Enzyme Production by the Mycoparasite Verticillium biguttatum against Rhizoctonia solani

Verticillium biguttatum, a mycoparasite of the ubiquitous soil-borne plant pathogen Rhizoctonia solani, excreted chitinase and beta-1,3-glucanase into liquid medium when grown on laminarin and chitin, respectively. Neither chitinase nor beta-1,3-glucanase was produced by the mycoparasite when grown on cell walls of two isolates of R. solani representing anastomosis groups (AG)-3 and AG-8. Extracellular protease was induced by growth on cell walls of the pathogen, whereas beta-1,3-glucanase and chitinase were produced bound to the cell wall of V. biguttatum. This is the first report of chitinase, beta-1,3-glucanase and protease production by V. biguttatum. These enzymes may play a previously unforeseen role in dissolving and penetrating the cell walls of R. solani.     

Production and catabolite repression of Penicillium italicum beta-glucanases.

The filamentous fungus Penicillium italicum, grown in a defined liquid medium, produced beta-1,3-glucanase, which remained essentially bound to the cells, and beta-1,6-glucanase, an essentially extracellular enzyme. When glucose was depleted from the medium, when a limited concentration of glucose (0.2%) was maintained, or when the carbon source was galactose (3%) or lactose (3%), a significant increase in the specific activity of beta-1,3-glucanase, in cell extracts, took place. This was paralleled by a very slow rate of growth, and under glucose limitation, the appearance of beta-1,3-glucanase in the medium was also observed. On the other hand, when an excess of glucose, fructose, or sucrose was present, the specific activity remained constant and active growth was promoted. Laminarin, cellobiose, gentiobiose, and isolated Penicillium italicum walls were not capable of significantly inducing beta-1,3-glucanase synthesis to a level beyond that attained by glucose limitation. A similar behavior was observed for beta-1,6-glucanase. beta-1,3-Glucanase and beta-1,6-glucanase are therefore constitutive enzymes subjected to catabolite repression. The results are discussed in the context of the possible functions that have been suggested for glucanases and related enzymes.     

Cloning of the Bacillus subtilis DLG beta-1,4-glucanase gene and its expression in Escherichia coli and B. subtilis.

The gene encoding beta-1,4-glucanase in Bacillus subtilis DLG was cloned into both Escherichia coli C600SF8 and B. subtilis PSL1, which does not naturally produce beta-1,4-glucanase, with the shuttle vector pPL1202. This enzyme is capable of degrading both carboxymethyl cellulose and trinitrophenyl carboxymethyl cellulose, but not more crystalline cellulosic substrates (L. M. Robson and G. H. Chambliss, Appl. Environ. Microbiol. 47:1039-1046, 1984). The beta-1,4-glucanase gene was localized to a 2-kilobase (kb) EcoRI-HindIII fragment contained within a 3-kb EcoRI chromosomal DNA fragment of B. subtilis DLG. Recombinant plasmids pLG4000, pLG4001a, pLG4001b, and pLG4002, carrying this 2-kb DNA fragment, were stably maintained in both hosts, and the beta-1,4-glucanase gene was expressed in both. The 3-kb EcoRI fragment apparently contained the beta-1,4-glucanase gene promoter, since transformed strains of B. subtilis PSL1 produced the enzyme in the same temporal fashion as the natural host B. subtilis DLG. B. subtilis DLG produced a 35,200-dalton exocellular beta-1,4-glucanase; intracellular beta-1,4-glucanase was undetectable. E. coli C600SF8 transformants carrying any of the four recombinant plasmids produced two active forms of beta-1,4-glucanase, an intracellular form (51,000 +/- 900 daltons) and a cell-associated form (39,000 +/- 400 daltons). Free exocellular enzyme was negligible. In contrast, B. subtilis PSL1 transformed with recombinant plasmid pLG4001b produced three distinct sizes of active exocellular beta-1,4-glucanase: approximately 36,000, approximately 35,200, and approximately 33,500 daltons. Additionally, B. subtilis PSL1(pLG4001b) transformants contained a small amount (5% or less) of active intracellular beta-1,4-glucanase of three distinct sizes: approximately 50,500, approximately 38,500 and approximately 36,000 daltons. The largest form of beta-1,4-glucanase seen in both transformants may be the primary, unprocessed translation product of the gene.     

Regulation of the beta-1,3-glucanase system in Penicillium italicum: glucose repression of the various enzymes.

