Biosci. Biotechnol. Biochem., 75 (9), 1778–1782, 2011
Purification and Characterization of a Novel (R)-Imine Reductase
from Streptomyces sp. GF3587
y
Koichi MITSUKURA, Mai SUZUKI, Sho SHINODA, Tatsuya KURAMOTO,
Toyokazu YOSHIDA, and Toru NAGASAWA
Department of Biomolecular Science, Faculty of Engineering, Gifu University,
1
-1 Yanagido, Gifu 501-1193, Japan
The (R)-imine reductase (RIR) of Streptomyces sp.
there have been no reports of stereospecific imine
reductase. It is not easy to carry out screening of imine
reductase using imines, because most imines are very
labile in aqueous media. Recently, however, asymmetric
reduction of aryl imines with whole cells of Candida
parapsilosis ATCC 7330, which show carbonyl reduc-
GF3587 was purified and characterized. It was found to
be a NADPH-dependent enzyme, and was found to be a
homodimer consisting of 32 kDa subunits. Enzymatic
reduction of 10 mM 2-methyl-1-pyrroline (2-MPN) re-
sulted in the formation of 9.8 mM (R)-2-methylpyrroli-
dine ((R)-2-MP) with 99% e.e. The enzyme showed not
only reduction activity for 2-MPN at neutral pH (6.5–
1
1)
tase activity, has been reported. Although the Candida
enzyme showed high (R)-selectivity, the productivity of
chiral amine using whole cells was very low.
8
.0), but also oxidation activity for (R)-2-MP under
1
2)
alkaline pH (10–11.5) conditions. It appeared to be a
sulfhydryl enzyme based on the sensitivity to sulfhydryl
specific inhibitors. It was very specific to 2-MPN as
substrate.
In a previous study, we attempted to survey a novel
imine reductase with high activity and excellent enan-
tioselectivity using a cyclic imine, 2-methyl-1-pyrroline
(2-MPN), which is extremely stable in neutral aqueous
media. We found (R)- and (S)-selective imine-reducing
activity in cells of Streptomyces sp. GF3587 and
GF3546 respectively. (R)-2-Methylpyrrolidine ((R)-2-
MP) and (S)-2-MP were synthesized from 2-MPN with
Key words: (R)-imine reductase; Streptomyces sp.
GF3587; 2-methyl-1-pyrroline; 2-methyl-
pyrrolidine
9
9.2% e.e and 92.3% e.e using the whole-cell catalysts
1
2)
Recently the catalytic synthesis of chiral amines has
received much attention due to wide application as
pharmaceutical and agrochemical intermediates. Vari-
ous catalysts have been developed for the asymmetric
reduction of imines, enamines, and oximes, and appli-
of strains GF3587 and GF3546 respectively. In the
present study, we focused on the purification and
characterization of the (R)-imine reductase (RIR) from
Stretptomyces sp. GF3587 (Fig. 1).
1
,2)
cations have been reported.
hydrolases and !-transaminases have been examined
As for biocatalysts,
Materials and Methods
3
,4)
for the synthesis of optically active primary amines.
Chemicals. 2-Methyl-1-pyrroline (2-MPN), racemic, (R)- and (S)-2-
methylpyrrolidine (2-MP), and an assay kit for dihydrofolate reductase
Recently, efficient production systems have been de-
signed using !-transaminase coupled with amine oxi-
dase, amino acid dehydrogenase, lactate dehydrogenase,
(DHFR) activity were purchased from Sigma-Aldrich (Tokyo). The
other chemicals used were of guaranteed reagent grade.
5
,6)
pyruvate decarboxylase, or ꢀ-transaminase.
As for
Cultivation. Streptomyces sp. GF3587 was cultivated for 24 h at
ꢀ
optically active cyclic secondary amines, optically
active pyrrolidine and piperidine derivatives were
synthesized from their corresponding racemic oxalamic
esters through steps of protease-catalyzed hydrolysis and
chemical deprotection. In the enantioselective syn-
thesis of cyclic tertiary amines, monoamine oxidase
variant N-5 (MAO-N-5), which oxidizes one enantiomer
of racemic cyclic tertiary amine to the corresponding
imine, was developed by directed evolution of MAO-N
from Aspergillus niger. The cyclic imine thus formed
was reduced to racemic amine with ammonia borane.
Through repetition of the consecutive reaction process,
the racemic cyclic tertiary amines was converted to
enantiomerically-pure tertiary amines.10)
2
8 C with reciprocal shaking (115 strokes/min) in a nutrient medium
(400 mL) containing 0.8% w/v glucose, 2% w/v malt extract (Difco,
New Jersey, USA), 1% w/v NZ amine (Wako Pure Chemical
.
Industries, Osaka, Japan), and 0.0005% w/v FeSO4 7H2O at pH 7.3.
ꢀ
7
)
Cells, harvested by centrifugation at 16;000 g for 10 min at 4 C, were
ꢀ
stored at ꢁ30 C until just before use.
Enzyme assay. Imine reductase activity was assayed in a quartz
cuvette (1-cm path) by measuring the oxidation of NADPH (molecular
ꢀ
extinction coefficient, 6,200/M/cm) at 340 nm (A340) at 30 C. The
8
,9)
reaction mixture (1 mL) consisted of 10 mM 2-MPN or an other tested
compound, 100 mM potassium phosphate buffer (pH 7.5), 0.1 mM
NADPH, and an appropriate amount of enzyme. The enzyme reaction
was started by the addition of the enzyme solution, and the value of
ꢀ
A340 was measured at 30 C for 2 min with UV1600PC (Shimadzu,
Tokyo, Japan). One unit of enzyme was defined as the amount that
catalyzes the oxidation of 1 mmol of NADPH per min. Reduction
activity for 2-MPN was also examined using human DHFR in a
Enzymatic asymmetric reduction of imine is a
promising alternative for chiral amine synthesis, but
y
To whom correspondence should be addressed. Tel: +81-58-293-2649; Fax: +81-58-293-2794; E-mail: mitukura@gifu-u.ac.jp
Abbreviations: 2-MPN, 2-methyl-1-pyrroline; 2-MP, 2-methylpyrrolidine; RIR, (R)-imine reductase