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Fruiting Body Production and Aroma Profile Analysis of Agrocybe aegerita Cultivated on Different Substrates  [PDF]
Vanessa Kleofas, Laura Sommer, Marco A. Fraatz, Holger Zorn, Martin Rühl
Natural Resources (NR) , 2014, DOI: 10.4236/nr.2014.56022

The industrial cultivated basidiomycete Agrocybe aegerita, also known as “Pioppino” in Italy or “Samthaube” in Germany, is a high quality mushroom with a delicious aroma. Cultivation of A. aegerita on wheat straw supplemented with different residues of the food industry showed the highest yields in fruiting body production with a biological efficiency of 36% when black tea pomace was added. The addition of other substrates resulted in biological efficiencies of 23% to 33%. Besides the mushroom yields, the number and size of fruiting bodies harvested on the different substrates were determined. A comparison of the aroma profiles of A. aegerita grown on wheat straw and on wheat straw supplemented with black tea by means of GC/MS/MS/O is presented.

Transcriptome and Proteome Exploration to Provide a Resource for the Study of Agrocybe aegerita  [PDF]
Man Wang, Bianli Gu, Jie Huang, Shuai Jiang, Yijie Chen, Yalin Yin, Yongfu Pan, Guojun Yu, Yamu Li, Barry Hon Cheung Wong, Yi Liang, Hui Sun
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0056686
Abstract: Background Agrocybe aegerita, the black poplar mushroom, has been highly valued as a functional food for its medicinal and nutritional benefits. Several bioactive extracts from A. aegerita have been found to exhibit antitumor and antioxidant activities. However, limited genetic resources for A. aegerita have hindered exploration of this species. Methodology/Principal Findings To facilitate the research on A. aegerita, we established a deep survey of the transcriptome and proteome of this mushroom. We applied high-throughput sequencing technology (Illumina) to sequence A. aegerita transcriptomes from mycelium and fruiting body. The raw clean reads were de novo assembled into a total of 36,134 expressed sequences tags (ESTs) with an average length of 663 bp. These ESTs were annotated and classified according to Gene Ontology (GO), Clusters of Orthologous Groups (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways. Gene expression profile analysis showed that 18,474 ESTs were differentially expressed, with 10,131 up-regulated in mycelium and 8,343 up-regulated in fruiting body. Putative genes involved in polysaccharide and steroid biosynthesis were identified from A. aegerita transcriptome, and these genes were differentially expressed at the two stages of A. aegerita. Based on one-dimensional gel electrophoresis (1-DGE) coupled with electrospray ionization liquid chromatography tandem MS (LC-ESI-MS/MS), we identified a total of 309 non-redundant proteins. And many metabolic enzymes involved in glycolysis were identified in the protein database. Conclusions/Significance This is the first study on transcriptome and proteome analyses of A. aegerita. The data in this study serve as a resource of A. aegerita transcripts and proteins, and offer clues to the applications of this mushroom in nutrition, pharmacy and industry.
Benzene oxygenation and oxidation by the peroxygenase of Agrocybe aegerita
Alexander Karich, Martin Kluge, René Ullrich and Martin Hofrichter
AMB Express , 2013, DOI: 10.1186/2191-0855-3-5
Abstract: Aromatic peroxygenase (APO) is an extracellular enzyme produced by the agaric basidiomycete Agrocybe aegerita that catalyzes diverse peroxide-dependent oxyfunctionalization reactions. Here we describe the oxygenation of the unactivated aromatic ring of benzene with hydrogen peroxide as co-substrate. The optimum pH of the reaction was around 7 and it proceeded via an initial epoxide intermediate that re-aromatized in aqueous solution to form phenol. Identity of the epoxide intermediate as benzene oxide was proved by a freshly prepared authentic standard using GC-MS and LC-MS analyses. Second and third [per]oxygenation was also observed and resulted in the formation of further hydroxylation and following [per]oxidation products: hydroquinone and p-benzoquinone, catechol and o-benzoquinone as well as 1,2,4-trihydroxybenzene and hydroxy-p-benzoquinone, respectively. Using H218O2 as co-substrate and ascorbic acid as radical scavenger, inhibiting the formation of peroxidation products (e.g., p-benzoquinone), the origin of the oxygen atom incorporated into benzene or phenol was proved to be the peroxide. Apparent enzyme kinetic constants (kcat, Km) for the peroxygenation of benzene were estimated to be around 8 s-1 and 3.6 mM. These results raise the possibility that peroxygenases may be useful for enzymatic syntheses of hydroxylated benzene derivatives under mild conditions.
茶树菇Agrocybe aegerita新泛素偶联酶蛋白表达及抗肿瘤活性研究  [PDF]
- , 2015, DOI: 10.13982/j.mfst.1673-9078.2015.12.001
Abstract: 大型真菌茶树菇活性蛋白组分Yt在动物模型中被发现具有抗肿瘤及免疫调节活性。为研究其蛋白组分,构建新鲜蘑菇子实体cDNA文库,对cDNA序列进行测序筛选,获得新型真菌泛素偶联酶(Ub-conjugating enzyme,UBC)的cDNA序列(GenBank登录代码:JQ362395),分析其含有UBC家族的保守残基。使用原核表达pET系统表达和纯化重组UBC蛋白,得率是10 mg/L大肠杆菌培养液。在细胞培养中加入重组UBC蛋白将显著改变细胞形态,通过流式细胞仪发现UBC(1.8、3.6、7.2 μM)均能诱导HeLa细胞凋亡,Annexin Ⅴ+/PI-百分比由对照组的1.4%上调至10.7%、15.8%和20.3%,呈浓度依赖性。本文是首次报道真菌UBC具有抗肿瘤活性。该结果暗示,真菌泛素系统成员可能通过改变细胞基本生理功能,成为潜在的候选药物,值得探讨。
The active protein component Yt from macrofungi Agrocybe aegerita has been found to have antitumor and immune regulation activity. For the investigation of the proteins, a cDNA library of the Agrocybe aegerita fruiting body was constructed and screened. The cDNA of a novel fungal Ub-conjugating enzyme (UBC) was identified (Genbank accession code: JQ362395), and sequence analysis revealed that it contained the conserved residues and belonged to the UBC family. Recombinant UBC was expressed using the pET system and purified, and obtained 10 mg from 1 L E coli. culture media. Exogenously applied UBC (1.8, 3.6 and 7.2 μM) in cell cultures altered HeLa cell morphology and induced cell apoptosis determined by flow cytometry, and the percentage of AnnexinV+/PI- cells was increased from 1.4% in control to 10.7%, 15.8% and 20.3%, which was concentration dependent. This is the first report on the effects of a fungal UBC on cancer cells, which suggests that the members of the fungal ubiquitin system might alter fundamental cellular processes and require further investigation.
Anti-oxidative responses of Agrocybe aegerita mycelia to cadmium stress

