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Y. FUKUTA et al.
G171A, F312Y, and R420Q) were introduced into pU224 with a
ꢁ
100 ml of the cell-free extract obtained from 1 liter of culture broth and
9.85 g (0.05 mol) or 19.7 g (0.1 mol) of !-laurolactam. It was stirred at
QuikChange Multi Site-Directed Mutagenesis Kit (Stratagene,
ꢁ
Tokyo, Japan) using pU224 as a template. The reaction mixture for
the mutagenesis contained 2.5 ml of QuikChangeꢁ Multi reaction
buffer, 1 ml of dNTP mix, 9.5 ml of distilled H2O, 50 ng of ds-DNA
template, pU224, four sense and four antisense primers each at 50 ng,
and 1 ml of QuikChangeꢁ Multi enzyme blend. The four primers were
35 C for 18 h in the flask. After the reaction, the product was filtrated
and washed with deionized water. The crystals formed were dried at
ꢁ
80 C and then in a vacuum desiccator night over.
Results
0
as follows: primer 1 5 -AAAACTAATACACCGGAGATGGGCAAT-
0
0
CAG-3 (for V118T and T125N), primer 2 5 -CGCACGGCAATGAC-
0
Comparison of the amino acid sequences of cloned
-laurolactam hydrolase with Acd hydrolase from
0
GCGGCAGGTTCCGTGC-3 (for G171A), primer 3 5 -CTTCCTC-
0
!
Arthrobacter sp. KI72
AAGGACTACTCGACGATTTGCGA-3 (for F312Y), and primer 4
0
0
5
-TCTCGGGCAGTCTGCAGATGCTGGCCTTCA-3 (for R420Q).
The deduced amino acid sequence of !-laurolactam
hydrolase from Rhodococcus sp. U224 and Acd hydro-
lase from Arthrobacter sp. KI72 has only five substitu-
These designed primers contained mutation points (double-under-
lined). Thirty cycles were performed, each consisting of a denaturing
ꢁ
ꢁ
step at 95 C for 1 min, an annealing step at 55 C for 1 min, and an
ꢁ
12)
elongation step at 65 C for 8 min. The amplified product was
ꢁ
tions. We designed primers for PCR based on the
incubated with 1 ml of Dpn I (10 units/ml) at 37 C for 1 hr, and then
sequence information, and successfully cloned genes
encoding !-laurolactam hydrolase from Cupriavidus sp.
T7, Acidovorax sp. T31, Cupriavidus sp. U124, and
Sphingomonas sp. U238. A 1,482-bp DNA fragment
starting at GTG and terminating at the TGA codon was
found in all of the nucleotide sequences encoding
!-laurolactam hydrolyzing enzymes from the four
strains, although the N-terminal and C-terminal gene
sequences of these genes were unclear. These open
reading frames encoded 493 amino acid residues. A
homology search using the BLAST program showed
that in primary structure, the !-laurolactam hydrolases
from the four strains were almost identical with the
F-nylA protein. Multiple alignments of the deduced
sequences of these six !-laurolactam hydrolases are
shown in Fig. 2. These enzymes were highly homolo-
gous with each other.
used for the transformation of E. coli JM109. The plasmid was
extracted and sequenced to make sure occurrences of each mutation,
V118T, T125N, G171A, F312Y, and R420Q, and the plasmid was
named pF-nylA.
Expression of recombinant enzymes. Recombinant E. coli JM109
strains harboring each of the expression vectors, pT7, pT31, pU124,
ꢁ
pU224, pU238, and pF-nylA, were cultured at 37 C for 8 h with
shaking (200 rpm) in a test tube containing 5 ml of LB broth containing
80 mg/ml of ampicillin. After 8 h of cultivation, IPTG (final concen-
tration, 0.5 mM) was added to the culture broth and the cultivation
ꢁ
temperature was shifted to 30 C. The E. coli transformants were
totally cultivated for 20 h. Cells of 1 ml culture were harvested by
ꢁ
centrifugation (15;000 ꢂ g, 5 min, 4 C) and suspended in 1 ml of
100 mM potassium phosphate buffer (pH 7.0). Cell suspensions were
then disrupted by Multi Beads Shocker (Yasui Instruments, Osaka,
Japan). For removal of intact cells and cell debris, the lysate was
ꢁ
centrifuged at 15;000 ꢂ g for 10 min at 4 C to yield the cell-free
extract. The reaction mixture (1 ml) contained 100 mM potassium
phosphate buffer (pH 7.0), 10 mM of !-laurolactam dissolved in
toluene, and an appropriate amount of the enzyme.
