Botanical Studies (2008) 49: 119-125.
Chuian-Fu KEN and Hsueh-Tai CHEN contributed equally
to this paper.
Corresponding author: E-mail:;
Phone: 886-2-24622192 ext. 5513; Fax: 886-2-24622320.
Biochemical characterization of a catalase from Antrodia
camphorata: expression in Escherichia coli and enzyme
Chuian-Fu KEN
, Hsueh-Tai CHEN
, Reny-Chang CHANG
, and Chi-Tsai LIN
Institute of Bioscience and Biotechnology and
Center for Marine Bioscience and Biotechnology, National Taiwan Ocean
University, Keelung 202, 2 Pei-Ning Rd, Taiwan
Institute of Biotechnology, National Changhua University of Education, Changhua 500, Taiwan
Department of Marine Biotechnology, National Kaohsiung Marine University, Taiwan
(Received July 31, 2007; Accepted November 13, 2007)
Catalase plays important roles in
antioxidation and cell signaling. One cDNA (1794 bp,
DQ021914) encoding the putative catalase was cloned from Antrodia camphorata. The deduced amino acid
sequence is conserved among the reported catalases. To characterize the A. camphorata catalase, the coding
region was subcloned into a vector pET-20b(+) and transformed into E. coli. The recombinant 6His-tagged
catalase was expressed and purified by Ni
-nitrilotriacetic acid sepharose. The purified enzyme showed one
band by 10% SDS-PAGE. The enzyme retained 50% activity at 60¢XC for 14 min. The enzyme was active
under a broad pH range from 7.8 to 11.2. The enzyme showed 67% activity after 4 h of incubation at 37¢XC
with trypsin. It was also proven able to protect intact supercoiled plasmid DNA from ¡POH-induced nicking.
Study of the enzyme¡¦s properties may prove beneficial for future applications in medicine or health food.
Keywords: Antrodia camphorata; Catalase; Expression.
Medicinal mushrooms have a long history of use
in folk medicine in Taiwan. Antrodia camphorata (A.
camphorata) is a unique medicinal mushroom species
found only in the forests of Taiwan which traditionally has
been used as a remedy for drug intoxication, abdominal
pain, and cancer. Antrodia camphorata has been shown to
exhibit antioxidative (Song and Yen, 2002), vasorelaxative
(Wang et al., 2003), and anti-inflammatory (Shen et
al., 2004) effects. Although A. camphorata shows
physiological activities with great potential for medical
applications, few scientific studies of it have appeared.
Recently, we established an EST (expressed sequence tag)
from fruiting bodies of A. camphorata in order to search
for physiologically active components for medicinal use.
Catalase (E.C.; H
: H
-oxidoreductase) is
one of the important antioxidative enzymes with a heme
structure that can catalyze the decomposition of 2 H
to 2 H
O and O
. It is found in most aerobic organisms,
including prokaryotes and eukaryotes (Kashiwagi et al.,
1997; Klotz et al., 1997), and it protects cells against the
toxic effects of reactive oxygen species. Catalase is usually
formed by four identical subunits (Wu and Shah, 1995) of
50-60 kDa (Klotz et al., 1997; Garcia et al., 2000). Though
some catalase sequences have been reported, many queries
regarding catalase function and structure remain unsolved.
Catalases from mammals have a binding site for NADPH,
but its function is not entirely known (Kirkman et al.,
1999). Catalases from E. coli-like hydroperoxidase do
not bind to NADPH but have an extra "flavodoxin-like"
domain of unknown function (Bravo et al., 1997).
There are three families of catalases: (1) A novel
manganese catalase that uses Mn as a cofactor in the
active center without a heme structure (Barynin et al.,
2001). It is 170-210 kDa in size with a subunit of 50-65
kDa and has been identified in bacteria, plants, fungi, and
animals. It may form unusual oligomeric structures. (2)
Bifunctional catalases with molecular masses that vary
from 120-340 kDa. In general, they are homodimers
(Obinger et al., 1997; Nagy et al., 1997). These catalases
have a heme structure that contains both catalase and
peroxidase activity, and are found mostly in fungi (Fraaije
et al., 1996). (3) The typical monofuctional catalase,
most of which are homotetramers, 200-400 kDa in size
with four prosthetic haem groups (Hirasawa et al., 1989;
Sheptovitsky and Brudvig, 1996).
