COMMUNICATION
Transaminations with isopropyl amine: equilibrium displacement with
yeast alcohol dehydrogenase coupled to in situ cofactor regenerationwz
Karim Engelmark Cassimjee,a Cecilia Branneby,b Vahak Abedi,c Andrew Wellsd and
Per Berglund*a
Received 21st February 2010, Accepted 28th April 2010
First published as an Advance Article on the web 12th May 2010
DOI: 10.1039/c0cc00050g
Enantiopure chiral amines synthesis using x-transaminases is
hindered by an unfavourable equilibrium, but when using isopropyl-
amine as the amine donor the equilibrium can be completely
displaced by using a specific dehydrogenase in situ for removal of
formed acetone.
remove highly soluble volatile substances like acetone from
aqueous mixtures, also raising the temperature to assist this
process requires a more heat stable enzyme. In this work, an
alternative method for removal of formed acetone with a
commonly available commercial dehydrogenase was used.
The substrate ketone was not affected by the dehydrogenase
due to its narrow substrate specificity, a good example of
effective use of enzyme selectivity in a cascade reaction.
Yeast alcohol dehydrogenase (YADH; Saccharomyces
cerevisiae) is a well described enzyme with a narrow substrate
range. The active site of this enzyme is unable to accommodate
larger groups, methanol/methanal and ethanol/ethanal are
effectively converted, as well as isopropyl alcohol/acetone,
although at lower rate. For substrates with four carbons the
reaction is extremely slow, and for bigger substrates no reac-
tion has been observed. YADH should therefore be effective
for removal of the acetone in a transamination reaction where
isopropyl amine is the donor (Fig. 1). The amine acceptor
(acetophenone in Fig. 1) will not be a substrate for YADH as
long as it is not small, which is rarely the case.15
The production of chiral amines of high enantiomeric excess is
of great interest since such motifs are present in a plethora of
pharmaceuticals and other fine chemicals. Use of o-transaminases
(o-TA) for this purpose has been shown to be feasible and
effective.1–5 All known transaminases use pyridoxal-5-phosphate
(PLP) as a cofactor, providing a route for conversion of an
amine to the corresponding ketone, yielding pyridoxamine-5-
phosphate (PMP). The PMP thereafter functions as an amine
donor by converting a ketone of choice to the corresponding
chiral amine.6 A thermodynamic analysis of such a system
shows that the equilibrium is most often shifted towards
the substrates since the ketone of choice is usually much more
stable than the corresponding amine, due to electron resonance
effects.7 Therefore, different methods for shifting the equili-
brium have been devised by coupling the reaction with
secondary irreversible reactions. Then the yield can theoreti-
cally reach 100%. This has been shown to be effective with an
amino acid, e.g. alanine, as the amine donor as the produced
ketoacid can be further reacted with effective enzymes such as
pyruvate decarboxylase.7–12
Conversion of acetone to isopropyl alcohol by YADH
requires NADH, which must be regenerated for a cost efficient
synthesis. In this work we used formate dehydrogenase
(FDH). Other reports include glucose dehydrogenase for the
same purpose.16
The synthesis with the designed equilibrium displacement
system was tested with the o-transaminase from Arthrobacter
citreus variant denoted S9 (Cambrex Karlskoga AB) in
purified form after cloning and overexpression, and with the
enzyme ATA-113 (Codexis). The gene for A. citreus variant S9
was cloned into the vector pET28a(+) with an N-terminal
His6-tag and effectively expressed in E. coli BL21(DE) at
The use of isopropyl amine as the amine donor is, from an
industrialist’s point of view, more desirable since this chemical
is easily obtainable and cost efficient. The formed acetone in
such a case can be removed by distillation, thus shifting the
equilibrium and good yields can be obtained.9,13,14 However,
in large scale equipment it is not always feasible to effectively
a Division of Biochemistry, School of Biotechnology,
Royal Institute of Technology (KTH), AlbaNova University Centre,
SE-106 91 Stockholm, Sweden. E-mail: per.berglund@biotech.kth.se;
Fax: +46 8 5537 8468; Tel: +46 8 5537 8366
b Cambrex Karlskoga AB, SE-691 85 Karlskoga, Sweden.
E-mail: cecilia.branneby@cambrex.com; Fax: +46 586 783129;
Tel: +46 586 783000
c AstraZeneca R&D, SE-151 85 Sodertalje, Sweden.
¨
¨
E-mail: vahak.abedi@astrazeneca.com; Fax: +46 8 5532 4276;
Tel: +46 8 5532 1766
Fig. 1 Effective synthesis of (S)-1-phenylethylamine through the
transamination of acetophenone with isopropylamine as donor, by
using an S-selective o-transaminase variant from A. citreus, denoted
o-TA S9 (Cambrex Karlskoga AB). The employment of an equili-
brium displacement system consisting of yeast alcohol dehydrogenase
(YADH, S. cerevisiae) and formate dehydrogenase (FDH, Candida
boidinii) drives the reaction to completion.
d AstraZeneca Global Process R&D, Bakewell Road, Loughborough,
Leics, UK LE11 5RH. E-mail: andrew.wells@astrazeneca.com;
Fax: +44 (0)1509 645588; Tel: +44 (0)1509 644439
w Electronic supplementary information (ESI) available: Materials
and methods. See DOI: 10.1039/c0cc00050g
z This article is part of the ‘Enzymes and Proteins’ web-theme issue for
ChemComm.
ꢀc
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 5569–5571 | 5569