Trends and innovations in industrial biocatalysis for the
production of fine chemicals
Sven Panke, Martin Held and Marcel Wubbolts
Biocatalysis has become an established technology for the
industrial manufacture of fine chemicals. In recent years, a
multitude of chemical companies have embraced biocatalysis
for the manufacture of desired stereoisomers, and new or
improved methods for the synthesis of enantiomerically pure
a- and b-amino acids, amines, amides, peptides, nitriles,
alcohols, organic acids and epoxides have emerged.
Furthermore, the selectivity and mild operational conditions of
biocatalysts are increasingly applied in industry to modify
complex target molecules. These recent innovations in the
manufacture of industrial fine chemicals using biocatalysis are
discussed from an industrial perspective.
Addresses
DSM Research Campus Geleen, Advanced Synthesis & Catalysis,
PO Box 18, 6160 MD Geleen, The Netherlands

e-mail: marcel.wubbolts@dsm.com
Current Opinion in Biotechnology 2004, 15:272–279
This review comes from a themed issue on
Protein technologies and commercial enzymes
Edited by Karl-Erich Jaeger
Available online 17th July 2004
0958-1669/$ – see front matter
ß 2004 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.copbio.2004.06.011
Abbreviations
DERA deoxyribose-5-phosphate aldolase
ee enantiomeric excess
LNT lacto-N-tetraose
NeuAc N-acetyl-D-neuraminic acid
PDF peptide deformylase
PEP phosphoenolpyruvate
Introduction
Enzymes and whole-cell biocatalysts, as a result of their
complex chiral constitution, are predominantly suited for
the manufacture of optically pure stereoisomers. The
production of optically active intermediates is an area
of growing demand in the fine chemical industry and here
biocatalysis has developed from a niche technology to a
widely used manufacturing method [1]. In this review
we discuss recent biocatalysis developments and innova-
tions in industry grouped by product class and we have
restricted ourselves to a- and b-amino acids, amines,
amides, peptides, nitriles, alcohols, organic acids, epox-
ides and complex multifunctional molecules. As the basis
Current Opinion in Biotechnology 2004, 15:272–279
for this review, we performed a survey of patents in the
area of biotransformations over the past few years with a
priority date after 2000 that were issued by companies in
the pharmaceutical, fine chemical, and flavour and fra-
grances area. In total, we judged 205 patents as relevant
to this review (Table 1). In the following, we discuss a
selection of these patents, based on the information
provided therein and supporting publications in the open
scientific literature.
The production of a- and b-amino acids
Aminotransferases that produce a-amino acids from
a-keto acids using an amino donor such as L-aspartic or
L-glutamic acid are — despite the 100% theoretical yield
— typically hampered by moderate yields owing to the
reaction equilibrium. In the case of L-aspartic acid, the
oxaloacetate that is generated can undergo decarboxyla-
tion to pyruvate, thereby pulling the reaction in the
desired direction. Similarly, Great Lakes (http://
www.greatlakesfine.com) has developed a biocatalyst that
combines L-glutamate-preferring a-amino transferases,
such as tyrosine aminotransferase (TAT) and branched-
chain aminotransferase (BCAT), with L-ornithine d-
amino transferase (Figure 1). The latter enzyme converts
2-ketoglutarate and L-ornithine into L-glutamic acid and
L-glutamic acid semialdehyde, which spontaneously
cyclizes to form D1-pyrroline-5-carboxylate. As a result
the reaction equilibrium of the coupled a-amino trans-
ferase reaction can be shifted towards the synthesis
direction. Thus, the synthesis of L-2-aminobutyrate from
2-ketobutyric acid (TAT, 92% yield) and L-tert-leucine
from trimethylpyruvic acid (BCAT, 73% yield) was
achieved [2].
Degussa (http://www.degussa.com) has described the use
of engineered whole-cell biocatalysts producing balanced
levels of the enzymes L-carbamoylase, L-hydantoinase
and hydantoin racemase to synthesize unnatural L-amino
acids [3,4]. The company has further improved this
dynamic kinetic resolution technology for the production
of D-amino acids as well, by including a D-specific carba-
moylase and a D-hydantoinase obtained by directed evo-
lution from the L-specific enzyme [3]. In another known
dynamic kinetic resolution process of Degussa, racemic
N-acetyl amino acids are resolved using an acylase in
combination with an N-acetyl amino acid racemase. A
novel N-acetyl amino acid racemase from Amycolatopsis
orientalis, which is no longer prone to substrate inhibition,
provides a significant improvement over the old process
[3].
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 Innovations in industrial biocatalysis Panke, Held and Wubbolts 273

