A novel aspartic protease with HIV-1 reverse transcriptase inhibitory activity from fresh fruiting bodies of the wild mushroom Xylaria hypoxylon.

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 35-60°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.

Full text

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Hindawi Publishing Corporation
Journal of Biomedicine and Biotechnology
Volume 2012, Article ID 728975, 8 pages
doi:10.1155/2012/728975
Research Article
ANovel Aspartic Protease withHIV-1 Reverse
TranscriptaseInhibitoryActivity fromFreshFruitingBodiesof
theWildMushroom Xylariahypoxylon
Qing-XiuHu,1,2Guo-QingZhang,3Rui-YingZhang,2Dan-DanHu,4He-XiangWang,1
andTzi Bun Ng5
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
Correspondence should be addressed to He-Xiang Wang, hxwang1957@gmail.com and
Tzi Bun Ng, b021770@mailserv.cuhk.edu.hk
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.
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 43kDa 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
35–60◦C. The purified enzyme exhibited HIV-1 RT inhibitory activity with an IC50value of 8.3μM, but was devoid of antifungal,
ribonuclease, and hemagglutinating activities.
1.Introduction
Mushrooms produce spectacular diversity of biologically
active biomolecules encompassing laccase, lectins, nucle-
ases, proteases, antifungal proteins, ribosome inactivat-
ing 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 pro-
teases useful and commercial [7]. The sale of industrial
enzymes, a significant percentage of which is used for deter-
gents,isabusinessinvolvingmillionsofdollars[8].Proteases

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2Journal of Biomedicine and Biotechnology
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.MaterialsandMethods
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 (500g) was prepared by homoge-
nizationindistilledwater(4mL/g).Followingcentrifugation
of the homogenate at 12000g for 20 minutes, Tris-HCl
buffer (pH 7.2) was added to the supernatant obtained
until the concentration of Tris was 10mM. Ion exchange
chromatography of the supernatant on a 5 × 20cm column
of DEAE-cellulose was then carried out in 10mM Tris-
HCl buffer (pH 7.2). After removal of the flow-through
fraction (D1), the column was eluted stepwise with 0.2M
NaCl and then with 1M NaCl in the starting buffer to yield
fractions D2 and D3, respectively. Fraction D3 was dialyzed,
lyophilized,andchromatographedonaQ-Sepharosecolumn
(2.5 × 20cm) in 10mM 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-
1M) gradient of NaCl added to 10mM Tris-HCl buffer (pH
7.2). The second and most strongly adsorbed fraction, Q3,
was dialyzed, lyophilized, and then applied to a 2.5 × 20cm
columnofCM-cellulose.Thecolumnwaselutedwith10mM
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 (0-1M)
gradient of NaCl in 10mM 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 0.2M NH4HCO3buffer (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 pro-
tocol 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 (94kDa), bovine serum albumin (67kDa), ovalbumin
(43kDa), soybean trypsin inhibitor (20kDa), and bovine α-
Lactalbumin (14.4kDa).
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: 0.1g casein was added into 10mL Mes buffer
(200mM, pH 7.0). Subsequently, the solution was incubated
at 60◦C for 30min. Following centrifugation at 12000g 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) (20μL)
was mixed with the above casein solution (180μL) and
then incubated at 37◦C for 15min. Subsequently, 400μL
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
10000g for 15min. The absorbance of the supernatant
was read at 280nm 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 0.1M NaOAc (pH 3–5), 0.1M Mes (pH 5–
7), and 0.1M Hepes (pH 7–9) were used to determine the
optimalpHvalue.Todeterminetheoptimaltemperature,the
reaction mixture was incubated at 20◦C, 30◦C, 37◦C, 45◦C,

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Journal of Biomedicine and Biotechnology3
50◦C, 60◦C, 70◦C, 80◦C, and 100◦C in 0.1M Mes buffer (pH
7.0) for 30min [18].
2.7. Assay for Mechanistic Class. In this assay, the puri-
fied protease was exposed to the following inhibitors:
phenylmethylsulfonyl fluoride (PMSF, 1.0mM), ethylene-
diaminetetraacetic acid disodium salt (EDTA, 1.0mM),
pepstatin A (0.2mM), and lima bean trypsin inhibitor
(0.25mM)for30minatroomtemperature.Residualenzyme
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 activ-
ity 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% suspen-
sion 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 chromatogra-
phy 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 unad-
sorbed 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
proteaseactivitythanthefollowingadsorbedfraction(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 43kDa
in SDS-PAGE (Figure 3) and as a single peak with the same
molecular mass upon rechromatography on Superdex 75
0
0.2
0.4
0.6
0.8
1
0100200300400 500600700
Elution volume (mL)
0
0.2
0.4
0.6
0.8
1
NaCl (M)
CM1
CM2
CM3
A280 nm
Figure 1: Ion exchange chromatography on CM-cellulose. Sample:
fraction Q3. Fraction size: 8mL. Flow rate: 2mL/min. Dotted
line across right-hand side of chromatogram indicates linear NaCl
concentration (0-1M) gradient used to CM2 and CM3.
0
0.2
0.4
0.6
0.8
048
Elution volume (mL)
12 162024
SU1
SU2
A280 nm
Figure 2: FPLC-gel filtration on Superdex 75 HR 10/30 column.
Sample: fraction CM2. Eluent: 0.2M NH4HCO3buffer (pH 8.5).
Fraction size: 0.8mL. Flow rate: 0.4mL/min.
(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 459.3U/mg (Table 1).
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

