Enzymes of carbohydrate metabolism of mycelial fungi from marine environments. beta-1,3-glucanase of the marine fungus Chaetomium indicum

Thirty samples of fungi belonging to 17 species living in marine environments were studied for their ability to produce extracellular enzymes. In the culture fluids, a variety of glycosidases (beta-glucosidases, N-acetyl-beta-glucosaminidase, beta-galactosidases, and alpha-mannosidases) and glucanases (amylases and beta-1,3-glucanases) were found. Several cultures were found that could be used as efficient producers of either individual enzymes or a whole complement of enzymes degrading carbohydrate-containing compounds. Optimal growth conditions for the fungus Chaetomium indicum and beta-1,3-glucanase biosynthesis were developed. beta-1,3-Glucanase was isolated by a combination of ion-exchange chromatography, ultrafiltration, and gel chromatography. The molecular mass of the enzyme determined by gel-filtration was 54 kD. The enzyme was stable at temperatures below 50 degrees C, had a temperature optimum for activity at 60 degrees C, and retained activity between pH 4.5 and 7.5. The pH dependence of the beta-1, 3-glucanase activity showed two maxima, at pH 4.4 and 5.6; this suggested the existence of two forms of the enzyme. Analysis of the products of enzymatic hydrolysis of laminaran, transglycosylating ability, and the effect of a specific natural inhibitor indicates that both forms are exo-beta-1,3-glucanases.     

A study of some biochemical and histopathological responses of wet-stored recalcitrant seeds of Avicennia marina infected by Fusarium moniliforme

Although fungi cause a recognized problem during storage of recalcitrant seeds of many tropical species, there are no data to date on defence strategies of these seeds against fungal attack. To ascertain whether recalcitrant seeds of Avicennia marina elaborate compounds that might suppress fungal proliferation during hydrated storage, the production and efficacy of beta-1,3-glucanase (EC 3.2.1.39) and chitinase (EC 3.2.1.14) were studied in relation to histopathological changes. Freshly harvested seeds had low beta-1,3-glucanase and chitinase activities and fluorescence microscopy revealed progressive deterioration of the internal tissues of these seeds associated with fungal infection during hydrated storage. In seeds treated to minimize associated fungi (clean seeds), beta-1,3-glucanase and chitinase activities increased significantly during 10 d of hydrated storage. Similar high levels of activity were observed when these seeds were experimentally infected with Fusarium moniliforme and subjected to further storage. The histopathological observations indicated delayed disease development in the 10-d clean-storage period, although the hypersensitive response was not observed. The results suggest that, although the recalcitrant seeds of A. marina elaborate some antifungal enzymes, there is a lack of effective defence strategies that might lead to successful responses against fungal infections.     

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.     

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.     

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.     

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.     

Molecular organization of the alkali-insoluble fraction of Aspergillus fumigatus cell wall

Physical and biological properties of the fungal cell wall are determined by the composition and arrangement of the structural polysaccharides. Cell wall polymers of fungi are classically divided into two groups depending on their solubility in hot alkali. We have analyzed the alkali-insoluble fraction of the Aspergillus fumigatus cell wall, which is the fraction believed to be responsible for fungal cell wall rigidity. Using enzymatic digestions with recombinant endo-beta-1,3-glucanase and chitinase, fractionation by gel filtration, affinity chromatography with immobilized lectins, and high performance liquid chromatography, several fractions that contained specific interpolysaccharide covalent linkages were isolated. Unique features of the A. fumigatus cell wall are (i) the absence of beta-1,6-glucan and (ii) the presence of a linear beta-1, 3/1,4-glucan, never previously described in fungi. Galactomannan, chitin, and beta-1,3-glucan were also found in the alkali-insoluble fraction. The beta-1,3-glucan is a branched polymer with 4% of beta-1,6 branch points. Chitin, galactomannan, and the linear beta-1, 3/1,4-glucan were covalently linked to the nonreducing end of beta-1, 3-glucan side chains. As in Saccharomyces cerevisiae, chitin was linked via a beta-1,4 linkage to beta-1,3-glucan. The data obtained suggested that the branching of beta-1,3-glucan is an early event in the construction of the cell wall, resulting in an increase of potential acceptor sites for chitin, galactomannan, and the linear beta-1,3/1,4-glucan.     