Table 1

Indication of the industrial focus reflected by selecteda patents published in the area of biocatalysis and biotransformation.

Compound class Subdivision/identified technologies Number of patents

Alcohols Ketone reduction 35b
(Dynamic) kinetic resolution 13
Aldol condensation 5
Oxygenases 3
Other 1
Amino acids/peptides Hydantoin/carbamoyl hydrolysis 17
Amide hydrolysis 7
Transamination 3
b-Amino acids 5
Peptide synthesis by kinetic coupling 8
Other 1
Acids Resolution of esters 14
Nitrile/cyanohydrin hydrolysis 5
Oxidation by oxygenases 4
Acids from lyase reactions 3
Resolution of amides 2
Other 1
Sugars Glycosyltransferases/oligosaccharides/glycoconjugates 10
Glycoprotein remodeling 9
Activated monosaccharides 3
Monosaccharides 1
Others 4
Complex natural products Antibiotics 9
Steroids 5
Other modification of complex scaffolds 2
Others Epoxides/peroxides/diols 9
Amines 9
Aldehydes 6
Cyanohydrins 6
Others 5
a
Patents with priority date after 2000 were retrieved from Chemical Abstracts by SciFinder using enzym*, biocatal* and biotransform* search keys
and were cross-checked for all companies known to be active in the field. Fermentation processes were excluded. bIncluding nine patents
that primarily deal with aspects of cofactor regeneration.

Figure 1

O O

α-Amino
HO R transferase HO R
O NH 2

2-Ketoacid L-Amino acid

L-Glutamic acid 2-Ketoglutarate

O O O

HO HO NH 2
HO O
N NH 2 Ornithine NH 2
δ-amino transferase
3,4-Dihydro-2 H-pyrrole- L-Glutamicacid (OAT) L-Ornithine
2-carboxylic acid semialdehyde
Current Opinion in Biotechnology

Great Lakes process for the combined utilization of a-amino tranferases with L-ornithine-d-amino transferase to produce a-amino acids [2].

www.sciencedirect.com Current Opinion in Biotechnology 2004, 15:272–279

274 Protein technologies and commercial enzymes

Figure 2

HOOC NH2 HOOC NH2

HOOC NH2
HOOC NH2 HOOC NH2
N
S N
N
HOOC NH2 N S
N N
1 N 2 3 4 SO3 H 5
OH
N
N HOOC NH2
HOOC NH2
N
HOOC NH2
COOH
S
S
13 6 7
SH
S
N
HOOC NH2
HOOC NH2
HOOC NH2 HOOC NH2
HOOC NH2
S
N
N O
S Se N
N O
N
N+ N
12 O H
11 10 9 8
N–

Current Opinion in Biotechnology

The Wacker Chemie process for L-cysteine (shown in box) production, which provides access to a wide range of unnatural (S)-a-amino acids [5].
Compounds that have been made include the (S)-enantiomers of tetrazole-2-yl alanine 1, hydroxyethylcysteine 2, pyrazole-1-yl-alanine 3,
sulfocysteine 4, cyanoalanine 5, phenylcysteine 6, thiazole-2-yl-cysteine 7, quisqualic acid 8, azidoalanine 9, phenylselenocysteine 10, thien-2-yl-
cysteine 11, triazole-1-yl-alanine 12 and 5-carboxybenzo-triazole-2-yl-alanine 13.

