Role of a cell-wall glucan-degrading enzyme in mating of Schizophyllum commune

Mycelial enzyme extracts of Schizophyllum commune were prepared during vegetative growth matings leading to common-A and common-B heterokaryons and the dikaryon, and were examined for hydrolytic activity against an alkaliinsoluble cell-wall glucan (R-glucan) isolated from this mushroom. In extracts from several individual homokaryotic mycelia the R-glucanase activity was low and did not increase when the cultures exhausted glucose in the medium. In common-A matings, a 30-fold increase in specific activity of intracellular R-glucanase was found even in the presence of glucose in the broth. An increase of this magnitude was not observed in the common-B mating nor in the fully compatible cross leading to the dikaryon. Extracts of the dikaryon did show elevated R-glucanase activity after exogenous glucose disappearance and subsequent fruiting. In none of these situations was an enzyme activity detected towards an alkali-soluble cell-wall glucan (S-glucan) prepared from S. commune. Changes in R-glucanase were not parallelled by identical changes in laminarinase, pustulanase, cellobiase, and p-nitrophenyl-beta-d-glucosidase, but comparable increases in specific activities were found for hydrolysis of glycogen and maltose. After interaction of the various mycelia in mating combinations, the S-glucan/R-glucan ratio of the cell wall of the dikaryon was found to be similar to that of the homokaryons, but increased in the common-B interaction and was elevated almost threefold in the common-A heterokaryon.     

Purification and characterization of chitinase A of Streptomyces cyaneus SP-27: an enzyme participates in protoplast formation from Schizophyllum commune mycelia

A culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune has an activity to form protoplasts from S. commune mycelia. alpha-1,3-Glucanase and chitinase I, which were isolated from the filtrate, did not form the protoplast by itself while a mixture of them showed protoplast-forming activity. Streptomyces cyaneus SP-27 was isolated based on the productivity of chitinase. The culture filtrate of S. cyaneus SP-27 did not form S. commune protoplasts, but addition of it to alpha-1,3-glucanase of B. circulans KA-304 brought about protoplast-forming activity. Chitinase A isolated from the S. cyaneus SP-27 culture filtrate was more effective than chitinase I of B. circulans KA-304 for the protoplast formation in combination with alpha-1,3-glucanase. The N-terminal amino acid sequence of chitinase A (MW 29,000) has a sequential similarity to those of several Streptomycete family 19 chitinases. Chitinase A adsorbed to chitinous substrate and inhibited the growth of Trichoderma reesei mycelia. Anomer analysis of the reaction products also suggested that the enzyme is a family 19 chitinase.     

Opposing developmental functions of Agrocybe aegerita galectin (AAL) during mycelia differentiation

Mycelia of basidiomycetes differentiating into fruiting body is a controlled developmental process, however the underlying molecular mechanism remains unknown. In previous work, a novel fungal Agrocybe aegerita galectin (AAL) was isolated from A. aegerita in our laboratory. AAL was shown to promote mycelial differentiation in A. aegerita and Auricularia polytricha, indicating that AAL might function as a conserved fruiting initiator during basidiomycete mycelia development. In the current work, we investigate the role of AAL in mycelia differentiation and fruiting body formation. First, the expression and localization of AAL in mycelia, primordium and fruiting body were assessed by Western blotting and immunohistochemistry. AAL was found to be ubiquitously expressed in the primordium and fruiting body but not in the mycelia. AAL facilitated mycelia congregation and promoted fruiting body production when AAL was applied on mycelia. At the same time, when AAL was spread on potato dextrose agar (PDA) medium prior to mycelia inoculation, mycelia exhibited slowed growth rates, resulting in mycelia cords formation and inhibition of fruiting body formation. The 5' regulatory sequence of aal was cloned by 'genome walking'. Here, we show that aal lack introns in the coding region and the upstream 740 bp sequence was characterized by the existence of core promoter elements, which included: two CCAAT boxes (-535/-280), a GC box (-145), a TATA box (-30) and a fungal leader intron within the 5' UTR. The identification of regulatory expression elements may provide an explanation to the stage-specific and high-level expression of aal during fruiting development.     

Phenoloxidase Activity in Mycelia Carrying Modifier Mutations That Affect Sporocarp Development in Schizophyllum commune

Phenoloxidase activity was studied in mycelia carrying mutations that affect sporocarp formation. The results suggest that phenoloxidase activity may be essential to, but is not sufficient for, the fruiting response in Schizophyllum.     

