Angewandte
Chemie
Tailor-Made Enzyme Catalyst
from low turnover frequencies (TOF), low stereoselectivity,
and rapid deactivation at high substrate concentrations.
(R)-2-Chloromandelic acid (3b) is a key intermediate for
the production of a widely administered anticoagulant that
reduces the risk of cardiovascular events in patients with
acute coronary syndromes. However, ortho-substituted sub-
strates perform poorly in all known syntheses of chiral
cyanohydrins, and their use results in low yields, low
TOF values, and/or low ee values.[4,6–9] Furthermore, the
conversion of aromatic aldehydes into cyanohydrins with
the only reported recombinant (R)-hydroxynitrile lyase
(LuHNL, cloned from Linum usitatissimum) does not reach
completion.[10] (R)-HNL from Prunus amygdalus (PaHNL),
which shows a broad substrate specificity, is only available
from very limited natural sources, and the recombinant
production of any active FAD (flavin adenine dinucleotide)-
containing HNL was previously unsuccessful.[11] Herein we
report the first recombinant PaHNL that retains its activity
under process conditions and can be used in emulsion systems
at low pHvalues. We also demonstrate the success of a
generally applicable engineering concept, which could be
used to improve the productivity of this enzyme during the
fermentation process, and which led to a high TOF value for
the challenging substrate 1b. Our goal was to develop a
commercially viable biocatalyst for the production of (R)-3b
by the HNL route, or in other words to make the production
of optically pure (R)-2b possible in high yield in the presence
of a small quantity of a robust enzyme.
Comprehensive Step-by-Step Engineering of an
(R)-Hydroxynitrile Lyase for Large-Scale
Asymmetric Synthesis**
Anton Glieder,* Roland Weis, Wolfgang Skranc,
Peter Poechlauer, Ingrid Dreveny, Sandra Majer,
Marcel Wubbolts, Helmut Schwab, and Karl Gruber
Optically active a-hydroxyacids, such as mandelic acid, and
their derivatives are important building blocks for the
production of pharmaceuticals.[1] Attractive synthetic routes
are made available by hydroxynitrile lyases (HNLs). These
enzymes catalyze the asymmetric addition of HCN to
aldehydes to provide cyanohydrins in high yields and with
high selectivities (Scheme 1).[2,3] The cyanohydrin products
Scheme 1. Enantioselective synthesis of (R)-a-hydroxycarboxylic acids
(R)-3 from aldehydes 1 by the nitrilase and the hydroxynitrile lyase
(HNL) routes. Other methods, such as the hydrolytic resolution of
cyanohydrins in the absence of an additional catalyst, are limited to a
maximum yield of 50%.
A considerable amount of data from preparative reactions
and from biochemical and structural studies[2,3,12–17] are
available for PaHNL. We cloned the Pa_hnl5 gene from
Prunus amygdalus to have unlimited access to PaHNL. This
gene was very similar to hnl genes from Prunus dulcis (mdl1,
99% identity) and Prunus serotina (mdl5, 94% identity),
which are expressed specifically in floral tissues. By over-
expressing the Pa_hnl5 gene, including its native plant
secretory-signal sequence, in the methylotrophic yeast
Pichia pastoris, we obtained 250 mg of the active secreted
enzyme per liter of culture supernatant. The highly glycosy-
undergo chemical hydrolysis under acidic conditions at
elevated temperature to give the corresponding a-hydroxy-
acids without racemization.[1] An alternative route, which
involves enantioselective nitrilases,[4,5] should also theoret-
ically lead to quantitative yields through substrate racemiza-
tion in situ at high pHvalues. However, many nitrilases suffer
lated enzyme showed
a
specific activity of 295 Æ
30 mmolminÀ1 mg for the cleavage of 2a, which is twice as
high as the specific activity of PaHNL isolated from almond
seeds (Sigma M-6782, Lot41H4016; 160 Æ 30 mmol-
minÀ1 mgÀ1). The activity of the recombinant isoenzyme
PaHNL5 did not change significantly over the range
pH2.5–6.5. PaHNL5 was surprisingly stable under acidic
conditions (Figure 1), under which the competitive chemical
reaction is suppressed[18,19] and the enantiomerically pure
cyanohydrin product is also stable. This extremely important
technical feature of HNL reactions in emulsion systems
makes the production of pure enantiomers possible even from
substrates that react slowly and facilitates effective enzyme
recycling. No other HNL is known to be so stable at low
pHvalues. Although the molecular foundation for this
property is still unclear, overglycosylation by Pichia can be
excluded as the only cause, as deglycosylation of recombinant
PaHNL5 by endoglycosidase H in a nondenaturing buffer at
378C to leave just one N-acetylglucosamine substituent on
each of the modified Asn residues did not lead to instability at
[*] Dr. A. Glieder, S. Majer, Prof. Dr. H. Schwab
Institut für Biotechnologie, Technische Universität Graz
Petersgasse 12, 8010 Graz (Austria)
Fax: (+43)316-873-8434
E-mail: glieder@glieder.com
R. Weis, Prof. Dr. K. Gruber
Research Centre Applied Biocatalysis
Steyrergasse 17, 8010 Graz (Austria)
Dr. I. Dreveny, Prof. Dr. K. Gruber
Institut für Chemie, Karl-Franzens-Universität Graz
Heinrichstrasse 28, 8010 Graz (Austria)
Dr. W. Skranc, Dr. P. Poechlauer, Dr. M. Wubbolts
R & D Center Linz
DSM Fine Chemicals Austria Nfg. GmbH & Co KG
St.-Peter-Strasse 25, 4021 Linz (Austria)
[**] We thank the SFB Biokatalyse (F0101), the TIG, the province of
Styria, SFG, and the city of Graz for financial support.
Supporting information for this article is available on the WWW
Angew. Chem. Int. Ed. 2003, 42, 4815 –4818
DOI: 10.1002/anie.200352141
ꢀ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4815