of soluble β-glucan content purified from the yeasts. Much larger amount of soluble β-glucan was obtained from the cell walls of C. bombicola (0.08 g g⁻¹ dried cell) than C. albicans (0.025 g g⁻¹ dried cell).     

Analysis of the β-1,3-Glucanolytic System of the Biocontrol Agent Trichoderma harzianum

The biocontrol agent Trichoderma harzianum IMI206040 secretes β-1,3-glucanases in the presence of different glucose polymers and fungal cell walls. The level of β-1,3-glucanase activity secreted was found to be proportional to the amount of glucan present in the inducer. The fungus produces at least seven extracellular β-1,3-glucanases upon induction with laminarin, a soluble β-1,3-glucan. The molecular weights of five of these enzymes fall in the range from 60,000 to 80,000, and their pIs are 5.0 to 6.8. In addition, a 35-kDa protein with a pI of 5.5 and a 39-kDa protein are also secreted. Glucose appears to inhibit the formation of all of the inducible β-1,3-glucanases detected. A 77-kDa glucanase was partially purified from the laminarin culture filtrate. This enzyme is glycosylated and belongs to the exo-β-1,3-glucanase group. The properties of this complex group of enzymes suggest that the enzymes might play different roles in host cell wall lysis during mycoparasitism.     

Medicinal importance of fungal β-(1→3), (1→6)-glucans

Non-cellulosic β-glucans are now recognized as potent immunological activators, and some are used clinically in China and Japan. These β-glucans consist of a backbone of glucose residues linked by β-(1→3)-glycosidic bonds, often with attached side-chain glucose residues joined by β-(1→6) linkages. The frequency of branching varies. The literature suggests β-glucans are effective in treating diseases like cancer, a range of microbial infections, hypercholesterolaemia, and diabetes. Their mechanisms of action involve them being recognized as non-self molecules, so the immune system is stimulated by their presence. Several receptors have been identified, which include: dectin-1, located on macrophages, which mediates β-glucan activation of phagocytosis and production of cytokines, a response co-ordinated by the toll-like receptor-2. Activated complement receptors on natural killer cells, neutrophils, and lymphocytes, may also be associated with tumour cytotoxicity. Two other receptors, scavenger and lactosylceramide, bind β-glucans and mediate a series of signal pathways leading to immunological activation. Structurally different β-glucans appear to have different affinities toward these receptors and thus generate markedly different host responses. However, the published data are not always easy to interpret as many of the earlier studies used crude β-glucan preparations with, for the most part, unknown chemical structures. Careful choice of β-glucan products is essential if their benefits are to be optimized, and a better understanding of how β-glucans bind to receptors should enable more efficient use of their biological activities.     

Evolution and differential expression of the (1→3)-β-glucan endohydrolaseencoding gene family in barley, Hordeum vulgare

The (1→3)-β-d-glucan glucanohydrolases [(1→ 3)-GGH; EC 3.2.1.39] of barley (Hordeum vulgare L., cv Clipper) are encoded by a small gene family. Amino acid sequences deduced from cDNA and genomic clones for six members of the family exhibit overall positional identities ranging from 44% to 78%. Specific DNA and oligodeoxyribonucleotide (oligo) probes have been used to demonstrate that the (1→3)-GGH-encoding genes are differentially transcribed in young roots, young leaves and the aleurone of germinated grain. The high degree of sequence homology, coupled with characteristic patterns of codon usage and insertion of a single intron at a highly conserved position in the signal peptide region, indicate that the genes have shared a common evolutionary history. Similar structural features in genes encoding barley (1→3,1→4)-β-glucan 4-glucanohydrolases [(1→3,1→4)-GGH; EC 3.2.1.73] further indicate that the (l→3)-GGHs and (l→3,1→4)-GGHs are derived from a single ‘super’ gene family, in which genes encoding enzymes with related yet quite distinct substrate specificities have evolved, with an associated specialization of function. The (1→3,1→4)-GGHs mediate in plant cell wall metabolism through their ability to hydrolyse the (1→3,1→4)-β-glucans that are the major constituents in barley walls, while the (1→3)-GGHs, which are unable to degrade the plant (1→3,1→4)-β-glucans, can hydrolyse the (1→3)- and (1→3,1→6)-β-glucans of fungal cell walls.     