The microscopic fungus Penicillium italicum when grown in a synthetic liquid medium produced at least three enzymes with beta-1,3-glucanase activity which were separated by diethylaminoethyl-Sephadex column chromatography. These were named beta-1,3-glucanases I, II, and III respective to their order of elution from the column. A tentative characterization of these three enzymes indicated that they have different modes of action; the first one is an endoglucanase, the second is an exoglucanase, and the third probably has both mechanisms of action. Glucose had a repressive effect on all three enzymes. Only small amounts of beta-1,3-glucanases II and III were present in the cells when they were actively growing in the presence of this sugar. However, when the cells were transferred to a medium low in glucose, a significant increase in the specific activity of beta-1,3-glucanase took place; this was due in part to a much more active production of beta-1,3-glucanases II and III and in part to the appearance of beta-1,3-glucanase I, which could only be detected after more than 12 h of incubation in this medium. The results are discussed in the context of possible beta-1,3-glucanase functions in the fungal cells.     

Whole cells of Bacillus subtilis AF 1 proved more effective than cell-free and chitinase-based formulations in biological control of citrus fruit rot and groundnut rust

In foliar and postharvest biocontrol systems, the use of active metabolites produced by antagonistic microorganisms is advantageous compared with the use of living microorganisms. Chitinases, a major group of hydrolytic enzymes produced by biocontrol agents, are involved in the lysis of cell walls of pathogenic fungi. In the present study, an attempt was made to test the partially purified beta-1,4-N-acetylglucosaminidase (NAGase) of a biocontrol strain Bacillus subtilis AF 1 for control of rust in groundnut (caused by Puccinia arachidis) and soft rot in lemons (caused by Aspergillus niger). Four proteins of molecular mass 67, 40, 37, and 32 kDa were isolated from the culture filtrates of AF 1 by affinity chromatography, of which the 67-kDa protein has detectable chitinolytic ability. This protein (NAGase) effectively inhibited the in vitro growth of A. niger in microtitre plates. In the presence of NAGase, germination of urediniospores of P. arachidis was reduced by 96% compared with the control. In a detached leaf bioassay, NAGase reduced the rust lesion frequency by >60%. NAGase significantly reduced the incidence of soft rot in harvested lemon fruits. However, fresh cells and (or) alginate formulation of AF 1 were more effective than NAGase in control of both of the test plant - pathogen systems.     

Purification and properties of a β-1,6-glucanase from Streptomyces sp. EF-14, an actinomycete antagonistic to Phytophthora spp.

Extracellular enzymes with glucanase activities are an important component of actinomycete-fungus antagonism. Streptomyces sp. EF-14 has been previously identified as one of the most potent antagonists of Phytophthora spp. A beta-1,6-glucanase (EC 3.2.1.75; glucan endo-1,6-beta-glucosidase) was purified by four chromatographic steps from the culture supernatant of strain EF-14 grown on a medium with lyophilized cells of Candida utilis as main nutrient source. The glucanase level in this medium followed a characteristic pattern in which the rise of beta-1,6-glucanase activity always preceded that of beta-1,3-glucanase. The molecular mass of the enzyme was estimated to be 65 kDa and the pI approximately 5.5. It hydrolyzed pustulan by an endo-mechanism generating gentiobiose and glucose as final products. Laminarin was not hydrolyzed indicating that the enzyme does not recognize beta-1,6-links flanked by beta-1,3-links. No significant clearing of yeast cell walls in liquid suspensions or in agar plates was observed indicating that this beta-1,6-glucanase is a non-lytic enzyme. This is the first beta-1,6-glucanase characterized from an actinomycete.     

Lysis of yeast cell walls. Lytic beta-(1 leads to 3)-glucanases from Bacillus circulans WL-12.

