An alpha-galactosidase was purified from Pseudobalsamia microspora (PMG) to 1224.1-fold with a specific activity of 11,274.5 units/mg by ion-exchange chromatography and gel filtration. PMG is a monomeric protein with a molecular mass of 62 kDa as determined by SDS-PAGE and by gel filtration. Chemical modification using N-bromosuccinimide (NBS) resulted in a complete abrogation of the activity of PMG, suggesting that Trp is an amino acid essential to its activity. The activity was strongly inhibited by Hg2+, Cd2+, Cu2+, and Fe3+ ions. Three inner peptide sequences for PMG were obtained by liquid chromatography–tandem mass spectrometry (LC–MS–MS) analysis. When 4-nitrophenyl α-d-glucopyranoside (pNPGal) was used as substrate, the optimum pH and temperature of PMG were 5.0 and 55 °C, respectively. The Michaelis constant (Km) value of the alpha-galactosidase on pNPGal was 0.29 mM, and the maximal velocity (Vmax) was 0.97 μmol ml−1 min−1. Investigation by thin-layer chromatography (TLC) demonstrated its ability to hydrolyze raffinose and stachyose. Hence, it can be exploited in degradation of non-digestible oligosaccharides from food and feed industries.

The aim of the present study was to investigate the antioxidant and hepatoprotective effects of water-soluble polysaccharides (RVLWP) and alkali-soluble polysaccharides (RVLAP) from Russula vinosa on carbon tetrachloride (CCl4)-induced acute liver damage in mice. For the in vitro antioxidant activities, RVLWP and RVLAP exhibited potent 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (IC50 = 1.55 ± 0.04 and 3.37 ± 0.21 mg/mL, respectively), hydrogen peroxide scavenging activity (IC50 = 6.07 ± 0.24 and 9.23 ± 0.54 mg/mL, respectively), lipid peroxidation inhibitory effect (IC50 = 0.52 ± 0.095 and 0.86 ± 0.043 mg/mL, respectively), and moderate reducing power and Fe2+ chelating activity (IC50 = 1.86 ± 0.0036 and 0.22 ± 0.0057 mg/mL, respectively). Ascorbic acid was employed as the standard antioxidant in the present study. For the in vivo hepatoprotective activity, administration of RVLWP and RVLAP (200 mg/kg) significantly prevented the elevation in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in acute liver damage induced by CCl4 and suppressed hepatic malondialdehyde (MDA) formation. Mice treated with RVLWP and RVLAP demonstrated a better profile of antioxidants with augmented activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the liver. The results suggest that RVLWP and RVLAP protect the liver from CCl4-induced hepatic damage via antioxidant mechanisms.

An acid-tolerant α-galactosidase (CVGI) was isolated from the fruiting bodies of Coriolus versicolor with a 229-fold of purification and a specific activity of 398.6 units mg−1. It was purified to electrophoretic homogeneity by ion exchange chromatography and gel filtration chromatography. The purified enzyme gave a single band corresponding to a molecular mass of 40 kDa in SDS-PAGE and gel filtration. The α-galactosidase was identified by MALDI-TOF-MS and its inner peptides were sequenced by ESI-MS/MS. The optimum temperature and pH of the enzyme were determined as 60 °C and 3.0, respectively. The enzyme was very stable at a temperature range of 4–50 °C and at a pH range of 2–5. Among the metal ions tested, Cu2+, Cd2+ and Hg2+ ions have been shown to partially inhibit the activity of α-galactosidase, while the activity of CVGI was completely inactivated by Ag+ ions. N-bromosuccinamide inhibited enzyme activity by 100 %, indicating the importance of tryptophan residue(s) at or near the active site. CVGI had wide substrate specificity (p-nitrophenyl galactoside, melidiose, raffinose and stachyose). After treatment with CVGI, raffinose family oligosaccharide was hydrolyzed effectively to yield galactose and sucrose. The results showed that the general properties of the enzyme offer potential for use of this α-galactosidase in several production processes.

A novel lectin (ORL), with a molecular mass of 30 kDa and a unique amino acid sequence, was purified from dried fruiting bodies of the mushroom Oudemansiella radicata (Relhan.: Fr.) Sing. ORL is a dimer made of two 15-kDa subunits. The purification procedure encompassed ion exchange chromatography on DEAE-cellulose and QSepharose and fast protein liquid chromatography-gel filtration on Superdex 75. The hemagglutinating activity of ORL was stable at temperatures up to 70°C and between pH 2.0 ∼ 11.0, and was reduced at higher temperatures, and under acidic and alkaline conditions. The activity was activated by Cu2+, Al3+, Fe3+, and Pb2+ ions, and inhibited by Hg2+ and Fe2+ ions. ORL showed no specificity toward the carbohydrates tested. Unlike previously reported mushroom lectins, ORL had no inhibitory effect on HIV-1 reverse transcriptase and proliferation of hepatoma HepG2 cells, as well as breast cancer MCF7 cells, when tested up to 500 μM.

