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Journal of Biomedicine and Biotechnology
Volume 2012, Article ID 728975, 8 pages
http://dx.doi.org/10.1155/2012/728975
Research Article

A Novel Aspartic Protease with HIV-1 Reverse Transcriptase Inhibitory Activity from Fresh Fruiting Bodies of the Wild Mushroom Xylaria hypoxylon

1State Key Laboratory for Agrobiotechnology and Department of Microbiology, China Agricultural University, Beijing 100193, China
2Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing 100081, China
3Key Laboratory of Urban Agriculture (North) of Ministry of Agriculture, Beijing University of Agriculture, Beijing 102206, China
4Beijing Academy of Science and Technology, Beijing 100089, China
5School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong

Received 18 January 2012; Revised 17 March 2012; Accepted 17 March 2012

Academic Editor: Guihua H. Bai

Copyright © 2012 Qing-Xiu Hu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

A novel aspartic protease with HIV-1 RT inhibitory activity was isolated and characterized from fruiting bodies of the wild mushroom Xylaria hypoxylon. The purification protocol comprised distilled water homogenization and extraction step, three ion exchange chromatographic steps (on DEAE-cellulose, Q-Sepharose, and CM-cellulose in succession), and final purification was by FPLC on Superdex 75. The protease was adsorbed on all the three ion exchangers. It was a monomeric protein with a molecular mass of 43 kDa as estimated by SDS-PAGE and FPLC. Its N-terminal amino acid sequence was HYTELLSQVV, which exhibited no sequence homology to other proteases reported. The activity of the protease was adversely affected by Pepstatin A, indicating that it is an aspartic protease. The protease activity was maximal or nearly so in the pH range 6–8 and in the temperature range 35– 6 0 C . The purified enzyme exhibited HIV-1 RT inhibitory activity with an IC50 value of 8.3 μM, but was devoid of antifungal, ribonuclease, and hemagglutinating activities.

1. Introduction

Mushrooms produce spectacular diversity of biologically active biomolecules encompassing laccase, lectins, nucleases, proteases, antifungal proteins, ribosome inactivating proteins, and polysaccharides, polysaccharide—peptides and polysaccharide—protein complexes [1]. Many of these proteinaceous and nonproteinceous molecules manifest potentially worthwhile activities including antitumor, HIV-1 reverse transcriptase inhibitory, and immunomodulatory activities [1, 2]. A variety of mushroom constituents are known to have health promoting activities and therapeutic potential. Some of them are under clinical investigations [3, 4]. Many mushrooms have a good taste and are highly nutritious. Thus mushrooms are popular in the diet of many people all over the world.

Proteases catalyze degradation with high (e.g., trypsine) or low (e.g., subtilisin) specificity. [5, 6]. They can be produced in sizeable quantities by employing microbial approaches. The requirements of brewing, dairy, meat, leather, detergent, and photographic industries make proteases useful and commercial [7]. The sale of industrial enzymes, a significant percentage of which is used for detergents, is a business involving millions of dollars [8]. Proteases have been reported from a number of mushrooms, including Agaricus bisporus [9], Armillariella mellea [10], Cordyceps sinensis [11], Flammulina velutipes [12], Grifola frondosa [13], Helvella lacunosa [14], Lyophyllum cinerascens [15], Pleurotus eryngii [16], Pleurotus ostreatus [17], Pleurotus citrinopileatus [18], and Tricholoma saponaceum [19].

Xylaria hypoxylon, commonly known as candlestick fungus, carbon antlers, or stag’s horn fungus, is an inedible mushroom belonging to the genus Xylaria [20]. In China, X. hypoxylon is a kind of competitor fungus found in the cultivation of straw mushroom (Volvariella volvacea). A variety of bioactive compounds have been isolated from the fungus. Two tetralone derivatives xylariol A and B, and two α-pyrone derivatives xylarone and 8,9-dehydroxylarone possessing cytotoxic activities were isolated from the culture broth of X. hypoxylon [21, 22]. Sex cytochalasins binding to actin in muscle tissue, a xylose-specific lectin with antiproliferative, and antimitogenic activities have also been found in the fungus [23, 24]. In view of the importance of proteases and the differences in characteristics of proteases from different sources and the dearth of information on protease from wild mushrooms, the present study was undertaken to purify and characterize a protease from the wild mushroom X. hypoxylon.