Bacillus circulans WL-12 when grown in a mineral medium with yeast cell walls or yeast glucan as the soli carbon source, produced five beta-glucanases. Two beta-(1 leads to 3)-glucanases (I and II), which are lytic to yeast cell walls, were isolated from the culture liquid by batch adsorption on yeast glucan, and separated by chromatography on hydroxylapatite. Lytic beta-(1 leads to 3)-glucanase I was further purified by carboxymethylcellulose chromatography. The specific activity of lytic beta-(1 leads to 3)-glucanase I on laminarin was 4.1 U per mg of protein. The enzyme moved as a single protein with a molecular weight of 40000 during sodium dodecylsulfate electrophoresis in slab gels. It was specific for the beta-(1 leads to 3)-glucosidic bond but the enzyme did not hydrolyze laminaribiose. Hydrolysis of laminarin went through a series of oligosaccharides, and laminaribiose and glucose accumulated till the end of the reaction. A small amount of gentibiose was also produced from laminarin. Products from yeast cell walls and yeast glucan included laminaripentaose, laminaritriose, laminaribiose, glucose and gentiobiose, but no laminaritetraose was detected. This glucanase has an optimum pH of 5.5.     

C4-dicarboxylate transport in Bacillus subtilis studied with 3-fluoro-L-erythro-malate as a substrate

Bacillus subtilis cells grown in yeast extract medium accumulated 3-fluoro-l-erythro-[1,2-(14)C(2)]malate more than 30-fold from the surrounding medium. No metabolic products derived from 3-fluoro-l-erythro-malate could be detected in these cells. l-Malate competitively inhibited transport of 3-fluoro-l-erythro-malate. This malate analogue was itself a competitive inhibitor of l-malate uptake. Cells that had been grown in yeast extract supplemented with 5 mM l-malate showed a 10-fold increased affinity towards 3-fluoro-l-erythro-malate relative to cells grown in yeast extract medium with no added malate. Our results suggest that two transport systems for l-malate can be induced in B. subtilis. The first of these systems seems to effect uptake of C(4)-dicarboxylates (l-malate, succinate, and fumarate) in yeast extract medium. The second transport system (or possibly a modification of the first transport system) seems to be induced by addition of l-malate to this medium and is also functioning in malate minimal medium.     

Derepression of beta-1,3-glucanases in Penicillium italicum: localization of the various enzymes and correlation with cell wall glucan mobilization and autolysis.

The localization of the derepressible beta-1,3-glucanases of Penicillium italicum and the cell wall autolysis under conditions of beta-1,3-glucanase derepression (24 h in a low-glucose medium) were studied. About 15% of the total activity was secreted into the culture medium during the 24-h period and consisted of similar amounts of each of the three beta-1,3-glucanases (I, II, III) produced by this species. Treatment of derepressed mycelia with periplasmic enzyme-inactivating agents resulted in a loss of 45% of the mycelium-bound beta-1,3-glucanase. Analysis of periplasmic enzymes solubilized by 2 M NaCl or by autolysis of isolated cell walls revealed that only beta-1,3-glucanases II and III were bound to the cell wall. These two enzymes were capable of releasing in vitro reducing sugars from cell walls, whereas beta-1,3-glucanase I was not. In addition, the autolytic activity of cell walls isolated from derepressed mycelium was greater than that of cell walls isolated from repressed mycelium. The incubation of the fungus in the low-glucose medium also resulted in the in vivo mobilization of 34% of the cell wall beta-1,3-glucan, and this mobilization was fully prevented by cycloheximide, which also blocked derepression of beta-1,3-glucanases. Derepression of beta-1,3-glucanase seems to be coupled to the mobilization of cell wall glucan.     

Regulation of beta-1,3-glucanase synthesis in Penicillium italicum.

The filamentous fungus Penicillium italicum produced a certain level of beta-1,3-glucanase during active growth in a glucose-supplemented medium; however, at a low glucose concentration (2 to 10 mM), derepression took place and the specific activity of the enzyme increased significantly. Derepressed cells (incubated in a glucose-limited medium) accumulated a capacity for the synthesis of beta-1,3-glucanase, which led to a subsequent increase in the specific activity even when the cells were transferred to a medium with an excess of glucose (180 mM). Two protein synthesis inhibitors, cycloheximide and trichodermin, immediately stopped the increase in specific activity when added to derepressed cells. On the other hand, 8-hydroxyquinoline, an RNA a synthesis inhibitor, acted differently, since it permitted the specific activity to increase for some time after being added to depressed cells. Moreover, the concentration of glucose did not affect the 8-hydroxyquinoline-insensitive synthesis of beta-1,3-glucanase. It is concluded that the glucose repression effect on beta-1,3-glucanase production must be exerted at a pretranslational level that could be either mRNA synthesis or some stage of the process involved in its maturation or stabilization.     

