Shouxian Wang

Cordysobin, a novel alkaline serine protease with HIV-1 reverse transcriptase inhibitory activity from the medicinal mushroom Cordyceps sobolifera

A novel serine protease, designated as cordysobin, was purified from dried fruiting bodies of the mushroom Cordyceps sobolifera. The isolation procedure utilized ion exchange chromatography on DEAE-cellulose and SP-Sepharose followed by gel filtration on Superdex 75. The protease did not adsorb on DEAE-cellulose but bound to SP-Sepharose. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the protease resolved as a single band with an apparent molecular mass of 31 kDa. Its optimal pH was 10.0, and the optimal temperature was 65°C. The protease displayed a K(m) value of 0.41 μM and 13.44 μM·min⁻¹ using Suc-Leu-Leu-Val-Tyr-MCA as substrate at pH 10.0 and 37°C. Protease activity was enhanced by the Fe²⁺ ion at low concentration range of 1.25-10 mM and was strongly inhibited by Hg²⁺ up to 1.25 mM. The protease was strongly inhibited by chymostatin and phenylmethylsulfonyl fluoride (PMSF), suggesting that it is a serine protease. It manifested significant inhibitory activity toward HIV-1 reverse transcriptase (RT) with an IC₅₀ value of 8.2×10⁻³ μM, which is the highest anti-HIV-1 RT activity of reported mushroom proteins.

Extraction of a soluble polysaccharide from Auricularia polytricha and evaluation of its anti-hypercholesterolemic effect in rats

Mushroom extracts are a new source of supplements for health and pharmaceutical due to their bioactivities. This study was to optimize the extraction parameters of a soluble polysaccharide from Auricularia polytricha (SPAP) by response surface methodology. The practical optimum parameters were an extraction time of 4h, an extraction temperature of 95 °C and a ratio of water to fruiting bodies of 28 mL/g, and the highest extraction rate was 19.77%. In vivo, male Sprague-Dawley (SD) rats were diet-induced hypercholesterolemic models and oral administration of SPAP to evaluate anti-hypercholesterolemic effects. The results showed that SPAP decreased the serum concentrations of blood lipid, made them close to the normal level. The total cholesterol in the SPAP consumption groups was significantly decreased 34.6 ± 7.6% and 33.3 ± 7.9% with dose of 4.5 and 9.0mg/kg BW in the 29th day. This study suggested that SPAP was a suitable natural agent and may be applied in therapy.

A Novel Laccase with Potent Antiproliferative and HIV-1 Reverse Transcriptase Inhibitory Activities from Mycelia of Mushroom Coprinus comatus

A novel laccase was isolated and purified from fermentation mycelia of mushroom Coprinus comatus with an isolation procedure including three ion-exchange chromatography steps on DEAE-cellulose, CM-cellulose, and Q-Sepharose and one gel-filtration step by fast protein liquid chromatography on Superdex 75. The purified enzyme was a monomeric protein with a molecular weight of 64 kDa. It possessed a unique N-terminal amino acid sequence of AIGPVADLKV, which has considerably high sequence similarity with that of other fungal laccases, but is different from that of C. comatus laccases reported. The enzyme manifested an optimal pH value of 2.0 and an optimal temperature of 60°C using 2,2′-azinobis(3-ethylbenzothiazolone-6-sulfonic acid) diammonium salt (ABTS) as the substrate. The laccase displayed, at pH 2.0 and 37°C, K m values of 1.59 mM towards ABTS. It potently suppressed proliferation of tumor cell lines HepG2 and MCF7, and inhibited human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) with an IC50 value of 3.46 μM, 4.95 μM, and 5.85 μM, respectively, signifying that it is an antipathogenic protein.

Evaluation of Genetic Diversity Among Chinese Pleurotus eryngii Cultivars by Combined RAPD/ISSR Marker

Pleurotus eryngii (DC. Ex. Fr.) Quél is a rare precious edible fungus which belongs to the family Pleurotaceae. This mushroom has highly nutritional, pharmaceutical, economic and ecological values. In the present study, combined randomly amplified polymorphic DNA (RAPD)/inter-simple sequence repeat (ISSR) was used to assess the genetic diversity of P. eryngii strains cultivated in China. For the RAPD and ISSR analyses, 404 and 392 polymorphic bands were obtained from 32 P. eryngii strains using 28 and 24 selected primers, respectively. A combined RAPD/ISSR dendrogram grouped the 32 strains into five clades with coefficient of 0.770. The comparison of RAPD and ISSR was also elucidated in the present study. The results of our study obtained by combined RAPD/ISSR analysis contributed to a better understanding of the genetic relationships among the P. eryngii strains and provide orientation for the strain improvement of P. eryngii species.