2. Materials and Methods

2.1. Fungal Material and Reagents

Fruiting bodies of X. hypoxylon were purchased from a company specializing on straw mushroom in Beijing and identified by Institute of Microbiology, Chinese Academy of Sciences. The sources of other biochemical and chemical reagents used in this work are as follows: DEAE-cellulose, CM-cellulose, Coomassie brilliant blue R-250, glycine, casein, trypsin, and yeast tRNA, were obtained from Sigma. Q-Sepharose, Superdex 75, molecular mass standards, and AKTA Purifier were purchased from GE Healthcare (USA). All other reagents were of reagent grade.

2.2. Isolation of Protease

A water extract of the fruiting bodies of X. hypoxylon (500 g) was prepared by homogenization in distilled water (4 mL/g). Following centrifugation of the homogenate at 12000 g for 20 minutes, Tris-HCl buffer (pH 7.2) was added to the supernatant obtained until the concentration of Tris was 10 mM. Ion exchange chromatography of the supernatant on a 5 × 2 0  cm column of DEAE-cellulose was then carried out in 10 mM Tris-HCl buffer (pH 7.2). After removal of the flow-through fraction (D1), the column was eluted stepwise with 0.2 M NaCl and then with 1 M NaCl in the starting buffer to yield fractions D2 and D3, respectively. Fraction D3 was dialyzed, lyophilized, and chromatographed on a Q-Sepharose column ( 2 . 5 × 2 0  cm) in 10 mM Tris-HCl buffer (pH 7.0). When all the unadsorbed proteins (collected as fraction Q1) had been eluted, the column was eluted with a linear concentration (0-1 M) gradient of NaCl added to 10 mM Tris-HCl buffer (pH 7.2). The second and most strongly adsorbed fraction, Q3, was dialyzed, lyophilized, and then applied to a 2 . 5 × 2 0  cm column of CM-cellulose. The column was eluted with 10 mM NH4OAc buffer (pH 4.5) until all the unadsorbed proteins had been eluted and collected as fraction CM1. Adsorbed proteins were desorbed with a linear concentration (0-1 M) gradient of NaCl in 10 mM NH4OAc buffer (pH 4.5) to yield fractions CM2 and CM3. Final purification was conducted by FPLC-gel filtration of fraction CM2 on a Superdex 75 HR 10/30 column in 0.2 M NH4HCO3 buffer (pH 8.5) using an AKTA Purifier. The second eluted peak represented purified protease. All the purification steps were carried out at 4°C.

2.3. Molecular Mass Determination by SDS-PAGE and by FPLC-Gel Filtration

SDS-PAGE was assayed using the protocol of Laemmli and Favre [25], using a 12% resolving gel and a 5% stacking gel. At the end of electrophoresis, the gel was dyed with 0.1% Coomassie brilliant blue R-250. FPLC-gel filtration was carried out using a Superdex 75 HR 10/30 column which had been calibrated with the undermentioned molecular mass standards [26]. The molecular mass of the protein was determined by comparison of the elution volume with those of molecular mass standards including blue dextran (to determine void volume), phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), soybean trypsin inhibitor (20 kDa), and bovine α-Lactalbumin (14.4 kDa).

2.4. Analysis of N-Terminal Amino Acid Sequences

Amino acid sequence analysis was carried out using an HP G1000A Edman degradation unit and an HP1000 HPLC system [27].

2.5. Assay for Protease Activity

In this assay, an improved method of Satake et al. [28] was used. In brief, a casein solution, which was used as substrate, was freshly prepared as follows: 0.1 g casein was added into 10 mL Mes buffer (200 mM, pH 7.0). Subsequently, the solution was incubated at 60°C for 30 min. Following centrifugation at 12000 g for 20 minutes, the precipitate was removed and the resulting solution could be used as the protease substrate. The test sample or trypsin solution (as positive control) (20 μL) was mixed with the above casein solution (180 μL) and then incubated at 37°C for 15 min. Subsequently, 400 μL of 5% trichloroacetic acid (TCA) was added for ending the enzymatic reaction. The reaction mixture was then cooled to the room temperature before centrifugation at 10000 g for 15 min. The absorbance of the supernatant was read at 280 nm against water as blank using a UV-spectrophotometer. Protease activity was calculated based on the activity of trypsin (7900 BAEE units/mg according to Sigma) in the protease assay using casein as substrate [29].