Botanical Studies, Vol. 49, 2008
Lots of fungi have catalases that have been identified
and showed different regulation (Navarro et al., 1996;
Kawasaki et al., 1997), but no catalases have been
reported from A. camphorata. Previously, we cloned and
characterized a themostable superoxide dismutase (Liau
et al., 2007), a 2-Cys peroxiredoxin (Huang et al., 2007),
and a 1-Cys peroxiredoxin (Wen et al., 2007) based on the
established EST from A. camphorata. Gems and McElwee
reported that the pro-longevity genes include those
encoding antioxidant enzymes that can restore misfolded
proteins to their active conformations (Gems and
McElwee, 2005). We continue to search for antioxidant
enzymes in A. camphorata with potential anti-oxidative
and anti-inflammation applications. Here, we report the
cloning of an antioxidant enzyme, catalase cDNA (Ac-
catalase), from A. camphorata on the basis of EST.
Understanding the properties of this Ac-catalase would be
beneficial for its applications in medicine or as a health
food. Thus, the coding region of the Ac-catalase gene was
introduced into an E. coli expression system. The active
enzyme was purified and its properties studied.
mAteriALS AND methoDS
Antrodia camphorata
Fruiting bodies of A. camphorata which had grown
in the hay of C. kanehirai were obtained from Asian-Bio
Company (
total rNA preparation and cDNA synthesis
Total RNA was prepared from fresh fruiting bodies (wet
weight 5 g) using Straight A¡¦s mRNA Isolation System
(Novagen, USA). The total RNA (22 £gg) was obtained.
Three £gg of the mRNA was used for cDNA synthesis
using a ZAP-cDNA kit from Stratagene (La Jolla, CA).
isolation of Ac-catalase cDNA
We previously established an EST database from
fruiting bodies of A. camphorata and sequenced all clones
with insert sizes greater than 0.2 kb (data not shown).
The identity of a catalase cDNA clone was assigned by
comparing the inferred amino acid sequence in various
databases using the basic local alignment search tool
Subcloning of Ac-catalase cDNA
The coding region of the Ac-catalase cDNA was
amplified using two gene-specific primers. The 5¡¦
upstream primer contains the NdeI recognition site (5¡¦
C AT AT G CCC TCT AAA CAG GTT T 3¡¦) and the 3¡¦
downstream primer contains the NotI recognition site (5¡¦
Using 0.1 £gg of A. camphorata cDNA as a template, and
10 pmole of each 5¡¦ upstream and 3¡¦ downstream primers,
a 1.5 kb fragment was amplified by PCR. The fragment
was ligated into pCR4.0 and transformed into E. coli
TOPO10. Plasmid DNA was isolated from the clone and
digested with NdeI and NotI. The digestion products were
separated on a 0.8% agarose gel. The 1.5 kb insert DNA
was gel purified and subcloned into NdeI and NotI sites
of pET-20b(+) vector (Novagen, USA). The recombinant
DNA was then transformed into E. coli BL21(DE3)pLysS.
expression and purification of the recombinant
The transformed E. coli containing the Ac-catalase
was grown at 30¢XC in 200 mL of Luria Bertani medium
containing 50 £gg/mL ampicillin until A
reached 0.6.
Protein expression was induced by addition of isopropyl
£]-D-thiogalactopyranoside (IPTG) to a final concentration
of 1 mM. The culture was incubated for an additional 4
h at 80 rpm, and then the bacterial cells were harvested
by centrifugation. Cells were suspended in 2 mL of PBS
containing 1% glycerol and 1 g glass beads. The content
was vortexed for 5 min and centrifuged at 10,000 g for 10
min. The extraction procedure was repeated thrice, and
the supernatants were combined. The Ac-catalase was
purified by Ni-NTA affinity chromatography as per the
manufacturer¡¦s instruction (Qiagen) and then dialyzed as
described before (Ken et al., 2005). The dialyzed sample
was either used directly for analysis or stored at -20¢XC
until use.
Protein concentration measurement
Protein concentration was determined by a Bio-Rad
Protein Assay Kit (Richmond, CA) using bovine serum
albumin as a reference standard.
Ac-catalase activity assay (ferrithiocyanate
The recombinant Ac-catalase (0.17 £gg protein) was
incubated in 45-47 £gL buffer (1 mM DTT in 0.33 ¡Ñ PBS
containing 5% glycerol) for 2 min at room temperature.