Effects of Temperature, Water Activity, and Incubation Time on Production of Aflatoxins and Cyclopiazonic Acid by an Isolate of Aspergillus flavus in Surface Agar Culture

An experiment with a full factorial design was used to study the effects of and interactions among temperature, water activity (a(infw)), incubation period, and substrate on coproduction of aflatoxins (AF) and cyclopiazonic acid (CPA) by an isolate of Aspergillus flavus. Analysis of variance showed that there was a complex interaction among all of these factors and that this influenced the relative concentrations of the mycotoxins produced. The optimum temperatures for the production of AF and CPA were 30(deg)C and 25(deg)C, respectively. Both mycotoxins were maximally produced (0.306 to 0.330 (mu)g of AF(middot)ml of medium(sup-1), 4.040 to 6.256 (mu)g of CPA(middot)ml of medium(sup-1)) at an a(infw) of 0.996 and after 15 days of incubation. No AF were produced in either yeast extract agar or Czapek yeast autolysate agar medium at an a(infw) of 0.90 at 20 or 37(deg)C after 15 days (minimum conditions), while 0.077 to 0.439 (mu)g of CPA(middot)ml of medium(sup-1) was produced under the same conditions. Yeast extract agar favored maximum AF production, and Czapek yeast autolysate agar favored maximum CPA production.     

Characterization of a chitinase and an endo-beta-1,3-glucanase from Trichoderma harzianum Rifai T24 involved in control of the phytopathogen Sclerotium rolfsii

Of 24 Trichoderma isolates, T harzianum Rifai (T24) showed a potential for control of the phytopathogenic basidiomycete Sclerotium rolfsii. When T24 was grown on different carbon sources, growth inhibition of S. rolfsii by the T24 culture filtrate correlated with the activity of extracellular chitinase and beta-1,3-glucanase. The 43-kilodalton (kDa) chitinase and the 74-kDa beta-1,3-glucanase were purified from the T24 culture filtrate in two and three steps, respectively, using ammonium sulphate precipitation followed by hydrophobic interaction chromatography (phenyl-Sepharose) and gel filtration (beta-1,3-glucanase). Km and Kcat were 3.8 g l(-1) and 0.71 s(-1) for the chitinase (chitin) and 1.1 g(-1) and 52 s(-1) for the beta-1,3-glucanase (laminarin). The chitinase showed higher activity on chitin than on less-acetylated substrate analogues (chitosan), while the beta-1,3-glucanase was specific for beta-1,3-linkages in polysaccharides. Both enzymes were stable at 30 degrees C, while at 60 degrees C the chitinase and the beta-1,3-glucanase were rapidly inactivated, showing half-lives of 15 and 20 min, respectively. The enzymes inhibited growth of S. rolfsii in an additive manner showing a promising ED50 (50% effective dose) value of 2.7 microg/ml.     

Nucleotide sequence of a beta-1,3-glucanase isoenzyme IIA gene of Oerskovia xanthineolytica LL G109 (Cellulomonas cellulans) and initial characterization of the recombinant enzyme expressed in Bacillus subtilis.

The nucleotide sequence of the betaglIIA gene, encoding the extracellular beta-1,3-glucanase IIA (betaglIIA) of the yeast-lytic actinomycete Oerskovia xanthineolytica LL G109, was determined. Sequence comparison shows that the betaglIIA enzyme has over 80% identity to the betaglII isoenzyme, an endo-beta-1,3-glucanase having low yeast-lytic activity secreted by the same bacterium. The betaglIIA enzyme lacks a glucan- or mannan-binding domain, such as those observed in beta-1,3-glucanases and proteases having high yeast/fungus-lytic activity. It can be included in the glycosyl hydrolase family 16. Gene fusion expression in Bacillus subtilis DN1885 followed by preliminary characterization of the recombinant gene product indicates that betaglIIA has a pI of 3.8 to 4.0 and is active on both laminarin and curdlan, having an acid optimum pH activity (ca. 4.0).     