2.6. Optimal pH and Temperature of Purified Protease

Protease activity was tested over the pH range (pH 3–9) and temperature range (20°C–100°C). Casein in different buffers including 0.1 M NaOAc (pH 3–5), 0.1 M Mes (pH 5–7), and 0.1 M Hepes (pH 7–9) were used to determine the optimal pH value. To determine the optimal temperature, the reaction mixture was incubated at 20°C, 30°C, 37°C, 45°C, 50°C, 60°C, 70°C, 80°C, and 100°C in 0.1 M Mes buffer (pH 7.0) for 30 min [18].

2.7. Assay for Mechanistic Class

In this assay, the purified protease was exposed to the following inhibitors: phenylmethylsulfonyl fluoride (PMSF, 1.0 mM), ethylenediaminetetraacetic acid disodium salt (EDTA, 1.0 mM), pepstatin A (0.2 mM), and lima bean trypsin inhibitor (0.25 mM) for 30 min at room temperature. Residual enzyme activity was measured using the standard assay above. Control, in which inhibitors were substituted by Mes buffer (pH 7.0), was taken as 100% [26].

2.8. Assay for HIV-1 Reverse Transcriptase Inhibitory Activity

The assay for HIV-1 reverse transcriptase inhibitory activity was carried out using the assay kit from Boehringer Mannheim (Germany) and the reaction protocol of Zhao et al. [30].

2.9. Assay for Antifungal Activity

The assay for antifungal activity toward Fusarium oxysporum, Rhizoctonia cerealis, Rhizoctonia solani, and Sclerotinia sclerotiorum was carried out using the method of Lam and Ng [31].

2.10. Assay for Ribonuclease Activity

The ribonuclease activity of the purified protease was assayed following the method of Mock et al. [32] and using yeast tRNA as substrate.

2.11. Assay for Hemagglutinating Activity

The assay for hemagglutinating activity was measured with a 2% suspension of rabbit red cells in phosphate-buffered saline (pH 7.2) at 20°C with the method of Zhang et al. [33].

3. Results

3.1. Isolation of Protease

After ion exchange chromatography of the fruiting body extract on DEAE-cellulose, protease activity resided only in fraction D3 which was most strongly adsorbed on the column. Both fractions D1 and D2 were devoid of protease activity (Table 1). Protease activity was again enriched in the most strongly adsorbed fraction, Q3, derived by ion exchange chromatography of fraction D3 on Q-Sepharose. The unadsorbed fraction Q1 lacked protease activity and the adsorbed fraction Q2 exhibited very low protease activity (Table 1). Further purification of the unadsorbed fraction Q3 on CM-cellulose allowed separation of a small unadsorbed peak from two adsorbed peaks (Figure 1). No protease activity was detected in the unadsorbed fraction CM1. The first adsorbed fraction (CM2) had much higher protease activity than the following adsorbed fraction (CM3) (Table 1). CM2 was resolved into two peaks of similar sizes upon gel filtration on Superdex 75 (Figure 2). The first fraction, SU1, was without protease activity. Protease activity was confined to the second fraction, SU2 (Table 1). SU2 appeared as a single band with a molecular mass of 43 kDa in SDS-PAGE (Figure 3) and as a single peak with the same molecular mass upon rechromatography on Superdex 75 (data not shown). The enzyme was purified 74.1-fold from the crude extract with 43.6% yield. The purified protease exhibited an activity of 459.3 U/mg (Table 1).

tab1
Table 1: Comparion of characteristics of Xylaria hypoxylon protease with other fungal proteases.
728975.fig.001
Figure 1: Ion exchange chromatography on CM-cellulose. Sample: fraction Q3. Fraction size: 8 mL. Flow rate: 2 mL/min. Dotted line across right-hand side of chromatogram indicates linear NaCl concentration (0-1 M) gradient used to CM2 and CM3.
728975.fig.002
Figure 2: FPLC-gel filtration on Superdex 75 HR 10/30 column. Sample: fraction CM2. Eluent: 0.2 M NH4HCO3 buffer (pH 8.5). Fraction size: 0.8 mL. Flow rate: 0.4 mL/min.
728975.fig.003
Figure 3: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results. Left lane: purified protease (fraction SU2, 10 μg). Right lane: molecular mass markers, from top downward: phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), and α-lactalbumin (14.4 kDa).
3.2. Characterization of Isolated Protease

