A novel S-amidase from Arthrobacter sp. S-2
3
saturated (NH4)2SO4, the enzyme was eluted with a
linear gradient of (NH4)2SO4 (30–0%) in 20 mM
buffer. The combined active fractions were dialyzed
same conditions as the first PCR with the primers F2
and C2. The two PCRs yielded a product that was
approximately 1.2 kb, which was cloned into the
Novagen® pT7 Blue T-vector (Merck KGaA, Darmstadt,
Germany) and sequenced. To obtain the complete
sequence of the gene coding S-amidase, inverse PCR
was carried out as follows. Genomic DNA was digested
with some appropriate restriction enzymes, incubated
for 24 h at 37 °C, and then circularized by T4 DNA
ligase (Takara Bio Inc., Shiga, Japan) for 1 h at 16 °C.
The primers InF (5′-CATCGTTCCGGTAGGTGAAG-
CCGTTC-3′) and InR (5′-GCAGCAACAGGAACG-
TCTGCCCTCATC-3′) were designed based on the
partial gene sequenced above. Inverse PCR was carried
out under the same PCR conditions described above.
The PCR product was cloned into the Novagen® pT7
Blue T-vector and sequenced. The gene sequence of
S-amidase from Arthrobacter sp. S-2 has been deposited
into GenBank with the accession number LC026945.
with 1.0 mM buffer and applied to
a Gigapite
(Seikagakukogyo Corp., Tokyo, Japan) column
(100 mL) equilibrated with 1.0 mM buffer, and the
flow-through was collected. This flow-through was
applied to a DEAE-Toyopearl® 650M (Tosoh Corp.,
Tokyo, Japan) column (20 mL) equilibrated with
20 mM buffer. After the column had been washed thor-
oughly with 20 mM buffer, the enzyme was eluted with
a linear gradient of NaCl (0–300 mM) in 20 mM buf-
fer. The combined active fractions were then brought to
30% (NH4)2SO4 saturation and applied to a Resource
PHE (GE Healthcare UK Ltd., Buckinghamshire,
England) column (1 mL) equilibrated with 20 mM buf-
fer containing 30% saturated (NH4)2SO4. After the col-
umn had been washed thoroughly with 20 mM buffer
containing 30% saturated (NH4)2SO4, the enzyme was
eluted with a linear gradient of (NH4)2SO4 (30–0%) in
20 mM buffer using the ÄKTA explorer 10S (GE
Healthcare UK Ltd., Buckinghamshire, England) at
0.20 mL/min. The combined active fractions were dia-
lyzed with 20 mM buffer, concentrated by Amicon®
Ultra-4 (Merck KGaA, Darmstadt, Germany), and
applied to a Superdex 200 HR 10/30 (GE Healthcare
UK Ltd., Buckinghamshire, England) column (24 mL)
equilibrated with 20 mM buffer. The enzyme was
eluted with 20 mM buffer using the ÄKTA explorer
10S at 0.20 mL/min. The active fraction was used to
analyze N-terminal amino acid sequence of the enzyme,
and its analysis was performed at APRO Life Science
Institute Inc. (Tokushima, Japan) with a Procise 494
HT protein sequence system.
Construction of a plasmid and transformation of
E. coli for the expression of the gene coding S-amidase
from Arthrobacter sp. S-2. NdeI-XhoI fragments con-
taining the gene coding S-amidase were amplified with
the primers Aas-F (5′-GATACATATGCAGGGACTG-
CCAACACGG-3′) and Aas-R (5′-GATACTCGAGT-
CAGACCGTCCGGCTTGATC-3′), and Aas-F and
AasH-R
(5′-GATACTCGAGGACCGTCCGGCTT-
GATC-3′, for the C-terminal His6-tag) including appro-
priate restriction sites (underlined sequence), and
genomic DNA as a template. Each of the amplified
PCR products was digested with NdeI and XhoI, and
was ligated into the expression vector Novagen®
pET23a(+) or pET15b (Merck KgaA, Darmstadt,
Germany) digested with the same restriction enzymes,
respectively. Each of the recombinant plasmids pET23a
(+)-S-amidase, pET23a(+)-S-amidase with the C-His6-tag,
and pET15b-S-amidase, named pEAAS23, pEAAS23-
CH, and pEAAS15, respectively, was used to transform
E. coli BL21(DE3) or E. coli BL21-CodonPlus®(DE3)-
RIPL (Agilent Technologies Inc., Santa Clara, CA,
USA), respectively.
Cloning of the gene coding S-amidase from
Arthrobacter sp. S-2.
Genomic DNA was prepared
from Arthrobacter sp. S-2 cells by the method of
Misawa12) and used as a template for PCR. A partial
gene coding S-amidase from Arthrobacter sp. S-2 was
amplified using genomic DNA as a template with two
combinations of primer F1 (5′-ATGCARGGNYTNCC-
NACNMG-3′) and cassette primer C1 (5′-GTACA-
TATTGTCGTTAGAACGCGTAATACGACTCA-3′), or
primer F2 (5′-WSNAAYGGNGCNATH-3′) and cassette
primer C2 (5′-CGTTAGAACGCGTAATACGACTCAC-
TATAGGGAGA-3′) using the LA PCR™ in vitro clon-
ing kit (Takara Bio Inc., Shiga, Japan) according to the
manufacturer’s protocol. The sequences of F1 and F2
were designed based on the N-terminal amino acid
sequence. The two cassette primers C1 and C2 corre-
sponded to the cassette in the LA PCR™ in vitro cloning
kit. The first PCR was carried out as follows. After ini-
tial denaturation of genomic DNA for 2 min 30 s at
94 °C, amplification was performed in 30 cycles of
denaturation for 30 s at 94 °C, annealing for 30 s at
55 °C, and extension for 2 min at 72 °C in 50 μL of the
reaction mixture containing 100 ng of genomic DNA,
0.20 μM each of the F1 and C1 primers, 0.40 mM
dNTPs, 1× LA Buffer II (Mg2+ plus), and 2.5 U of
TaKaRa LA Taq® DNA polymerase (Takara Bio Inc.,
Shiga, Japan). The second PCR was carried out using
50 ng of the first PCR product as a template under the
Production and purification of recombinant
S-amidase in E. coli BL21-CodonPlus®(DE3)-RIPL
transformants. E. coli BL21-CodonPlus®(DE3)-RIPL
harboring pEAAS23 was inoculated into 5.0 mL of
Luria–Bertani (LB) medium consisting of 10 g of
Hipolypepton (Nihon Pharmaceutical Co., Ltd., Tokyo,
Japan), 5.0 g of yeast extract, and 10 g of NaCl in
1000 mL of water containing 80 μg/mL of ampicillin
and 50 μg/mL chloramphenicol and then incubated
with reciprocal shaking at 300 strokes/min for 12 h at
37 °C. The cells that grew were added to 500 mL of
the same medium and incubated with shaking at
150 rpm for 12 h at 37 °C. When the optical density
at 610 nm of the medium reached 0.5–1.0, isopropyl
β-D-thiogalactopyranoside (IPTG) was added to a final
concentration of 1.0 mM, and the culture was further
incubated for 10 h at 30 °C. Cells were harvested by
centrifugation (6000 × g, 10 min, 4 °C) and washed
with 20 mM KPB (pH 7.0). Unless otherwise stated,