WANG Song-hua,ZHANG Hua,FU Man-qin,CHEN Qing-yu,ZHOU Zheng-yi,LU Xiao-min,

应用生态学报 , 2007,
Abstract: This paper studied the effects of different concentration Cd on the anti-oxidative enzyme activities and glutathione content in Agrocybe aegerita cultivated in liquid medium. The results indicated that at low concentrations of Cd, the test enzyme activities increased with increasing Cd concentration, being the maximum at 0.1 mmol x L(-1) for CAT, at 0.2 mmol x L(-1) for POD, GR and LOX, and at 0.4 mmol x L(-1) for SOD. At 1.6 mmol x L(-1) of Cd, the activities of POD, CAT and SOD were inhibited markedly. 0.4-1.6 mmol x L(-1) of Cd resulted in an increase of glutathione content, but glutathione disulfide content was less affected. The ascorbate acid content and APX activity were too low to be detectable. The PAGE analysis revealed that 0.1-0.8 mmol x L(-1) of Cd induced the additional isozyme bands of POD, EST and LOX, and increased the intensity of the constitutive isozymes of CAT and SOD. 1.6 mmol x L(-1) of Cd decreased the intensity of the isozymes of POD, CAT and SOD significantly.
Changes of extracellular enzymes activities during development of Agrocybe aegerita