Expression of recombinant !-laurolactam hydrolase
and Acd hydrolase in E. coli
Purification of Acd hydrolase from E. coli JM109/pF-nylA. The
subculture of E. coli JM109/pF-nylA was inoculated into 500 ml of LB
medium containing ampicillin in a 2-liter Sakaguchi flask and the
For expression of the genes from Cupriavidus sp. T7,
Acidovorax sp. T31, Cupriavidus sp. U124, Rhodococ-
cus sp. U224, and Sphingomonas sp. U238, the upstream
regions of the start codons were modified (GTG to
ATG). The six E. coli JM109 transformants (E. coli
JM109 harboring pT7, pT31, pU124, pU224, pU238, or
pF-nylA) were used for the expression of the respective
recombinant enzymes. These cell-free extracts of the
E. coli JM109 transformants were prepared as described
in ‘‘Materials and Methods,’’ and were used to assay
ꢁ
culture broth was shaken (96 stroke/min) at 37 C, and after 12 h of
cultivation, IPTG (final concentration, 0.5 mM) was added to the
ꢁ
culture broth and the cultivation temperature was shifted 30 C to
induce the enzyme (total cultivation time, 24 h). Cells from 5 liters of
culture were suspended in the buffer described above and the
suspensions were disrupted by sonication for 15 min (19 kHz, Insonator
model 201M; Kubota, Tokyo). The cell-free extract was produced
ꢁ
by centrifugation (15;000 ꢂ g, 15 min, 4 C). It was fractionated with
ammonium sulfate (30–60% saturation). The active pellet was
dialyzed, applied to a DEAE-Toyopearl (Tosoh, Tokyo) column, and
eluted with 100 mM of potassium phosphate buffer (pH 7.0) containing
!-laurolactam hydrolase activity. All of them expressed
-laurolactam hydrolase successfully, and their activity
!
levels were different. The cell-free extracts of recombi-
nant enzymes from Cupriavidus sp. T7 and Cupriavidus
sp. U124 showed similar activity (total activity, 0.66
and 0.66 units/ml culture; specific activity, 1.22 and
0
.5 M NaCl. The active fraction was dialyzed, and applied to MonoQ
HR 5/5 (GE Healthcare, Buckinghamshire, UK) equilibrated with
0 mM buffer. The enzyme was eluted with a gradient of 0–0.5 M NaCl
2
in 20 mM buffer with A¨ kta FPLCꢀ (GE Healthcare) at 0.8 ml/min, and
applied to a column of Superdex 200 HR 10/30 (GE Healthcare)
equilibrated with 20 mM buffer containing 150 mM NaCl. The column
was eluted by FPLC at 0.5 ml/min, and the active fractions were
collected. The dialyzed active fractions were combined and concen-
trated with the Centricon (Amicon, Beverly, MA).
1
.29 units/mg protein, respectively). E. coli JM109/
pU224 and pU238 gave lower hydrolyzing activity
total activity; 0.14 and 0.12 units/ml culture, specific
(
activity; 0.36 and 0.29 units/mg protein, respectively)
than the others, although these activities were 2 times
1
2)
Enzymatic transcrystallization of !-laurolactam to 12-aminolauric
acid using overexpressed !-laurolactam hydrolase from Acidovorax
sp. T31. The cultivation conditions for E. coli JM109/pT31 were
carried out by the same method as for E. coli JM109/pF-nylA. Cells
from 5 liters of culture were suspended in the buffer described above,
and the suspensions were disrupted by sonication for 15 min. The cell-
higher than the wild strains.
E. coli JM109/pT31
showed the highest activity, of 1.14 units/ml of culture
and 2.77 units/mg protein. The cultivation conditions
for E. coli JM109/pT31 for induction of !-laurolactam
hydrolase were optimized. The highest enzyme activity
(8.26 units/mg) was obtained when the recombinant
ꢁ
free extract was produced by centrifugation (15;000 ꢂ g, 15 min, 4 C).
ꢁ
strain was grown at 37 C after 12 h of the cultivation,
IPTG (final concentration, 0.5 mM) was added to the
Enzymatic transcrystallization of !-laurolactam using !-laurolac-
tam hydrolase from Acidovorax sp. T31 was carried out with 0.5 M and
ꢁ
1.0 M of the substrate concentrations. The reaction mixture contained
culture and the temperature was lowered to 30 C (total