Glucans from fruit bodies of cultivated mushrooms Pleurotus ostreatus and Pleurotus eryngii: Structure and potential prebiotic activity

Cultivated oyster mushrooms (genus Pleurotus) are interesting as a source of biologically active glucans. Partially, β-glucan from Pleurotus sp. (pleuran) has been used as food supplements due to its immunosuppressive activity. Like other dietary fibre components, oyster mushroom polysaccharides can stimulate the growth of colon microorganisms (probiotics), i.e. act as prebiotics. Specific glucans were isolated from stems of Pleurotus ostreatus and Pleurotus eryngii by subsequent boiling water and alkali extraction. Obtained water soluble (L1), alkali soluble (L2) and insoluble (S) fractions were characterised by various analytical methods. Spectroscopic analysis detected glucans in all the fractions: branched 1,3-1,6-β-d-glucan predominated in L1 and S, while linear 1,3-α-d-glucan in L2. Fractions L1 also contained marked amount of proteins partially in complex with glucans; protein content in L2 was insignificant. Effective deproteinisation of L1 and separation of α- and β-glucans in L2 was achieved by the treatment with phenolic reagent. Small amount of chitin was found in S as a component of cell wall chitin–glucan complex. Potential prebiotic activity of extracts L1 and L2 was testing using nine probiotic strains of Lactobacillus, Bifidobacterium and Enterococcus. These probiotics showed different growth characteristics dependently on used extract and strain specificity due to the presence of structurally diverse compounds. The extracts L1 and L2 can be applied to synbiotic construction only for carefully selected probiotic strains. This exploitation of fruit body extracts extends the use of mushrooms P. ostreatus and P. eryngii for human health.     

The Cell Wall of Fusarium oxysporum

Sugar analysis of isolated cell walls from three formae speciales of Fusarium oxysporum showed that they contained not only glucose and (N-acetyl)-glucosamine, but also mannose, galactose, and uronic acids, presumably originating from cell wall glycoproteins. Cell wall glycoproteins accounted for 50–60% of the total mass of the wall. X-ray diffraction studies showed the presence of α-1,3-glucan in the alkali-soluble cell wall fraction and of β-1,3-glucan and chitin in the alkali-insoluble fraction. Electron microscopy and lectin binding studies indicated that glycoproteins form an external layer covering an inner layer composed of chitin and glucan.     

Cloning and Targeted Disruption of MLG1, a Gene Encoding Two of Three Extracellular Mixed-Linked Glucanases of Cochliobolus carbonum

Mixed-linked glucanases (MLGases), which are extracellular enzymes able to hydrolyze β1,3-1,4-glucans (also known as mixed-linked glucans or cereal β-glucans), were identified in culture filtrates of the plant-pathogenic fungus Cochliobolus carbonum. Three peaks of MLGase activity, designated Mlg1a, Mlg1b, and Mlg2, were resolved by cation-exchange and hydrophobic-interaction high-performance liquid chromatography (HPLC). Mlg1a and Mlg1b also hydrolyze β1,3-glucan (laminarin), whereas Mlg2 does not degrade β1,3-glucan but does degrade β1,4-glucan to a slight extent. Mlg1a, Mlg1b, and Mlg2 have monomer molecular masses of 33.5, 31, and 29.5 kDa, respectively. The N-terminal amino acid sequences of Mlg1a and Mlg1b are identical (AAYNLI). Mlg1a is glycosylated, whereas Mlg1b is not. The gene encoding Mlg1b, MLG1, was isolated by using PCR primers based on amino acid sequences of Mlg1b. The product of MLG1 has no close similarity to any known protein but does contain a motif (EIDI) that occurs at the active site of MLGases from several prokaryotes. An internal fragment of MLG1 was used to create mlg1 mutants by transformation-mediated gene disruption. The total MLGase and β1,3-glucanase activities in culture filtrates of the mutants were reduced by approximately 50 and 40%, respectively. When analyzed by cation-exchange HPLC, the mutants were missing the two peaks of MLGase activity corresponding to Mlg1a and Mlg1b. Together, the data indicate that Mlg1a and Mlg1b are products of the same gene, MLG1. The growth of mlg1 mutants in culture medium supplemented with macerated maize cell walls or maize bran and the disease symptoms on maize were identical to the growth and disease symptoms of the wild type.     

