Cloning and Molecular Characterization of beta-1,3-Glucan Synthase from Sparassis crispa

A beta-glucan synthase gene was isolated from the genomic DNA of polypore mushroom Sparassis crispa, which reportedly produces unusually high amount of soluble beta-1,3-glucan (beta-glucan). Sequencing and subsequent open reading frame analysis of the isolated gene revealed that the gene (5,502 bp) consisted of 10 exons separated by nine introns. The predicted mRNA encoded a beta-glucan synthase protein, consisting of 1,576 amino acid residues. Comparison of the predicted protein sequence with multiple fungal beta-glucan synthases estimated that the isolated gene contained a complete N-terminus but was lacking approximately 70 amino acid residues in the C-terminus. Fungal beta-glucan synthases are integral membrane proteins, containing the two catalytic and two transmembrane domains. The lacking C-terminal part of S. crispa beta-glucan synthase was estimated to include catalytically insignificant transmembrane alpha-helices and loops. Sequence analysis of 101 fungal beta-glucan synthases, obtained from public databases, revealed that the beta-glucan synthases with various fungal origins were categorized into corresponding fungal groups in the classification system. Interestingly, mushrooms belonging to the class Agaricomycetes were found to contain two distinct types (Type I and II) of beta-glucan synthases with the type-specific sequence signatures in the loop regions. S. crispa beta-glucan synthase in this study belonged to Type II family, meaning Type I beta-glucan synthase is expected to be discovered in S. crispa. The high productivity of soluble beta-glucan was not explained but detailed biochemical studies on the catalytic loop domain in the S. crispa beta-glucan synthase will provide better explanations.

Sparassis crispa Attenuates Carbon Tetrachloride-Induced Hepatic Injury in Rats / 체질인류학회지

Sparassis crispa is an edible mushroom with various medicinal properties. Here we demonstrate the effect of Sparassis crispa on carbon tetrachloride (CCl4)-induced hepatotoxicity and the underlying mechanism. To evaluate the hepatoprotective effects of Sparassis crispa ethanol extract (SCE), 50 male Sprague-Dawley rats were equally divided into 5 groups. Group I is the normal control rats with an intraperitoneal (i.p.) 0.5% carboxy methyl cellulose (CMC) pretreatment and olive oil treatment. Group II is the model group with an i.p. 0.5% CMC and 0.5 mL/kg CCl4 treatment. Group III and IV is the CCl4-administered rats pretreated with an i.p. 100 and 200 mg/kg SCE, respectively. Group V includes the silymarin group with an i.p. 50 mg/kg silymarin and CCl4 treatment. At 16 h after the CCl4 treatment, the levels of serum aminotransferases, TNF-alpha, and lipid peroxidation were substantially increased, whereas the activity of hepatic antioxidative enzymes, such as superoxide dismutase and catalase, was decreased. These changes were attenuated by SCE. The histological studies also showed that SCE inhibited the CCl4-induced liver injury. Furthermore, the contents of hepatic nitrite, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) were elevated after CCl4 treatment, while the cytochrome P450 2E1 (CYP2E1) expression was suppressed. SCE treatment inhibited the formation of liver nitrite, reduced the over-expression of iNOS and COX-2 proteins, but restored the liver CYP2E1 content compared with the CCl4-treated model group. The present data elucidate that SCE protects the liver against CCl4-induced acute hepatotoxicity, which might be due to its ability to restore the CYP2E1 function and suppress the inflammatory responses, in combination with its capacity to reduce oxidative stress.

Generation and Evaluation of High beta-Glucan Producing Mutant Strains of Sparassis crispa

A chemical mutagenesis technique was employed for development of mutant strains of Sparassis crispa targeting the shortened cultivation time and the high beta-glucan content. The homogenized mycelial fragments of S. crispa IUM4010 strain were treated with 0.2 vol% methyl methanesulfonate, an alkylating agent, yielding 199 mutant strains. Subsequent screening in terms of growth and beta-glucan content yielded two mutant strains, B4 and S7. Both mutants exhibited a significant increase in beta-glucan productivity by producing 0.254 and 0.236 mg soluble beta-glucan/mg dry cell weight for the B4 and S7 strains, respectively, whereas the wild type strain produced 0.102 mg soluble beta-glucan/mg dry cell weight. The results demonstrate the usefulness of chemical mutagenesis for generation of mutant mushroom strains.

Sawdust Media Affecting the Mycelial Growth and the Fruiting Body Formation of Sparassis crispa

Six strains of Sparassis crispa such as S. crispa DUM-01, DUM-02, DUM-03, DUM-04, DUM-05, and DUM-06 were tested for their mycelial growth on 6 different kinds of sawdust media and primordial formation on 10 different compositions of larch sawdust media. The highest mycelial growth was recorded on the larch sawdust. Of the 6 strains of S. crispa, S. crispa DUM-04 recorded the favorable formation of primordia. The primordial formation of S. crispa DUM-04 was more favorable on L-3 medium than 9 kinds of larch sawdust media. When S. crispa DUM-04 was cultured on the media of larch sawdust+pine sawdust, the formation of its fruiting bodies was more outstanding on the media of larch sawdust+pine sawdust than those of larch sawdust.