Purification and characterization of a novel serine protease from the mushroom Pholiota nameko

Purification and characterization of a novel serine protease from the mushroom Pholiota nameko

Journal of Bioscience and Bioengineering VOL. 111 No. 6, 641 – 645, 2011 www.elsevier.com/locate/jbiosc Purification and characterization of a novel ...

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Journal of Bioscience and Bioengineering VOL. 111 No. 6, 641 – 645, 2011 www.elsevier.com/locate/jbiosc

Purification and characterization of a novel serine protease from the mushroom Pholiota nameko Gui-Ping Guan,1,2 Guo-Qing Zhang,3 Ying-Ying Wu,1 He-Xiang Wang,1 and Tzi-Bun Ng4,⁎ State Key Laboratory for Agrobiotechnology and Department of Microbiology, China Agricultural University, Beijing 100193, China, 1 College of Biosciences and Biotechnology, Hunan Agricultural University, Changsha 410128, China, 2 Key Laboratory of Urban Agriculture (North) of Ministry of Agriculture, Beijing University of Agriculture, Beijing 102206, China, 3 and School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China 4 Received 13 October 2010; accepted 17 February 20112 Available online 8 March 2011

A novel serine protease, with a molecular mass of 19 kDa and the N-terminal sequence of ARTPEAPAEV, was isolated from dried fruiting bodies of the mushroom Pholiota nameko. The purification protocol comprised ion exchange chromatography on DEAE-cellulose, Q-Sepharose and SP-Sepharose, and gel filtration on Superdex 75. It was unadsorbed on DEAE-cellulose and Q-Sepharose but adsorbed on SP-Sepharose. It exhibited an optimum temperature at 50°C, an optimum pH at pH 8.8, a Km of 5.64 mg/mL and a Vmax of 0.98 μmol/min/mL against substrate casein. A number of metal ions inhibited the enzyme including Pb2+, Mn2+, Ca2+, Hg2+, Zn2+, Cu2+, Co2+, Fe3+ and Al3+, with the inhibition of the last two cations being the most potent. K+ and Mg2+ slightly enhanced, while Li+ moderately potentiated the activity of the protease. The protease was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), suggesting that it is a serine protease. © 2011, The Society for Biotechnology, Japan. All rights reserved. [Key words: Protease; Fruiting bodies; Mushroom; Pholiota nameko]

Protease represents one of the three largest groups of industrial enzymes and account for about 60% of the total worldwide sale of enzymes (1). They are the proteolytic enzymes which participate in many pathological processes such as protein turnover, sporulation and conidial discharge, germination, enzyme modification, nutrition, regulation of gene expression, and part of them also are considered as important virulence factors of many pathogens, including viruses, bacteria, fungi and parasites (2,3). As well as their metabolic functions, proteases play a vital role in commercial fields, i.e., in the food, leather, detergent, pharmaceutical industries and in ecological bioremediation processes. Proteases have been isolated from different organisms including animals (4,5), plants (6–8), and microorganisms, e.g., mushrooms such as Agaricus bisporus (9,10), Armillariella mellea (11,12), Flammulina velutipes (13), Grifola frondosa (14), Helvella lacunose (15), Lyophyllum cinerascens (16), Pleurotus eryngii (17), Pleurotus ostreatus (18), Pleurotus citrinopileatus (2), and Tricholoma saponaceum (19). A. bisporus protease is a serine protease, proteases from Irpex lacteus and P. eryngii are aspartic proteinases, L. cinerascens protease is an aminopeptidase (20), P. citrinopileatus protease is an alkaline protease, while the rest are metalloproteases. They exhibit a range of molecular masses and different thermostability. Regarding the mushroom Pholiota nameko, only information on polysaccharides and several proteins including a tyrosinase, ribonucleases, an acid phosphatase, a glucose-1-phosphatase, and a hydro-

⁎ Corresponding author. Tel./fax: +852 2609 8031. E-mail address: [email protected] (T.-B. Ng).