- , 2018,
Abstract: 【目的】研究3株茶树菇菌株栽培过程中7种胞外酶活性的变化规律,了解茶树菇在不同生长发育阶段利用营养物质的规律。【方法】以茶树菇菌株ZK08、JX09、Ag16为材料,将其接种于组分为棉籽壳39%、木屑30%、麸皮20%、玉米粉8%、蔗糖1.5%、石膏1.5%(均为质量分数),含水量62%的栽培袋中。25 ℃条件下遮光培养50~60 d,分别在菌丝生长半袋、满袋、现蕾、出一潮菇、一潮菇子实体成熟、出二潮菇、二潮菇子实体成熟和二潮菇采收1周后,测定羧甲基纤维素酶、淀粉酶、滤纸纤维素酶、半纤维素酶、漆酶、多酚氧化酶和过氧化物酶的活性,分析其活性变化规律。【结果】3株茶树菇菌株在栽培过程中的7种胞外酶活性变化规律基本一致,但表现出明显的阶段性。其中淀粉酶、羧甲基纤维素酶、滤纸纤维素酶、半纤维素酶活性均表现出先升后降的趋势,峰值出现在一潮菇子实体成熟前;而漆酶、多酚氧化酶和过氧化物酶活性则随着栽培时间延长一直呈下降趋势。【结论】漆酶、多酚氧化酶和过氧化物酶的高峰值比羧甲基纤维素酶、半纤维素酶来得早;淀粉酶的高峰出现在一潮幼菇期到子实体成熟期。因此在培养基中添加适量淀粉类物质可促进茶树菇的生殖发育。
【Objective】We studied the changes in activities of seven extracellular enzymes during the cultivation of three different Agrocybe aegerita strains and studied the utilization of nutrients at different growth stages to provide basis for selection of high yield cultivation substrate.【Method】Strains ZK08,JX09 and Ag16 were cultured in bags consisted of cotton seed hulls 39%,sawdust 30%,bran 20%,corn flour 8%,sucrose 1.5%,gypsum 1.5%,and water 62%.They were cultured at 25 ℃ under shade for 50-60 d.The activities of enzymes including carboxymethyl cellulase,amylase,filter paper cellulase,hemicellulase,laccase,polyphenol oxidase and peroxidase at different growth stages including hypha growing half bag,full bag,budding,first mushroom,fruiting bodies mature of first mushroom,second fruiting bodies mature of the second mushroom and one week after picking second mushroom were measured and analyzed.【Result】The three Agrolybe aegerita strains had basically same changes in activities of extracellular enzyme with obvious stage characteristics.The activities of amylase,carboxymethyl cellulase,filter cellulase and hemicellulase increased first with peaks before the maturing of first fruiting body and then decreased.Activities of laccase,polyphenol oxidase and peroxidase presented downward trend along with the cultivation time.【Conclusion】The peak values of laccase,polyphenol oxidase and peroxidase activates appeared earlier than that of carboxymethyl celluase and hemicellulase.The peak of amylase occurred at stages from first young mushroom to the mature of first fruiting body.So the addition of starch in medium can promote the reproductive development of tea mushroom
The Molecular Cloning and Bioactivity Characterization of Unknown Gene 1C10 from Medicinal Fungus Agrocybe aegerita

梁一, 郭莲仙, 孙慧
Medical Diagnosis (MD) , 2012, DOI: 10.12677/md.2012.21001
目的:克隆杨树菇抗肿瘤相关蛋白基因,初步鉴定其活性。方法:构建cDNA文库,随机测序得到杨树菇基因1C10。高通量测序分析其在菌丝体和子实体的差异表达。构建真核表达质粒检测其在HeLa细胞中表达引起的细胞凋亡及衰老的变化,初步鉴定其抗肿瘤活性。结果:得到全长为728 bp的杨树菇基因1C10,编码107个氨基酸,高通量测序分析发现其在子实体中为高表达。在真核细胞中表达会引起肿瘤细胞的凋亡而非衰老。结论:通过构建cDNA文库及测序初步鉴定出具抗肿瘤活性的杨树菇未知基因1C10,为进一步研究其活性及机理奠定基础。
Objective: The antitumor-associated gene of Agrocybe aegerita was cloned to identify the bioactivity. Method: cDNA library was constructed and the gene 1C10 was obtained by random sequencing. The differential ex- pression of the gene was analyzed by high-throughout sequencing. The eukaryotic expression vector was constructed to detect cell apoptosis and senescence activity. Result: The gene of 1C10 was cloned with 728 bp and 107 amino acid residues. The high-throughout sequencing analyse showed that 1C10 was highly expressed in the fruiting body. The expression in HeLa cells could induce the cell apoptosis instead of senescence. Conclusion: 1C10 was identified to have antitumor activity by cDNA library and sequencing, which was fundation for further study.