A production method for the manufacture of cysteine by
fermentation — to replace cysteine produced by extrac-
tion from human hair — has been developed by Wacker
Chemie (http://www.wacker.com/). Building on this tech-
nology in an engineered Escherichia coli strain, the enzyme
O-acetylserine sulfhydrylase, which accepts a broad range
of substrates, was used to produce a stunning number of
unnatural amino acids, such as triazole-1-yl-alanine, (S)-
hydroxyethylcysteine and phenylselenocysteine, by feed-
ing a fermentation with unnatural precursor molecules
(Figure 2) [5]. These novel unnatural amino acids may
prove useful for the synthesis of combinatorial (peptide)
libraries and pharmaceutical intermediates.
Peptide deformylase (PDF) catalyzes the hydrolysis of
the N-formyl group from the N-terminal N-formylmethio-
nine group from nascent polypeptides. Interestingly, the
enzyme is also active with an almost complete enantios-
electivity (E ratio >500) on several N-formylated a- and
b-amino acids, a-amino acid amides and a-amino nitriles
[6]. PDF was used for the enantioselective formylation of
a-amino nitriles, such as phenylalanine nitrile, to yield
(S)-N-formyl-phenylalanine nitrile with an enantiomeric
excess (ee) of over 99.5%. In addition, the enzyme can be
used for the mild and selective deprotection of N-formyl-
protected peptides [6]. In addition to the above-men-
tioned use of PDF [6] and more established resolution
methods using N-acylases and lipases, a novel route for
the production of b-amino acids from b-amino nitriles has
been described that uses nitrilases from Rhodococcus sp.
R312 and Rhodococcus erythropolis NCIMB 11540 [7].
Amines
Enantiomerically pure amines are important chiral inter-
mediates with both pharmaceutical and agricultural appli-
cations. BASF (http://www.basf.com/) has developed a
very versatile Burkholderia plantarii lipase platform for
the resolution of amines using methoxy-acetic acid ethyl
ester as acylating agent [8]. The B. plantarii lipase
has been improved by mutagenesis [9] and other lipases

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 Innovations in industrial biocatalysis Panke, Held and Wubbolts 275

Figure 3

OH OH O

R
OR
15

OC(O)OMe OC(O)OMe

O α-Chymotrypsin O Steps
(a) 16
HO 15
EtO 98.1% ee

O OEt O OEt

O O O
OH O O 17
rec. L. brevis ADH Steps
(b) Cl > 99.5% ee
Cl 15
OC(CH3)3
OC(CH3)3

O O OH OH O O OH
DERA Cl
(c) Cl + Cl Steps
15
2 equivalents
18, 96.6% de
OH

O OH OH
KCN Nitrilase Steps
(d) 19
Cl NC CN NC COOH 15
>95% ee

Current Opinion in Biotechnology

Various biocatalytic routes to (R,R)-3,5-dihydroxy-hexanoate ester derivatives (shown in box; 15), that serve as precursors for the synthesis of statins.
(a) Prochiral 3-substituted glutarates undergo desymmetrization by a-chymotrypsin to (R)-3-methoxyacetylglutaryl monoethyl ester 16 [12]. (b) The
reduction of 3,5-dioxo-hexanoic acid tert-butyl ester to (S)-6-chloro-5-hydroxy-3-oxo-hexanoic acid tert-butyl ester 17 [13]. The reaction is catalysed
by L. brevis ADH. (c) The condensation of chloroacetaldehyde with two molecules of acetaldehyde to form (3R,5S)-6-chloro-3,5-dihydroxy-hexanal
(18) is catalyzed by DERA [16]. The hexanal spontaneously cyclizes with retention of the stereochemistry to the hemi-acetal, which drives the
equilibrium in the desired direction. (d) Nitrilase-catalyzed desymmetrization of 3-hydroxy-glutarodinitrile, which is easily accessible via
epichlorohydrin, yields (R)-4-cyano-3-hydroxy-butyric acid 19 [19]. The enantiomeric excess (ee) values are given as percentages.