Comparison of poly(A)-containing RNAs in different cell types of the lower eukaryote Schizophyllum commune

Poly(A)-containing RNAs were isolated from morphologically different cells of the fungus Schizophyllum commune. Using mRNA markers the number-average length of poly(A)-containing RNA in total RNA and in purified poly(A)-containing RNA was estimated as 1100 nucleotides. Number-average length of poly(A)-tracts was 33 nucleotides. 2.5% of total RNA is poly(A)-containing RNA and probably up to 7.5% are non-polyadenylated polydisperse RNA sequences. Saturation hybridization of poly(A)-containing RNA to gap-translated [3H]DNA resulted in 16% of the reactive single-copy DNA to become S1 nuclease resistant. It was found that purified poly(A)-containing RNA represented the entire RNA complexity, i.e. 10 000 different RNA sequences in S. commune. RNA sequences isolated from morphologically different mycelia and from fruiting and non-fruiting mycelia were identical for at least 90%.     

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.     

Cell wall degradation in the autolysis of filamentous fungi

A systematic study on autolysis of the cell walls of fungi has been made on Neurospora crassa, Botrytis cinerea, Polystictus versicolor, Aspergillus nidulans, Schizophyllum commune, Aspergillus niger, and Mucor mucedo. During autolysis each fungus produces the necessary lytic enzymes for its autodegradation. From autolyzed cultures of each fungus enzymatic precipitates were obtained. The degree of lysis of the cell walls, obtained from non-autolyzed mycelia, was studied by incubating these cell walls with and without a supply of their own lytic enzymes. The degree of lysis increased with the incubation time and generally was higher with a supply of lytic enzymes.Cell walls from mycelia of different ages were obtained. A higher degree of lysis was always found, in young cell walls than in older cell walls, when exogenous lytic enzymes were present.In all the fungi studied, there is lysis of the cell walls during autolysis. This is confirmed by the change of the cell wall structure as well as by the degree of lysis reached by the cell wall and the release of substances, principally glucose and N-acetylglucosamine in the medium.     

蜜环菌对镁的耐性和富集特性

研究了镁对蜜环菌生长的影响, 蜜环菌对于镁的耐性和富集规律, 以及高浓度镁胁迫下蜜环菌的抗氧化酶的变化情况。3?15 g/L的Mg浓度对于蜜环菌菌体的生长有促进作用。Mg浓度19 g/L以上时, 蜜环菌菌体的生长受到抑制。蜜环菌的子实体形成和子实体生物量在Mg的浓度为11 g/L以下时不受影响, 超过11 g/L则子实体不能萌发。皮壳状菌丝和菌索中Mg的含量随培养基中Mg浓度的增大而增大, 培养基Mg浓度达到16 g/L后, 菌丝、菌索中Mg的含量都不再上升。子实体对Mg的富集量比菌丝体小的多, 在培养基Mg浓度在9、10 g/L时, 子实体中Mg的含量与对照组有显著差异。随着培养基中Mg浓度的提高, 菌丝和菌索POD、CAT、SOD活性都有增加, 而且菌丝与菌索之间抗氧化酶活力的差异随着培养基中Mg浓度的提高而增大。     

Effect of glucose on the fruiting body formation and adenosine 3',5'-cyclic monophosphate levels in Coprinus macrorhizus

The formation of fruiting bodies in the monokaryotic fis(c) strain and a dikaryon of Coprinus macrorhizus was inhibited by growth in high-glucose media. In high-glucose media the characteristic burst of adenosine 3',5'-cyclic monophosphate (cAMP) accumulation during fruiting-body formation was absent. Enzymatic activity assays revealed that mycelia grown in high-glucose media contained relatively lower amounts of adenylate cyclase and cAMP-phosphodiesterase than mycelia grown in low-glucose media. The synthesis of inducible d-serine deaminase and tryptophanase was repressed in high-glucose media. A mutant (gluR) in which the glucose repression of fruiting-body formation is affected was isolated by selection in high-glucose media. The mutation caused the cAMP levels to be no longer affected by glucose and affected ability to synthesize the inducible d-serine deaminase and tryptophanase. The gluR mutant was partially dominant in dikaryons. It is suggested that cAMP may play important roles in inducing fruiting bodies and in controlling inducible enzyme synthesis in C. macrorhizus.     

