Ganodermasides C and D, two new anti-aging ergosterols from spores of the medicinal mushroom Ganoderma lucidum

Two new anti-aging compounds, ganodermasides C and D, were isolated and their structures elucidated. They are novel ergosterols possessing a 4,6,8(14),22-tetraene-3-one unit with unique hydroxylation at C-9. Both of them significantly extended the replicative lifespan of the K6001 yeast strain. Ganodermasides C and D regulated the expression of the gene for UTH1 to prolong the replicative lifespan of yeast.     

A distinctive ribonuclease from fresh fruiting bodies of the medicinal mushroom Ganoderma lucidum

A ribonuclease with an N-terminal sequence distinct from other mushroom ribonucleases was isolated from fresh fruiting bodies of the medicinal mushroom Ganoderma lucidum. The ribonuclease was adsorbed on DEAE-cellulose and Q-Sepharose, and unadsorbed on CM-Sepharose. It possessed a molecular mass of 42 kDa as judged by gel filtration by fast protein liquid chromatography on Superdex 75 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its molecular mass was similar to that of straw mushroom ribonuclease but much higher compared with those of other mushroom ribonucleases. The ribonuclease was unique among mushroom ribonucleases in that it exhibited the highest potency toward poly(U), followed by poly(A). Its activity toward poly(G) and poly(C) was about one-half of that toward poly(A) and one-quarter of that toward poly(U). A pH of 4.0 and a temperature of 60 degrees C were required for optimal activity of the enzyme. The optimum pH was low compared with those reported for other mushroom ribonucleases.     

Ganodermin, an antifungal protein from fruiting bodies of the medicinal mushroom Ganoderma lucidum

A 15-kDa antifungal protein, designated ganodermin, was isolated from the medical mushroom Ganoderma lucidum. The isolation procedure utilized chromatography on DEAE-cellulose, Affi-gel blue gel, CM-Sepharose and Superdex 75. Ganodermin was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and CM-Sepharose. Ganodermin inhibited the mycelial growth of Botrytis cinerea, Fusarium oxysporum and Physalospora piricola with an IC50 value of 15.2 microM, 12.4 microM and 18.1 microM, respectively. It was devoid of hemagglutinating, deoxyribonuclease, ribonuclease and protease inhibitory activities.     

Ganodermasides C and D,Two New Anti-Aging Ergosterols from Spores of the Medicinal Mushroom Ganoderma lucidum

Two new anti-aging compounds, ganodermasides C and D, were isolated and their structures elucidated. They are novel ergosterols possessing a 4,6,8(14),22-tetraene-3-one unit with unique hydroxylation at C-9. Both of them significantly extended the replicative lifespan of the K6001 yeast strain. Ganodermasides C and D regulated the expression of the gene for UTH1 to prolong the replicative lifespan of yeast.     

Effects of extracts from sporoderm-broken spores of Ganoderma lucidum on HeLa cells

The effects of extracts from Ganoderma lucidum spores on the growth of human cervix uteri tumor HeLa cells as well as on the cell cycle and intracellular calcium level were investigated. Alcohol extracts were prepared from sporoderm-broken and sporoderm-nonbroken spores (termed extract I and extract II) of G. lucidum. Extract I was then subjected to silica gel chromatography to obtain extract III. Cytotoxicity was examined by means of trypan blue exclusion and MTT tests. It was found that extract I and extract III, but not extract II strongly inhibited the growth of HeLa cells, and that extract III was more effective than extract I. Moreover, extract III was shown to be capable of blocking the cell cycle at the transition from G₁ to S phase and inducing a marked decrease of intracellular calcium level, determined by flow cytometry and the specific fluorescent calcium probe Fura-2, respectively. These results imply that (1) the breaking of G. lucidum spores improves the release of cytotoxic activity and (2) the effective extract might influence the cell cycle and cellular signal transduction by altering the calcium transport system. This revised version was published online in July 2006 with corrections to the Cover Date.     

