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T. Nakamura et al. / Biochemical and Biophysical Research Communications 394 (2010) 470–475
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enzymes. In the present study, we focused on two vanillin-respon-
sive ALDHs, PcALDH1 and PcALDH2, from P. chrysosporium. We
investigated their catalytic properties using heterologously ex-
pressed recombinant enzymes. The recombinant ALDHs showed
catalytic activity against several aryl-aldehydes including vanillin,
suggesting that PcALDH1 and PcALDH2 have key roles in lignin
degradation. To the best of our knowledge, this is the first report
on functional characterization of ALDHs from basidiomycetes.
Kit (QIAGEN) and sequenced with a CEQTM 2000XL DNA sequenc-
ing system (Beckman Coulter) after the sequencing reaction using
the GenomeLab DTCS Quick Start Kit (Beckman Coulter). The
nucleotide and deduced amino acid sequences were analyzed
using BLAST and BLASTP search programs.
Expression of recombinant protein and preparation of cell-free ex-
tracts. Expression vectors were constructed using the pET-22
expression system. The coding regions for ALDHs were amplified
from plasmid DNAs. cDNA fragments were amplified using specific
primers that included restriction enzyme-recognition sequences
(NdeI, upstream primer; HindIII, downstream primer). The PCR
Materials and methods
mixture (50 lL) contained 0.1 lg plasmid DNA, 2.5 U Pyrobest
Chemicals. All chemicals were of analytical grade and were pur-
chased from Wako Pure Chemicals.
Organism and culture conditions. P. chrysosporium (ATCC 34541)
was grown from hyphae-inocula at 37 °C in a stationary culture
(20 mL medium) under air. The medium (pH 4.5) used in this study
contained 28 mM D-glucose and 1.2 mM ammonium tartrate (low
nitrogen; LN) as the carbon and nitrogen sources, respectively, as
described previously [11,15]. After a 2-day preincubation, vanillin
DNA polymerase (TaKaRa), 1Â Pyrobest buffer II, 100 pmol each
of the upstream and downstream primers, and dNTPs at a final
concentration of 200 lM. The PCR conditions were as follows:
94 °C for 3 min followed by 94 °C for 20 s, 63 °C for 30 s, and
72 °C for 2 min for 28 cycles, and then a final 3-min extension at
72 °C. The amplified cDNA fragments were digested with NdeI
and HindIII, and ligated into the pET-22 vector that had been trea-
ted with the same endonucleases. The recombinant plasmids con-
taining the ALDH coding regions were transferred to into E. coli
BL21 (DE3) pLysS (TaKaRa). The transformants were grown in TB
(4-hydroxy-3-methoxybenzaldehyde) in acetonitrile (80
added to a final concentration of 2 mM. For the control culture,
only acetonitrile (80 L) was added.
lL) was
l
broth containing ampicillin (10
lg/mL) with shaking (180 rpm) at
Proteomic analysis. The mycelial mat was incubated with vanil-
lin for 24 h at 37 °C. The mat was separated from the medium by
filtration, washed with chilled water, frozen under liquid nitrogen,
and ground into a fine powder using a mortar and pestle. Intracel-
lular proteins were extracted using SDS buffer (4% SDS, 2% DTT,
20% glycerol, 20 mM PMSF, and 100 mM Tris–HCl) [16]. After 2 h
incubation at room temperature, the extracted proteins were pre-
cipitated by addition of four volumes of cold acetone (À20 °C). The
resultant pellet was solubilized in urea buffer containing 7 M urea,
2 M thiourea, 4% CHAPS, 0.12% DeStreak reagent (GE Healthcare),
0.5% IPG buffer (pH 3–10 NL), and a trace amount of bromophenol
blue.
Two-dimensional gel electrophoresis (2-DE), in-gel tryptic
digestion, and MALDI-TOF-MS analysis were performed as de-
scribed previously [11]. Identification of proteins by peptide mass
fingerprinting analysis was carried out using MASCOT search (Ma-
trix Science) against a P. chrysosporium in silico protein database
generated from genomic annotation data, which was used in com-
bination with our own annotation data.