An amylase with a molecular mass of 55 kDa and an N-terminal sequence exhibiting similarity to enzyme from Bacteroides thetaitaomicron was isolated from fruiting bodies of the monkey head mushroom Hericium erinaceum. The purification scheme included extraction with distilled water, ion exchange chromatography on DEAE-cellulose and SP-sepharose, and gel filtration by FPLC on Superdex 75. The amylase of H. erinaceum was adsorbed on DEAE-cellulose in 10 mM Tris-HCl buffer (pH 7.4) and eluted with 0.2 M NaCl in the same buffer. The enzyme was subsequently adsorbed on SP-Sepharose in 10 mM ammonium acetate buffer (pH 4.5) and eluted with 0.3 M NaCl in the same buffer. This fraction was subsequently subjected to gel filtration on Superdex 75. The first peak eluted had a molecular mass of 55 kDa in SDS-PAGE. The amylase of H. erinaceum exhibited a pH optimum of 4.6 and a temperature optimum of 40°C. The enzyme activity was enhanced by Mn2+ and Fe3+ ions, but inhibited by Hg2+ ions.

A 14.6-kDa RNase, with a pH optimum of 5.5 and a temperature optimum of 70 °C, was isolated from dried fruiting bodies of the edible mushroom Lactarius flavidulus. The purification procedure involved, in succession, ion exchange chromatography on DEAE-cellulose, CM-cellulose and SP-Sepharose, and finally FPLC-gel filtration on Superdex 75. The RNase was adsorbed on all three ion exchangers. The ranking of its activity toward various polyhomoribonucleotides was poly(C) > poly(G) > poly(A) > poly(U). It suppressed proliferation of HepG2 cells and L1210 cells with an IC50 of 3.19 μM and 6.52 μM, respectively. It also inhibited the activity of HIV-1 reverse transcriptase with an IC50 of 2.55 μM.

The isolation of a dimeric 29.8-kDa lectin (LFL) from dried Lactarius flavidulus fruit bodies is reported herein. The chromatographic procedure utilized comprised anion-exchange chromatography on DEAE-cellulose, cation-exchange chromatography on CM-cellulose, anion-exchange chromatography on SP-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The hemagglutinating activity of LFL was inhibited by a variety of simple sugars, such as lactose, p-nitrophenyl α-d-glucopyranoside, p-nitrophenyl β-d-glucopyranoside and inositol, and by the polysaccharide inulin. The activity of LFL was stable up to 40 °C. There was a precipitous drop in activity when the temperature was elevated to 50 °C. Hemagglutinating activity was retained when LFL was exposed to 6.25–12.5 mM HCl and NaOH. The activity was potently inhibited by Fe2+ and Fe3+ ions, and slightly inhibited by Al3+ and Mn2+ ions. LFL suppressed the proliferation of hepatoma (HepG2) and leukemic (L1210) cells with an IC50 of 8.90 and 6.81 μM, respectively. It inhibited the activity of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) with an IC50 of 5.68 μM. However, LFL did not exhibit antifungal activity.N-terminal sequence of the dimeric 29.8-kDa Lactarius flavidulus lectin is SGTYTIFNSAFDNSVID.

A 20-kDa ribonuclease (RNase) was purified from fresh fruiting bodies of cultured Schizophyllum commune mushrooms. The RNase was not adsorbed on Affi-gel blue gel but adsorbed on DEAE-cellulose and CM-cellulose. It exhibited maximal RNase activity at pH 6.0 and 70°C. It demonstrated the highest ribonucleolytic activity toward poly (U) (379.5 μ/mg), the second highest activity toward poly (C) (244.7 μ/mg), less activity toward poly (A) (167.4 μ/mg), and much weaker activity toward poly (G) (114.5 μ/mg). The RNase inhibited HIV-1 reverse transcriptase with an IC50 of 65 μM. No effect on [3H-methyl]-thymidine uptake by lymphoma MBL2 cells and leukemia L1210 cells was observed at 100 μM concentration of the RNase. A comparison of RNases from S. commune and Volvariella volvacea revealed that they demonstrated some similarities in N-terminal amino acid sequence, optimum pH and polyhomoribonucleotide specificity. However, some differences in chromatographic behavior and molecular mass were observed.

