M. Chiriac et al.
polymerases were purchased from Roche Applied Science, New 36.8 (a-ketoisocaproate), 17.4 (a-ketocaproate), 7.8 (a-keto-g-
England Biolabs, or Qbiogene Inc.
methylthiobutyrate) and 4.7 (a-ketobutyrate). In the crude
bacterial extract, the specific activity of LeuDH with these a-
keto acids is approximately four times lower. Since the enzyme
activity is dependent on the chemical nature of the a-keto acid,
the number of units used in each synthesis is calculated
according to the corresponding a-keto acid. GlucDH activity was
assayed in a medium containing 50 mM Tris HCl (pH 8.0), 10 mM
glucose and 2 mM NAD. The specific activity of a crude bacterial
extract in which GlucDH is overproduced is of about 200 U/mg
of protein. GalM activity was determined in 50 mM Tris-HCl (pH
8.0), 2 mM NAD, 1 U of GlucDH and appropriately diluted
bacterial extract containing overproduced GalM. Then freshly
dissolved glucose in Tris-HCl buffer was added to the medium
and the initial rate of NADH formation recorded at 340 nm. The
rate of NADH formation in the absence of GalM was subtracted
to determine the enzyme-catalysed anomerization. LeuDH,
GlucDH and GalM can be stored at ꢀ20 or ꢀ801C for months
without significant loss of activity.
Recombinant DNA technology
General DNA manipulations were performed as described by
Sambrook et al.11 The gene encoding LeuDH from Bacillus
stearothermophillus was amplified using the corresponding
genomic DNA as template and the oligonucleotides listed in
Table 1 as primers in a standard polymerase chain reaction (PCR)
experiment. The amplified DNA was then digested with BamHI
and XhoI restriction enzymes, purified and inserted by ligation in
the pET21b vector (Novagen) digested with the same restriction
enzymes.
The genes encoding GlucDH from B.subtilis and GalM from
Escherichia coli were amplified by PCR using chromosomal DNA
from the corresponding bacteria as templates and the
oligonucleotides indicated in Table 1 as primers. The amplified
products were then digested with NdeI and BamHI restriction
enzymes, purified and inserted by ligation in the pET24a
(Novagen) digested with the same restriction enzymes. After
transformation of strain DH5a, the plasmid DNA was purified
from several colonies and digested to check for the presence of
an insert of the correct size. The plasmid with the good insert
was used to transform strain BL21 (DE3). For overexpression of
proteins, BL21 (DE3) containing either pET24a GlucDH, pET24a
GalM or pET21b LeuDH was grown at 371C in 2YT medium
supplemented with kanamycin (30 mg/ml) and chloramphenicol
(30 mg/ml) for the first two strains, and in Luria–Bertani medium
supplemented with ampicillin (100 mg/ml) for the third one.
When OD600 value 1.3 was reached isopropyl b-D-thiogalacto-
pyranoside (IPTG) was added at a final concentration of 1 mM,
and the cultures were further incubated at 371C for 3 h, before
being harvested by centrifugation.
Synthesis and purification of L [15N]-labelled amino acids
A typical synthesis of L[15N]-labelled amino acid was performed
as follows: 8 mmol glucose, 8 mmol a-keto acid (Na-salt) and
8 mmol [15N] ammonium chloride (ꢁ99 at.% 15N) were
dissolved in 65 ml of bidistilled water maintained at 301C and
the pH adjusted to 8.0 with 1 N NaOH. Then 0.2 mmol NAD1,
followed by LeuDH (200 U), GlucDH (100 U) and GalM (100 U)
were added and the volume adjusted to 70 ml. Under these
conditions the formation of L[15N]-labelled amino acid is
accompanied by the release of gluconic acid and the pH
decreases accordingly. By adding small volumes of 1 N NaOH the
pH is restored to 8.0–8.2. The consumed NaOH indicates the
amount of amino acid formed. Routinely after 2–3 h the reaction
was complete and no further change in pH was observed. The
disappearance of ammonia was tested colorimetrically; then the
reaction mixtures were heated to 851C for 15 min. After chilling,
the denaturated proteins were removed by filtration or
centrifugation. The solution was passed onto a 120 ml column
(2.5 ꢂ 25 cm) of Dowex 50WX-8 in H1 form. After washing the
column with water, the adsorbed amino acid was eluded with
1 M ammonium hydroxide.
Preparation of bacterial extracts and determination of enzyme
activities
The bacterial pellet was stored at ꢀ801C until used for amino
acid synthesis. Bacteria were defrosted in 50 mM Tris-HCl (pH
7.4), then sonicated at 41C and centrifuged at 10 000g for
30 min. The supernatant was tested for enzyme activity at 301C
and 340 nm by following either the formation or the consump-
tion of NADH. One unit (1 U) of enzyme activity corresponds to
1 mmole of NADH formed or consumed in 1 min. LeuDH activity
was determined in a medium containing 50 mM Tris-HCl (pH
8.0), 10 mM a-keto acid, 0.15 mM NADH and 1 M NH4Cl. Under
these conditions the specific activity of a purified enzyme
preparation (U/mg protein) corresponds to the following
numbers : 168 (a-ketovalerate), 46.5 (a-keto-b-methylvalerate),
The fractions of the eluate containing the labelled amino acid
were concentrated in a rotary vacuum evaporator at 501C to
approximately 5 ml. Ninety millilitre of 50/50 (v/v) and anhy-
drous ethanol/ether were added and the mixture was kept
overnight in the refrigerator, while the amino acid crystallized.
The precipitate was filtered off and dried. Table 2 shows the
yield for various amino acids synthesized.
Table 1.
Enzyme
LeuDH
Primer
Template
50-GGTGGATCCGATGGAATTGTTCAAATATATGG-3’
50-TATTCTCGAGTATTGCCGAAGCACCTGC-3’
B. stearothermophilus
GlucDH
GalM
50-GGAATTCCATATGTATCCGGATTTAAAAGGAAAAG-30
50-CGCGGATCCTCAACCGCGGCCTGCCTGG-3’
50-GGAATTCCATATGCTGAACGAAACTCCCGCAC-3’
50-CGCGGATCCTCACTCAGCAATAAACTGATATTCCG-30
B. subtilis
E. coli
Copyright r 2008 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2008, 51 171–174
Chiriac, Maria