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4Journal of Biomedicine and Biotechnology
Table 1: Yields and protease activities of various chromatographic
fractions of Xylaria hypoxylon (from 500g fresh fruiting bodies.
Assay conditions: 37◦C/15min, 0.1M Mes buffer, pH 7.0).
Fraction
Yield
(mg)
Protease
activity
(u/mg)
6.2
<0. 5
<0. 5
20.3
<0. 5
1.9
57.9
<0. 5
219.4
2.8
<0. 5
459.3
Total
activity
(u)
12636
—
—
10840
—
310
8569
—
6801
115
—
5512
Recovery
of activity
(%)
100
—
—
85.8
—
2.5
67.8
—
53.8
0.9
—
43.6
Purification
fold
Extract
D1
D2
D3
Q1
Q2
Q3
CM1
CM2
CM3
SU1
SU2
2038
506
405
534
68
163
148
30
31
41
9
12
1
—
—
3.3
—
0.3
9.3
—
35.4
0.5
—
74.1
—: no protease activity observed.
Protease-enriched fractions are highlighted in boldface.
Protease marker (kDa)
94
67
43
30
20
14.4
Figure 3: Sodium dodecyl sulfate-polyacrylamide gel electrophore-
sis results. Left lane: purified protease (fraction SU2, 10μg). Right
lane: molecular mass markers, from top downward: phosphorylase
b (94kDa), bovine serum albumin (67kDa), ovalbumin (43kDa),
carbonic anhydrase (30kDa), and α-lactalbumin (14.4kDa).
(Figure 5).Ataproteaseconcentrationof1μM,5μM,15μM,
and 25μM, 17.5%, 42.3%, 65.6%, and 79.8% inhibition,
respectively of HIV-1 reverse transcriptase were observed
(Figure 6). The IC50value was estimated to be 8.3μM. It did
not exhibit antifungal, ribonuclease, and hemagglutinating
activities (data not shown).
4.Discussion
The present study constitutes the first report on the purifica-
tion 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,
40
60
80
100
120
246810
pH
AA
Mes
Hepes
a
c
d
d
d
Relative activity (%)
b
b
e
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 < 0.05) when the data are analyzed by analysis of
variance followed by Duncan’s multiple range test.
10
30
50
70
90
110
130
1030 507090110
b
cc
d
d
e
f
g
Relative activity (%)
a
Temperature (◦C)
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 < 0.05) when the data are analyzed by analysis of
variance followed by Duncan’s multiple range test.
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
manifestsaclosetemperatureoptima(60◦Cand65◦C,resp.),
while C. sinensis protease possesses a quite low optimal
temperature of 30◦C. X. hypoxylon and C. sinensis proteases
exhibitasamemolecularmassof43kDawhichishigherthan

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Journal of Biomedicine and Biotechnology5
Table 2: Comparion of characteristics of Xylaria hypoxylon protease with other fungal proteases.
SpeciesPhylumN-terminal sequence
Chromatography
behavior
Molecular
mass
(kDa)
pH
optimum
Temperature
optimum
(◦C)
Xylaria hypoxylon (This study) Ascomycota 1 HYTEL LSQVV 10
Adsorbed on
DEAE-,
CM-cellulose,
and Q-Sepharose
436–8 60
Aspergillus clavatus [34]Ascomycota 1 ALTTQ SGAPW GLGSI 15
Adsorbed on
CM-Sepharose
328.550
Aspergillus nidulans [35] Ascomycota——195.5 65
Cordyceps sinensis [11]Ascomycota X DNLMR AVGAL LR X
Adsorbed on
HiTrap Q XL
43 9.5 30
Helvella lacunosa [14] Ascomycota 1 ANVVQ WPVPC 10
Adsorbed on
DEAE-,
CM-cellulose,
and Q-Sepharose
33.511 65
Agaricus bisporus [9, 36]Basidiomycota 1 MHFSL SFATL ALLVA 15
Adsorbed on
DEAE-, and
CM-cellulose
276.5–11.5—
Armillariella mellea [10]Basidiomycota1 XXYNG XTXSR QTTLV 15
Adsorbed on
DEAE-cellulose
557 55
Coprinus 7N [37] Basidiomycota—
Adsorbed on
DEAE-Sepharose
338.5 37
Grifola frondosa [38] Basidiomycota 1 AQTNA PWGLA 10—209-10—
Hypsizigus marmoreus [26]Basidiomycota1 VTQTN APWGL ARLSQ 15
Adsorbed on
CM-cellulose;
Unadsorbed on
DEAE-cellulose
287.5 50
Pleurotus citrinopileatus [18]Basidiomycota 1 VCQCN APWGL 10
Adsorbed on
CM-cellulose;
Unadsorbed on
DEAE-cellulose
and Q-Sepharose
2810 50
Pleurotus eryngii [16]Basidiomycota 1 GPQFP EA 7
Adsorbed on
Affi-gel Blue gel
and
CM-Sepharose;
Unadsorbed on
DEAE-cellulose
11.55.045
—: no data available. Identical corresponding amino acid residues are underscored.
that of H. lacunose (33.5kDa). 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 18kDa to 126kDa [39]. X. hypoxylon protease exhibits
an intermediate molecular mass (43kDa), which is the same
as C. sinensis protease, but higher than those of proteases
from P. eryngii (11.5kDa) [16], T. saponaceum (about
18kDa) [19], A. mellea (18.5kDa) [10], G. frondosa (20kDa)
[38], P. citrinopileatus (28kDa) [18], and H. lacunose
(33.5kDa) [14] and lower than P. ostreatus (75kDa) [17].