Variations of SSU rDNA group I introns in different isolates of Cordyceps militaris and the loss of an intron during cross-mating.

Cordyceps militaris, the type species of genus Cordyceps, is one of the most popular mushrooms and a nutraceutical in eastern Asia. It is considered a model organism for the study of Cordyceps species because it can complete its life cycle when cultured in vitro. In the present study, the occurrence and sequence variation of SSU rDNA group I introns, Cmi.S943 and Cmi.S1199, among different isolates of C. militaris were analyzed. Based on the secondary structure predictions, the Cmi.S943 intron has been placed in subgroup IC1, and the Cmi.S1199 intron has been placed in subgroup IE. No significant similarity between Cmi.S943 and Cmi.S1199 suggested different origins. Three genotypes, based on the frequency and distribution of introns, were described to discriminate the 57 surveyed C. militaris strains. It was found that the genotype was related to the stroma characteristics. The stromata of all of the genotype II strains, which possessed only Cmi.S943, could produce perithecium. In contrast, the stromata of all genotype III strains, which had both Cmi.S943 and Cmi.S1199, could not produce perithecium. Cmi.S1199 showed the lowest level of intra-specific variation among the tested strains. Group I introns can be lost during strain cross-mating. Therefore, we presumed that during cross-mating and recombination, intron loss could be driven by positive Darwinian selection due to the energetic cost of transcribing long introns.

Reliable reference gene selection for Cordyceps militaris gene expression studies under different developmental stages and media.

Cordyceps militaris is considered a model organism for the study of Cordyceps species, which are highly prized in traditional Chinese medicine. Gene expression analysis has become more popular and important in studies of this fungus. Reference gene validation under different experimental conditions is crucial for RT-qPCR analysis. In this study, eight candidate reference genes, actin, cox5, gpd, rpb1, tef1, try, tub, and ubi, were selected and their expression stability was evaluated in C. militaris samples using four algorithms, genorm, normfinder, bestkeeper, and the comparative ∆Ct method. Three sets of samples, five different developmental stages cultured in wheat medium and pupae, and all the samples pool were included. The results showed that rpb1 was the best reference gene during all developmental stages examined, while the most common reference genes, actin and tub, were not suitable internal controls. Cox5 also performed poorly and was less stable in our analysis. The ranks of ubi and gpd were inconsistent in different sample sets by different methods. Our results provide guidelines for reference gene selection at different developmental stages and also represent a foundation for more accurate and widespread use of RT-qPCR in C. militaris gene expression analysis.

Process optimization for extraction of carotenoids from medicinal caterpillar fungus, Cordyceps militaris (Ascomycetes).

Natural carotenoids have attracted great attention for their important beneficial effects on human health and food coloring function. Cordyceps militaris, a well-known edible and medicinal fungus, is a potential source of natural carotenoids. The present study aimed to optimize the process parameters for carotenoid extraction from this mushroom. The effects of different methods of breaking the fungal cell wall and organic solvents were studied by the one-factor-at-a-time method. Subsequently, the process parameters including the duration of the extraction time, the number of extractions, and the solvent to solid ratio were optimized by using the Box-Behnken design. The optimal extraction conditions included using an acid-heating method to break the cell wall and later extracting three times, each for a 1 h duration, with a 4:1 mixture of acetone: petroleum ether and a solvent: solid ratio of 24:1. The carotenoid content varied from 2122.50 to 3847.50 µg/g dry weights in different commercially obtained fruit bodies of C. militaris. The results demonstrated that the C. militaris contained more carotenoid content in its fruit bodies than other known mushrooms. Stability monitoring by HPLC demonstrated that the carotenoids could be stored at 4°C for 40 d. It is suggested that the carotenoid content should be considered as the quality standard of commercial products of this valued mushroom. These findings will facilitate the exploration of carotenoids from C. militaris.

Three types of geranylgeranyl diphosphate synthases from the medicinal caterpillar fungus, Cordyceps militaris (Ascomycetes).