Immunogold localization of beta-1,3-glucanases in two plants infected by vascular wilt fungi.

An antiserum raised against a purified tobacco beta-1,3-glucanase (PR-N) was used to study the subcellular localization of enzyme in fungus-infected plant tissues by means of post-embedding immunogold labeling. In susceptible tomato plants, the enzyme accumulation was found to occur as a result of successful tissue colonization, whereas it appeared to be an early event associated with limited spread of the fungus in resistant tissues. Although marked differences between susceptible and resistant tomato cultivars were observed in the rate of production of beta-1,3-glucanase, the pattern of enzyme distribution was similar. The enzyme was found to accumulate predominantly in host cell walls and secondary thickenings of xylem vessels. By contrast, a very low amount of enzyme was associated with compound middle lamellae. The occurrence of beta-1,3-glucanase at the cell surface of invading fungi was an indication of their possible antifungal activity. A low enzyme concentration was detected in vacuoles of both healthy and infected tissues. In infected eggplant tissue, the pattern of beta-1,3-glucanase distribution was similar to that observed with tomato. Whether these hydrolases accumulate first in vacuoles and are subsequently conveyed toward the outside to participate in fungal wall lysis remains to be determined.     

Characterization of a 29-kDa beta-1,3-glucanase from Trichoderma harzianum

A beta-1,3-glucanase, from culture filtrates of Trichoderma harzianum, was purified in sequential steps by gel filtration, hydrophobic interaction and ion exchange chromatography. A typical procedure provided 69-fold purification with 0.32% yield. The molecular mass of the protein was found to be approximately 29 kDa, as estimated by SDS-PAGE on a 10% slab gel. The K(M) and V(max) values for beta-1,3-glucanase, using laminarin as substrate, were 1. 72 mg ml(-1) and 3.10 U ml(-1), respectively. The pH optimum for the enzyme was pH 4.4 and maximum activity was obtained at 50 degrees C. The enzyme was strongly inhibited by HgCl(2) and SDS. These results suggest that each beta-1,3-glucanase produced by T. harzianum is different and is probably encoded by different genes.     

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.     

Purification and characterization of an exo-beta-1,3-glucanase produced by Trichoderma asperellum

Trichoderma asperellum produces at least two extracellular beta-1,3-glucanases upon induction with cell walls from Rhizoctonia solani. A beta-1,3-glucanase was purified by gel filtration and ion exchange chromatography. A typical procedure provided 35.7-fold purification with 9.5% yield. The molecular mass of the purified exo-beta-1,3-glucanases was 83.1 kDa as estimated using a 12% (w/v) SDS-electrophoresis slab gel. The enzyme was only active toward glucans containing beta-1,3-linkages and hydrolyzed laminarin in an exo-like fashion to form glucose. The K(m) and V(max) values for exo-beta-1,3-glucanase, using laminarin as substrate, were 0.087 mg ml(-1) and 0.246 U min(-1), respectively. The pH optimum for the enzyme was pH 5.1 and maximum activity was obtained at 55 degrees C. Hg(2+) strongly inhibited the purified enzyme.     

Hydrolytic enzymes and antifungal compounds produced by Tilletiopsis species, phyllosphere yeasts that are antagonists of powdery mildew fungi