The reaction was initiated by addition of 3-5 £gL 1 mM
(60-100 £gM). At 0 and 10 min reaction times, 50
£gL of the reaction mixture was taken, and 20 £gL of 26%
trichloroacetic acid was added to stop the reaction. The
peroxidase activity was determined by following
disappearance of the peroxide substrate (the total peroxide,
at the beginning of the reaction minus the remaining
amount of the 10 min). The remaining peroxide content
was determined as a red-colored ferrithiocyanate complex
formed by addition of 20 £gL 10 mM Fe(II)(NH
and 10 £gL 2.5 M KSCN to the 70 £gL reaction mixture,
which was quantified by measurement of the absorbance at
475 nm (Thurman et al., 1972).
Ac-catalase activity assay on the 10% native
Duplicate samples containing the Ac-catalase were
electrophoresed on a 10% native gel for 2.5 h at 100 V.
The duplicate lanes were sliced into two parts. One part
was stained for catalase activity, the other for protein
staining. For catalase activity staining, the gel was
KEN et al. ¡X Characterization of Ac-catalase
incubated in 0.005% (v/v) hydrogen peroxide for 10 min,
then rinsed with water, followed by an immersion in 1%
(w/v) FeCl
and 1% (w/v) K
with gentle shaking,
the gel became uniformly green except at the position
of Ac-catalase, which showed achromatic zones which
revealed that catalase had prevented the formation of the
insoluble green precipitation.
enzyme characterization
The enzyme sample was tested for stability under
various conditions. Aliquots of the Ac-catalase sample
were treated as follows: (1) Thermal stability. Enzyme
sample was heated to 60¢XC for 2, 4, 8 or 16 min. (2) pH
stability. Enzyme sample was adjusted to desired pH by
adding a half volume of buffer at different pHs: 0.2 M
citrate buffer (pH 2.2, 5.4), 0.2 M Tris-HCl buffer (pH 7.8,
or 9.0), or 0.2 M glycine-NaOH buffer (pH 10.4, or 11.2).
Each sample was incubated at 37¢XC for 1 h. (3) SDS effect.
SDS, a denaturing reagent, was added to the enzyme
sample to the levels of 0.5, 1, or 2% and incubated at 37¢XC
for 0.5 h. (4) Imidazole effect. During protein purification,
the Ac-catalase enzyme was eluted with imidazole.
Therefore, the effect of imidazole on protein activity was
examined. Imidazole was added to the enzyme sample
to the levels of 0.2, 0.4, or 0.8 M and incubated at 37
¢XC for 1 h. (5) Proteolytic susceptibility. The enzyme
was incubated with one-tenth its weight of trypsin or
chymotrypsin at pH 8.0, 37¢XC for a period of 60, 120 or
240 min. In the chymotrypsin digestion, CaCl
was added
to 5 mM. Aliquots were removed at various time intervals
for analysis. After each treatment, two-thirds of the sample
was electrophoresed onto a 10% native polyacrylamide
gel electrophoresis (PAGE) to determine any changes in
protein levels. The other one-third of the sample was used
for ferrithiocyanate assay to determine any changes in
enzyme activity.
thiol mixed-function oxidation (mFo) assay
Ac-catalase-dependent inactivation of DNA cleavage
evaluated by MFO assay (Kwon et al., 1993). A reaction
mixture (15 £gL) containing 40 £gM FeCl
, 10 mM
dithiothreitol (DTT), 25 mM HEPES (pH 7.0), and 0.69
£gg of pUC19 plasmid DNA was incubated with or without
the Ac-catalase protein (0.17 or 0.34 £gg) at 25¢XC for 0.5 or
1 h. After incubation, nicking of the supercoiled plasmids
by the MFO was evaluated on 1% agarose gels stained
with ethidium bromide.
cloning and characterization of a cDNA
encoding Ac-catalase
By sequencing about 20,000 A. camphorata cDNA
clones, these nucleotide sequences and the inferred amino
acid sequences were compared to the NCBI (www.ncbi. data banks by using the FASTN and FASTP
programs. We found a putative Ac-catalase cDNA clone
was identified by sequence homology to the published
Figure 1. Optimal alignment
of the am ino a cid se quenc es
of Ac-cata las e wi th sel ected
s e q ue nc e s . T h e c a t a l y t i c
am ino a cids of Ac-c ata las e,
including H
, N
, and Y
are i ndi cated with a steris ks.
T h e c a ta l y t ic do m a in w a s
between F
to G
, a nd t he
h ae m -b in di ng do m ai n wa s
between R
to H
. Bo th
are boxed. Ac-ca tala se (thi s
study), yeas t (Saccharomyces
cerevisiae) , Hu (H o m o
sapiens), Mus (Mus musculus),
and Ara (Arabidopsis thaliana).