The N-terminal amino acid sequence of purified X. hypoxylon protease was HYTELLSQVV. A comparison of characteristics of X. hypoxylon and other fungal proteases is listed in Table 2. The protease was strongly inhibited by Pepstatin A, but not significantly affected by PMSF, EDTA, and Trypsin inhibitor (Table 3). The protease activity increased steadily as the pH was raised from 3.0 to 6.0 and then remained high when the pH was further raised to 8.0. There was an approximately 12% decrease in activity as the pH reached 9.0 (Figure 4). The protease activity escalated as the ambient temperature was raised from 20°C to 40°C. There was very little change in activity between 40°C and 60°C. As the temperature was raised to 100°C, there was a quick fall in activity. However, about 30% of the maximal activity was remained at 100°C (Figure 5). At a protease concentration of 1 μM, 5 μM, 15 μM, and 25 μM, 17.5%, 42.3%, 65.6%, and 79.8% inhibition, respectively of HIV-1 reverse transcriptase were observed (Figure 6). The IC50 value was estimated to be 8.3 μM. It did not exhibit antifungal, ribonuclease, and hemagglutinating activities (data not shown).

tab2
Table 2: Effects of protease inhibitors on Xylaria hypoxylon protease activity.
tab3
Table 3: Yields and protease activities of various chromatographic fractions of Xylaria hypoxylon (from 500 g fresh fruiting bodies. Assay conditions: 37°C/15 min, 0.1 M Mes buffer, pH 7.0).
728975.fig.004
Figure 4: Effect of pH on activity of the purified protease. Results are presented as mean ± SD ( 𝑛 = 3 ). Different letters (e.g., a, b, c, and d) next to the data points indicate statistically significant difference ( 𝑃 < 0 . 0 5 ) when the data are analyzed by analysis of variance followed by Duncan’s multiple range test.
728975.fig.005
Figure 5: Effect of temperature on activity of the purified protease. Results are presented as mean ± SD ( 𝑛 = 3 ). Different letters (e.g., a, b, c, and d) next to the data points indicate statistically significant difference ( 𝑃 < 0 . 0 5 ) when the data are analyzed by analysis of variance followed by Duncan’s multiple range test.
728975.fig.006
Figure 6: Inhibitory activity of purified protease on HIV-1 RT activity. Results represent mean ± SD ( 𝑛 = 3 ). The IC50 value toward HIV-1 RT was 1 estimated to be 8.3 μM. Different letters (a, b, c, etc.) next to the data points indicate statistically significant difference ( 𝑃 < 0 . 0 5 ) when the data are analyzed by analysis of variance followed by Duncan’s multiple range test.

4. Discussion

The present study constitutes the first report on the purification of a protease from Xylaria hypoxylon. A comparison of chromatography behavior of X. hypoxylon and other fungal proteases is listed in Table 2. X. hypoxylon protease, just like H. lacunose protease, adsorbs on DEAE-, CM-cellulose, and Q-Sepharose [14]. A protease from P. citrinopileatus unadsorbs on DEAE-cellulose and Q-Sepharose, but adsorbs on CM-cellulose [18]. So far a few ascomycetous proteases from genus Aspergillus, Cordyceps, Helvella, and Xylaria have been reported, and only C. sinensis, H. lacunose proteases, and the present one are from mushroom species. Compared with each other, the three ascomycetous proteases displayed different characters. X. hypoxylon and H. lacunose proteases manifests a close temperature optima (60°C and 65°C, resp.), while C. sinensis protease possesses a quite low optimal temperature of 30°C. X. hypoxylon and C. sinensis proteases exhibit a same molecular mass of 43 kDa which is higher than that of H. lacunose (33.5 kDa). C. sinensis and H. lacunose proteases are both alkaline proteases (with optimal pH of 9.5 and 11, resp.), while X. hypoxylon proteases has a fairly stable optimal activity in a pH range of 6–8.