Isolates of five species of the yeast-like fungus Tilletiopsis Derx (Tilletiopsis albescens Gokhale, Tilletiopsis fulvescens Gokhale, Tilletiopsis minor Nyland, Tilletiopsis pallescens Gokhale, and Tilletiopsis washingtonensis Nyland) were screened for exo- and endo--beta-1,3-glucanase and chitinase production in a liquid broth used to produce inoculum for biological control studies. There were significant differences among the species, and highest overall enzyme activity was present in T albescens and T. pallescens and lowest in T. washingtonensis. A time-course study of beta-1,3-glucanase and chitinase production in T pallescens ATCC 96155 in broth culture with 2.5% glucose as the carbon source showed that enzyme activity gradually increased over a 3- to 21-day period. Maximum enzyme activity was found between pH 4.0 and 5.0. SDS-PAGE of beta-1,3-glucanase isozymes revealed a range of molecular masses from 18 to 29 kDa. Five isozymes were present in both T albescens and T. pallescens and two in T washingtonensis. Antifungal compounds were also detected in ethyl acetate extracts of culture filtrates of T. pallescens ATCC 96155 after 6 days of incubation, while no activity was detected at 14 days. One active fraction was selected following fractionation and preparative chromatography and was bioassayed against Podosphaera (sect. Sphaerotheca) xanthii (Castagne) U. Braun & N. Shishkoff and a number of other fungi. A concentration of 130 microg/mL inhibited germ tube development in P. xanthii, and mildew spores appeared plasmolyzed. Other fungi were inhibited at higher concentrations. Collapse of hyphae and conidiophores was also observed on mildewed leaves treated with the active fraction. Proton NMR analysis indicated that the inhibitory compound was a fatty acid ester. In 3- to 6-day-old cultures of T pallescens ATCC 96155 demonstrating biological control activity, antifungal compound production may have a primary role in restricting growth of mildew fungi and other competitors when applied to leaves.     

Role of chitinase and beta-1,3-glucanase activities produced by a fluorescent pseudomonad and in vitro inhibition of Phytophthora capsici and Rhizoctonia solani

A study was conducted to investigate the possibility of involvement of chitinase and beta-1,3-glucanase of an antagonistic fluorescent Pseudomonas in growth suppression of phytopathogenic fungi, Phytophthora capsici and Rhizoctonia solani. Fluorescent Pseudomonas isolates GRC(3) and GRC(4) were screened for their antifungal potential against phytopathogenic fungi by using dual culture technique both on solid and liquid media. The percent inhibition was calculated. Various parameters were monitored for optimization of enzyme activities by fluorescent Pseudomonas GRC(3). The involvement of chitinases, beta-1,3-glucanases, and antifungal metabolites of nonenzymatic nature was correlated with the inhibition of P. capsici and R. solani. The results provide evidence for antibiosis as a mechanism for antagonism. The study also confirms that multiple mechanisms are involved in suppressing phytopathogens as evidenced by the involvement of chitinase and beta-1,3-glucanase in inhibition of R. solani but not P. capsici by isolate GRC3.     

Purification and properties of a beta-1,6-glucanase from Penicillium brefeldianum.

An inducible endo-beta-1,6-glucanase was purified from Penicillium brefeldianum by DEAE-cellulose, Bio-Gel P-150 and high-pressure liquid chromatography. The final preparation was essentially free from beta-1,3-glucanase and beta-glucosidase activities. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed one protein band with an Mr of 44000. The Vmax. and Km values were calculated to be 624 units (mumol/min)/mg and 2.78 mg/ml respectively. The glucanase had lytic activity against mycelial cells of the yeast Candida albicans. The yield of purified beta-1,6-glucanase from 100 mg dry weight of freeze-dried culture filtrate varied from 60 to 180 units.     

Extracellular hydrolases of strain Bacillus sp. 739 and their involvement in the lysis of micromycete cell walls

The mycolytic bacterial strain Bacillus sp. 739 produces extracellular enzymes which degrade in vitro the cell walls of a number of phytopathogenic and saprophytic fungi. When Bacillus sp. 739 was cultivated with Bipolaris sorokiniana, a cereal root-rot pathogen, the fungus degradation process correlated with the levels of the beta-1,3-glucanase and protease activity. The comparative characteristic of Bacillus sp. 739 enzymatic preparations showed that efficient hydrolysis of the fungus cell walls was the result of the action of the complex of enzymes produced by the strain when grown on chitin-containing media. Among the enzymes of this complex, chitinases and beta-1,3-glucanases hydrolyzed most actively the disintegrated cell walls of B. sorokiniana. However, only beta-1,3-glucanases were able to degrade the cell walls of native fungal mycelium in the absence of other hydrolases, which is indicative of their key role in the mycolytic activity of Bacillus sp. 739.     

