Mannitol Metabolism in Lentinus edodes, the Shiitake Mushroom

Mannitol metabolism was evaluated in fruiting bodies of Lentinus edodes. Cell extracts were prepared from fruiting bodies, and key enzymes involved in mannitol metabolism were assayed, including hexokinase, mannitol dehydrogenase, mannitol-1-phosphate dehydrogenase, mannitol-1-phosphatase, and fructose-6-phosphatase. Mannitol dehydrogenase, fructose-6-phosphatase, mannitol-1-phosphatase, and hexokinase activities were found in extracts of fruiting bodies. However, mannitol-1-phosphate dehydrogenase activity was not detected. Mycelial cultures were grown in an enriched liquid medium, and enzymes of the mannitol cycle were assayed in cell extracts of rapidly growing cells. Mannitol-1-phosphate dehydrogenase activity was also not found in mycelial extracts. Hence, evidence for a complete mannitol cycle both in vegetative mycelia and during mushroom development was lacking. The pathway of mannitol synthesis in L. edodes appears to utilize fructose as an intermediate.     

Characterization of Ribonucleases from Culture Medium of Lentinus edodes

Lentinus edodes (shiitake) cultivated in potato dextrose medium produced five RNases in the culture filtrate. The two major RNases (RNase Le37 and RNase Le45) were highly purified and their molecular masses, base specificities, N-terminal amino acid sequences, and amino acid compositions were analyzed and compared to RNase Le2 isolated from the fruit bodies of the same mushroom. RNase Le37 and RNase Le45 are base non-specific and adenylic acid preferential RNases like RNase Le2 and their N-terminal sequences are very similar to RNase Le2, but they are glycoproteins and their amino acid compositions are significantly different from that of RNase Le2. In addition to these enzymes, a guanylic acid-specific RNase with a molecular mass 13 kDa was partially purified. Since RNase Le2, which has very similar N-terminal sequence to RNase Le 37 and RNase Le 45, was not excreted from the mycelia, the analysis of the structures of these two excreted RNase may shade a light on the mechanism of excretion of RNases in this organism.     

Lignocellulolytic Enzymes Produced by Volvariella volvacea, the Edible Straw Mushroom

Volvariella volvacea, commonly known as the straw or paddy mushroom, had the following growth characteristics: minimum temperature, 25°C; optimal temperature, 37°C; maximal temperature, 40°C; pH optimum 6.0. Optimal pH for cellulase production was 5.5. The optimal initial pH for cellulase production and mycelial growth was found to be 6.0. The pH and temperature optima for cellulolytic activity were 5.0 and 50°C, respectively. Maximal cellulolytic activity was obtained within 5 days in shake-flask culture. The cellulases were found to be partly cell free and partly cell bound during growth on microcrystalline cellulose. The endoglucanase activity was primarily extracellular, and β-glucosidase activity was found exclusively extracellularly. Weak cellulase activity was detected when cells were grown on cellobiose and lactose. V. volvacea could not digest the lignin portion of newspaper in shake-flask cultivation. Phenol oxidase, an important enzyme in lignin biodegradation, also was lacking in the cell-free filtrate. However, the organism oxidized phenolic compounds when it was cultured on agar plates containing commercial lignin.     

香菇栽培料新工艺

在国内香菇栽培料常规配方基础上,设计了添加2.5mL盐酸/kg取代常规配方中1%蔗糖的新工艺,经测定,2种栽培料灭菌后还原糖与蔗糖浓度、发菌期与子实体产量基本一致,新配方栽培料灭菌后pH值为5.6,是香菇菌丝生长最适pH值。新配方工艺可降低生产成本10%左右。     

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.     

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.     

Purification and Characterization of Extracellular Pectinolytic Enzymes Produced by Sclerotinia sclerotiorum

An exopolygalacturonase (exoPG) and an exopolymethylgalacturonase (exoPMG) produced by Sclerotinia sclerotiorum have been purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. The exoPG and the exoPMG were purified 66- and 50-fold, respectively, by using a series of separation procedures that included ammonium sulfate precipitation and gel chromatography. Molecular masses of the native proteins were 68 kDa for exoPG and 140 kDa for exoPMG. The pH optima of the enzymes were about pH 5, and their optimum temperature was 45°C. Activities of both enzymes were inhibited by Hg²⁺, Zn²⁺, Cu²⁺, and p-chloromercuribenzoate. ExoPMG activity, in contrast to exoPG activity, was stimulated by Mn²⁺ and Co²⁺. ExoPMG hydrolyzed only citrus pectin, while exoPG degraded sodium polygalacturonate and, to a lesser extent, citrus pectin. The exo mode of action of the enzymes was revealed by thin-layer chromatography of substrate hydrolysates. Antibodies raised against each purified protein exhibited no cross-reaction, thus confirming the biochemical specificities of the enzymes.     