Scale-up of a liquid static culture process for hyperproduction of ganoderic acid by the medicinal mushroom Ganoderma lucidum

Scale-up of a liquid static culture process was studied for hyperproduction of ganoderic acid (GA) by a famous Chinese traditional medicinal mushroom, Ganoderma lucidum. Initial volumetric oxygen transfer coefficient (K(L)a) and area of liquid surface per liquid volume (A(s)) were identified as key factors affecting cell growth and GA accumulation in liquid static cultures of G. lucidum, on the basis of which a multilayer static bioreactor was designed. At a low initial K(L)a level of 2.1 h(-1), a thick layer of white mycelia was formed on the liquid surface, and an optimal production of total GA (i.e., GA production in the liquid and on the liquid surface) was obtained. Both the formation of white mycelia and production of GA on the liquid surface were enhanced with an increase of A(s) within the range as investigated (0.24-1.53 cm(2)/mL). At an A(s) value of 0.90 cm(2)/mL, the total GA production reached maximum. A successful scale-up from a 20-mL static T-flask to a 7.5-L three-layer static bioreactor was achieved based on initial K(L)a. The maximum biomass (20.8 +/- 0.1 g DW/L), GA content (4.96 +/- 0.13 mg/100 mg DW), and total GA production (976 +/- 35 mg/L) were attained in static bioreactors. Not only GA content but also its production obtained in this work were the highest ever reported.     

Structural characterization and immunomodulating activity of a complex glucan from spores of Ganoderma lucidum.

A polysaccharide with a molecular weight of 1.26 x 10(5), obtained from the sporoderm-broken spores of Ganoderma lucidum, was purified by anion-exchange and gel-permeation chromatography. This polysaccharide had a strong effect on suppressing the antibody production and the Con A or LPS induced lymphocyte proliferation in mice. Chemically, the structure of the polysaccharide was identified by methylation analysis, 1 D, 2 D NMR and ESI-MS spectroscopic studies of the native one and of the oligosaccharide fragments generated by partial acid hydrolysis, Smith degradation, and acetolysis. It was concluded that the intact polysaccharide was a complex beta-D-glucan consisting of a (1-->6)-linked backbone chain, in which every other glucosyl residue was substituted at C-3 or C-4 with mono-, di- and trisaccharide branches.     

Cloning and characterization of a gene encoding HMG-CoA reductase from Ganoderma lucidum and its functional identification in yeast

A gene encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) was isolated from a triterpene-producing fungus, Ganoderma lucidum (Reishi or Lingzhi). This report provides the complete nucleotide sequence of the full-length cDNA encoding HMGR and its genomic DNA sequence. The cDNA of the HMGR (GenBank Accession no., EU263989) was found to contain an open reading frame (ORF) of 3,681 bp encoding a 1,226-amino-acid polypeptide, whereas the HMGR genomic DNA sequence (GenBank Accession no., EU263990) consisted of 4,262 bp and contained seven exons and six introns. The deduced amino acid sequence of G. lucidum HMGR showed significant homology to the known HMGRs from Ustilago maydis and Cryptococcus neoformans, and contained four conserved domains. Gene expression analysis showed that the expression level was relatively low in mycelia incubated for 10, 12, and 14 d, and reached the highest level in the primordia. Functional complementation of Gl-HMGR in a HMGR-deficient mutant yeast strain indicated that the cloned cDNA encoded a HMG-CoA reductase.     

Performance analyses of a pH-shift and DOT-shift integrated fed-batch fermentation process for the production of ganoderic acid and Ganoderma polysaccharides by medicinal mushroom Ganoderma lucidum

Investigations on Ganoderma lucidum fermentation suggested that the responses of the cell growth and metabolites biosynthesis to pH and dissolved oxygen tension (DOT) were different. The ganoderic acid (GA) production of 321.6 mg/L was obtained in the pH-shift culture by combining a 4-day culture at pH 3.0 with the following 6-day culture at pH 4.5, which was higher by 45% and 300% compared with the culture at pH 3.0 and 4.5, respectively. The GA production of 487.1 mg/L was achieved in the DOT-shift culture by combining a 6-day culture at 25% of DOT with a following 6-day culture at 10% of DOT, which was higher by 43% and 230% compared with the culture at 25% and 10% of DOT, respectively. A fed-batch fermentation process by combining the above-mentioned pH-shift and DOT-shift strategies resulted in a significant synergistic enhancement of GA accumulation up to 754.6 mg/L, which is the highest reported in the submerged fermentation of G. lucidum in stirred-tank bioreactor.     