Geranylgeranyl diphosphate synthase (GGPPS) is a key enzyme in the carotenoid biosynthetic pathway, catalyzing the synthesis of its C20 precursor. In the present study, three types of ggpps genes were cloned and analyzed from the Caterpillar Medicinal Fungus Cordyceps militaris, a valued carotenoid-producing species. The sequences were named as ggpps727, ggpps191, and ggpps595. The open reading frame codes for predicted polypeptides of 464, 550, and 431 aa. Three predicted GGPPSs had a high similarity to that from Beauveria bassiana ARSEF 2860 with identity of 73%, 71%, and 56%, respectively. Homology comparison of the deduced peptide sequences of the various GGPPSs revealed highly conserved domains. Both GGPPS727 and GGPPS191 from C. militaris contained all five domains highly conserved among prenyltransferases as well as two aspartate-rich DDXX(XX)D motifs in domains II and V, which have been proven essential for prenyltransferase activity. By constructing the phylogenetic tree of fungal GGPPSs, it was found that fungi-derived GGPPSs could be divided into three clusters, suggesting there were three types of GGPPSs in fungi. Each type may be responsible for a different metabolism. Three types of GGPPSs from C. militaris belonged to the different clusters separately. Expression analysis of three ggpps genes during the fruit body cultivation of C. militaris by real-time polymerase chain reaction (PCR) suggested the ggpps 191 gene may be involved in the synthesis of carotenoids and ggpps 727 may be responsible for primary metabolism. This is the first report of the GGPPS from C. militaris, a valued edible and medicinal fungus.

Extraction of water-soluble polysaccharide and the antioxidant activity from Semen cassiae.

Water-soluble polysaccharide was isolated from Semen cassiae using water for extraction and ethanol for deposition. The optimized conditions for polysaccharide isolation by orthogonal experiments were a sample to liquid ratio of 1:30 at 80°C for 3.5 hours; the yield of polysaccharide from Semen cassiae under these conditions was 5.46%. Different polysaccharides (SCPW-1, SCPW-2, SCPW-3, SCPW-4, SCPW-5, SCPS-1, SCPS-2) were obtained from the extract (i.e., crude polysaccharide) by DEAE-cellulose column chromatography. The polysaccharides obtained showed different structures by Fourier transform infrared therein the five elected from the seven kinds separated. The antioxidant activities of the extract were evaluated. The scavenging rates of the present extract on hydroxyl and superoxide were 43.32% and 64.97%, respectively, at a concentration of polysaccharide of 94.03 µg/mL, which was better than vitamin C at the same concentration. The scavenging rate of the present extract on 1,1-diphenyl-2-picrylhydrazyl was 13.33% at a polysaccharide concentration of 94.03 µg/mL, which was less than vitamin C at the same concentration.

Production, purification and characterization of novel beta glucosidase from newly isolated Penicillium simplicissimum H-11 in submerged fermentation.

ß-Glucosidase is an important component of the cellulase complex. It not only hydrolyzes cellobiose and short-chain cellooligosaccharides to glucose, but also removes the inhibitory effect of cellobiose on the ß-1, 4-endoglucanase and exoglucanase, thereby increasing the overall rate of cellulose biodegradation. ß-glucosidasefrom culture supernatant of a fungus Penicillium simplicissimum was purified to homogeneity, by using ammonium sulfate fraction, Sephadex G-100 chromatography, and its properties were studied. The molecular mass of the enzyme was about 126.0 kDa, as identified by 12% SDS-PAGE. The optimum pH and temperature were 4.4 ~ 5.2 and 60 °C, respectively. The enzyme was stable in pH 5.2 ~ 6.4 and under 40 °C. Metal profile of the enzyme showed that Mn(2+) enhances its activity, while Cu(2+), Co(2+)and Fe(3+) cause obvious inhibition. The K m and V max was 14.881 mg/ml and 0.364 mg ml/min against salicin as a Substrate. This enzyme had secondary protein structure as evidenced by FTIR spectrum.

[Identification and degradation capability of three pyrene-degrading Gordonia sp. strains].