A 20-kDa Kunitz-type trypsin inhibitor was isolated from Gymnocladus chinensis (Yunnan bean) seeds. The isolation procedure involved ion exchange chromatography on diethylaminoethyl cellulose (DEAE-cellulose), affinity chromatography on Affi-gel blue gel, ion exchange chromatography on sulfopropyl sepharose (SP-sepharose), and gel filtration by FPLC on Superdex 75. The trypsin inhibitor was adsorbed on DEAE-cellulose, unadsorbed on Affi-gel blue gel, and adsorbed on SP-Sepharose. It dose-dependently inhibited trypsin with an IC50 value of 0.4 μM. Dithiothreitol reduced its trypsin inhibitory activity, suggesting that an intact disulfide bond is indispensable to the activity. It suppressed [methyl-3H] thymidine incorporation by leukemia L1210 cells and lymphoma MBL2 cells with an IC50 value of 4.7 and 9.4 μM, respectively. There was no effect on human immunodeficiency virus4-1 reverse transcriptase activity and fungal growth when the trypsin inhibitor was tested up to 100 μM.

Living organisms produce a myriad of molecules to protect themselves from fungal pathogens. This review focuses on antifungal proteins from plants and mushrooms, many of which are components of the human diet or have medicinal value. Plant antifungal proteins can be classified into different groups comprising chitinases and chitinase-like proteins, chitin-binding proteins, cyclophilin-like proteins, defensins and defensin-like proteins, deoxyribonucleases, embryo-abundant protein-like proteins, glucanases, lectins, lipid transfer proteins, peroxidases, protease inhibitors, ribonucleases, ribosome-inactivating proteins, storage 2S albumins, and thaumatin-like proteins. Some of the aforementioned antifungal proteins also exhibit mitogenic activity towards spleen cells, nitric oxide inducing activity toward macrophages, antiproliferative activity toward tumor cells, antibacterial activity, and inhibitory activity toward HIV-1 reverse transcriptase. In contrast to the large diversity of plant antifungal proteins, only a small number of mushroom antifungal proteins have been reported. Mushroom antifungal proteins are distinct from their plant counterparts in N-terminal sequence. Nevertheless, some of the mushroom antifungal proteins have been shown to inhibit HIV-1 reverse transcriptase activity and tumor cell proliferation.

A 14.5-kDa ribonuclease, with an optimum pH of 6 and a temperature optimum at 70°C, was isolated from fresh fruiting bodies of the edible mushroom Lyophyllum shimeiji. It was purified by ion exchange chromatography on DEAE cellulose, Q Sepharose, and SP Sepharose, followed by FPLC gel filtration on Superdex 75, and was adsorbed on all three ion exchangers. It showed the highest ribonucleolytic potency toward poly (U), 25% as much activity toward poly (C), and undetectable activity toward poly (A) and poly (G). Its ribonucleolytic activity at 100°C was similar to that at 20°C. It suppressed proliferation of hepatoma HepG2 cells and breast cancer MCF7 cells with an IC50 of 10 and 6.2 μM, respectively. It inhibited the activity of HIV-1 reverse transcriptase with an IC50 of 7.2 μM.

A dimeric lectin with a molecular weight of 60 kDa and high hemagglutinating activity was isolated from fresh fruiting bodies of the wild mushroom Russula delica. The lectin was composed of two identical subunits, each with a molecular weight of 30 kDa. It was adsorbed on both SP-Sepharose and Q-Sepharose and unadsorbed on DEAE-cellulose. Its hemagglutinating activity was stable up to 70°C, and in HCl and NaOH solutions of concentrations up to 25 and 12.5 mM, respectively. The activity was inhibited by inulin and o-nitrophenyl-β-D-galactopyranoside. Al3+, Fe3+ and Zn2+ ions, but not by Ca2+, Mg2+ and Mn2+ ions. Mg2+ ions at 10 mM concentration potentiated the hemagglutinating activity of the lectin. Russula delica lectin was devoid of mitogenic activity toward mouse splenocytes, but potently inhibited proliferation of HepG2 hepatoma and MCF 7 breast cancer cells, with an IC50 value of 0.88 µM and 0.52 µM, respectively. It potently inhibited HIV-1 reverse transcriptase activity with an IC50 of 0.26 μM.