phobin are available (21–24). In view of the importance of proteases and the differences in characteristics of proteases from different sources, the present investigation was undertaken to isolate and characterize a protease from the edible mushroom P. nameko. MATERIALS AND METHODS Materials Dried fruiting bodies of the edible mushroom P. nameko were purchased from a local supermarket. The sources of other materials and chemicals used in this work are as follows: DEAE-cellulose was from Sigma. Q-Sepharose, SP-Sepharose and Superdex 75 were obtained from GE Healthcare. Substrate casein, protease inhibitors, agarose, glycine, ammonium sulfate, metal ions, buffers and all other chemicals were from Sigma. Isolation of protease Dried fruiting bodies of P. nameko (20 g) were homogenized in 0.15 M NaCl (10 mL/g) at 4°C followed by centrifugation at 8000×g for 25 min. Proteins in the supernatant were precipitated by 80% (NH4)2SO4. Centrifugation at 8000×g for 25 min was carried out. The precipitate was collected, dissolved in water, and dialyzed extensively against distilled water to remove (NH4)2SO4. NH4HCO3 buffer (pH 9.4) was added until the concentration of NH4HCO3 reached 10 mM. Ion exchange chromatography on a column of DEAE-cellulose (Sigma) (2.5×20 cm) in 10 mM NH4HCO3 buffer (pH 9.4) was carried out. After removal of unadsorbed proteins in fraction D1, adsorbed proteins were desorbed and eluted into three fractions D2, D3 and D4, by addition of 50 mM NaCl, 150 mM NaCl and 1 M NaCl, respectively, to the NH4HCO3 buffer. Ion exchange chromatography of fraction D1 on a column of Q-Sepharose (GE Healthcare) (1.5×20 cm) in 10 mM NH4HCO3, (pH 9.4), was carried out. After removal of unadsorbed proteins Q1, adsorbed proteins were desorbed and eluted into three fractions Q2, Q3 and Q4 by addition of 50 mM NaCl, 150 mM NaCl, and 1 M NaCl to the NH4HCO3 buffer. Fraction Q1 was next subjected to ion exchange chromatography on a column of SP-Sepharose (GE Healthcare) (1.5×15 cm) in 10 mM phosphate buffer (pH 6.2). Unadsorbed proteins were eluted in fraction SP1. Adsorbed proteins were eluted into fractions SP2 and SP3 using a gradient of 0–0.5 M NaCl. FPLC of fraction SP2 on Superdex 75 (GE Healthcare) in 0.2 M NH4HCO3 (pH 8.5), at a flow rate of 0.4 mL/min and with a fraction size of 0.8 mL, was used as the final purification step. The first fraction SU1 represented the purified enzyme.

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Determination of molecular mass and N-terminal sequence The active peak (SU1) was subsequently analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) (25). The molecular mass of the purified protein was determined in SDS–PAGE as well as in FPLC-gel filtration as described above. Nterminal sequencing of the protein was carried out using an HP G-1000A Edman degradation unit and an HP 1000 HPLC system (2). Assay for protease activity Proteolytic activity was assayed according to the method of Satake et al. (26). A solution of casein, which could be used in the protease assay, was freshly prepared as follows. To 0.1 g casein, 10 mL 50 mM phosphate buffer (pH 7.5) were added. Subsequently, the solution was incubated at 4°C for overnight. The precipitate was removed and the resulting solution could be used. The test sample (25 μL) was mixed with 140 μL of the above casein solution, and the reaction mixture was incubated at 37°C for 15 min. Subsequently, 600 μL 5% trichloroacetic acid (TCA) was added. The reaction mixture was allowed to stand at room temperature for 30 min, before centrifugation at 8000×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 expressed in units, where 1 U represented a 0.001 absorbance increase per minute in the supernatant per milliliter of reaction mixture under specified conditions (15). Determination of optimum pH and temperature In the determination for optimum pH and temperature, a solution of casein, which was used as substrate, was freshly prepared as described above (15). The assay buffers were prepared in 50 mM NaH2PO4–citric acid (pH 4.0–7.0), 50 mM Tris–HCl (pH 7.0–8.6), 50 mM glycine–NaOH (pH 8.8–9.8), 50 mM NaHCO3–NaOH (pH 9.8–11.0) (Buffer A–D, respectively). The purified protease (10 μL) was incubated at 37°C for 15 min with 90 μL 1% casein solution (pH 7.5) and 100 μL assay buffer as described above. The reaction was subsequently ended by addition of 600 μL 5% trichloroacetic acid (TCA). The reaction mixture was allowed to stand at room temperature for 30 min before centrifugation at 8000×g for 15 min. The absorbance of the supernatant was read at 280 nm against water as blank. The protease activity tested at pH 7.5 (50 mM Tris–HCl) was regarded as 100%. To determine the optimum temperature, the reaction mixture was incubated at 20–85°C for 15 min. The assay buffer was 50 mM phosphate buffer (pH 7.5). The protease activity tested at 37°C was regarded as 100%. Assay of the enzyme thermostability In order to check thermal stability, the purified protease was incubated at 37°C and 50°C for 0, 30, 60, 90 and 120 min, respectively, cooled to 37°C and then assayed using the standard protease assay above. The protease activity tested at 37°C was regarded as 100%. Assay of enzyme kinetics Casein at different concentrations (2%, 1%, 0.5%, 0.2%, 0.1%, 0.08%, 0.06%, 0.04%, 0.02%) was used as substrate. The Km and Vmax of the enzyme were calculated based on the Lineweaver–Burk plot constructed by plotting the reciprocal of substrate concentration on the x-axis, and reciprocal of the enzyme reaction velocity on the y-axis (27). Assay of mechanistic class In the determination of mechanistic class for the purified protease, the protease was exposed to protease inhibitors including ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis (β-aminoethylether)tetraacetic acid (EGTA), lima bean trypsin inhibitor, Pepstatin A, and phenyl methyl sulfonyl fluoride (PMSF) with different concentrations of 0.04 mM–1.0 mM for 30 min. The residual enzyme activity was measured (28). Assay of metal ions and effects of chemical reagents The enzyme solution (10 μL) was preincubated, at 37°C for 30 min at pH 7.5, with 10 μL of different metal ions and chemical reagents at concentrations ranging from 5 mM to 200 mM (15). The protease activity was assayed as described above.