Lectin activity of Lentinus edodes

The hemagglutinating activity of submerged mycelium and culture liquid for four strains of Lentinus edodes (Berk.) Sing [L. edodes (Berk.) Pegler] was studied in the search for lectins. The hemagglutinating activity of culture liquid was substantially higher, compared with mycelium. The carbohydrate-binding capacity of the agglutinins was established, and the lectin activity of extracts from mycelia grown on several agar media was elucidated in relation to fruiting. The lectin activity of L. edodes was examined at different morphogenetic steps: mycelium, brown mycelial film, primordium, and fruiting body. Hemagglutination titers at the brown film step were higher than in the mycelium, whereas activity at the primordial and fruiting bodies steps decreased. Lectins seem to be involved in the formation of hyphal aggregates of brown mycelial film.     

桑黄菌丝体和子实体中次级代谢产物及其活性的比较

研究桑黄发酵菌丝体次级代谢产物及活性与子实体的差异性,探讨其替代子实体的可能性。研究通过高效液相色谱分析和化学法比较菌丝体和子实体石油醚、氯仿、乙酸乙酯和正丁醇4个萃取相中的成分差异,以二苯基三硝基苯肼自由基（DPPH）清除率和Trolox当量抗氧化能力（TEAC）作为抗氧化活性的指标、HepG2和MCF-7癌细胞的抑制率作为抑制肿瘤细胞生长的指标,比较其活性差异。结果表明,菌丝体和子实体4个萃取相在化学成分上存在差异;在活性方面,菌丝体各萃取相的抗氧化活性高于子实体,而子实体抗肿瘤活性优于菌丝体。菌丝体醇提取的总黄酮含量高于子实体醇提物,抗氧化活性和总黄酮含量有显著相关性,发酵菌丝体在抗氧化活性方面具有替代子实体的可行性。     

灵芝子实体、菌丝体及孢子粉中多糖成分差异比较研究

为探讨灵芝子实体、菌丝体和孢子粉3种材料中多糖成分的差异,分别运用苯酚硫酸法进行多糖含量测定,运用离子色谱分析其酸水解后单糖组成,并运用HPLC分析各多糖图谱及经α-淀粉酶和β-1,3-葡聚糖酶处理后HPLC图谱的变化,结果发现,灵芝菌丝体中多糖含量最高,达到3.81%,孢子粉多糖含量为1.8%,灵芝子实体中多糖含量最低,仅为0.59%;水解后的单糖组成及摩尔比也有差异,子实体的单糖主要为葡萄糖和半乳糖,菌丝体和孢子粉的单糖主要为葡萄糖;HPLC图谱显示3种多糖出峰位置和分子量也不同,酶解效果表明多糖结构也相差较大。各样品多糖对小鼠巨噬细胞RAW264.7释放NO的产量的影响上,菌丝体与子实体多糖都表现出了很好的活性,而孢子粉多糖却呈现出较低活性。实验结果表明灵芝子实体、菌丝体和孢子粉3种材料的多糖成分差异大,在医药保健品使用中应区分使用。     

Post-harvest sugarcane residue degradation by autochthonous fungi

Several fungal species were isolated from different sources: post-harvest sugarcane residue, soil, decomposing forest litter and from mycelia obtained from the inner parts of fresh fungal fruiting bodies collected in Las Yungas region (Argentina). These isolates were first screened for their ability to produce carboxymethyl cellulose (CMC) degradation and guaiacol oxidation. After primary screening, seventeen isolates were further tested for their ligninolytic ability by assessing polyphenoloxidase, laccase, manganese peroxidase and endoxylanase activities. Based on their lignocellulolytic activities, five isolates (named Bjerkandera sp. Y-HHM2, Phanerochaete sp. Y-RN1, Pleurotus sp. Y-RN3, Hypocrea nigricans SCT-4.4 and Myrothecium sp. S-3.20) were selected for liquid and solid-state fermentation assays in culture media including sugarcane debris. Lignocellulolytic enzymes production, dry mass loss and phenol concentration in the water soluble fraction were then evaluated. Results suggest that native strains with lignocellulolytic activity are suitable to increase post-harvest sugarcane residue decomposition and support the use of these strains as an alternative to pre and post-harvest burning. Biological treatments using Phanerochaete sp. Y-RN1, Pleurotus sp. Y-RN3 and Myrothecium sp. S-3.20 could be used to degrade and increase the accessibility to lignocellulose components of sugarcane residue.     