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.     

Molecular characterization and expression in Escherichia coli of three beta-1,3-glucanase genes from Lysobacter enzymogenes strain N4-7

Lysobacter enzymogenes strain N4-7 produces multiple biochemically distinct extracellular beta-1,3-glucanase activities. The gluA, gluB, and gluC genes, encoding enzymes with beta-1,3-glucanase activity, were identified by a reverse-genetics approach following internal amino acid sequence determination of beta-1,3-glucanase-active proteins partially purified from culture filtrates of strain N4-7. Analysis of gluA and gluC gene products indicates that they are members of family 16 glycoside hydrolases that have significant sequence identity to each other throughout the catalytic domain but that differ structurally by the presence of a family 6 carbohydrate-binding domain within the gluC product. Analysis of the gluB gene product indicates that it is a member of family 64 glycoside hydrolases. Expression of each gene in Escherichia coli resulted in the production of proteins with beta-1,3-glucanase activity. Biochemical analyses of the recombinant enzymes indicate that GluA and GluC exhibit maximal activity at pH 4.5 and 45 degrees C and that GluB is most active between pH 4.5 and 5.0 at 41 degrees C. Activity of recombinant proteins against various beta-1,3 glucan substrates indicates that GluA and GluC are most active against linear beta-1,3 glucans, while GluB is most active against the insoluble beta-1,3 glucan substrate zymosan A. These data suggest that the contribution of beta-1,3-glucanases to the biocontrol activity of L. enzymogenes may be due to complementary activities of these enzymes in the hydrolysis of beta-1,3 glucans from fungal cell walls.     

Involvement of secondary metabolites and extracellular lytic enzymes produced by Pseudomonas fluorescens in inhibition of Rhizoctonia solani, the rice sheath blight pathogen

Fourteen strains of Pseudomonas fluorescens isolated from rhizosphere soil of rice were tested for their antagonistic effect towards Rhizoctonia solani, the rice sheath blight fungus. Among them, PfMDU2 was the most effective in inhibiting mycelial growth of R. solani in vitro. Production of chitinase, beta-1,3-glucanase, siderophores, salicylic acid (SA) and hydrogen cyanide (HCN) by P. fluorescens strains was evaluated. The highest beta-1,3-glucanase activity, siderophore production, SA production and HCN production were recorded with PfMDU2. A significant relationship between the antagonistic potential of P. fluorescens against R. solani and its level of beta-1,3-glucanase, SA and HCN was observed.     

Purification of a chitinase from Trichoderma sp. and its action on Sclerotium rolfsii and Rhizoctonia solani cell walls

Trichoderma harzianum is an effective biocontrol agent of several important plant pathogenic fungi. This Trichoderma species attacks other fungi by secreting lytic enzymes, including beta-1,3-glucanase and chitinolytic enzymes. Superior biocontrol potential may then be found in strains having a high capacity to produce these enzymes. We have therefore evaluated the capacity of six unidentified Trichoderma spp. isolates to produce chitinolytic enzymes and beta-1,3-glucanases in comparison with T. harzianum 39.1. All six isolates demonstrated substantial enzyme activity. However, while the isolates hereafter called T2, T3, T5, and T7 produced lower amounts of enzymes, the activity of isolates T4 and T6 were 2-3 fold higher than that produced by T. harzianum 39.1. A chitinase produced by the T6 isolate was purified by a single ion-exchange chromatography step and had a molecular mass of 46 kDa. The N-terminal amino-acid sequence showed very high homology with other fungal chitinases. Its true chitinase activity was demonstrated by its action on chitin and the failure to hydrolyze laminarin and p-nitrophenyl-beta-N-acetylglucosaminide. The hydrolytic action of the purified chitinase on the cell wall of Sclerotium rolfsii was convincingly shown by electron microscopy studies. However, the purified enzyme had no effect on the cell wall of Rhizoctonia solani.