Activation of B lymphocytes by GLIS,a bioactive proteoglycan from Ganoderma lucidum

A bioactive fraction (GLIS) was isolated from the fruiting body of the fungus Ganoderma lucidum using successive chromatographic steps. GLIS is a proteoglycan and has a carbohydrate: protein ratio of 11.5 : 1. The carbohydrate portion is composed of seven different monosaccharides, predominantly D-glucose, D-galactose and D-mannose in the molar ratio of 3.0 : 1 : 1.GLIS stimulated the proliferation of mouse spleen lymphocytes, resulting in a three to four-fold increase in the percentage of B cells. GLIS also activated mouse spleen lymphocytes, and most of the activated cells were B cells. The B cells were enlarged, expressed CD71 and CD25 on the cell surface, and showed an increase in the secretion of immunoglobulin. Lymphocytes also showed a slightly increased production of IL-2, whereas the secretion of IL-4 was not influenced by GLIS. Furthermore, GLIS did not influence the intracellular Ca2+ concentration of lymphocytes, but it enhanced the expression of protein kinase C alpha and protein kinase C gamma in B cells. According to our results GLIS is a new B cell-stimulating factor.     

Purification and characterization of a novel proteinase A inhibitor from Ganoderma lucidum by submerged fermentation

A novel proteinase A inhibitor was purified from Ganoderma lucidum. The purification was carried out by ethanol precipitation (50–80%), ACA44 gel filtration and Source 30Q anion exchange, respectively. The molecular mass of the inhibitor was 38 kDa as estimated via SDS-PAGE and gel filtration. Its carbohydrate content was up to 70%. β-Elimination revealed that the linkage between the glycan and the core protein backbone might be O-linkage. This inhibitor showed a remarkable heat stability. By investigating the interaction between this inhibitor and a variety of proteinases, it is indicated that the inhibitor was more specific against yeast proteinase A than other proteinases. The dissociation constants (Ki) and concentration required for 50% inhibition (IC₅₀) for proteinase A were 2.7 × 10⁻⁶ M and 0.16 mg/ml, respectively.     

Structural and immunological studies of a major polysaccharide from spores of Ganoderma lucidum (Fr.) Karst

A polysaccharide isolated from spores of the fungus, Ganoderma lucidum, was found to be a complex glucan. On the basis of compositional and methylation analyses, periodate oxidation, Smith degradation, 1D and 2D NMR, and ESIMS experiments of the native polysaccharide and its degraded products, the polysaccharide was shown to have a backbone of beta-(1-->3)-linked D-glucopyranosyl residues, with branches of mono-, di- and oligosaccharide side chains substituting at the C-6 of the glucosyl residues in the main chain. Conformational analysis in aqueous solution and immunological activities of the native and degraded glucans were also investigated. The results suggested that the degree of substitution on the main chain and the length of side chains may be very important factors in determining the conformation and the biological activities of beta-(1-->3)-linked glucans.     

Ganoderiol A and B,New Triterpenoids from the Fungus Ganoderma lucidum (Reishi)

Two new lanostane-type triterpenoids, ganoderiol A (1) and ganoderiol B (2) were isolated from the fruiting bodies of Ganoderma lucidum, together with known ganodermanontriol (3) and ganodermatriol (4). The compounds were identified as 5α-lanosta-7,9(11)-dien-3β,24,25,26-tetraol (1), 15α,26,27-trihydroxy-5α-lanosta-7,9(11),24-trien-3-one (2), 24,25,26-trihydroxy-5α-lanosta-7,9(11)-dien-3-one (3) and 5α-lanosta-7,9(ll),24-trien-3β,26,27-triol (4), respectively.     

灵芝免疫调节蛋白LZ-8的制备和晶体分析

LZ-8蛋白是从灵芝菌丝中分离到的真菌免疫调节蛋白,具有多种免疫调节功能,然而这一蛋白的作用机制尚不清楚。通过蛋白质晶体结构的解析,能够得到蛋白质空间结构特点,从而阐述蛋白质功能的机制。旨在得到LZ-8蛋白的晶体,并获得空间结构数据。以pET21a为表达载体,获得诱导表达的rLZ-8,通过亲和层析、分子筛凝胶层析和阴离子交换层析纯化,蛋白纯度在98%以上,采用悬滴气相扩散法得到蛋白晶体,并获得3.2?数据,为进一步对真菌免疫调节蛋白功能和结构的研究奠定了基础。     

药用昆虫蜣螂对灵芝多糖生物合成的影响

采用液体深层发酵方式,研究了几种药用昆虫对灵芝多糖生物合成的影响。结果表明,药用昆虫蜣螂在添加量为5g/L时能显著促进灵芝胞内多糖(IPS)和胞外多糖(EPS)的形成(P<0.05)。胞内多糖和胞外多糖的产量分别由对照的(1.93±0.09)g/L和(520.3±20.2)mg/L提高到(2.41±0.12)g/L和(608.9±20.2)mg/L。灵芝胞内多糖和胞外多糖在DEAE纤维素柱上都可分离得到5种主要组分,其中IPS-1和EPS-1分别为2类多糖的主要组分。进一步用凝胶柱分离显示,IPS-1由3个单个的组分组成,EPS-1由2个单个的组分组成。添加蜣螂发酵后,灵芝胞内多糖和胞外多糖中没有出现新的组分,且各组分的相对含量也没有显著变化(P>0.05),提示添加蜣螂发酵后,灵芝胞内多糖和胞外多糖主要组分的合成途径并未改变。     