Keratinolytic activity of Bacillus subtilis AMR using human hair

Aims:  To determine the ability of a novel Bacillus subtilis AMR isolated from poultry waste to hydrolyse human hair producing peptidases including keratinases and hair keratin peptides.
Methods and Results:  The Bacillus subtilis AMR was identified using biochemical tests and by analysis of 16S rDNA sequence. The isolate was grown in medium containing human hair as the sole source of carbon and nitrogen. The supplementation of hair medium (HM) with 0·01% yeast extract increased the keratinolytic activity 4·2-fold. B. subtilis AMR presented high keratinase production on the 8th day of fermentation in hair medium (HM) supplemented with 0·01% yeast extract (HMY) at pH 8·0. Keratinase yield was not correlated with increase in biomass. Zymography showed keratin-degrading peptidases migrating at c. 54, 80 and 100 kDa and gelatin-degrading bands at c. 80, 70 63, 54 32 and 15 kDa. Keratinases were optimally active at 50°C and pH 9·0 and was fully inhibited by the serine proteinase inhibitor (PMSF). Scanning electron microscopy showed complete degradation of the hair cuticle after exposure to B. subtilis AMR grown in HMY. MALDI-TOF analysis of culture supernatant containing peptides produced during enzymatic hydrolysis of hair by B. subtilis AMR revealed fragments in a range of 800–2600 Da.
Conclusions:  This study showed that B. subtilis AMR was able to hydrolyse human hair producing serine peptidases with keratinase and gelatinase activity as well as hair keratin peptides.
Significance and Impact of the Study:  This is the first report describing the production and partial characterization of keratinases by a B. subtilis strain grown in a medium containing human hair . These data suggest that peptides obtained from enzymatic hair hydrolysis may be useful for future applications on pharmaceutical and cosmetic formulations.     

Wide-range antifungal antagonism of Paenibacillus ehimensis IB-X-b and its dependence on chitinase and beta-1,3-glucanase production

Previously, we isolated a strain of Bacillus that had antifungal activity and produced lytic enzymes with fungicidal potential. In the present study, we identified the bacterium as Paenibacillus ehimensis and further explored its antifungal properties. In liquid co-cultivation assays, P. ehimensis IB-X-b decreased biomass production of several pathogenic fungi by 45%-75%. The inhibition was accompanied by degradation of fungal cell walls and alterations in hyphal morphology. Residual medium from cultures of P. ehimensis IB-X-b inhibited fungal growth, indicating the inhibitors were secreted into the medium. Of the 2 major lytic enzymes, chitinases were only induced by chitin-containing substrates, whereas beta-1,3-glucanase showed steady levels in all carbon sources. Both purified chitinase and beta-1,3-glucanase degraded cell walls of macerated fungal mycelia, whereas only the latter also degraded cell walls of intact mycelia. The results indicate synergism between the antifungal action mechanisms of these enzymes in which beta-1,3-glucanase is the initiator of the cell wall hydrolysis, whereas the degradation process is reinforced by chitinases. Paenibacillus ehimensis IB-X-b has pronounced antifungal activity with a wide range of fungi and has potential as a biological control agent against plant pathogenic fungi.     

Functional analysis of a beta-1,3-glucanase gene (Tag1) with anther-specific RNA and protein accumulation using antisense RNA inhibition

A critical stage in pollen development is the dissolution of tetrads into free microspores. Tetrads are surrounded by a wall composed primarily of beta-1,3-glucan. At the completion of meiosis, tetrads are released into the anther locule after hydrolysis of the callose by a beta-1,3-glucanase complex. The cDNA corresponding to a beta-1,3-glucanase cloned from tobacco (Tag 1) represents a gene that is highly similar to other beta-1,3-glucanases and is expressed exclusively in anthers from the tetrad to free microspore stage of pollen development. Tag 1 protein was overexpressed in E. coli, accumulating in insoluble inclusion bodies. Polyclonal antibodies against Tag 1 recombinant protein identify a single 33 kD protein accumulating only in anthers at tetrad and free microspore stages where beta-1,3-glucanase activity is present. Transgenic plants expressing Tag 1 antisense RNA were produced. Although Tag 1 RNA and protein levels were greatly reduced, tetrad dissolution and pollen development were normal. These data indicate that under the conditions these tobacco plants were grown, wild type levels of Tag 1 protein are not necessary for male fertility.     

