Hemagglutinating activity of Lentinus edodes (Berk.) Sing [Lentinula edodes (Berk.) Pegler]

The hemagglutinating (HA) activity of the submerged mycelium and the culture liquid (CL) of four strains of Lentinus edodes was studied. The HA activity of CLs proved to be much higher than that of mycelia. The carbohydrate specificity of fungal agglutinating factors was determined. HA activity was investigated as a function of the inoculum size, cultivation temperature, and culture age. The agglutinating activity of different morphogenetic structures of L. edodes F-249, including mycelium, brown mycelial mat (MM), primordia, and fruiting bodies, was studied, MM was found to possess the maximum HA activity, which can be explained by the possible involvement of agglutinins in the formation of MM, which is composed of glued hyphae.     

Relationship between the Carbohydrate Specificity of Lectins and the Carbohydrate Composition of the <Emphasis Type="Italic">Lentinus edodes</Emphasis> (Berk.) Sing [<Emphasis Type="Italic">Lentinula edodes</Emphasis> (Berk.) Pegler] Mycelium at Different Stages of Its Morphogenesis

The specificity of lectins from the basidiomycete Lentinus edodes (Berk.) Sing [Lentinula edodes (Berk.) Pegler] during solid-state cultivation was found to be maximum to carbohydrates detected in the pyri-dine-soluble fraction of mycelium during the brown mycelial film stage. The carbohydrate composition of the mycelium (i.e., its content of maltose, rhamnose, mannitol, and inositol) was found to be different upon normal fruiting body formation and when the nonpigmented mycelium produced defective fruiting bodies.     

Intracellular lectins of Lentinus edodes at various developmental stages of the fungus

A number of lectins varying in polypeptide composition and carbohydrate specificity were isolated from Lentinus edodes at different stages of its morphogenesis: nonpigmented mycelium, brown mycelium film, and fruiting body. Three lectins were identified at the nonpigmented mycelium stage, two of them being dimers consisting of 16 and 45 kDa and 16 and 42 kDa subunits; the third is a tetramer of 16, 39, 42, and 45 kDa subunits. The fractions with lectin activity obtained at the brown mycelium film stage contained polypeptides of 24, 30, and 38 kDa, characteristic of this morphological structure. The fruiting body was shown to contain two lectins of 43 and 55 kDa. All of the isolated lectins expressed the highest affinity towards L,D-melibiose, D-lactose, and D-galactose.     

Hemagglutinating activity of the fungusLentinus edodes (Berk.) sing [Lentinus edodes (Berk.) Pegler]

The hemagglutinating (HA) activity of the submerged mycelium and the culture liquid (CL) of four strains ofLentinus edodes was studied. The HA activity of the CLs proved to be much higher than that of mycelia. The carbohydrate specificity of fungal agglutinating factors was determined. HA activity was investigated as a function of the inoculum size, cultivation temperature, and culture age. The agglutinating activity of different morphogenetic structures ofL. edodes F-249, including mycelium, brown mycelial mat (MM), primordia, and fruiting bodies, was studied. MM was found to possess the maximum HA activity, which can be explained by the possible involvement of agglutinins in the formation of MM, which is composed of glued hyphae.     

Auxin synthesis by the higher fungus Lentinus edodes (Berk.) Sing in the presence of low concentrations of indole compounds

The auxin formation in a submerged culture of the xylotrophic basidiomycete Lentinus edodes (Berk.) Sing (Lentinula edodes (Berk.) Pegler) (shiitake) is studied. Biologically active substances of an indole nature are identified, "the effect of small doses" of which lies in not only the stimulation of growth of the mycelium (indole-3-acetic acid, 2 x 10(-7)-2 x 10(-4) g/l), but also in the induction of tryptophan-independent paths of auxin biosynthesis. The above-mentioned path is realized in the presence of exogenous indole (1 x 10(-3)-1 x 10(-4) g/l), as well as while inducing the biosynthesis of indole-3-acetic acid by its microadditives (1 x 10(-5)-1 x 10(-8) g/l), and is accompanied by the formation of anthranilic acid (up to 1.5 mg/l). Induction of the generative development stage ofshiitake by indole derivatives is revealed. It was found that among the studied compounds only indoleacetamide at a concentration of an order of x 10(-4) g/l in the culture fluid of L. edodes had a pronounced stimulatory effect on the formation of shiitake's brown mycelial film.     

Activity of Lentinus edodes intracellular lectins at various developmental stages of the fungus

We studied changes of the hemagglutinating activity of intracellular lectins of the basidiomycete Lentinus edodes (shiitake) at various stages of its morphogenetic development depending on erythrocyte type, growth medium, and lectin purification degree. Under certain experimental conditions, the specific lectin activity at the brown mycelium film stage exceeded the corresponding value for nonpigmented mycelium. The sensitivity of the lectins towards trypsin-treated rabbit erythrocytes was no less than a hundredfold higher than towards any other erythrocyte type studied. The general regularities of specific activity change did not depend on nutrient medium composition. With purification of intracellular shiitake lectins, their sensitivity to human erythrocytes decreased seventyfold or more, whereas their sensitivity to rabbit erythrocytes increased by the same factor.     