Ide ntic al a mi no ac ids in a ll
s equences are s haded black,
and conservative replacements
are shaded gray.
Botanical Studies, Vol. 49, 2008
catalases in NCBI data bank. The coding region of Ac-
catalase cDNA was 1,527 bp that encodes a protein of
509 amino acid residues with calculated molecular mass
of 57 kDa (EMBL accession no. DQ021914). Figure 1
shows the optimal alignment of the amino acid sequences
of Ac-catalase with four selected sequences. This Ac-
catalase shared 55% identity and 71% similarity with
yeast catalase (Saccharomyces cerevisiae, accession no.
CAA31443.1), shared 54% identity and 67% similarity
with human catalase (Homo sapiens, NP001743.1), shared
54% identity and 66% similarity with mouse catalase
(Mus musculus, NP033934.1), shared 47% identity
and 64% similarity with Arabidopsis thaliana catalase
(NP001031791.1) by using the BLAST 2 SEQUENCES
program (Tatusova and Madden, 1999). All the catalytic
amino acids, including H
, N
and Y
, are identified in
each selected sequence and are indicated by an asterisk in
Figure 1. The active site was between F
to G
, and the
haem-binding site was between R
to H
. Both are also
highly conserved and boxed in Figure 1. As compared
with yeast catalase, the crystal structure of which was
determined by X-ray diffraction (Berthet et al., 1997), the
amino acids from Ala
to Tyr
of this Ac-catalase appear
conserved as yeast catalase. This conserved core was
identified to be a typical catalase because it contains the
essential distal catalytic domain
and the proximal haem-
ligand domain as described in a previous report (Zamocky
and Koller, 1999).
expression and purification of the recombinant
The coding region of Ac-catalase (1.5 kb) was
amplified by PCR and subcloned into an expression
vector, pET-20b(+) as described in Materials and Methods.
Figure 2. E xpr es s i on an d p uri fi ca t io n o f re c om bi na nt
Ac-catalase in E. coli. Fifteen £gL (loading buffer with
mercaptoethanol and without boiling) of each fraction wa s
loaded into each lane of the 10% SDS-PAGE. Lane 1, crude
extract from E. coli expressing Ac-catalas e; 2, flow-through
proteins from the Ni-NTA column; 3, purified Ac-catalase eluted
from Ni-NTA column. A, Coomassie blue-stained; B, Activity
stained. Molecular masses (in kDa) of standards are shown at
left. Arrow indicates the target protein.
Figure 3. Effect of temperature on the purified Ac-catalase. The
enzyme sample was heated at 60¢XC for various time intervals.
Aliquots of the sample were taken at 0, 2, 4, 8 or 16 min and
analyzed by a 10 % native-PAGE or assayed for enzyme activity.
A, Staining for protein (0.82 £gg protein/ lane) after separation on
a 10% native-PAGE; B, Plot of thermal inactivation kinetics. Ac-
catalase activity assay (0.17 £gg protein/ time interval). E
and E
are original activity and residual activity after being heated for
different time intervals. Data are means of three experiments.
Positive clones were verified by DNA sequence analysis.
The recombinant Ac-catalase was expressed, and the
proteins were analyzed by a 10% SDS-PAGE in the
presence a reducing agent without boiling (Figure 2). The
recombinant Ac-catalase was expressed as a 6His-tagged
fusion protein and was purified by affinity chromatography
with nickel chelating Sepharose. A band with a molecular
mass of ~114 kDa (expected size of Ac-catalase dimer)
was detected in Ni-NTA eluted fractions by SDS-PAGE
(Figure 2A, B, lane 3). The Ni-NTA eluted fractions
containing pure protein were pooled and characterized
further. The yield of the purified 6His-tagged Ac-catalase
was 80 £gg from 200 mL of culture. Functional Ac-catalase
was detected by activity assay as described below.
characterization of the purified Ac-catalase
To examine the heat stability, the purified Ac-catalase
was heat-treated as described in Materials and Methods
and then analyzed by 10% native gel or activity assay. The
enzyme appeared to be heat stable. Approximately 50% of
the Ac-catalase activity was lost at 60¢XC for 14 min (Figure
3). The Ac-catalase was stable in a broad range of pH from
7.8 to 11.2 as shown in Figure 4A. The enzyme retained
20% activity in 2% SDS (Figure 4B). The enzyme retained
about 38% activity in 0.8 M imidazole (Figure 4C). The
KEN et al. ¡X Characterization of Ac-catalase
enzyme showed 70% activity after 4 h of incubation at 37
¢XC with one-tenth its weight of trypsin (Figure 4D). The
enzyme showed 20% activity after 3 h of incubation at 37¢X
C with chymotrypsin (data not shown).