Production of Major Extracellular Enzymes during Lignocellulose Degradation by Two Streptomycetes in Agitated Submerged Culture

[This corrects the article on p. 1167 in vol. 55.].     

Production of Major Extracellular Enzymes during Lignocellulose Degradation by Two Streptomycetes in Agitated Submerged Culture

Streptomyces viridosporus T7A and S. badius 252 were grown in 1 to 2% (wt/vol) slurry cultures with mineral salts solution containing 0.6% yeast extract and 100/200 mesh ground and extracted corn lignocellulose at 37°C. Enzyme activities rapidly increased in the first 3 to 4 days and then declined and remained at a relatively constant level. Concentrations of endoglucanase and xylanase produced by S. badius were lower than those produced by S. viridosporus. However, the lignin-peroxidase peak concentration was threefold higher than with S. viridosporus and was obtained at 9 to 10 days of incubation. By polyacrylamide gel analysis, it was determined that peroxidases from both species consisted of four enzymes, with only one, the lignin peroxidase, having high activity. A culture pH of 8.5 was preferable for lignocellulose degradation by S. badius.     

节木代料香菇子实体蛋白质的营养评价

对不同节木代料香菇子实体蛋白质的营养价值进行了研究。结果表明 ,2种节木代料香菇子实体蛋白质的必需氨基酸含量与木屑代料香菇子实体蛋白质的必需氨基酸含量没有明显差异 ,且前者蛋白质的氨基酸系数分均高于后者 ,这说明节木代料香菇子实体蛋白质具有较高的营养价值。     

Alliinase-like Enzymes in Fruiting Bodies of Lentinus edodes: Their Purification and Substrate Specificity

3-Chloro-d-alanine chloride-lyase, which occurs in the cells of Pseudomonas putida CR 1-1, catalyzes not only the α,β-elimination reaction of 3-chloro-d-alanine to form pyruvate, but also its β-replacement reaction in the presence of a high concentration of sodium hydrosulfide to form d-cysteine. Using the β-replacement reaction, the enzymatic synthesis of d-cysteine by resting cells was investigated. The culture conditions for cell production of the bacterium with high d-cysteine-producing activity and the reaction conditions for d-cysteine production were optimized. Under these optimal reaction conditions, 100% of the added 3-chloro-d-alanine could be converted to d-cysteine and, as the highest yield, 20.6 mg of d-cysteine per 1.0 ml of reaction mixture could be synthesized.     

Laccase and Lectin Activities of Intracellular Proteins Produced in a Submerged Culture of the Xylotrophic Basidiomycete Lentinus edodes

The white-rot fungus Lentinus edodes produced D: -melibiose-specific lectins and two laccase forms in a lignin-containing medium. The maxima of laccase and lectin activities coincided, falling within the period of active mycelial growth. The enzymes and lectins were isolated and purified by gel filtration followed by anion-exchange chromatography. The L. edodes lectins were found to be able to stabilize the activity of the fungus's own laccases. Lectin activity during the formation of lectin-enzyme complexes remained unchanged.     

Tolerance of tannin by the shiitake mushroom,Lentinus edodes

Summary Tannin at 1% (w/v) did not inhibit the growth ofLentinus edodes, but did inhibitPleuroius florida, P. sajor-caju, P. cystidosus, Agaricus bisporus andVolvariella volvacea. The inhibition was not due to its acidity.

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Tolérance de Lentinus edodes aux ajouts de tannin

Résumé Le tannin à la concentration de 1% (p/v) n'inhibe pas la croissance deLentinus edodes, mais inhibe celle dePleurotus florida, P. sajor-caju, P. cystidosus, Agaricus bisporus, etVolvariella volvacea. L'inhibition n'est pas due à son acidité.