Microbial proteases display a range of molecular masses from 18 kDa to 126 kDa [39]. X. hypoxylon protease exhibits an intermediate molecular mass (43 kDa), which is the same as C. sinensis protease, but higher than those of proteases from P. eryngii (11.5 kDa) [16], T. saponaceum (about 18 kDa) [19], A. mellea (18.5 kDa) [10], G. frondosa (20 kDa) [38], P. citrinopileatus (28 kDa) [18], and H. lacunose (33.5 kDa) [14] and lower than P. ostreatus (75 kDa) [17]. Its N-terminal sequence is largely different from previously reported fungal protease sequences, suggesting that it is a new protein.

Among the protease inhibitors tested, PMST, EDTA, and Trypsin inhibitor have no significant effect on the protease activity, suggesting that it is neither a serine protease, nor a metalloprotease. The activity of the protease was adversely affected by Pepstatin A, indicating that it is an aspartic protease. To date, many serine proteases have been isolated from mushrooms, such as A. bisporus [40], Cordyceps sobolifera [41], H. lacunose [14], and H. marmoreus [26]. Other mushroom proteases are metalloproteases, including A. mellea [10], G. frondosa [38], and T. saponaceum [19]. Only a few mushroom proteases are belonging to aspartic proteases, such as P. eryngii protease [16]. The present report of X. hypoxylon protease thus adds to the literature.

X. hypoxylon protease is relatively thermostable because its activity remains within the range of 65%–100% of its maximal activity at 60°C as the temperature varies from 20°C to 70°C. At a high temperature of 80°C, a significant portion (about 50%) of enzyme activity remains and at 100°C there is still 30% residual activity. A. mellea protease is stable up to 55°C, but its activity rapidly declines at higher temperatures [10]. The optimal temperature of X. hypoxylon protease is very close to that of H. lacunose protease (65°C) [14], and higher than that of P. eryngii (45°C) [16], A. clavatus (50°C) [34] H. marmoreus (50°C) [26], and T. saponaceum (55°C) [19].

The protease activity remains essentially unaltered in the pH range 6–8 and 60%–100% of the maximal activity is present even of the pH is changed from 3–9. P. eryngii protease demonstrates an acidic pH optimum of 5.0 [16], while several proteases of other mushrooms manifest optimal pH in the alkaline range, such as proteases from C. sinensis (pH 9.5) [11], P. citrinopileatus (pH 10) [18], and H. lacunose (pH 11) [14]. Protease from H. marmoreus exhibits a pH optimum of 7.5. Rapid decline in activity is noted, when the pH value is lower or higher than pH 7.5 [26]. Hence, the isolated protease from X. hypoxylon appears to be relatively stable with regard to variations in ambient pH and temperature, compared with H. marmoreus protease.

X. hypoxylon protease demonstrates HIV-1 reverse transcriptase inhibitory activity, with an IC50 value of 8.3 μM. Many mushroom proteins, such as lectins, ribonucleases, and proteases, manifest inhibitory activity toward HIV-1 reverse transcriptase. The IC50 values of I. umbrinella lectin, L. shimeji ribonuclease, P. eryngii protease, H. erinaceum lectin, are 4.7 μM, 7.2 μM, 30 μM, and 31.7 μM, respectively [16, 30, 42, 43]. X. hypoxylon protease is the first ascomycetous protease reported manifesting HIV-1 RT inhibitory activity. Up till now, only a basidiomycetous protease from P. eryngii has been reported to inhibit HIV-1 RT. Inhibitory activity of the purified protease is higher than those of P. eryngii protease and H. erinaceum lectin, but lower than those of I. umbrinella lectin and L. shimeji ribonuclease. The inactivity of X. hypoxylon protease in hydrolyzing tRNA, inhibiting fungal growth and agglutinating rabbit red blood cells indicates that it is free of contamination with ribonucleases, antifungal proteins, and lectins that are commonly produced by mushrooms.

In summary, X. hypoxylon protease is a unique enzyme with a distinctive N-terminal sequence, relatively high thermostability and the first report of HIV-1 reverse transcriptase inhibitory activity from Ascomycota mushrooms.

Acknowledgments

The authors thank Professor Xiao-Lan Mao, Institute of Microbiology, Chinese Academy of Sciences for fungi identification and National Grants of China (2010CB732202 and 2012BAD14B09).