A protease was purified from fresh fruiting bodies of the edible mushroom Pleurotus citrinopileatus. The isolation procedure included ion exchange chromatography on DEAE-cellulose, CM-cellulose, and Q-Sepharose and fast protein liquid chromatography-gel filtration on Superdex 75. The protease was unadsorbed on DEAE-cellulose and Q-Sepharose, but adsorbed on CM-cellulose. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protease demonstrated a single band with a molecular mass of 28 kDa. The protease showed an optimal pH at 10 and an optimal temperature at 50°C. The activity of the protease was not affected by EDTA, indicating that it is not a metalloprotease. The protease exhibited a higher activity in the presence of K+ and Li+, but its activity was potently inhibited by Al3+, Cu2+, and Hg2+ ions. It manifested a Km of 3.44 mg/ml and a Vmax of 0.139 mg ml−1 min−1. It was devoid of ribonuclease and antifungal activities.

•An extracellular laccase was purified from sanghuang mushroom Inonotus baumii.•It was a monomeric protein with a molecular mass of 66 kDa.•Its N-terminal amino acid sequence was AIGPVDEV.•It manifested antiproliferative activities toward HepG2 and L1210 cells.We described the purification and characterization of a novel extracellular laccase from the traditional Chinese medicinal mushroom Inonotus baumii with antiproliferative activity. The laccase (IBL) was purified from fermentation broth of I. baumii by employing initial filtration and centrifugation steps, followed by three ion-exchange chromatography steps comprising DEAE-cellulose, CM-cellulose, and Q-Sepharose, and a final gel-filtration step by fast protein liquid chromatography (FPLC) on Superdex 75. The purified enzyme was a monomeric protein with a molecular mass of 66 kDa calculated by FPLC and SDS-PAGE. It possessed an N-terminal amino acid sequence of AIGPVDEV (SPIN: C0HJB2), a temperature optimum of 20 °C, pH optima of 2.4 and 3.2 toward ABTS and guaiacol respectively, and Km values of 1.31 mM and 2.27 mM toward ABTS and guaiacol respectively at pH 2.4 and 30 °C. The ranking of its oxidative activity toward various aromatic substrates was ATBS > guaiacol > 4-methylcatechol > 4-hydroxyindole > catechol > hydroquinone > 2,6-dimethoxy-phenol (19.6%) > pyrogallol > ferulic acid > N, N-dimethyl-1, 4-phenylenediamine. Cu2+ can enhance the enzyme activity of 10.8–14.6 fold in the ion concentration range of 1.25–10 mM. IBL manifested antiproliferative activities toward HepG2 and L1210 cells with IC50 values of 2.4 μM and 3.2 μM, respectively, but is devoid of inhibitory activity toward HIV-1 reverse transcriptase.Download full-size image

An isolation procedure that comprised 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 was utilized to isolate a laccase with a molecular mass of 65 kDa from fresh fruiting bodies of the mycorrhizal fungus Lepiota ventriosospora. Laccase activity was adsorbed on both DEAE-cellulose and Q-Sepharose but unadsorbed on CM-cellulose. An overall 26.3-fold of purification was obtained. The enzyme demonstrated an optimum temperature at 60 °C and an optimum pH 4.0. The purified laccase was quite stable at pH range of 3.6–4.4, but only 17.8% of total activity left after 1 h incubating at 60 °C. The ranking of its degradative activity toward aromatic substrates was catechol > hydroquinone > ABTS > 2,6-dimethoxy-phenol. It demonstrated the highest inhibitory activity toward HIV-1 reverse transcriptase with an IC50 value of 0.60 μM among fungal laccaes reported up to now.Graphical abstractDownload full-size imageHighlights► A laccase from mycorrhizal not white-rot fungus is reported. ► We evaluate its physical, chemical, and bioactive properties. ► It manifests unique amino acid sequence comparing with other laccases. ► It is stable at pH 4.0 after an incubation of 1 h. ► It possesses a very high inhibitory activity toward HIV-1 RT.

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 Km 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.

To date only a ribonuclease and a protein with anti-HIV-1 reverse transcriptase activity have been isolated from mushrooms of the genus Russula. In this study a novel lectin, with a molecular weight of 32 kDa, and a unique N-terminal sequence different from other lectins, was isolated from the mushroom Russula lepida. It represents the first lectin isolated from Russula mushrooms. The purification scheme involved (NH4)2SO4 precipitation, ion exchange chromatography on diethylaminoethyl DEAE-cellulose and SP-Sepharose, and fast protein liquid chromatography-gel filtration on Superdex 75. The hemagglutinating activity of the lectin (RLL) was inhibited by inulin and O-nitrophenyl-β-D-galacto-pyranoside. The lectin was stable at temperatures up to 70 °C (half of the activity was preserved at 80 °C), and in the presence of NaOH or HCl solutions up to a concentration of 12.5 mM. Its hemagglutinating activity was reduced in the presence of Mn2+, Co2+, and Hg2+ ions, and enhanced by Cu2+ ions. It exhibited antiproliferative activity toward hepatoma Hep G2 cells and human breast cancer MCF-7 cells with an IC50 of 1.6 μM and 0.9 μM, respectively. Daily intraperitoneal injections of RLL (5.0 mg/kg body weight/day for 20 days) brought about 67.6% reduction in the weight of S-180 tumor. RLL was devoid of antifungal, ribonuclease, and HIV-1 reverse transcriptase inhibitory activities.

