Botanical Studies (2008) 49: 119-125.
Chuian-Fu KEN and Hsueh-Tai CHEN contributed equally
to this paper.
Corresponding author: E-mail: B0220@mail.ntou.edu.tw;
Phone: 886-2-24622192 ext. 5513; Fax: 886-2-24622320.
Biochemical characterization of a catalase from Antrodia
camphorata: expression in Escherichia coli and enzyme
, 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. 188.8.131.52; H
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).