References

  1. T. B. Ng, “Peptides and proteins from fungi,” Peptides, vol. 25, no. 6, pp. 1055–1073, 2004. View at Publisher · View at Google Scholar · View at Scopus
  2. E. F. Fang and T. B. Ng, “Ribonucleases of different origins with a wide spectrum of medicinal applications,” Biochimica et Biophysica Acta, vol. 1815, no. 1, pp. 65–74, 2011. View at Publisher · View at Google Scholar · View at Scopus
  3. C. Takizawa, “Clinical study on immunological activation response at the gastric cancer lesion by polysaccharide systemic administration,” Journal of Tokyo Medical College, vol. 44, no. 2, pp. 177–185, 1991. View at Google Scholar · View at Scopus
  4. T. B. Ng, “A review of research on the protein-bound polysaccharide (polysaccharopeptide, PSP) from the mushroom Coriolus versicolor (basidiomycetes: polyporaceae),” General Pharmacology, vol. 30, no. 1, pp. 1–4, 1998. View at Publisher · View at Google Scholar · View at Scopus
  5. A. Anwar and M. Saleemuddin, “Alkaline proteases: a review,” Bioresource Technology, vol. 64, no. 3, pp. 175–183, 1998. View at Publisher · View at Google Scholar · View at Scopus
  6. C. G. Kumar and H. Takagi, “Microbial alkaline proteases: from a bioindustrial viewpoint,” Biotechnology Advances, vol. 17, no. 7, pp. 561–594, 1999. View at Publisher · View at Google Scholar · View at Scopus
  7. H. M. Kalisz, “Microbial proteinases,” Advances in Biochemical Engineering / Biotechnology, vol. 36, pp. 1–65, 1988. View at Google Scholar · View at Scopus
  8. J. Hodgson, “The changing bulk biocatalyst market,” Nature Biotechnology, vol. 12, no. 8, pp. 789–790, 1994. View at Publisher · View at Google Scholar · View at Scopus
  9. K. S. Burton, D. A. Wood, C. F. Thurston, and P. J. Barker, “Purification and characterization of a serine proteinase from senescent sporophores of the commercial mushroom Agaricus bisporus,” Journal of General Microbiology, vol. 139, part 6, pp. 1379–1386, 1993. View at Google Scholar · View at Scopus
  10. J. H. Kim and Y. S. Kim, “A fibrinolytic metalloprotease from the fruiting bodies of an edible mushroom, Armillariella mellea,” Bioscience, Biotechnology, and Biochemistry, vol. 63, no. 12, pp. 2130–2166, 1999. View at Google Scholar
  11. B. Bi, X. Wang, H. Wu, and Q. Wei, “Purification and characterisation of a novel protease from Cordyceps sinensis and determination of the cleavage site motifs using oriented peptide library mixtures,” Food Chemistry, vol. 126, no. 1, pp. 46–53, 2011. View at Publisher · View at Google Scholar · View at Scopus
  12. H. X. Wang and T. B. Ng, “Flammulin: a novel ribosome-inactivating protein from fruiting bodies of the winter mushroom Flammulina velutipes,” Biochemistry and Cell Biology, vol. 78, no. 6, pp. 699–702, 2000. View at Publisher · View at Google Scholar · View at Scopus
  13. T. Nishiwaki, S. Asano, and T. Ohyama, “Properties and substrate specificities of proteolytic enzymes from the edible basidiomycete Grifola frondosa,” Journal of Bioscience and Bioengineering, vol. 107, no. 6, pp. 605–609, 2009. View at Publisher · View at Google Scholar · View at Scopus
  14. G. Zhang, H. Wang, X. Zhang, and T. Ng, “Helvellisin, a novel alkaline protease from the wild ascomycete mushroom Helvella lacunosa,” Journal of Bioscience and Bioengineering, vol. 109, no. 1, pp. 20–24, 2010. View at Publisher · View at Google Scholar · View at Scopus
  15. S. A. Abdus, T. Yoshimoto, and D. Tsuru, “Lyophyllum cinerascens aminopeptidase: purification and enzymatic properties,” Archives of Biochemistry and Biophysics, vol. 274, no. 1, pp. 241–250, 1989. View at Google Scholar