37 °C until the OD600 reached 0.5. Isopropyl-1-thio-b-
D-galactopy-
ranoside (IPTG) was then added to the culture at a final concentra-
tion of 0.5 mM, and bacterial cells were incubated for 24 h with
shaking (180 rpm) at 27 °C. Cell-free extracts were prepared as fol-
lows: bacterial cells were centrifuged at 7000g for 5 min at 4 °C.
The cells in the pellet were washed with 50 mM HEPES buffer
(pH 7.4), and resuspended in 30 mL HEPES buffer. Cells were dis-
rupted by freeze–thaw cycles and sonication, and the mixture
was centrifuged at 15,000g for 20 min at 4 °C. The supernatants
were used as cell-free extracts.
Purification of recombinant protein. Recombinant proteins were
purified from cell-free extracts using a histidine-tag (His-tag)-
based purification procedure. Extracts were applied to a His Gravi-
Trap (GE Healthcare) column that had been equilibrated with bind-
ing buffer containing 20 mM imidazole. Recombinant proteins
containing a six-repeat histidine sequence at the C-terminal bound
to the column. Unbound proteins were removed from the column
by washing with binding buffer. The bound recombinant proteins
were eluted from the column using elution buffer containing
500 mM imidazole. Eluted recombinant proteins were separated
by electrophoresis on a 12.5% SDS–polyacrylamide gel and visual-
ized with SYPRO Red protein gel stain (TaKaRa). After purification,
the elution buffer was replaced with storage buffer using a PD-10
desalting column (GE Healthcare). The purified protein solutions
were stored at À80 °C until further analyses.
Isolation and cloning of cDNA encoding PcALDH1 and PcALDH2. A
mycelial mat of P. chrysosporium was incubated with 2 mM vanillin
for 1 day, and then total RNA was isolated using an RNeasy Plant
Mini Kit (QIAGEN). The reverse transcription reaction (total vol-
ume, 20
(Invitrogen) in 1Â RT buffer containing 10 mM dithiothreitol,
0.5 mM dNTPs, 0.5 g total RNA, and 10 pmol oligo(dT)18 primer.
lL) contained 200 U SuperScript III reverse transcriptase
l
Enzyme assay. Protein concentrations were determined using
the DC Protein Assay Kit (Bio-Rad), which is based on the Lowry
method. Enzymatic activity of recombinant ALDH (rALDH) was
measured in a 1 mL reaction mixture consisting of recombinant
The reaction mixture was incubated at 37 °C for 30 min, and then
at 42 °C for 60 min. Target cDNA fragments were amplified with
1 U Phusion High-Fidelity DNA Polymerase (NEB) in a 50
tion mixture containing 1Â Phusion HF Buffer, 200 M dNTPs,
L template cDNA, and 100 pmol each of the upstream and
lL reac-
l
protein (100
(20% w/v), and 25 mM glycine–NaOH buffer (pH 10.0). The reaction
was initiated by the addition of 10
L tNAD+ solution. The rALDH
lg), substrates (100 l lM), glycerol
M), tNAD+ (200
2
l
downstream primers. Primer sequences are shown in Table S1.
The PCR conditions were as follows: 98 °C for 30 s followed by
98 °C for 10 s, 60 °C for 30 s, and 72 °C for 60 s for 30 cycles, and
then a final 3-min extension at 72 °C. The PCR products were sep-
arated on a 1.5% agarose gel, visualized with a UV transilluminator,
and purified using QIAquick PCR Purification Kit (QIAGEN). After
purification, PCR products were treated with 1 U Ex Taq (TaKaRa)
l
activity was determined as the initial tNADH production rates by
monitoring the formation of tNADH at 400 nm using a UV–vis
spectrophotometer.
Results and discussion
in 1Â Ex Taq buffer and 200
lM dNTPs at 72 °C for 30 min, cloned
into a pGEM-T Easy vector (Promega), and transformed into com-
petent cells ofEscherichia coli strain JM109. Positive clones were se-
lected by blue/white screening and colony-direct PCR. Plasmids
were isolated from 10 mL LB broth using a QIAprep Spin Miniprep
Proteomic differential display analysis
Expression profiles of cytosolic proteins were analyzed using a
proteomic differential display technique. As shown in Fig. 1,