Biosci. Biotechnol. Biochem., 75 (1), 159–161, 2011
Note
Characterization of an Aspergillus oryzae Cysteinyl Dipeptidase Expressed
in Escherichia coli
Ryota HATTORI,1 Mayumi MATSUSHITA-MORITA,1 Junichiro MARUI,1 Sawaki TADA,1
Satoshi SUZUKI,1 Ikuyo FURUKAWA,1 Youhei YAMAGATA,2 Hitoshi AMANO,3 Hiroki ISHIDA,4
y
1;
Michio TAKEUCHI,2 and Ken-Ichi KUSUMOTO
1National Food Research Institute, National Agriculture and Food Research Organization (NARO),
2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
2Department of Agriscience and Bioscience, Tokyo University of Agriculture and Technology,
3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan
3Gifu R&D Center, Amano Enzyme Inc., 1-6 Technoplaza, Kakamigahara, Gifu 509-0109, Japan
4Research Institute, Gekkeikan Sake Company Ltd., 101 Simotobakoyanagi-cho, Fushimi-ku, Kyoto 612-8385, Japan
Received August 20, 2010; Accepted October 19, 2010; Online Publication, January 7, 2011
Cysteinyl dipeptidase from Aspergillus oryzae (CdpA)
was produced in Escherichia coli and purified. The
enzyme showed activity specific toward cysteine-
containing dipeptides, but its substrate specificity was
distinct from those of other cysteinyl dipeptidases of the
M20 family. It was optimally active at pH 7–8 and
stable at pH 6–9 and at up to 40 ꢀC.
cdpA cDNA was PCR amplified using primers 50-
GCTCGGTACCCTCGAGATGGCACCACAGCTGG-
AACCATTT-30 and 50-GCAGAGATTACCTATCTA-
TGCCGCCACCATGGGCTCTTCT-30. The underlined
sequences are specific to the multi cloning site of pCold
I. The cDNA pool synthesized from A. oryzae grown in
YPD at 30 ꢀC for 20 h7) was used as template. The
amplified fragment was cloned into pCold I using an In-
Fusion Advantage PCR Cloning Kit (Takara Bio). Cells
of E. coli BL21 harboring pCold-cdpA were harvested
by centrifugation, washed with buffer A (20 mM Tris–
HCl buffer pH 7.5 containing 300 mM NaCl and 20 mM
imidazole), and sonicated in the same buffer. The lysate
was centrifuged, and the supernatant was absorbed into
Ni-IMAC gel (Bio-Rad Japan, Tokyo, Japan). The resin
was washed with buffer A, followed by elution of the
protein with buffer B (20 mM Tris–HCl buffer pH 7.5
containing 300 mM NaCl and 100 mM imidazole). The
purified protein was collected, concentrated, and desalted.
The protein was confirmed to be CdpA by peptide mass
fingerprinting (data not shown). SDS–PAGE analysis
using 10% w/v polyacrylamide gels revealed that the
molecular mass of the monomeric CdpA was 53 kDa.
This is consistent with the size of the monomer as
predicted from the cDNA sequence. The molecular mass
of purified CdpA was determined using a Superose 12
gel filtration column (column size, 1:0 ꢁ 30 cm; flow
rate, 0.5 mL/min) (GE Healthcare, Buckinghamshire,
UK) pre-equilibrated with buffer D (20 mM Tris–HCl
pH 7.5, 150 mM NaCl). A gel filtration calibration kit
(GE Healthcare) was used for calibration. The gel
filtration chromatography profile of CdpA showed a
single peak at 109 kDa, suggesting that purified CdpA
exists as a homodimer.
Key words: Aspergillus oryzae; cysteinyl dipeptidase;
metallopeptidase; characterization
Glutathione (gamma-glutamyl-cysteinylglycine) is a
major thiol compound that plays various roles in vivo.1)
Glutathione metabolism has been investigated because
of its physiological importance, and it is thought to be
conducted via the gamma-glutamyl cycle. The degrada-
tion of cysteinylglycine (Cys-Gly) is one of the steps in
glutathione metabolism.1) To date, several enzymes have
been reported to have hydrolyzing activity toward Cys-
Gly. For example, Dug1p, which belongs to the metal-
lopeptidase M20 family and has Cys-Gly hydrolyzing
activity, was recently found in Saccharomyces cerevi-
siae.2) Other enzymes that are members of the M1, M17,
and M19 families also have this function.3–6) Few studies
have examined the degradation of glutathione, and there
are no reports of Cys-Gly dipeptidase in filamentous
fungi. In this study, we found that the CdpA of A. oryzae
was active toward cysteine-containing dipeptides, and
that its characteristics were distinct from those of other
cysteinyl dipeptidases.
Using a BLAST search against the genome database
view/AO), we found an ortholog of DUG1 and desig-
nated it cdpA (cysteinyl dipeptidase in A. oryzae)
(AO090020000015). The length of the coding sequence
was 1,437 bp, encoding 478 amino acids with a
calculated molecular mass of 52.9 kDa. Expression
plasmid pCold I (Takara Bio, Otsu, Japan) was used
for the production of CdpA carrying an N-terminal His6
tag in E. coli strain BL21 cells (Takara Bio). Expression
plasmid pCold-cdpA was constructed as follows:
CdpA activity was determined using various peptide
substrates. For the standard reaction, 1.5 mU CdpA
(0.5 mg in a reaction volume of 150 mL) and 1 mM Cys-
Gly were incubated in 20 mM Tris–HCl pH 7.0, for
15 min at 30 ꢀC. The amount of L-cysteine was measured
as described previously.8) L-Cysteine concentrations
(measured by the optical density at 560 nm) were
y
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