  16. H. Wang and T. B. Ng, “Pleureryn, a novel protease from fresh fruiting bodies of the edible mushroom Pleurotus eryngii,” Biochemical and Biophysical Research Communications, vol. 289, no. 3, pp. 750–755, 2001. View at Publisher · View at Google Scholar · View at Scopus
  17. G. Palmieri, C. Bianco, G. Cennamo et al., “Purification, characterization, and functional role of a novel extracellular protease from Pleurotus ostreatus,” Applied and Environmental Microbiology, vol. 67, no. 6, pp. 2754–2759, 2001. View at Publisher · View at Google Scholar · View at Scopus
  18. L. Cui, Q. H. Liu, H. X. Wang, and T. B. Ng, “An alkaline protease from fresh fruiting bodies of the edible mushroom Pleurotus citrinopileatus,” Applied Microbiology and Biotechnology, vol. 75, no. 1, pp. 81–85, 2007. View at Publisher · View at Google Scholar · View at Scopus
  19. J. H. Kim and Y. S. Kim, “Characterization of a metalloenzyme from a wild mushroom, Tricholoma saponaceum,” Bioscience, Biotechnology, and Biochemistry, vol. 65, no. 2, pp. 356–362, 2001. View at Google Scholar
  20. W. Sundberg and A. Bessette, Mushrooms: A Quick Reference Guide to Mushrooms of North America (Macmillan Field Guides), Collier Books, New York, NY, USA, 1987.
  21. W. Gu and H. Ding, “Two new tetralone derivatives from the culture of Xylaria hypoxylon AT-028,” Chinese Chemical Letters, vol. 19, no. 11, pp. 1323–1326, 2008. View at Publisher · View at Google Scholar · View at Scopus
  22. A. Schuffler, O. Sterner, H. Anke et al., “Cytotoxic alpha-pyrones from Xylaria hypoxylon,” Zeitschrift für Naturforschung C, vol. 62, no. 3-4, pp. 169–172, 2007. View at Google Scholar
  23. A. Espada, A. Rivera-Sagredo, J. M. de la Fuente, J. A. Hueso-Rodriguez, and S. W. Elson, “New cytochalasins from the fungus Xylaria hypoxylon,” Tetrahedron, vol. 53, no. 18, pp. 6485–6492, 1997. View at Publisher · View at Google Scholar · View at Scopus
  24. Q. Liu, H. Wang, and T. B. Ng, “First report of a xylose-specific lectin with potent hemagglutinating, antiproliferative and anti-mitogenic activities from a wild ascomycete mushroom,” Biochimica et Biophysica Acta, vol. 1760, no. 12, pp. 1914–1919, 2006. View at Publisher · View at Google Scholar · View at Scopus
  25. U. K. Laemmli and M. Favre, “Maturation of the head of bacteriophage T4. I. DNA packaging events,” Journal of Molecular Biology, vol. 80, no. 4, pp. 575–599, 1973. View at Google Scholar · View at Scopus
  26. X. Q. Zhang, Q. H. Liu, G. Q. Zhang, H. X. Wang, and T. B. Ng, “Purification and molecular cloning of a serine protease from the mushroom Hypsizigus marmoreus,” Process Biochemistry, vol. 45, no. 5, pp. 724–730, 2010. View at Publisher · View at Google Scholar · View at Scopus
  27. P. H. Ngai and T. B. Ng, “A hemolysin from the mushroom Pleurotus eryngii,” Applied Microbiology and Biotechnology, vol. 72, no. 6, pp. 1185–1191, 2006. View at Publisher · View at Google Scholar · View at Scopus
  28. M. Satake, Y. Murata, and T. Suzuki, “Studies on snake venom: XIII. chromatographic separation and properties of three proteinases from Agkistrodon, halys blomhoffii venom,” Journal of Biochemistry, vol. 53, no. 6, pp. 438–447, 1963. View at Google Scholar · View at Scopus
  29. L. Keay and B. S. Wildi, “Proteases of the genus Bacillus. I. neutral proteases.,” Biotechnology and Bioengineering, vol. 12, no. 2, pp. 179–212, 1970. View at Google Scholar · View at Scopus
  30. J. K. Zhao, H. X. Wang, and T. B. Ng, “Purification and characterization of a novel lectin from the toxic wild mushroom Inocybe umbrinella,” Toxicon, vol. 53, no. 3, pp. 360–366, 2009. View at Publisher · View at Google Scholar · View at Scopus