protease activity eluted by 0.2 M NaCl, and a very small inactive adsorbed fraction SP3 (Fig. 1A). Fraction SP2 was resolved on Superdex 75 into a main fraction SU1 in which protease activity resided and a very small fraction SU2 (Fig. 1B). Fraction SU1, which represents purified protease, appeared as a single band with a molecular mass of 19 kDa (Fig. 2). The enzyme was purified 37.9-fold from ammonium sulfate precipitate with 31.2% yield, and 10,679 U/mg of the purified protease (Table 1). The N-terminal sequence of the purified protease was ARTPEAPAEV, which showed no similarity to purified fungal protease sequences previously reported (Table 2). Physiochemical properties of P. nameko protease The purified protease demonstrated an optimum pH of 8.8 (Fig. 3A) and an optimum temperature of 50°C (Fig. 3B). Maximal activity was detected at pH 8–9. There was an abrupt fall in activity (about 60% reduction) when the pH was lowered to 7 or increased to 11. The protease activity continued to decline as the pH was further lowered until negligible activity remained at pH 4 (Fig. 3A). Maximal activity of the protease depended on a temperature of 50°C. A slight decrement in activity was observed at 55°C and 60°C. About 60% of the activity remained when the temperature was reduced to 20°C or elevated to 70°C. A further fall in activity was seen at or above 70°C (Fig. 3B). The isolated enzyme showed strong thermal stability at 37°C and its optimal temperature (50°C). When the enzyme was incubated at 37 or 50°C for 2 h, the protease activity almost maintained its primal levels (Fig. 4). From the Lineweaver–Birk-plot, Vmax and Km were estimated to be 0.98 μmol·min− 1·ml− 1, and 5.64 mg·mL− 1, respectively (Fig. 5). The Km of P. nameko protease is lower than that of proteinase K, which is 29.6 mg·mL− 1 in our experiment. The Vmax of proteinase K is 5.01 μmol·min− 1·mL− 1 higher than that of P. nameko protease (0.98 μmol·min− 1·mL− 1) (28). The effects of various

RESULTS Isolation of P. nameko protease and determination of molecular mass and N-terminal sequence The unadsorbed fraction D1 from DEAE-cellulose, but not the adsorbed fractions, exhibited protease activity (Table 1). Similarly, the unadsorbed fraction Q1 was the only fraction with protease activity from the Q-Sepharose column (Table 1). Fraction Q1 was fractionated on SP-Sepharose into a large inactive unadsorbed fraction SP1, a large adsorbed fraction SP2 exhibiting

TABLE 1. Yields and protease activities of various chromatographic fractions derived from Pholiota nameko dried fruiting body extract (20 g). Fraction

Ammonium sulfate precipitate D1 Q1 SP2 SU1

Yield (mg)

Specific activity (U/mg)

Total activity (U/104)

1386.6

282

39.1

124.1 51.7 16.6 11.4

1862 4028 8754 10679

23.1 20.8 14.5 12.2

Recovery of activity (%) 100 59.1 53.2 37.1 31.2

Enzyme assay conditions: 37°C/15 min, in 0.1 M Tris–HCl, pH 7.5.