The separation of β-glucanases produced by Cytophaga johnsonii and their role in the lysis of yeast cell walls

1. When Cytophaga johnsonii was grown in the presence of suitable inducers the culture fluid was capable of lysing thiol-treated yeast cell walls in vitro. 2. Autoclaved or alkali-extracted cells, isolated cell walls and glucan preparations made from them were effective inducers, but living yeast cells or cells killed by minimal heat treatment were not. 3. Chromatographic fractionation of lytic culture fluids showed the presence of two types of endo-beta-(1-->3)-glucanase and several beta-(1-->6)-glucanases; the latter may be induced separately by growing the myxo-bacterium in the presence of lutean. 4. Extensive solubilization of yeast cell walls was obtained only with preparations of one of these glucanases, an endo-beta-(1-->3)-glucanase producing as end products mainly oligosaccharides having five or more residues. Lysis by the other endo-beta-(1-->3)-glucanase was incomplete. 5. The beta-(1-->6)-glucanases produced a uniform thinning of the cell walls, and mannan-peptide was found in the solution. 6. These results, and the actions of the enzyme preparations on a variety of wall-derived preparations made from baker's yeast, are discussed in the light of present conceptions of yeast cell-wall structure.     

Regulation of 36-kDa beta-1,3-glucanase synthesis in Trichoderma harzianum

The effect of carbon sources on the level of beta-1,3-glucanases in the culture filtrates of Trichoderma harzianum (Tc) was investigated. Enzyme activity was detected in all carbon sources, but highest levels were found when laminarin and purified cell walls were used. Three isoforms of beta-1,3-glucanase were produced during growth of the fungus on purified cell walls. Two isoforms were produced on chitin, chitosan, N-acetylglucosamine and laminarin, while only one was detected when the fungus was grown on cellulose and glucose. A 36-kDa beta-1,3-glucanase (GLU36) was secreted from T. harzianum (Tc) grown on all carbon sources tested as demonstrated by Western blot analysis. We found that a significant increase in the level of GLU36 in the culture filtrate follows glucose exhaustion, suggesting that this enzyme is controlled by carbon catabolite repression.     

Production of Chitinases and beta-1,3-Glucanases by Stachybotrys elegans, a Mycoparasite of Rhizoctonia solani

The in vitro production of chitinases and beta-1,3-glucanases by Stachybotrys elegans, a mycoparasite of Rhizoctonia solani, was examined under various culture conditions, such as carbon and nitrogen sources, pH, and incubation period. Production of both enzymes was influenced by the carbon source incorporated into the medium and was stimulated by acidic pH and NaNO(3). The activity of both enzymes was very low in culture filtrates from cells grown on glucose and sucrose compared with that detected on chitin (for chitinases) and cell wall fragments (for beta-1,3-glucanases). Protein electrophoresis revealed that, depending on the carbon source used, different isoforms of chitinases and beta-1,3-glucanases were detected. S. elegans culture filtrates, possessing beta-1,3-glucanase and chitinase activities, were capable of degrading R. solani mycelium.     

Lysis of yeast cell walls. Lytic beta-(1 leads to 6)-glucanase from Bacillus circulans WL-12.

When grown in a mineral medium with yeast cell walls or yeast glucan as the sole carbon source, Bacillus circulans WL-12 produces wall-lytic enzymes in addition to non-lytic beta-(1 leads to 3) and beta-(1 leads to 6)-glucananases. The lytic enzymes were isolated from the culture liquid by adsorption on insoluble yeast glucan in batch operation. After digestion of the glucan, the mixture of enzymes was chromatographed on hydroxylapatite on which the lytic activity could be resolved into one lytic beta-(1 leads to 6)glucanase and two lytic beta-(1 leads to 3)-glucanase was further purified by chromatography over diethylamino-ehtyl-agarose and carboxymethyl cellulose. Its specific activity on pustulan was 6.2 units per mg of protein. The enzyme moved as a single protein with a molecular weight of 54000 during sodium dodecylsulphate electrophoresis in slab gels. Hydrolysis of pustulan went thorugh a series of oligosaccharides, leading to a mixture of gentiotriose, gentiobiose and glucose. The enzyme also produced small amounts of gentiobiose from laminarin and pachyman and on this basis its lytic activity on yeast cell walls,was attribut beta-(1 leads to 3)-linked oligosaccharides were not detected. The lytic beta-(1 leads to 6)-glucanase has an optimum pH of 6.0. Pustulan hydrolysis followed Michaelis-Menten kinetics. A Km of 0.29 mg pustulan per ml and a V of 9.1 micro-equivalents of glucose released/min per mg of enzyme were calculated. The enzyme has no metal ion requirement. The lytic beta-(1 leads to 6)-glucanase differs in essence from the non-lytic beta-(1 leads to 6)-glucanase of the same organism by its positive action on yeast cell walls and yeast glucan and its much lower specific activity on soluble pustulan.