     

HPLC法测定香菇中有机酸含量的检测技术研究

本实验建立了HPLC法同时测定香菇中酒石酸、苹果酸、乙酸和柠檬酸的方法。在酸提条件下,利用Agela Venusll MP C18色谱柱,以2%CH3OH-20 mM(NH4)2HPO4(pH 2.40)为流动相,流速为1.0 mL/min,柱温为35℃,检测波长为210 nm,进样量为10μL。四种有机酸均在8 min内出峰。本法准确、可靠、快速、简便,检出限较低,适合于大批量样品的分析与检测。     

Extracellular Laccase Produced by an Edible Basidiomycetous Mushroom,Grifola frondosa: Purification and Characterization

A major laccase isozyme (Lac 1) was isolated from the culture fluid of an edible basidiomycetous mushroom, Grifola frondosa. Lac 1 was revealed to be a monomeric protein with a molecular mass of 71 kDa. The N-terminal amino acid sequence of Lac 1 was highly similar to those of laccases of some other white-rot basidiomycetes. Lac 1 showed the typical absorption spectrum of a copper-containing enzyme. The enzyme was stable in a wide pH range (4.0 to 10.0), and lost no activity up to 60 °C for 60 min. The optimal pH of the enzyme activity varied among substrates. The K _m values of Lac 1 toward 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), 2,6-dimethoxyphenol, guaiacol, catechol, and 3,4-dihydroxy-L-phenylalanine were 0.0137 mM, 0.608 mM, 0.531 mM, 2.51 mM, and 0.149 mM respectively. Lac 1 activity was remarkably inhibited by the chloride ion, in a reversible manner. Lac 1 activity was also inhibited by thiol compounds.     

Decreased zinc ion accumulation by the basidiomycete Lentinus edodes over-expressing L. edodes PriA gene

The information that the deduced expression product of Lentinus edodes priA gene consists of N-terminal hydrophobic sequence, putative zinc-binding motifs and C-terminal membrane-binding-promoting unique sequence led us to analyze its function in L. edodes. Here L. edodes monokaryotic cells over-expressing priA gene were found to exhibit a remarkably decreased accumulation of zinc ion, indicating the involvement of the priA gene in regulation of the intracellular zinc concentration.     

香菇双单杂交后代不同发育阶段酯酶同工酶研究

选用香菇野生株分别与栽培株及杂交株进行双单杂交 ,得到 8个杂交后代 ,且具有结实能力 ,分别对液培 2 0d杂交后代菌丝与液培原基酯酶同工酶进行了比较研究。结果表明 ,液培 2 0d菌丝菌株间酯酶同工酶酶谱显示出多样性 ,可以作为鉴定菌株的辅助遗传标记 ,而原基菌株间酯酶同工酶酶谱谱带较少 ,呈现趋同效应 ,不宜作为鉴定香菇菌株的辅助遗传标记。     

Characteristics of Wine Produced by Mushroom Fermentation

Saccharomyces cerevisiae is the main microorganism used in wine brewing, because this microbe has potent ability to produce alcohol dehydrogenase. We have recently discovered that some genera of mushroom produced alcohol dehydrogenase, and made wine by using a mushroom in place of S. cerevisiae. The highest alcohol concentration in this wine was achieved with Pleurotus ostreatus (2.6 M, 12.2%). In the case of Agaricus blazei, the same alcohol concentration (1.7 M, 8%) was produced under both aerobic and anaerobic conditions. This wine produced by A. blazei contained about 0.68% β-D-glucan, which is known to have a preventive effects against cancer. The wine made by using Flammulina velutipes showed thrombosis-preventing activity, giving a prolonged thrombin clotting time 2.2-fold that of the control. Thus, the wine made by using mushroom seems to be a functional food which can be expected to have preventive effects against cancer and thrombosis     

Some Properties of an Extracellular Proteolytic Enzyme of Verticillium fungicola, a Pathogen of the Cultivated Mushroom Agaricus bisporus

In a casein nutrient solution, Verticillium fungiocla, the causal agent of the dry bubble disease of the cultivated mushroom Agaricus bisporus, produces a proteolytic enzyme. The effects of the pH and of inhibitors on the protease activity and the heat stability of the enzyme are described. The protease is of interest in connection with the attacking mechanism of Verticillium fungicola.     

Analysis of mfbA alleles of the eubasidiomycete Lentinus edodes

A fruiting-body-specific mfbA cDNA derived from Lentinus edodes FMC2 has been shown to encode a high-molecular-weight protein, MFBA, containing the cell-adhesion-promoting Arg-Gly-Asp (RGD) sequence. Southern-blot analysis showed that all L. edodes strains tested have the mfbA gene (homologue). Nucleotide sequence analysis of the 1-kb mfbA fragments containing the RGD-coding sequence showed that each L. edodes strain has two types of mfbA homologues. It was found in FMC2 that two mfbA homologues are derived from different nuclei and these mfbA alleles are transcribed with similar frequencies in the fruiting bodies.