WANG Nan,SHEN Feng,TAN Qi,CHEN Ming-Jie,PAN Ying-Jie,

菌物学报 , 2000,
Abstract: 柱状田头菇Agrocybe aegerita作为一种新开发的食用菌,越来越受到消费者的喜爱和研究者的关注,但在其栽培生产中遇到的产量相对较低的问题,一直未能得到很好的解决,致使柱状田头菇的栽培难以得到大规模的推广。本研究对不同来源的柱状田头菇在其6个不同生长发育阶段中,9种与培养料中主要组分分解相关的胞外酶活性的变化进行了测定,结果表明:柱状田头菇属褐腐菌,对非木质纤维素的利用能力最强,对纤维素的利用能力较强,对木质素的利用能力较差,但柱状田头菇具有漆酶活性。
The Molecular Cloning and Bioactivity Characterization of Calmodulin from Medicinal Fungus Agrocybe aegerita

梁一, 孙慧
Medical Diagnosis (MD) , 2012, DOI: 10.12677/md.2012.22002
目的:克隆杨树菇抗肿瘤相关蛋白基因,初步鉴定其活性。方法:构建cDNA文库,随机测序得到杨树菇钙调素(calmodulin,CaM)基因,并对其进行生物信息学分析,通过检测其在HeLa细胞中表达引起的细胞凋亡及衰老的变化,初步鉴定其抗肿瘤活性。结果:得到全长为588 bp的杨树菇钙调素cDNA序列,编码150个氨基酸,其氨基酸序列在整个钙调素家族中十分保守。钙调素基因在真核细胞中表达没有引起肿瘤细胞的凋亡和衰老。结论:杨树菇钙调素序列具有CaM保守序列模式,是CaM家族的成员。
Objective: The antitumor-associated gene of Agrocybe aegerita was cloned to identify the bioactivity. Method: cDNA library was constructed and the gene calmodulin was obtained by random sequencing. The eukaryotic expression vector was constructed to detect cell apoptosis and senescence activity. Result: The calmodulin gene was cloned with 588 bps code for 150 amino acid residues, and the amino acid sequence was very conserved in calmodulin family. The expression of cloned gene in HeLa cells did not induce the cell apoptosis or senescence. Conclusion: The sequence of calmodulin contained conserved motif and belonged to calmodulin family.
The Molecular Cloning and Antitumor Activity Characterization of SUMO from Medicinal Fungus Agrocybe aegerita

梁一, 白娟, 孙慧
Medical Diagnosis (MD) , 2012, DOI: 10.12677/md.2012.22003
目的:克隆杨树菇抗肿瘤相关蛋白基因,初步鉴定其活性。方法:构建cDNA文库,随机测序得到SUMO (Small Ubiquitin-like Modifier)基因,通过构建真核表达质粒,并检测其在HeLa细胞中表达引起的细胞凋亡及衰老的变化,初步鉴定其抗肿瘤活性。结果:得到全长为606 bp的SUMO cDNA序列,编码103个氨基酸,将表达SUMO的真核表达质粒转染入HeLa细胞中,发现可引起肿瘤细胞的凋亡,但对细胞衰老无影响。结论:药用真菌中SUMO等小泛素类蛋白酶系统值得进一步研究,有助于新型抗肿瘤药物前体的开发。
Objective: The antitumor-associated gene of Agrocybe aegerita was cloned to identify the bioactivity. Method: cDNA library was constructed and the gene SUMO was obtained by random sequencing. The eukaryotic ex- pression vector was constructed to detect cell apoptosis and senescence activity. Result: The gene of SUMO was cloned with 606 bp and 103 amino acid residues. The expression in HeLa cells could induce the cell apoptosis instead of se- nescence. Conclusion: SUMO from medicinal fungus worth to be investigated for the discovery of new antitumor medicine.
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