Purification fold 1 6.6 14.3 31.0 37.9

FIG. 1. Elution profiles of protease from Pholiota nameko. (A) Ion exchange chromatography on SP-Sepharose. Sample: Fraction of P. nameko fruiting body extract unadsorbed on DEAE-cellulose and Q-Sepharose. Buffer: 10 mM phosphate buffer (pH 6.2). Broken line across right half of chromatogram indicates linear NaCl concentration gradient used to desorb adsorbed proteins. (B) FPLC-gel filtration on Superdex 75.

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FIG. 2. SDS–PAGE of Pholiota nameko protease. Left lane: molecular mass marker from GE Helthcare. Right lane: fraction SU1 representing purified protease (15 μg).

cations on the activity of the isolated process are shown in Table 3. All of the cations examined except K+, Li+ and Mg2+ exerted a dosedependent inhibitory action on protease activity. K+, Li+ and Mg2+ had a mild stimulatory effect on protease activity (Table 3). The protease activity is not affected to any major extent by metal ions at 25 mM concentration except for Zn2+ and Pb2+ ions. However, a number of metal ions (Pb2+, Ca2+, Zn2+, Cu2+, Co2+, Fe3+, and Al3+) begin to exert an inhibitory action at 50 mM. When the concentration reaches to 100 mM, most of metal ions tested showed strong inhibitory activity except K+, Li+ and Mg2+. The activity of P. nameko protease was adversely affected by PMSF with a concentration of 1.00 mM, indicating that it is a serine protease. Pepstatin A, trypsin lima bean trypsin inhibitor, EDTA, and EGTA exerted no striking effect, suggesting that it is neither an aspartic protease nor a metalloprotease (Table 4).

TABLE 2. N-terminal sequence of Pholiota nameko protease compared to other fungal alkaline serine proteases, aspartic proteinase and metallo-endopeptidases (results of BLAST search). Protein Pholiota nameko alkaline serine protease Pleurotus eryngii alkaline serine protease Pleurotus citrinopileatus alkaline serine protease Agaricus bisporus alkaline serine protease Cordyceps chlamydosporia (EMBL bank entry: AJ427454.1) alkaline serine protease Paecilomyces lilacinus alkaline serine protease Penicillium chrysogenum alkaline serine protease Aspergillus oryzae alkaline serine protease Irpex lacteus aspartic protease Pleurotus ostreatus metallo-endopeptidase Armillariella mellea metallo-endopeptidase Grifola frondosa metallo-endopeptidase Tricholoma saponaceum metallo-endopeptidase

Phylum

N-terminal sequence Reference

Basidiomycota

ARTPEAPAEV

Basidiomycota

GPQFPEA

17

Basidiomycota

VCQCNAPWGL

2

Basidiomycota MHFSLSFATL

10

Ascomycota

A I V EQQGAPW

Ascomycota

ARAPLLTPRG

34

Ascomycota

MGFLKLLSTS

35

Ascomycota

TQTNAPWGLA

36

Basidiomycota

AAGSVPATNQ

37

Basidiomycota

ATFVGCSATRQTQLN

18

Basidiomycota

XXYNGXTXSRQTTLV

11

Basidiomycota

TYNGCSSSEQSALA

14

Basidiomycota

ALYVGXSPXQQSLLV

19

Identical amino acid residues are underscored.

FIG. 3. pH and temperature dependence of P. nameko protease. (A) pH dependence of enzymatic activity of P. nameko protease. Buffer A (open circles): NaHPO4-citric acid (pH 4.0– 7.0). Buffer B (closed diamonds): Tris–HCl buffer (pH 7.0–8.6). Buffer C (open triangles): glycine–NaOH (pH 8.8–9.8). Buffer D (open squares): NaHCO3–NaOH (pH 9.8–11.0). Final concentration of buffer: 0.1 M. Incubation time: 15 min followed by addition of 5% trichloroacetic acid. Results represent mean±SD (n=3). Different letters (a, b, c, d) next to the data points indicate statistically significant difference (pb 0.05) when the data are analyzed by analysis of variance followed by Duncan's multiple range test. The protease activity tested at pH 7.5 (50 mM Tris–HCl) was regarded as 100%. (B) Temperature dependence of enzymatic activity of P. nameko protease. Incubation at different temperatures for 15 min. Results represent mean±SD (n=3). Different letters (a, b, c, d) next to the data points indicate statistically significant difference (pb 0.05) when the data are analyzed by analysis of variance followed by Duncan's multiple range test. The protease activity tested at 37°C was regarded as 100%.