Evaluation of edible mushroom Oudemansiella canarii cultivation on different lignocellulosic substrates

In this study, the mycelial growth rate, mycelial colonization time, yield, and biological efficiency of the edible mushroom Oudemansiella canarii were determined, and the effects of different substrate combinations on productivity, chemical contents and amino acids were evaluated. Lignocellulosic wastes, such as cottonseed hull, sawdust, corncob, and their combinations supplemented with 18% wheat bran and 2% lime, were used for the cultivation of O. canarii. The biological efficiency (BE) and essential amino acid content of treatment T1, which consisted of 80% cottonseed hull, were the highest among all the tested treatments. Mixtures that included sawdust, such as treatments T2 (80% sawdust), T4 (40% sawdust + 40% cottonseed hull), and T6 (40% sawdust + 40% corncob), exhibited lower yield and BE. Corncob was good for O. canarii production in terms of yield and BE, whereas the mycelial growth rate and colonization time were lower compared to those on other substrates. Comparing the BE, essential amino acids, and other traits of the six treatments, treatment T1 (80% cottonseed hull) was the best formula for O. canarii cultivation and should be extended in the future.

Evaluation of genetic diversity of Chinese Pleurotus ostreatus cultivars using DNA sequencing technology

Pleurotus spp. are well-known and economically important cultivated mushrooms in China. Knowledge of the genetic relationship between the Chinese cultivars is essential to the improvement of P. ostreatus strains. Sequence analysis of the internal transcribed spacers (ITS), translation elongation factor (EF1α) and the second largest subunit of RNA polymerase II (RPB2) was performed to assess the genetic diversity of Pleurotus ostreatus strains cultivated in China. The phylogenetic tree constructed using the combined results of the ITS, EF1α and RPB2 sequence analyses showed the genetic relationships between the studied strains. Our phylogenetic analyses therefore provided valuable information on the relationships among the P. ostreatus strains used in this study and that was useful for examining genetic diversity among these strains.

Polyethylene glycol-mediated transformation of fused egfp-hph gene under the control of gpd promoter in Pleurotus eryngii

Pleurotus eryngii was transformed using a polyethylene glycol-mediated method. A plasmid, pEPUGH, containing a reporter gene (enhanced green fluorescent protein gene, egfp) and a positive selectable marker gene (hygromycin phosphotransferase gene, hph) was constructed. The fused egfp-hph gene was placed under the control of the strong and constitutive native gpd promoter from P. eryngii. The recombinant plasmid was used to transform of P. eryngii protoplasts. Successful transformation was demonstrated by molecular analyses. Moreover, the mycelia of the transformants showed green epipolic dispersion on fluorescence microscopy. About 90–210 transformants were produced per μg plasmid DNA per 107 viable protoplasts.

Development of SCAR Markers to Determine the Mating Types of Lepista nuda Protoplast Monokaryons

Lepista nuda (Bull. ex Fr.) Cooke belongs to Tricholomataceae and is an edible fungus with both economic and medical value. Mycelia were isolated from the fruiting bodies of L. nuda and were used to prepare the protoplast monokaryons. One hundred and fifteen monokaryons were obtained and their mating types were determined using somatic incompatibility tests. Protoplast monokaryons segregated into either the A1B1 or the A2B2 mating types. Inter-simple sequence repeats and sequence-related amplified polymorphism fingerprinting were used to analyse the mating types of these protoplast monokaryons and 16 sequence-characterised amplified region primers were developed to efficiently differentiate between the monokaryon mating types. Multiplex PCR analyses were also established. The data presented here outline a method for the precise and rapid identification of protoplast monokaryon mating types, which has the promise to shorten the period required for conventional crossbreeding.

Cloning of the A Mating-Type Locus from Lepista nuda and Characterization of Its Genetic Structure

AbstractLepista nuda (Bull. ex Fr.) Cooke (Tricholomataceae) is an edible fungus with both economic and medical value. Identification of its mating-type loci is important for promoting breeding programs in L. nuda. The A mating-type locus of L. nuda and its flanking region were cloned and characterized in the present study. It contained two homeodomain transcription factor genes (called lna1 and lna2). Lna1 and Lna2 protein harbored conserved motif of homeodomain transcription factor protein. The novel finding of this study was that the gene order around the A locus was mip, lna2, lna1, and β-fg in L. nuda, which was differed from other edible fungi. In addition, lna1 and lna2 showed divergent, inward transcriptional direction. The phylogenetic tree of HD proteins showed that L. nuda Lna1 and Lna2 were phylogenetically related with Laccaria bicolor. Our results revealed that the A mating-type locus had been subjected to gene rearrangements relative to all other basidiomycetes.

Use of a culture independent method to analyze the diversity of soil fungi surrounding Chroogomphus rutilus in the Beijing region of China

Large-scale cultivation of Chroogomphus rutilus is too inefficient to be commercially feasible. In addition, isolating C. rutilus mycelia in the wild is difficult. Thus, determining the natural habitat of its fruiting body is important. The present study focused on the ecology of the C. rutilus habitat to facilitate its large-scale cultivation. A culture-independent molecular approach—a powerful technology for microbiological ecology studies—was used to investigate the diversity of soil fungal communities in samples surrounding C. rutilus from the Beijing region of China. Metagenomic DNA was isolated from soil samples collected around C. rutilus, and an internal transcribed spacer (ITS) gene library was constructed. Subsequently, polymerase chain reaction products were digested with HinfI, HaeIII, MspI, TaqI, or MboI. Clones were selected and sequenced based on their restriction fragment length polymorphisms. The diversity of the fungi represented by their ITS sequences was analyzed. Our results indicate the presence of numerous fungi in the C. rutilus habitat. This study is the first demonstration of the fungal ecology surrounding C. rutilus using a culture independent method.