Enzymes of the xylotrophic basidiomycete Lentinus edodes F-249 in the course of morphogenesis

It has been shown that the fungus Lentinus edodes grown on a solid wort agar substrate produces intracellular enzymes, including Mn-dependent peroxidase, laccase, and tyrosinase as a family of isoforms. The composition of the complex (containing one to four forms of each enzyme) varied during the basidiomycete life cycle. The activity of oxidases was maximal at the stage of nonpigmented mycelium and at the stages of a brown mycelial mat and a fruit body. The activity of tyrosinase increased in the course of mycelium pigmentation and had two maxima: at the stage of a brown mycelial mat and at the stage of a fruit body. Laccase and tyrosinase activities were shown to increase sharply upon addition of oak sawdust extract to the culture medium as compared with the enzyme activities of mycelium grown on wort agar alone. It was established that the effect of phenol oxidase substrates on the growing mycelium consists in a twofold acceleration of the process of morphogenesis in the fungus L. edodes.     

Isolation and characterization of <Emphasis Type="Italic">Lentinus edodes</Emphasis> (Berk.) singer extracellular lectins

Lectin preparations have been isolated and purified from the culture liquid of the xylotrophic basidiomycete Lentinus edodes (Berk.) Singer [Lentinula edodes (Berk.) Pegler]. The culture of L. edodes F-249 synthesizes two extracellular lectins different in composition and physicochemical properties. Extracellular lectin L1 from L. edodes is a glycoprotein of mono-subunit structure with molecular weight of 43 kD. L1 is comprised of 10.5 +/- 1.0% (w/w) carbohydrates represented by glucose (Glc). Extracellular lectin L2 is a proteoglycan of mono-subunit structure with molecular weight of 37 kD. L2 is comprised of 90.3 +/- 1.0% (w/w) carbohydrates represented by Glc (73% of the total mass of the carbohydrate moiety of the lectin molecule) and galactose (Gal) (27% of the total mass of the carbohydrate part of the lectin molecule). The content of Asn in L2 is high, i.e. 42% (w/w) of total amino acids. This fact along with the composition of the carbohydrate part of the molecule (Glc + Gal) allows one to assign L2 to N-asparagine-bound proteins. Both lectins are specific to D-Gal and lactose (Lac) at an equal for L1 and L2 minimal inhibiting concentration of these carbohydrates (2.08 mM Gal and 8.33 mM Lac). Other carbohydrates to which the lectins show affinity are different for the two lectins: Rha (4.16 mM) for L1 and Ara (4.16 mM) and mannitol (8.33 mM) for L2. The purified extracellular lectins of L. edodes are highly selective at recognition of definite structures on the surface of trypsinized rabbit erythrocytes and do not react with the erythrocytes of other animals and humans.     

Intracellular lectins of <Emphasis Type="Italic">Lentinus edodes</Emphasis> at various developmental stages of the fungus

A number of lectins varying in polypeptide composition and carbohydrate specificity were isolated from Lentinus edodes at different stages of its morphogenesis: nonpigmented mycelium, brown mycelium film, and fruiting body. Three lectins were identified at the nonpigmented mycelium stage, two of them being dimers consisting of 16 and 45 kDa and 16 and 42 kDa subunits; the third is a tetramer of 16, 39, 42, and 45 kDa subunits. The fractions with lectin activity obtained at the brown mycelium film stage contained polypeptides of 24, 30, and 38 kDa, characteristic of this morphological structure. The fruiting body was shown to contain two lectins of 43 and 55 kDa. All of the isolated lectins expressed the highest affinity towards L,D-melibiose, D-lactose, and D-galactose.     

Relationship between the molecular structure of nitrogen source and the activity of the extracellular lectins of Lentinus edodes (Berk.) Sing [Lentinula edodes (Berk.) Pegler] upon submerged cultivation

The activity of the extracellular lectins of Lentinus edodes (Berk.) Sing [Lentinula edodes (Berk.) Pegler] and the formation of a pigmented mycelial film by this fungus upon submerged cultivation in a synthetic medium were found to depend on the presence of some amino acids (particularly, asparagine) and Ca2+ and Mn2+ ions in the medium. Quantum chemical calculations suggest that the different character of the interaction of amino acids with the aforementioned ions is due to differences in the hydrophobicity of the amino acids rather than to differences in the electron structure of the amino acid zwitterions.     