FIG. 4. Thermal stability of the purified protease. The enzyme was incubated at 37°C and 50°C for 0, 30, 60, 90, and 120 min, followed by measurement of the residual protease activity using the standard assay. The protease activity tested at 37°C was regarded as 100%. Each value in both panels represents mean ± SD (n = 3).

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FIG. 5. Lineweaver–Burk plot. Buffer for enzyme reaction: 0.1 M glycine–NaOH (pH 8.8). Reaction temperature: 37°C. Reaction time: 15 min.

EDTA 1.00 mM 0.20 mM 0.04 mM Lima bean trypsin inhibitor 1.00 mM 0.20 mM 0.04 mM PMSF 1.00 mM 0.20 mM 0.04 mM

Remaining protease activity (%) 95.1 ± 1.8a 95.7 ± 2.4a 98.3 ± 3.9a 99.1 ± 4.2a 98.7 ± 5.7a 99.2 ± 3.3a

Inhibitors EGTA 1.00 mM 0.20 mM 0.04 mM Pepstatin A 1.00 mM 0.20 mM 0.04 mM

Remaining protease activity (%) 99.0 ± 4.2a 99.2 ± 3.6a 99.3 ± 6.4a 102.7 ± 6.5a 99.1 ± 5.3a 99.8 ± 6.7a

37.2 ± 2.7a 82.1 ± 4.8b 98.1 ± 4.4c

Results represent mean ± SD (n = 3). Different superscripts (e.g., a, b, c) indicate statistically significant difference (p b 0.05) when the data corresponding to different concentrations of a cation are analyzed by analysis of variance followed by Duncan's multiple range test.

DISCUSSION In the present study, we have purified a novel serine protease from P. nameko with an elution procedure of DEAE-cellulose, Q-Sepharose, SP-Sepharose, and gel filtration on Superdex 75. The purified protease is a monomeric protein with a molecular mass of 19 kDa. Fungal proteases exhibit a variety of molecular masses from 18.5 kDa to 78 kDa (29). P. nameko protease demonstrates a low molecular mass (19 kDa), similar to proteases from A. mellea (18.5 kDa) (11,12), G. frondosa (20 kDa) (14), and T. saponaceum (about 18 kDa) (19). Its N-terminal sequence is dissimilar to previously reported sequences, suggesting that it is a novel protein. P. nameko protease is most active at around pH 8–9, similar to a number of other mushrooms, including A. mellea (11), A. bisporus (9,10), Coprinus sp. (30), G. frondosa (14), L. cinerascens (16) and P. ostreatus (18). The optimum pH of P. citrinopileatus protease is pH 10 (2). P. nameko protease exhibits a higher optimal pH and a higher optimal temperature than some reported mushroom proteases (31–33). The activity of P. nameko protease does not vary much in the range of pH 7.5–9.5. Although the activity drops rapidly when the pH is lowered from pH 7.5 or raised from pH 9.5, over 30% of the activity still remains at pH 7 and pH 11. Nevertheless, the isolated protease appears to be fairly stable with regard to variations in ambient pH and temperature, like the aspartic proteinase from P. eryngii (17). The optimum temperature of P. nameko protease is around 50°C, similar to that of P. citrinopileatus (2), but different from those of A. bisporus (35°C) (9,10), and Coprinus sp. (37°C) (30). P. nameko protease is a fairly thermostable enzyme since its activity varies from 70% to 100% of its maximal activity at 50°C when the temperature changes from 20°C to 70°C. At a high temperature of 85°C, a significant amount (about 43%) of enzyme activity still remains.

TABLE 3. Effect of metal ions on protease activity (100 represents protease activity in absence of metal ion). Cation

25 mM

50 mM

100 mM

K+ Li+ Pb2+ Mn2+ Ca2+ Mg2+ Hg2+ Zn2+ Cu2+ Co2+ Fe3+ Al3+

110.7 ± 6.8a 114.3 ± 7.4a 72.8 ± 4.3a 102.6 ± 6.5a 91.0 ± 5.0a 103.6 ± 5.9a 89.9 ± 4.4a 63.6 ± 4.1a 85.4 ± 5.2a 86.9 ± 4.6a 104.5 ± 8.2a 102.1 ± 6.7a