  31. S. K. Lam and T. B. Ng, “Isolation of a small chitinase-like antifungal protein from Panax notoginseng (sanchi ginseng) roots,” International Journal of Biochemistry and Cell Biology, vol. 33, no. 3, pp. 287–292, 2001. View at Publisher · View at Google Scholar · View at Scopus
  32. J. W. Mock, T. B. Ng, R. N. Wong, Q. Z. Yao, H. W. Yeung, and W. P. Fong, “Demonstration of ribonuclease activity in the plant ribosome-inactivating proteins alpha- and beta-momorcharins,” Life Sciences, vol. 59, no. 22, pp. 1853–1859, 1996. View at Publisher · View at Google Scholar · View at Scopus
  33. G. Q. Zhang, J. Sun, H. X. Wang, and T. B. Ng, “A novel lectin with antiproliferative activity from the medicinal mushroom Pholiota adiposa,” Acta Biochimica Polonica, vol. 56, no. 3, pp. 415–421, 2009. View at Google Scholar · View at Scopus
  34. M. Hajji, S. Kanoun, M. Nasri, and N. Gharsallah, “Purification and characterization of an alkaline serine-protease produced by a new isolated Aspergillus clavatus ES1,” Process Biochemistry, vol. 42, no. 5, pp. 791–797, 2007. View at Publisher · View at Google Scholar · View at Scopus
  35. T. Emri, M. Szilagyi, K. Laszlo, M. M-Hamvas, and I. Pocsi, “PepJ is a new extracellular proteinase of Aspergillus nidulans,” Folia Microbiologica, vol. 54, no. 2, pp. 105–109, 2009. View at Google Scholar
  36. K. S. Burton, M. D. Partis, D. A. Wood, and C. F. Thurston, “Accumulation of serine proteinase in senescent sporophores of the cultivated mushroom, Agaricus bisporus,” Mycological Research, vol. 101, no. 2, pp. 146–152, 1997. View at Publisher · View at Google Scholar · View at Scopus
  37. K. A. Shaginyan, I. A. Alekhina, and N. P. Denisova, “Serine proteinase from the higher basidiomycetes of Coprinus genus,” Biokhimiya, vol. 55, no. 8, pp. 1387–1395, 1990. View at Google Scholar · View at Scopus
  38. T. Nonaka, H. Ishikawa, Y. Tsumuraya, Y. Hashimoto, and N. Dohmae, “Characterization of a thermostable lysine-specific metalloendopeptidase from the fruiting bodies of a basidiomycete, Grifola frondosa,” Journal of Biochemistry, vol. 118, no. 5, pp. 1014–1020, 1995. View at Google Scholar · View at Scopus
  39. M. B. Rao, A. M. Tanksale, M. S. Ghatge, and V. V. Deshpande, “Molecular and biotechnological aspects of microbial proteases,” Microbiology and Molecular Biology Reviews, vol. 62, no. 3, pp. 597–635, 1998. View at Google Scholar · View at Scopus
  40. A. K. Sattar, N. Yamamoto, T. Yoshimoto, and D. Tsuru, “Purification and characterization of an extracellular prolyl endopeptidase from Agaricus bisporus,” Journal of Biochemistry, vol. 107, no. 2, pp. 256–261, 1990. View at Google Scholar · View at Scopus
  41. S. X. Wang, Y. Liu, G. Q. Zhang et al., “Cordysobin, a novel alkaline serine protease with HIV-1 reverse transcriptase inhibitory activity from the medicinal mushroom Cordyceps sobolifera,” Journal of Bioscience and Bioengineering, vol. 113, no. 1, pp. 42–47, 2012. View at Google Scholar
  42. R. Y. Zhang, G. Q. Zhang, D. D. Hu, H. X. Wang, and T. B. Ng, “A novel ribonuclease with antiproliferative activity from fresh fruiting bodies of the edible mushroom Lyophyllum shimeiji,” Biochemical Genetics, vol. 48, no. 7-8, pp. 658–668, 2010. View at Publisher · View at Google Scholar · View at Scopus
  43. Y. Li, G. Zhang, T. B. Ng, and H. Wang, “A novel lectin with antiproliferative and HIV-1 reverse transcriptase inhibitory activities from dried fruiting bodies of the monkey head mushroom Hericium erinaceum,” Journal of Biomedicine and Biotechnology, vol. 2010, Article ID 716515, 9 pages, 2010. View at Publisher · View at Google Scholar