Activity of <Emphasis Type="Italic">Lentinus edodes</Emphasis> intracellular lectins at various developmental stages of the fungus

We studied changes of the hemagglutinating activity of intracellular lectins of the basidiomycete Lentinus edodes (shiitake) at various stages of its morphogenetic development depending on erythrocyte type, growth medium, and lectin purification degree. Under certain experimental conditions, the specific lectin activity at the brown mycelium film stage exceeded the corresponding value for nonpigmented mycelium. The sensitivity of the lectins towards trypsin-treated rabbit erythrocytes was no less than a hundredfold higher than towards any other erythrocyte type studied. The general regularities of specific activity change did not depend on nutrient medium composition. With purification of intracellular shiitake lectins, their sensitivity to human erythrocytes decreased seventyfold or more, whereas their sensitivity to rabbit erythrocytes increased by the same factor.     

Characterization, occurrence, and molecular cloning of a lectin from Grifola frondosa: jacalin-related lectin of fungal origin

A lectin named GFL was isolated from the fruiting body of the basidiomycete mushroom Grifola frondosa, which belongs to Aphyllophorales. The lectin had a molecular mass of 24 kDa on SDS-PAGE. The hemagglutinating activity of GFL was not inhibited by any monosaccharide, and inhibited only by porcine stomach mucin so far as tested. The occurrence of GFL was studied at three stages during fruiting body formation. The largest quantity of hemagglutinating activity was found in the fruiting body, and lesser amounts in the mycelial mat and the primordium. The 24-kDa band of GFL was found at all three stages, and the band-intensity corresponded to the level of activity in each sample. By cloning and sequencing the GFL-cDNA, the primary structure of this lectin was determined. GFL is composed of 181 amino acids, having no signal peptide. The amino acid sequence was found to be homologous to those of so-called jacalin-related plant lectins, suggesting that GFL is the first example of a jacalin-related lectin of fungal origin.     

Erratum to: Extracellular protein production and morphogenesis of <Emphasis Type="Italic">Lentinula edodes</Emphasis> in submerged culture

Along with a brief review of Lentinula edodes (shiitake mushroom) submerged cultivation history within the framework of important extracellular proteins biosynthesis, this study contains the authors’ own results. The possibility of regulating the lectin activity of shiitake using the synthetic components is shown. The time course of lectin production in culture liquid of L. edodes in different media under submerged culture conditions was studied. The activity of agglutinins depended on the ratio between carbon and nitrogen sources and the pH of the culture medium. A relationship between the chemical composition of nutrient medium, the activity of extracellular lectins of L. edodes, and the formation of pigmented mycelial film in liquid culture has been found. The formulation of medium, on which the brown mycelial film appears in several days of submerged cultivation, is proposed. The results obtained make a contribution to the present notion of biochemical processes that give rise to the occurrence of the aforesaid morphological structure of shiitake. Finally, two extracellular lectins from the submerged culture of L. edodes have been isolated and purified to homogeneity. Their physicochemical properties and composition have been studied.     

Extracellular protein production and morphogenesis of <Emphasis Type="Italic">Lentinula edodes</Emphasis> in submerged culture

Along with a brief review of Lentinula edodes (shiitake mushroom) submerged cultivation history within the framework of important extracellular proteins biosynthesis, this study contains the authors’ own results. The possibility of regulating the lectin activity of shiitake using the synthetic components is shown. The time course of lectin production in culture liquid of L. edodes in different media under submerged culture conditions was studied. The activity of agglutinins depended on the ratio between carbon and nitrogen sources and the pH of the culture medium. A relationship between the chemical composition of nutrient medium, the activity of extracellular lectins of L. edodes, and the formation of pigmented mycelial film in liquid culture has been found. The formulation of medium, on which the brown mycelial film appears in several days of submerged cultivation, is proposed. The results obtained make a contribution to the present notion of biochemical processes that give rise to the occurrence of the aforesaid morphological structure of shiitake. Finally, two extracellular lectins from the submerged culture of L. edodes have been isolated and purified to homogeneity. Their physicochemical properties and composition have been studied.     

Characterization of a shiitake mutant strain producing ballshaped fruiting bodies

Growth characteristics of a spontaneous mutant of shiitake Lentinula edodes (Berk.) Pegler were studied. The mutant was first detected as a result of changes in the growth habit of the normal strain in the liquid medium. Abundant formation of aerial hyphae was distinctive. In sawdust logs the mutant strain produced abnormal basidiocarps, lacking stipe, gill and spore formation.
Growth rates of the normal and the mutant strain were compared in two liquid media: malt-yeast extract and Leatham's medium. The increase in dry weight of the mutant's mycelium was much higher than that of the wild type in both media, which indicated better adaptation to liquid culture. In the sawdust, however, growth of the mutant was slower than that of the normal strain. The mutant's intracellular protein content was lower than that of the normal strain. The pH of the liquid cultures differed: the wild type decreased the pH during growth, while the mutant increased the pH. Comparison of the protein and esterase isoenzyme profiles of the vegetative hyphae of both strains indicated profound differences. One protein (pI 6.5, 39 kDa), which in earlier studies has been found to be typical of L. edodes species, was absent from the mutant's profile. Differences in the esterase profile were also clear.     

