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Supplementary material
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Table S1. Oligonucleotide primers used in this study
Primer Nucleotide sequencea (5´ → 3´)
Remarks
KH45
For
CCATGGTGAAAGTAGTCCTAGTGCTA
amplification
of
apu
gene,
of
apu
gene,
containing NcoI
KH46
CCATGGTTTCAGGAACCTCCTTACGGT
For
amplification
containing NcoI
KH89
GGGCTGCGTTATCCAGATT
For disruption of apu, specific for
upstream of apu
KH90
TGGAGGATCCTCGTCACTGCTAGGTAGTC
For disruption of apu, specific for
CAG
upstream of apu, introducing BamH1
and sequence of downstream of apu
KH91
ACGAGGATCCTCCAAAATAACTCTAACGA
For disruption of apu, specific for
AAGCA
downstream of apu, introducing BamH1
and sequence for upstream of apu
KH92
TTCGAAGGATGGAGGACATC
For disruption of apu, specific for
downstream of apu
KH71
KH72
PyrEF
GGATCCAATGAAACTACTTTCCCTGATAG
For amplification of pyrE from S.
ATAA
solfataricus P2, introducing BamH1
GGATCCCTACTTTTCAACATTCTTCACCAA
For amplification of pyrE from S.
A
solfataricus P2, introducing BamH1
TTTCGTTTTAACATCAGGTAAGG
Specific for internal region of pyrE from
S. solfataricus P2
PyrER TGTGAAGCCCCTTCTTGTCT
Specific for internal region of pyrE from
S. solfataricus P2
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a
5
PCR are underlined.
The restriction enzyme sites are written in bold and complementary sequences for overlap extension
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Table S2. Effects of metal ions and reagents on the activity of Apu
Reagents
Specific activity (U/mg)
Relative activity (%)
Metal ions
None
0.9
100
2+
1.0
112
Co2+
1.3
146
Cu2+
0.9
102
2+
Ca
Fe
1.0
113
2+
Mg
1.2
133
Mn2+
0.9
103
1.0
109
2-mercaptoethanol
1.3
140
Dithiothreitol
1.3
137
1.0
104
2+
Zn
Chemical reagents
Dimethylaminopropyl
ethylcarbodiimide
EDTA
1.0
SDS
ND
Urea
1.0
111
a
109
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The enzyme was pre-incubated with the above reagents at room temperature for 1 h. Aliquots were tested
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for Apu activity by incubating the samples with 0.1% soluble starch in 20 mM sodium citrate buffer (pH
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3.0) at 95 oC for 4 h. The metal ions and other reagents were added at a final concentration of 1 mM except
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for SDS (1%). The relative values shown are the percentage of the activity without additives.
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a
ND, not detected.
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Table S3. Relative amylolytic activity of Apu on different substrates
Substrates
Relative activity (%)
Polysaccahrides (0.1%)
Starch
77.8
Amylose
46.2
Amylopectin
71.8
Glycogen
52.8
Pullulan
100
β-cyclodextrin
2.8
Oligosaccharides (1 mM)
Maltose
7.8
Maltotriose
13.8
Maltotetraose
55.2
Maltopentaose
100
Maltohexaose
94.5
Maltoheptaose
86.2
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The substrates (1 mM or 0.1%) were digested with recombinant Apu (0.2 U) in 20 mM sodium citrate
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buffer (pH 3.0) for 12 h at 95 oC. The hydrolyzed products were separated on a silica gel TLC plate and
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quantified using a densitometer (ImageJ software, ver. 1.46, National Institute of Mental Health, Bethesda,
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MD).
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