<Emphasis Type="Italic">Mucor rouxii</Emphasis> Rho1 protein; characterization and possible role in polarized growth

We have previously shown that protein kinase A of the medically important zygomycete Mucor rouxii participates in fungal morphology through cytoskeletal organization. As a first step towards finding the link between protein kinase A and cytoskeletal organization we here demonstrate the cloning of the Rho1 gene and the characterization of its protein product. The RHO1 protein primary sequence shows 70–85% identity with fungal RHO1 or mammalian RhoA. Two protein kinase A phosphorylation sequences in adequate context are predicted, Ser73 and Ser135. The peptide IRRNSQKFV, containing Ser135 proved to be a good substrate for M. rouxii protein kinase A catalytic subunit. The over-expressed Rho1 fully complements a Saccharomyces cerevisiae null mutant. The endogenous protein was identified by western blot against a developed antibody and by ADP-ribosylation. Localization in germlings was visualized by immunofluorescence; the protein was localized in patches in the mother cell surface and excluded from the germ tube. Measurement of Rho1 expression during germination indicates that Rho1, at both the mRNA and protein levels, correlates with differentiation and not with growth. Rho1 has been shown to be the regulatory protein of the β-1,3-glucan synthase complex in fungi in which β-1,3-glucans are major components of the cell wall. Even though glucans have not been detected in zygomycetes, caspofungin, an echinochandin known to be an inhibitor of β-1,3-glucan synthase complex, is shown here to have a negative effect on growth and to produce an alteration on morphology when added to M. rouxii growth culture medium. This result has an important impact on the possible participation of β-1,3-glucans on the regulation of morphology of zygomycetes.     

Structure of a novel highly branched α-glucan enzymatically produced from maltodextrin

The bacterial strain PP710, isolated from soil and identified as Paenibacillus species, produced a low-digestibility α-glucan containing a large amylase-resistant portion. This α-glucan was obtained in high yields from maltodextrin (dextrose equivalent 3) by using the condensed culture supernatant of the strain as the enzyme preparation. The water-soluble dietary fiber content of the low-digestibility α-glucan was 80.2%, and showed resistance to a rat intestinal enzyme preparation. The α-glucan was found to be a novel highly branched α-glucan by acid hydrolysis, NMR analysis, gel permeation chromatography, methylation analysis, and enzymatic digestion.     

Mutants of the white-rot fungus Sporotrichum pulverulentum with increased cellulase and β- -glucosidase production

This paper reports the isolation of mutants of the white-rot fungus Sporotrichum pulverulentum and the results of a survey of enzymic activity among these mutants. The strains were screened for extracellular cellulase [see 1,4-(1,3;1,4)-β- -glucan 4-glucanohydrolase, EC 3.2.1.4] and β- -glucosidase (β- -glucoside glucohydrolase, EC 3.2.1.21) production in shake flask experiments. Apart from strain 63-2, strains 6, 63, 9, L5, E-1 and UV-18 showed equal or higher endo-1,4-β- -glucanase (cellulase), filter paper-degrading and β- -glucosidase activities than S. pulverulentum. The cellulase activity obtained, measured as filter paper activity, was comparable to that reported for Trichoderma reesei QM9414. However, the β- -glucosidase activity was about six times higher than for the QM9414 strain. The pH and temperature-activity profiles of crude β- -glucosidase preparations from the various strains were determined and were found to be identical. The thermal stability at pH 4.5 and 40°C was 5 days for all these preparations.     

Ultrastructure and Chemical Analysis of the Cell Wall of Pythium debaryanum1

The ultrastructure and component polysaccharides of the cell wall of Pythium debaryanum IFO-5919 were investigated. From results obtained by means of acid, alkali, Schweitzer reagent and β-1, 3-glucanase treatments and electron microscopy, it was concluded that 1) the acid-extracted fraction was a 1,3-linked branched glucan, 2) the alkali-extracted fraction was a mixture of 1,3-, 1,6-, and 1,3,6-linked highly branched two glucans, 3) the Schweitzer reagent-extracted fraction was a β-1, 4-linked glucan, 4) the cell wall was constructed from two types of cullulosic microfibrils, as a frame and as a finer network, and amorphous β-1, 3-glucan including β-1, 6-linkage, 5) cellulosic microfibrils were covered by matrix material consisting of a mixture of amorphous β-1, 3-linked and β-1, 6-linked branching glucans.     

Facile synthesis of glucose-1-phosphate from starch by Thermus caldophilus GK24 α-glucan phosphorylase

The glgP gene encoding α-glucan phosphorylase (α-GP) from the thermopile Thermus caldophilus GK24 has been identified, cloned, and overexpressed in Escherichia coli and used to synthesize d-glucose-1-phospate (G1P) from an inexpensive starch. The enzyme, purified 6.5-fold, was isolated in 31% yield from the transformed E. coli, and gave a single band. The purified enzyme may exist as a homohexamer with an apparent molecular mass of a 550 kDa molecule, consisting of 90 kDa per subunit. The optimal pH and temperature were 7.0 and 70 °C in the α-GP reaction with starch producing G1P. Soluble starch (amylopectin, amylose) turned out to be a better substrate giving a higher yield of G1P than α-1,6-branched α-1,4-glucans (glycogen, potato starch, etc.). As a result, G1P was obtained in a good yield (47%, w/w) from the reaction containing 5% (w/v) soluble starch in 0.7 M potassium phosphate at pH 7.0. T. caldophilus α-GP shows a high tolerance (up to 0.7 M) of potassium phosphate and plays a critical role in shifting the reaction equilibrium in favor of G1P synthesis. The G1P product can be purified simply by ethanol precipitation, after removing the unreacted starch and inorganic phosphate by activated charcoal and magnesium acetate precipitation. It is concluded that T. caldophilus α-GP readily utilized in large scale synthesis of G1P.     