蝉虫草子实体与发酵菌丝体胞内多糖成分分析

比较蝉虫草子实体、孢梗束、虫体以及蝉虫草发酵菌丝体的胞内多糖含量、单糖组成和分子量差异,结果表明,蝉虫草子实体与发酵菌丝中胞内多糖的含量和组成均存在差异。葡萄糖、半乳糖和甘露糖是蝉虫草发酵菌丝体和子实体粗多糖中主要的3种单糖,阿拉伯糖和木糖是子实体粗多糖中的特征性单糖,而果糖属于菌丝体的特征性单糖。子实体粗多糖中高分子量组分（>1×10 ⁶Da）的相对含量明显高于菌丝体粗多糖。本研究对提升蝉虫草发酵多糖产品品质、促进蝉虫草开发应用具有参考价值。     

Enzymatic activity in basidiomycetes

The oxidation-reduction activity of the basidiomyceteOudemansiella mucida was studied in relation to its growth in a laboratory fermentor. Cytochromes were detected in the mycelium destroyed by sonication, flavin diaphorase, polyphenoloxidase, peroxidase and catalase were found to be present in the mycelial homogenate from Waring-blendor. The obtained values of the enzymatic activity were dependent on the method of preparation of the mycelium. Homogenization in a Waring-blendor was the most appropriate. Certain interrelationships between the appearance and activity of the enzymes followed and the phase of submerged growth of the fungus were shown. The results document the succession of cytochromes, flavins and polyphenoloxidase and the specific time differences of the activity of catalase and that of peroxidase.     

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.     

蛹虫草对锌的耐性与富集特征

在培养基内添加不同量的锌,研究其对蛹虫草子实体的形成、子实体和菌丝体生物量、子实体多糖含量和葡萄糖含量的影响,以及蛹虫草子实体和菌丝体对锌的富集能力。结果表明锌对上述各项都有影响。液体培养条件下,锌浓度在453906mg/L范围内可以促进菌丝体生长,锌浓度超过4077mg/L时,菌丝生长受到抑制。培养基锌的浓度在4077mg/L以下时,蛹虫草菌丝体锌的富集量随着液体培养基锌浓度的提高而提高。固体培养条件下,锌含量在226453mg/kg范围内可以促进蛹虫草子实体生长,并且在此含量范围内,蛹虫草子实体中葡萄糖含量较高。培养基锌含量在680906mg/kg时,子实体多糖含量较高。培养基锌含量在2038mg/kg以下时,蛹虫草子实体中锌的富集量随着培养基锌含量的提高而提高,在培养基锌含量为2038mg/kg时,子实体中锌的含量达到28570mg/kg(干重)。     

沪农灵芝一号生长过程中子实体各部位糖醇和海藻糖积累及其代谢酶的表达变化

食用菌子实体通常会在生长过程中积累较高含量的糖醇及海藻糖,这些碳水化合物的积累能够促进食用菌的生长,而在灵芝中的同类研究较少,本研究通过高效阴离子-脉冲安培法对沪农灵芝一号子实体发育过程中不同部位的糖类成分的含量变化进行分析,发现灵芝子实体中主要的可溶性糖类成分是阿拉伯糖醇、甘露醇和海藻糖,甘露醇在子实体成熟时的菌盖中的含量达到最高值,阿拉伯糖醇在产孢子期的子实体中含量较高,两种糖醇的含量呈现相反的变化趋势,一种糖醇积累的同时会消耗利用另一种糖醇,而海藻糖在灵芝子实体的整个生长过程中含量处于较低水平,仅在子实体初期的菌基部位检测到较高的含量;同时通过qRT-PCR技术检测灵芝子实体不同部位中这几种糖类的主要代谢酶基因的表达变化,发现这些代谢酶在子实体的菌基部位的表达水平相对其他部位较高,且随着子实体生长这一差异更加显著,这一结果表明灵芝中的糖醇和海藻糖分布差异可能是先由菌基的菌丝体中合成产物并转运到子实体不同部位,再经过一段时间的积累和代谢之后产生。     

巴西蘑菇菌体深层发酵培养条件的优化及成分分析*

对巴西蘑菇深层发酵条件进行了详细的研究,确定了巴西蘑菇菌体发酵的最优方案是:7%葡萄糖,1.5%酵母膏,0.3%磷酸氢二钾,0.1%硫酸镁,VB1 10mg/100ml,自然pH,接种量为15%,装液量为100ml/250ml三角瓶,150r/min,25℃恒温培养9天,菌丝干重达到1.8g每100ml发酵液。通过对巴西蘑菇子实体与深层培养菌丝体中蛋白质营养成分及多糖含量进行了分析比较,发现它们均是极好的营养食品之源。