The Ac-catalase as shown in Figure 2 contains
44 potential trypsin cleavage sites and 53 potential
chymotrypsin-high specificity (C-term to [FYW], not
before P) cleavage sites. However, the enzyme appeared
susceptible to chymotrypsin but resistant to digestion by
trypsin, even at a high enzyme/substrate (w/w) ratio of
1/10 (Figure 4D).
To test peroxidase activity of the Ac-catalase, we
used the assay of plasmid DNA protection against ROS
generated by the Fenton reaction induced by this metal-
catalyzed system. In the presence of Ac-catalase, the
alteration of the plasmid DNA from the supercoiled to
the nicked form was prevented. The inhibition was dose-
dependent (Figure 5). The assay employs a system that
generates hydroxyl radicals to damage DNA. In the
absence of Ac-catalase, the hydroxyl radicals produced
in the system caused nicking of the DNA, as evidenced
by a shift in gel mobility of the supercoiled plasmid. The
addition of purified Ac-catalase to the MFO system at
concentrations of 0.17 £gg and 0.34 £gg prevented nicking of
the supercoiled DNA, demonstrating antioxidant activity
of Ac-catalase.
Figure 5. Functional activity of recombinant Ac-catalase. The
reaction was performed in a mixture consisting of 40 £gM FeCl
10 mM DTT, 25 mM HEPES (pH 7.0) and 0.69 £gg of pUC19
plasmid DNA. The reaction was stopped after incubation for 0.5
h (A), 1 h (B), and the plasmid DNA was analysed on ethidium
bromide stained agarose gel. Lane 1, only DNA; 2, Fe
+ DTT;
3, Fe
+ DTT + 0.17 £gg Ac-catalase; 4, Fe
+ DTT + 0.34 £gg
Ac-catalase; 5, F e
+ DTT + 0.34 £g g BSA. SF, supercoiled
form; NF, nicked form.
F igu re 4. E ffect of pH, SDS, imidazole and trypsin on the
purified Ac-catalase. A, The enzyme samples were incubated
with different pH buffer at 37
C for 1 h and then assayed for
Ac-catalase activity (0.17 £gg protein/ pH point); B, The enzyme
samples were incubated with various concentration of SDS at
37¢XC for 30 min and then checked for activity (0.17 £gg protein/
SDS level); C, The enzyme samples were incubated with various
concentration of imidazole at 37¢XC for 1 h and then checked for
Ac-catalase activity (0.17 £gg protein / imidazole level); D, The
enzyme samples were incubated with trypsin at 37¢XC for various
times and then checked for Ac-catalase activity (0.17 £gg protein
/ time interval). Data are means of three experiments.
Heat stability and imidazole effects were tested
because the information is useful for developing enzyme
purification protocols. Protease tests were useful in
understanding the effect of the digestive enzymes on the
Ac-catalase and its suitability as health food.
The fruiting body of A. camphorata is known in Taiwan
for treating cancer and inflammation, yet little is known
about its biological effects. This Ac-catalase may be also
one of the important physiological components in A.
camphorata responsible for its medicinal efficacy. We are
particularly interested in the antioxidant effects of the A.
camphorata. To aid us in understanding them, we cloned
the antioxidant enzyme catalase from A. camphorata,
expressed in E. coli and characterized it. This enzyme
appears to be stable under various conditions. The most
significant finding in this paper is that the recombinant Ac-
catalase can enzymatically detoxify ¡POH. Using a function
oxidation (MFO) system to generate ¡POH in vitro, we have
demonstrated the ability of Ac-catalase to protect intact
supercoiled plasmid DNA from ¡POH-induced nicking.
Further investigation is required to evaluate the possible
medical applications of Ac-catalase, including the removal
of peroxide (especially hydroxyl radicals) from wounded
tissue to promote healing or its use as a health food.
Acknowledgments. This work was supported by the
National Science Council of the Republic of China under
Grant NSC95-2313-B-019-007 to C-T. L.
Botanical Studies, Vol. 49, 2008
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