香菇菌丝深层培养的碳、氮源和生长因子

本文分别设计了生长刺激因子、碳、氮源试验基本培养基。结果表明在深层培养时,废糖蜜、粗制蔗糖、玉米浆、酵母粉、麸皮浸汁中含有刺激香菇菌丝生长的物质。而各种维生素(包括 B_1)、氨基酸、有机酸、植物生长激素等均无明显的作用。在上述生长因子的促进下,香菇菌丝可利用无机或有机氮源。无机氮源以 NH_4NO_3,最好,其次为(NH_3)H_2PO_4、NH)4Cl 等。有机氮源以玉米浆、酵母粉最好。碳源以玉米淀粉最好。本文并介绍了一种廉价的、良好的香菇菌丝液体培养配方,可以稳定地培养出细小均匀的菌丝球、生长丰满稠密的液体菌种。     

Extracellular enzyme activities in six Lentinula edodes strains during cultivation in wheat straw

Lentinula edodes (Berk.) Pegler is found in nature on dead broadleaf trees, but it is commercially produced on different substrates. The question of adaptation to different lignocellulosic substrates was addressed by measuring enzyme activities produced by six strains that were cultivated on wheat straw and that were able to produce sporophores. Despite quantitative variations, each strain of L. edodes had similar patterns of enzyme secretion into the wheat straw log matrix. Two peaks of carbohydrase activities were observed, the first relating to the early mycelial growth during the first days after spawning and the second during sporophore extension. Laccase activity in the early stage of colonization was related to the degradation of soluble phenolic compounds present in wheat straw. Manganese peroxidase activity was associated with mycelia th. The strains with the earlier production and higher yield were able to hydrolyse and utilize straw cell wall components soon aft er inoculation, and developed high metabolic activities.     

用灭活原生质体融合进行高温香菇育种——Ⅰ.融合产物的选出及其鉴定

用灭活的近裸香菇(Lentinus subnudus Berk.)双核菌株原生质体与香菇[L. edodes(Berk.)Sing.]双核菌株原生质体融合,在35℃的条件下选得融合子。融合频率为0—4.3×10~(-5)。融合子与双亲有明显的拮抗性。融合子的菌丝形态、氨基酸含量,子实体的形态,以及酸性磷酸酶同功酶的测定都与双亲不同。     

Characterization of brown film formed by Lentinula edodes

Lentinula edodes is a widely-produced mushroom in China that forms a brown film via pigment accumulation on mature mycelial surfaces to ensure high-quantity and high-quality fruiting body formation. Here, ultraviolet–visible, infrared spectra, and elemental analyses predicted that the pigment in the brown film was melanin. Electron microscopy revealed the size, morphological characteristics, accumulation, and morphogenesis of electron-dense material, which were similar to those of melanin, as well as subcellular structural changes during brown film formation. The electron-dense material appeared as granules, vesicles, and polymers. The accumulation of electron-dense materials on the cell wall was followed plasmolysis, plasma membrane disruption, electron-dense material accumulation in the interstitial space, and gradual accumulation on the outer cell wall. Dolipore septa degradation and morphogenetic cell death occurred during browning. In the final stage of browning, the dolipore septum disappeared and the cell was nearly empty. This study provides a cytological foundation for evaluating the regulation of brown film formation in L. edodes.     

The antagonistic action of Trichoderma sp. hyphae to Lentinula edodes hyphae changes lignocellulotytic activities during cultivation in wheat straw

Lentinula edodes (Berk.) Pegler was cultivated in sterilized or pasteurized wheat straw both with and without inoculation with Trichoderma sp. Enhancements of -mannosidase and laccase activities and lowering of Mn-dependent peroxidase activity were observed seven days after inoculation in substrates inoculated with Trichoderma sp. These enzymes were not produced by Trichoderma sp. Most of the polysaccharidase activities were higher in substrates with Trichoderma sp. than in absence of Trichoderma sp. The area of the substrate contaminated with T. harzianum significantly correlated with cellulase, laccase and Mn-dependent peroxidase activities measured in the substrate. The increase of cellulase activity was due to enzymes produced by Trichoderma sp. and the decrease of Mn-dependent peroxidase activity was due to diminished growth of L. edodes. The stimulation of laccase activity was linked with the formation of brown lines (oxidation of polyphenols) at the contact between the mycelia of the two antagonists.