•An α-galactosidase was highly purified from Pleurotus citrinopileatus fruiting bodies.•It exhibited remarkable resistance to protease.•It could efficiently hydrolyze RFOs.•Galactose (Ki = 0.92 mM) and melibiose (Ki = 7.13 mM) competitively inhibited the enzyme.•It was strongly inhibited by Cd2+, Cu2+, Hg2+, Al3+, Fe3+ and Ag+ ions.An acidic α-galactosidase designated as PCGI was isolated from the fruiting bodies of Pleurotus citrinopileatus with 264-fold purification and a specific activity of 7.92 units/mg. It was purified to homogeneity by ion exchange chromatography and gel filtration chromatography. PCGI is a heterodimeric protein consisting of a 33 kDa and a 27 kDa subunit in SDS-PAGE. The purified enzyme was identified by MALDI-TOF-MS. It belongs to the GH27 family. The optimum pH and temperature of the enzyme with pNPGal as substrate were 4.4 and 50 °C, respectively. Besides, it displayed remarkable resistance to acid protease, neutral protease, α-chymotrypsin, and trypsin. It was strongly inhibited by Cd2+, Cu2+, Hg2+, Al3+, Fe3+ and Ag+ ions. Diethypyrocarbonate (DEPC) doubled the activity of PCGI whereas N-bromosuccinimide (NBS) drastically decreased it. PCGI displayed wide substrate diversity with activity toward substrates such as stachyose, raffinose, melibiose. The Km values for hydrolysis of pNPGal, stachyose, raffinose, and melibiose were 0.2, 16.7, 18.9, and 6.3 mM, respectively. Galactose (Ki = 0.92 mM) and melibiose (Ki = 7.13 mM) competitively inhibited the enzymes. Futhermore, it completely degraded raffinose and sthachyose. These results suggest that PCGI has great potential for removal of the non-digestible and flatulence-causing oligosaccharides stachyose and raffinose from legumes.Download high-res image (181KB)Download full-size image

An antifungal peptide with a defensin-like sequence and exhibiting a molecular mass of 7.3 kDa was purified from dried seeds of Phaseolus vulgaris ‘Cloud Bean’. The isolation procedure entailed anion exchange chromatography on DEAE-cellulose, affinity chromatography an Affi-gel blue gel, cation exchange chromatography on SP-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. Although the antifungal peptide was unadsorbed on DEAE-cellulose, it was adsorbed on both Affi-gel blue gel and SP-Sepharose. The antifungal peptide exerted antifungal activity against Mycosphaerella arachidicola with an IC50 value of 1.8 μM. It was also active against Fusarium oxysporum with an IC50 value of 2.2 μM. It had no inhibitory effect on HIV-1 reverse transcriptase when tested up to 100 μM. Proliferation of L1210 mouse leukemia cells and MBL2 lymphoma cells was inhibited by the antifungal peptide with an IC50 of 10 μM and 40 μM, respectively.

From the dried fruiting bodies of the toxic mushroom Inocybe umbrinella, a novel lectin with a molecular mass of 17 kDa has been isolated with about 160-fold purification. The purification protocol comprised ion exchange chromatography on DEAE-cellulose, and CM-cellulose, and gel filtration on Superdex 75. Among the carbohydrates tested, raffinose, d-melibiose, α-lactose and d(+)-galactose could inhibit the hemagglutinating activity of the lectin. The hemagglutinating activity was stable between 10 and 60 °C, in 12.5–100 mM HCl, and in 50 mM NaOH. The hemagglutinating activity was inhibited by Ca2+, Mn2+and Mg2+ ions, but was unaffected by Fe3+, Zn2+ and Al3+ ions. The lectin inhibited HIV-1 reverse transcriptase with an IC50 of 4.7 ± 0.2 μM. Proliferation of tumor cells including hepatoma HepG2 cells and breast cancer MCF7 cells was inhibited by the lectin with an IC50 of 3.5 ± 0.2 μM and 7.4 ± 0.3 μM, respectively. The lectin has a unique N-terminal amino acid sequence, DGVLATNAVA. It did not exhibit antifungal activity. The present report is the first on an Inocybe lectin and represents one of the very few reports on lectins from toxic mushrooms.