2
Y. Imabayashi et al.
temperatures differ. In addition, the enzyme activity
needs to be strengthened, if it is to be used in efficient
conversion in industrial production.
12 g/L peptone, 24 g/L yeast extract, 2.3 g/L KH2PO4,
and 12.5 g/L K2HPO4 with 100 µg/mL ampicillin.
pUC118 (TaKaRa Bio Inc., Shiga, Japan) was used
for most of the cloning experiments. The plasmid
ptrp4,14) an expression plasmid that contain the trypto-
phan promoter and the rrnB terminator of E. coli,
was used for the expression of (R)-enantioselective
N-acetyl-β-Phe acylase. The plasmid pSFN_Sm_Aet,15)
an expression plasmid that contain the mutated acid-
phosphatase promoter of Enterobactor aerogenes,
was used for the expression of (S)-enantioselective
N-acetyl-β-Phe acylase.
To solve these problems, we sought here to purify,
clone, and characterize both N-acetyl-(R)- and (S)-β-
Phe acylases from Burkholderia sp. AJ110349. In addi-
tion, both (R)- and (S)-enantioselective N-acetyl-β-Phe
acylases were expressed in Escherichia coli. Using
these recombinant strains in the resting cell reaction,
enantiomerically pure (R)- and (S)-β-Phe were obtained
from the racemic substrate.
Materials and methods
Preparation of cell-free extract.
For enzyme
Materials.
(R,S)-3-amino-3-phenylpropionic acid
purification, the cells of Burkholderia sp. AJ110349
were collected through a 6800 g, 10-min centrifugation
operation, from 2000 mL of culture broth. Since precip-
itation of the bacterial cells was not adequately
observed after centrifugation, about 1600 mL of super-
natant was removed and the remainder was rendered
uniform by pipetting. The concentrated culture broth
thus prepared, without being washed with a buffer, and
was then disrupted by ultrasonication for 20 min at
200 W. The sonicate was centrifuged at 200,000 g for
30 min, and the obtained supernatant was used as a
cell-free extract.
The extract from recombinant E. coli JM109/
pSFN_BS was similarly prepared. The cells obtained
from 250 mL of culture broth were collected via
10 min of 8000 g centrifugation and re-suspended of
the buffer containing Tris–HCl (pH 7.6). The cell
suspension was ultrasonicated; the supernatant obtained
by centrifugation at 15,000 g for 10 min was used as a
cell-free extract.
[(R,S)-β-Phe], DL-β-leucine, DL-β-homophenylalanine, L-
β-leucine hydrochloride, and L-β-homophenylalanine
hydrochloride were purchased from Sigma-Aldrich (St.
Louis, MO, USA). (R)-3-amino-3-phenylpropionic acid
and (S)-3-amino-3-phenylpropionic acid were pur-
chased from Watanabe Chemical Industries (Hiroshima,
Japan). (R,S)-3-amino-5-methyl-hexanoic acid (DL-β-ho-
moleucine) and (R)-3-amino-5-methyl-hexanoic acid (D-
β-homoleucine) were purchased from AstaTech (Bristol,
PA, USA). (R,S)-3-amino-3-(4-hydroxyphenyl)-propa-
noic acid (DL-β-tyrosine) and (R,S)-3-amino-3-(4-fluo-
rophenyl)-propanoic acid (DL-4-fluoro-β-phenylalanine)
were purchased from Bionet (Cornwall, UK). β-valine
was purchased from Apollo Scientific (Cheshire, UK).
(R)-3-amino-3-(4-hydroxyphenyl)-propanoic acid (L-β-
tyrosine) and (R)-3-amino-3-(4-fluorophenyl)-propanoic
acid (L-4-fluoro-β-phenylalanine) were purchased from
PepTech (Bedford, MA, USA).
N-acetyl-β-Ala was purchased from Watanabe
Chemical Industries. N-acetyl-DL-α-phenylalanine was
purchased from Bachem AG (Bubendorf, Switzerland).
N-acetyl-(R,S)-β-Phe, N-acetyl-DL-4-fluoro-β-phenylala-
nine, N-acetyl-DL-β-tyrosine, N-acetyl-DL-β-homoleucine,
N-acetyl-DL-β-homophenylalanine, N-acetyl-DL-β-leucine,
and N-acetyl-β-valine were acetylated with acetic
anhydride from each corresponding racemic amino acid,
as previously described.13)
Enzyme
purification
from
Burkhordelia
sp
AJ110349. Protein purification was conducted at tem-
peratures <4 °C. The cell-free extract obtained above
was a resultant supernatant applied to ammonium sul-
fate fractionation. (NH4)2SO4 was added to the cell-free
extract, to a final concentration of 40% saturation. The
mixture was stirred on ice for 1 h and centrifuged for
15 min at 9200 g. The precipitate obtained was dis-
solved in a small quantity of 25 mM Tris–HCl (pH
7.6) and dialyzed against 25 mM Tris–HCl (pH 7.6).
After dialysis, the protein solution was used as a
sample in chromatography, as further described below.
Bacterial strain, culture media, and plasmid. The
isolation and characterization of Burkholderia sp.
AJ110349 has been reported previously.11) For the
purification of acylases, AJ110349 was grown in
enzyme-producing medium comprising 10 g/L D-glu-
cose, 10 g/L (NH4)2SO4, 10 g/L (R,S)-β-Phe, 2 g/L
casamino acid, 1 g/L KH2PO4, 0.4 g/L MgSO4·7H2O,
1 g/L NaCl, 19.5 g/L 2-(N-morpholino)ethanesulfonic
acid (MES), 5 mg/L nicotinamide, 0.2 mg/L thiamin,
10 mg/L FeSO4·7H2O, and 10 mg/L MnSO4·4–5H2O.
Cultivation was carried out with shaking at 120 rpm in
a 500-mL Sakaguchi flask, for 66 h at 30 °C.
E. coli JM109 was selected as the host strain for the
recombinant DNA studies. For the cloning experiments,
E. coli strains were routinely cultured at 37 °C in LB
medium supplemented with ampicillin (100 µg/mL).
For the purification and expression experiments, E. coli
JM109 was cultured at 37 °C in LB medium or terrific
broth (TB) medium composed of 4 g/L glycerol,
(1) Phenyl Sepharose 26/10 (GE Healthcare UK
Ltd., Buckinghamshire, England).
The ammonium sulfate fractions obtained as
described above were dialyzed against a buffer solution
comprising 25 mM Tris–HCl (pH 7.6) and 0.6 M
(NH4)2SO4; they were then placed on Phenyl Sephar-
ose 26/10 that had been equilibrated with the same buf-
fer solution. Following non-adsorptive protein elution,
the adsorptive protein was eluted by linearly varying
the (NH4)2SO4 concentration in the buffer solution,
from 0.6 to 0 M. This operation resulted in the
N-acetyl-(R)-β-Phe acylase activity being detected when
the (NH4)2SO4 concentration was about 0.2 M, and the