of isopropylamine (1 M) as the amino donor, instead of L-alanine,
with a substrate concentration of 20 mM (Fig. 3).
cofactor, 0.5 g/L pyridoxal-5-phosphate cofactor, 45 g/L alanine
(500 mM), 20 mM acetopohenone. The reactions were run in 2 mL
Eppendorf tubes and placed in a shaking, temperature controlled
Although these reactions proceeded more slowly, probably as
a result of enzyme inactivation from the high concentration
of isopropylamine, both (S)- and (R)-2 were produced in 95%
conversion and with >99% e.e. using transaminases ATA-113
and ATA-117 respectively. Finally in Method 3 an amino acid
dehydrogenase (AADH) is added to the reaction, with NADH
cofactor recycling, to convert the pyruvate back to L-alanine.
Under these conditions, the ultimate amine donor now becomes
ammonia and hence the process is equivalent to a reductive
amination. To demonstrate the feasibility of this approach, a sub-
stoichiometric amount (25 mM) of pyruvate was added to the
reaction to generate the L-alanine in situ. The catalytic nature of
the system is evident, as a reaction with 50 mM acetophenone
proceeded to 96% conversion (Fig. 3).
◦
incubator (Thermomixer) at 30 C. 40 uL samples were taken
every hour to determine enzyme activity. Samples for reverse phase
HPLC were diluted 1 : 10 with acetonitrile, filtered and run using
the method described above. Samples for normal phase HPLC
were extracted with methyl tertbutyl ether (MTBE), dried down,
re-suspended in the mobile phase (90% hexanes/10% 2-propanol),
and run according to the method described above.
Colorimetric transaminase activity assay
1
00 uL reactions were run in a 96 well microtiter plate using
the following conditions and concentrations: 10 mM potassium
phosphate buffer with 5% v/v MeOH, 0.036 g/L phenol red
(
100 mM), 1 g/L NADH, 0.5 g/L pyridoxal-5-phosphate, 9 g/L
glucose (50 mM), 45 g/L alanine (500 mM), 20 mM acetophenone,
g/L glucose dehydrogenase (GDH), 1 g/L lactate dehydrogenase
Conclusions
1
In summary, we have developed a convenient and inexpensive
(LDH), and 2 g/L transaminase (ATA). The reactions were run at
30 C in the plate spectrophotometer. Absorbance was measured
at a wavelength of 560 nm every 30 seconds.
19
◦
assay for rapid screening of transaminase activity followed by
scale-up to 25 mL scale under essentially identical conditions.
This system, which should be applicable to any ketone substrate
of interest, also exhibited high rates of reaction, as well as being
tolerant of the high substrate charge. An alternative approach
LDH/GDH transamination system
(
Method 2), involving isopropylamine as the amine donor, has
Transaminations of acetophenone were conducted at 25 mL scale
using the LDH/GDH system under the following conditions:
been demonstrated at 25 mL scale and has the dual advantage
of using an inexpensive amine donor and also being a single
enzyme system. Finally, an AADH system that generates catalytic
L-alanine amine donor in situ, was demonstrated. This system,
while exhibiting a slower rate than Method 1, has the advantage
1
00 mM potassium phosphate buffer, 1 g/L NAD, 0.5 g/L
pyridoxal-5-phosphate, 18 g/L glucose (100 mM), 45 g/L alanine
(
(
500 mM), 50 mM acetophenone, 1 g/L glucose dehydrogenase
GDH), 1 g/L lactate dehydrogenase (LDH), and 5 g/L transam-
20
of using inexpensive ammonia as the effective amine donor.
◦
inase (ATA). Reactions were run at 30 C and pH 7.5 in a
ꢀ
R
Multimax reactor system with overhead mechanical stirring at
400 rpm. Reaction pH was controlled through the automated
addition of 2M NaOH.
Experimental
Commercial grade reagents and solvents were purchased from
Sigma-Aldrich and used without further purification. All enzymes
including transaminases (ATAs), glucose dehydrogenase (GDH)
and lactate dehydrogenase (LDH), were generously supplied by
Codexis (Redwood City, CA).
Reaction conversion was monitored using reverse phase high
performance liquid chromatography (HPLC) at 210 nm using
an Agilent 1100 series HPLC and a Zorbax Eclipse XDB-C18
Isopropylamine transamination system
Transaminations of acetophenone were run at 25 mL scale using
the isopropylamine amine donor system under the following con-
ditions: 100 mM potassium phosphate buffer, 0.5 g/L pyridoxal-5-
phosphate, 1 M isopropylamine, 20 mM acetophenone, and 5 g/L
◦
transaminase (ATA). Reactions were run at 30 C and pH 7.5 in
(
50 ¥ 4.6 mm) column with a flow rate of 1 mL/min (60%
ꢀ
R
a Multimax reactor system with overhead mechanical stirring at
00 rpm.
acetonitrile/40% water) for 3 minutes. Enantiomeric excess was
determined by normal phase high performance liquid chromatog-
raphy (HPLC) at 210 nm using an Agilent 1100 series HPLC
and a Chiralpak OD-H (250 ¥ 4.6 mm) column with a flow rate of
4
AADH/GDH transamination system
1
mL/min (90% hexanes/10% 2-propanol) for 12 minutes. Specific
Transaminations of acetophenone were run at 25 mL scale
using the amino acid dehydrogenase/catalytic alanine system
under the following conditions: 100 mM potassium phosphate
buffer, 100 mM ammonium chloride, 100 mM glucose, 1 g/L
NAD, 0.5 g/L pyridoxal-5-phosphate, 25 mM pyruvate, 50 mM
acetophenone, 1 g/L glucose dehydrogenase (GDH), 1 g/L L-
amino acid dehydrogenase (LAADH-117), and 5 g/L transam-
rotation of the methylbenzylamine product was established by
comparison to known standards.
Conventional enzyme assay
Conventional screening reactions were run at 1 mL scale in
1
00 mM potassium phosphate buffer using the following con-
◦
◦
ditions and concentrations: 30 C, pH 7.5, 2 g/L transaminase
inase (ATA-103). Reactions were run at 30 C and pH 7.5 in a
ꢀ
R
(
ATA) enzyme, 1 g/L lactate dehydrogenase (LDH), 1 g/L glucose
Multimax reactor system with overhead mechanical stirring at
400 rpm.
dehydrogenase (GDH), 9 g/L glucose (50 mM), 1 g/L NAD
This journal is © The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 395–398 | 397