115.8 ± 5.5a 128.4 ± 7.3b 71.3 ± 4.2a 84.5 ± 4.6b 71.9 ± 3.8b 115.2 ± 6.0b 87.1 ± 5.5a 20.9 ± 1.2b 39.4 ± 2.1b 67.2 ± 2.8b 0b 4.8 ± 0.2b

119.4 ± 5.9a 135.5 ± 6.3b 46.9 ± 2.2b 77.0 ± 3.7c 58.8 ± 2.1c 122.1 ± 7.0b 34.9 ± 2.3b 17.9 ± 1.1b 16.1 ± 1.0c 55.5 ± 2.1c 0b 4.1 ± 0.2b

Results represent mean ± SD (n = 3). Different superscripts (e.g., a, b, c) indicate statistically significant difference (p b 0.05) when the data corresponding to different concentrations of a cation are analyzed by analysis of variance followed by Duncan's multiple range test.

Among the mushroom proteases reported, the proportion of serine proteases are higher than that of aspartic proteases or metalloprotease. Proteases from H. lacunose (15), H. marmoreus (28), and P. citrinopileatus (2) are serine proteases, while the protease from P eryngii is an aspartic protease (17). P. nameko protease is also a serine protease with Km and Vmax towards casein of 5.64 mg/mL and 0.98 μmol/min/mL, respectively. In summary, we report the purification and characterization of a novel serine protease from the mushroom P. nameko with some distinctive characteristics. ACKNOWLEDGMENTS This work was financially supported by National Grants of China (2010CB732202). References 1. Rao, M. B., Tanksale, A. M., Ghatge, M. S., and Deshpande, V. V.: Molecular and biotechnological aspects of microbial proteases, Microbiol. Mol. Biol. Rev., 62, 597–635 (1998). 2. Cui, L., Liu, Q. H., Wang, H. X., and Ng, T. B.: An alkaline protease from fresh fruiting bodies of the edible mushroom Pleurotus citrinopileatus, Appl. Microbiol. Biotechnol., 75, 81–85 (2007). 3. Sabotic, J., Trcek, T., Popovic, T., and Brzin, J.: Basidiomycetes harbour a hidden treasure of proteolytic diversity, J. Biotechnol., 128, 297–307 (2007). 4. Gotoh, T., Ono, H., Kikuchi, K., Nirasawa, S., and Takahashi, S.: Purification and characterization of aspartic protease derived from Sf9 insect cells, Biosci. Biotechnol. Biochem., 74, 2154–2157 (2010). 5. Vaiyapuri, S., Harrison, R. A., Bicknell, A. B., Gibbins, J. M., and Hutchinson, G.: Purification and functional characterisation of rhinocerase, a novel serine protease from the venom of Bitis gabonica rhinoceros, PLoS ONE, 5, e9687 (2010). 6. Khan, H., Ali, I., Khan, A. U., Ahmed, M., Shah, Z., Saeed, A., Naz, R., Mustafa, M. R., and Abbasi, A.: Purification and biochemical characterization of alkaline serine protease from Caesalpinia bonducella, Nat. Prod. Commun., 5, 931–934 (2010). 7. Saxena, L., Iyer, B. K., and Ananthanarayan, L.: Purification of a bifunctional amylase/protease inhibitor from ragi (Eleusine coracana) by chromatography and its use as an affinity ligand, J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci., 878, 1549–1554 (2010). 8. Nagarathnam, R., Rengasamy, A., and Balasubramanian, R.: Purification and properties of cysteine protease from rhizomes of Curcuma longa (Linn.), J. Sci. Food Agric., 90, 97–105 (2010). 9. Burton, K. S., Partis, M. D., Wood, D. A., and Thurston, C. F.: Accumulation of serine proteinase in senescent sporophores of the cultivated mushroom, Agaricus bisporus, Mycol. Res., 101, 146–152 (1997). 10. Burton, K. S., Smith, J. F., Wood, D. A., and Thurston, C. F.: Mycelial proteinases of the cultivated mushroom Agaricus bisporus, Mycol. Res., 101, 1341–1347 (1997). 11. Kim, J. H. and Kim, Y. S.: A fibrinolytic metalloprotease from the fruiting bodies of an edible mushroom, Armillariella mellea, Biosci. Biotechnol. Biochem., 63, 2130–2136 (1999). 12. Lee, S. Y., Kim, J. S., Kim, J. E., Sapkota, K., Shen, M. H., Kim, S., Chun, H. S., Yoo, J. C., Choi, H. S., Kim, M. K., and Kim, S. J.: Purification and characterization of fibrinolytic

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