beta-Glucan Synthetases of Plasma Membrane and Golgi Apparatus from Onion Stem

Biosynthesis of glucans occurred in cell-free fractions isolated from onion stem (Allium cepa L.) enriched in either dictyosomes or plasma membranes. β-1,3- and β-1, 4-Glucans were synthesized in differing proportions and at different rates as the concentration of uridine diphosphoglucose or the proportion of dictyosomes or plasma membrane varied. At low (1.5 μm) UDP-glucose concentrations synthesis of alkali-insoluble glucan was correlated with abundance of dicytosomes; most of the substrate utilized by plasma membrane was for glycolipid synthesis. At high (1 mm) UDP-glucose concentration, the synthesis of alkali-insoluble glucans correlated with the abundance of plasma membrane. Substrate enhancement of β-1, 4-glucan synthesis in dictyosome fractions was less than proportional to increases in substrate concentration. In contrast, β-1, 4-glucan synthesis by plasma membrane was more than proportionately increased. At high substrate concentrations the synthesis of β-1, 3-glucans predominated in both dictyosome and plasma membrane fractions. The results show that the capacity to synthesize glucans resides in both Golgi apparatus and plasma membranes of onion stem, but that the plasma membrane has the greatest capacity for synthesis of alkali-insoluble glucans at high UDP-glucose concentrations.     

Loss of the Plasma Membrane-Bound Protein Gas1p in Saccharomyces cerevisiae Results in the Release of β1,3-Glucan into the Medium and Induces a Compensation Mechanism To Ensure Cell Wall Integrity

Deletion of GAS1/GGP1/CWH52 results in a lower β-glucan content of the cell wall and swollen, more spherical cells (L. Popolo, M. Vai, E. Gatti, S. Porello, P. Bonfante, R. Balestrini, and L. Alberghina, J. Bacteriol. 175:1879–1885, 1993; A. F. J. Ram, S. S. C. Brekelmans, L. J. W. M. Oehlen, and F. M. Klis, FEBS Lett. 358:165–170, 1995). We show here that gas1Δ cells release β1,3-glucan into the medium. Western analysis of the medium proteins with β1,3-glucan- and β1,6-glucan-specific antibodies showed further that at least some of the released β1,3-glucan was linked to protein as part of a β1,3-glucan–β1,6-glucan–protein complex. These data indicate that Gas1p might play a role in the retention of β1,3-glucan and/or β-glucosylated proteins. Interestingly, the defective incorporation of β1,3-glucan in the cell wall was accompanied by an increase in chitin and mannan content in the cell wall, an enhanced expression of cell wall protein 1 (Cwp1p), and an increase in β1,3-glucan synthase activity, probably caused by the induced expression of Fks2p. It is proposed that the cell wall weakening caused by the loss of Gas1p induces a set of compensatory reactions to ensure cell integrity.     

Chemical Organization of the Cell Wall Polysaccharide Core of Malassezia restricta

Malassezia species are ubiquitous residents of human skin and are associated with several diseases such as seborrheic dermatitis, tinea versicolor, folliculitis, atopic dermatitis, and scalp conditions such as dandruff. Host-Malassezia interactions and mechanisms to evade local immune responses remain largely unknown. Malassezia restricta is one of the most predominant yeasts of the healthy human skin, its cell wall has been investigated in this paper. Polysaccharides in the M. restricta cell wall are almost exclusively alkali-insoluble, showing that they play an essential role in the organization and rigidity of the M. restricta cell wall. Fractionation of cell wall polymers and carbohydrate analyses showed that the polysaccharide core of the cell wall of M. restricta contained an average of 5% chitin, 20% chitosan, 5% β-(1,3)-glucan, and 70% β-(1,6)-glucan. In contrast to other yeasts, chitin and chitosan are relatively abundant, and β-(1,3)-glucans constitute a minor cell wall component. The most abundant polymer is β-(1,6)-glucans, which are large molecules composed of a linear β-(1,6)-glucan chains with β-(1,3)-glucosyl side chain with an average of 1 branch point every 3.8 glucose unit. Both β-glucans are cross-linked, forming a huge alkali-insoluble complex with chitin and chitosan polymers. Data presented here show that M. restricta has a polysaccharide organization very different of all fungal species analyzed to date.     