Three pyrene-degrading bacterial strains named D44, D82S and D82Q were isolated from PAHs-contaminated soil in Shenfu Irrigation Area of Shenyang, Northeast China. The strains were identified as Gordonia sp., based on the morphological observation, physiological and biochemical identification, and phylogenetical analysis of 16S rDNA sequences. For all the three stains, their optimal pH was 7, and their growth was obviously inhibited when the pH was lower than 5 or higher than 9. The three strains were capable of utilizing pyrene, benzo[a] pyrene, anthracene, naphthalene, phenanthrene, and fluoranthene as the sole source of carbon and energy. After seven days incubation, the three strains could degrade more than 65% of pyrene with an initial concentration 100 mg x L(-1), and the D44, D82S, and D82Q could degrade 79.6%, 91.3%, and 62.8% of benzo[a] pyrene with an initial concentration 50 mg x L(-1), respectively. PCR amplification indicated that the strains D82Q and D82S possessed alkane monooxygenase gene alkB.

[Isolation and identification of a highly efficient pyrene-degrading Mycobacterium sp. strain N12].

By using selective enrichment method, a highly efficient pyrene-degrading bacterium strain N12 was isolated from an oil-contaminated soil collected from Shenfu irrigation area of Shenyang. Based on the physiological and biochemical characteristics and the phylogenetic similarity of 16S rDNA gene sequence, the strain N12 was identified as Mycobacterium sp. , which could utilize phenanthrene, acenaphthene, fluorine, and pyrene, but not anthracene, naphthalene, and benzo (a)pyrene as the sole carbon and energy source. However, when the strain N12 was cultured with pyrene and phenanthrene, 79.0% of benzo(a)pyrene could be co-metabolized within 9 days. The degradation rate of 100 mg x L(-1) of pyrene by the strain N12 was 94.4% within 7 days and 100% after 14 days, and that of 600 mg x L(-1) of pyrene was 56.1% within 7 days and 95.5% within 14 days. The addition of glucose promoted the degradation of pyrene. It was suggested that the strain N12 was an efficient PAHs-degrading bacterium, being a potential candidate for the bioremediation of PAHs-contaminated soils.

[Optimum of polysaccharide distillation on scrap Cordyceps militaris medium].

A mass of scrap Cordyceps militaris solid culture medium could not be utilized better. In this test, using orthogonal design the optimal technique parmeter of extracting polysaccharide was 80 degrees C, two times, in twenty times of water, and 120 minutes each time. Temperature was the most important factor. The referenced data could be provided to depurative production of Cordyceps militaris and resource utilization.

[Study on the immunogenicity of major leptospiral genus-specific protein antigens and the distribution of antigens in different serogroups of Leptospira interrogans].

OBJECTIVE: The determination of antigenicity and immunogenicity of Leptospira interrogans genus-specific outer envelope proteins (OEPs) will offer evidence for developing universal leptospiral genetic engineering vaccine and detection kit. METHODS: In this study, Ni-NTA affinity chromatography is used to purify the recombinant products rLipL21, rOmpL1/1, rOmpL1/2, rLipL32/1, rLipL32/2, rLipL41/1 and rLipL41/2 expressed by the major genotypes of four leptospiral OEPs of 15 serogroups. SDS-PAGE is applied to examine the expression and purity of the recombinant proteins. Rabbits are intracutaneously immunized with the recombinant proteins to obtain antisera. Microscope agglutination test (MAT) is used to measure the cross inmmunoagglutination titers of antisera. The OMPs of the reference standard strains belonging to 15 serogroups of L. interrogans in China and L. biflexa strain Patoc I are prepared using salt-denature method. By each of the antisera as the first antibody, Western blot assay is established to detect the natural expressions and immunoreactivity of the four OEPs. RESULTS: The outputs of rLipL21, rLipL32/1, rLipL32/2, rLipL41/1l, rLipL41/2, rOmpL1/1 and rOmpL1/2 are 10%, 40%, 35%, 15%, 10%, 30% and 15%, respectively. Each the purified recombinant proteins shows a single fragment after SDS-PAGE. Each the rabbit antisera displays extensive cross immunoreactivity between the products expressed by different genotypes of the same gene and the MAT titers ranging from 1:2-1:128. All the four OEPs can be detectable in the OEPs preparations. However, LipL21 is found to exist only in L. interrrogans. CONCLUSION: The results of this study indicate that all the four OEPs are superficial genus-specific antigens of Leptospira which can be used as the candidate antigens of leptospiral universal vaccine and detection kit.