Potential of waste reduction of agro-biomasses through Reishi medicinal mushroom (Ganoderma lucidum) production using different substrates and techniques

One of the most important priorities in the medicinal mushrooms cultivation is the selection and preparation of the substrate, which depends on factors such as price and easy access to different lignocellulosic compounds. In order to investigate the effect of mixed and non-mixed agro-waste substrates on some vegetative, reproductive, nutritional and medicinal properties of two Iranian and Chinese strains of Ganoderma lucidum, the factorial experiment was conducted as a completely randomized design with three replications. The experimental substrates of different poplar wood chip (PWC), wheat bran (WB), soybean meal (SM), date palm leaf (DPL) and olive oil extraction waste (OOEW) were used. They were enriched with chemical supplements (ammonium nitrate, manganese sulfate and Nano manganese oxide) using two cultivation techniques (open bag door and Polika-Cotton) for fruiting body production.The results showed that various substrates significantly affect dry weight, moisture content quantity, vegetative and fruit yield, total ganoderic acids content, total polysaccharide value and mineral elements amount (nitrogen, potassium and calcium) in fruiting body. The highest amount of N content in the fruiting body of both Iranian and Chinese strains (5.88 and 6.25 mg/100 g dry matter) was dedicated to the combined PWC + SM (70:30) substrate enriched with Nano manganese oxide supplement and Polika-Cotton method. The highest total fresh of Iranian and Chinese strains weight (109.60 and 127.66 g, respectively) obtained in the combined PWC + WB (70:30) substrate enriched with manganese sulfate. The highest amount of total ganoderic acids in fruiting organs in both strains (0.52 and 0.42 μg/mg dry matter) is achieved using a combined PWC + SM (70:30) enriched with manganese sulfate.     

Cloning and sequence analysis of a glyceraldehyde-3-phosphate dehydrogenase gene from Ganoderma lucidum

A cDNA library of Ganoderma lucidum has been constructed using a Zap Express cloning vector. A glyceraldehyde-3-phosphate dehydrogenase gene (gpd) was isolated from this library by hybridization of the recombinant phage clones with a gpd-specific gene probe generated by PCR. By comparison of the cDNA and the genomic DNA sequences, it was found that the complete nucleotide sequence encodes a putative polypeptide chain of 338 amino acids interrupted by 6 introns. The predicted amino acid sequence of this gene shows a high degree of sequence similarity to the GPD proteins from yeast and filamentous fungi. The promoter region contains a CT-rich stretch, two CAAT boxes, and a consensus TATA box. The possibility of using the gpd promoter in the construction of new transformation vectors is discussed.     

A novel membrane-surface liquid co-culture to improve the production of laccase from Ganoderma lucidum

In this work, a laccase producer, Ganoderma lucidum, was separated and identified according to its morphological characteristics and phylogenetic data. A 4000 U/l and 8500 U/l of laccase activity was obtained in 500 ml flask by submerged culture and biomembrane-surface liquid culture (BSLC), respectively. Furthermore, the novel biomembrane-surface liquid co-culture (BSLCc) was developed by adding Saccharomyces cerevisiae to reactor in order to shorten the fermentation period and improve laccase production. Laccase activity obtained by BSLCc, 23 000 U/l, is 5.8 and 2.7 times of that obtained by submerged culture and BSLC, respectively. In addition, laccase production by BSLCc was successfully scaled-up to 100 l reactor, and 38 000 U/l of laccase activity was obtained on day 8. The mechanism of overproducing laccase by BSLCc was investigated by metabolism pathway analysis of glucose. The results show glucose limitation in fermentation broth induces the secretion of laccase. The addition of S. cerevisiae, on one hand, leads to an earlier occurrence of glucose limitation state, and thus shortens the fermentation time; on the other hand, it also results in the appearance of a series of metabolites of the yeast including organic acids, ethanol, glycerol and so forth in fermentation broth, and both polyacrylamide gel electrophoresis analysis and enzyme activity detection of laccase show that these metabolites contribute to the improvement of laccase activity.