Isolation, Properties, Function, and Regulation of Endo-(1 → 3)-β-Glucanases in Schizosaccharomyces pombe

Cell-free extracts, membranous fractions, and cell wall preparations from Schizosaccharomyces pombe were examined for the presence of (1 → 3)-β-, (1 → 3)-α-, and (1 → 6)-β-glucanase activities. The various glucanases were assayed in cells at different growth stages. Only (1 → 3)-β-glucanase activity was found, and this was associated with the cell wall fraction. Chromatographic fractionation of the crude enzyme revealed two endo-(1 → 3)-β-glucanases, designated as glucanase I and glucanase II. Glucanase I consisted of two subunits of molecular weights 78,500 and 82,000, and glucanase II was a single polypeptide of 75,000. Although both enzymes had similar substrate specificities and similar hydrolytic action on laminarin, glucanase II had much higher hydrolytic activity on isolated cell walls of S. pombe. On the basis of differential lytic activity on cell walls, glucanase II was shown to be present in conjugating cells and highest in sporulating cells. Glucanase II appeared to be specifically involved in conjugation and sporulation since vegetative cells and nonconjugating and nonsporulating cells did not contain this enzyme. The appearance of glucanase II in conjugating cells may be due to de novo enzyme synthesis since no activation could be demonstrated by combining extracts from vegetative and conjugating cells. Increased glucanase activity occurred when walls from conjugating cells were combined with walls from sporulating cells. Studies with trypsin and proteolytic inhibitors suggest that glucanase II exists as a zymogen in conjugating cells. A temperature-sensitive mutant of S. pombe was isolated which lysed at 37°C. Glucanase activity was higher in vegetative cells held at 37°C than cells held at 25°C. Unlike the wild-type strain, this mutant contained glucanase II activity during vegetative growth and may be a regulatory mutant.     

β-1, 3-Glucanase from trichoderma viride. Purification and characterization

The extracellular complex of β-glucanases produced by the mould Trichoderma viride hydrolyzes β-1,3-; β-1,4- and β-1, 6-bonds of β-glucans, as well as mixed β-1,3- and β-1,4-glycosidic linkages. This complex contains also xylanase. The enzymes were isolated from liquid culture medium by centrifuge techniques, concentration and precipitation with acetone. Isolation and purification of β-1,3-glucanase was carried out according to a procedure involving filtration on Bio-Gel P-100, DEAE-Sephadex A 50 and CM-cellulose C-11 chromatography, ultrafiltration and selective adsorption on xylan. The homogeneity of the enzyme was determined by polyacrylamidegel electrophoresis. The purified homogeneous preparation of the isolated β-1,3-glucanase from Tr. riride was subjected to detailed characterization. Amino acid composition, molecular weight and optimum conditions for the enzymatic activity of the protein were determined. The isolated enzyme was shown to be highly specific to substrates with β-1,3-glycosidic linkages; the rate of degradation was found to be proportional to the degree of polymerization of the substrate.     

Characterization and <Emphasis Type="Italic">in vitro</Emphasis> expression patterns of extracellular degradative enzymes from non-pathogenic binucleate <Emphasis Type="Italic">Rhizoctonia</Emphasis> AG-G

Many filamentous fungi produce an array of extracellular enzymes that acting in cell walls release elicitors of the plant defense response These enzymes may therefore be important in biocontrol applications. The aim of this study was to characterize extracellular degradative enzymes produced by a non-pathogenic binucleate isolate of Rhizoctonia AG-G. The fungus was grown in liquid culture supplemented with pectin, polygalacturonic acid or glucose as a carbon sources and filtrates of the culture media were analyzed for the detection of pectinolytic and glucan hydrolytic enzymes. Using only pectin as a carbon source, secretion of polygalacturonases and methylesterases was found. When the liquid medium was supplemented with polygalacturonic acid, only polygalacturonase activity was detected. However, when glucose was used as carbon source -1,3 and -1,6 glucanases activities were detected, using laminarin and pustulan as substrates, but none of the pectinolytic activities were found. These enzymes were partially purified and characterized. The -(1,3)(1,6) glucanase and polygalacturonase enzymes showed to be active against cell wall polysaccharides from potato sprouts. These enzymes may have an important role in fungus-plant cell wall interaction. This is the first study about the production of extracellular enzymes by non-pathogenic binucleate Rhizoctonia AG-G.