and acylating agents have also been successfully used
for amine resolutions by BASF, as reviewed in Breuer
et al. [1].
Diversa (http://www.diversa.com/) has explored the
potential of nitroreductases for the production of (chiral)
amines and has performed high-throughput screening to
obtain suitable enzymes for this purpose [10].
Alternative methods for amine resolutions, based on
penicillin acylases that act by either hydrolyzing N-acyl
amines (hydrolysis mode) or by N-acylation of the amine
starting compounds (synthesis mode), have been
described by DSM (http://www.dsm.com/) [11].
Alcohols and acids
The manufacture of optically active alcohols is an impor-
tant activity in biotransformation. For example, optically
pure alcohols are a key intermediate in the manufacture
of the sidechains of synthetic 3-hydroxy-3-methylglu-
taryl-CoA (HMG-CoA)-inhibiting (cholesterol lowering)
drugs such as atorvastatin, cerivastatin, fluvastatin or
rosuvastatin. Several approaches have recently been dis-
cussed in the academic and patent literature (Figure 3).
One approach involves the desymmetrization of a
methoxyacetyl-ester of glutaric acid diethyl ester with
a-chymotrypsin (Figure 3a) [12]. Alternatively, a 3,5-
dioxocarboxylate can be reduced with excellent enantio-
selectivity employing a recombinant NADPH-dependent
Lactobacillus brevis alcohol dehydrogenase (Figure 3b)
[13]. Another promising route was reported in patent
and open literature by both DSM and Diversa [14,15].
This route employs a deoxyribose-5-phosphate aldolase
(DERA) that catalyzes a tandem aldol condensation in
which two equivalents of acetaldehyde are added to
chloroacetaldehyde to produce a lactol derivative that

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276 Protein technologies and commercial enzymes
is similar to the 3,5-dihydoxy sidechain of synthetic
statins (Figure 3c). Diversa screened for novel DERAs
that were both tolerant to high substrate concentrations
and that were more active than DERA from E. coli [14]. A
DERA variant with less than 30% identity to the E. coli
enzyme led to a significant improvement over the E. coli
wild-type DERA based process originally described by
Wong et al. [16]. The application of rational mutagenesis,
based on the available crystal structure of the E. coli
enzyme, expanded the range of suitable acceptor sub-
strates for DERA to azido-substituted aldehydes, further
facilitating access to, for example, the atorvastatin side-
chain [17].
An interesting variation on the desymmetrisation
approach discussed above is presented by Diversa’s nitri-
lase technology, applied again for the manufacture of the
key intermediate for atorvastatin [18]. Here, a prochiral
dinitrile is hydrolyzed selectively to (R)-3-hydroxy-4-
cyanobutyrate (Figure 3d), which in several subsequent
steps can be converted into 6-cyano-3R,5R-dihydroxy-
hexanoic acid ethyl ester as an advanced intermediate to
atorvastatin [19].
The most prominent method to produce optically active
alcohols by biocatalysis is the enantioselective reduction
of ketones. Novel industrially relevant alcohol dehydro-
genases continue to become available and novel process
engineering concepts have also been developed. For
example, an NAD+-dependent (S)-alcohol dehydrogen-
ase from R. erythropolis DSM43297 has recently been
expressed in E. coli [20]. This enzyme displays excellent
enantioselectivity and accepts aliphatic and aromatic
ketones as well as b-keto esters [21]. Such reactions
typically suffer from the low solubility of the organic
substrate in aqueous buffer. The addition of a second
liquid — organic — phase as a substrate reservoir is an
attractive approach, but has been hampered by the sen-
sitivity of the cofactor recycling enzyme formate dehy-
drogenase from Candida boidinii to such systems.
However, conditions have been identified where an
engineered variant of formate dehydrogenase [22] and
R. erythropolis dehydrogenase remained stable — a two-
phase system consisting of aqueous buffer and 20%
hexane or heptane [23] — thus enabling simple batch
processes with substrate concentrations between 100 mM
and 200 mM.
Another industrially very suitable approach is a mono-
phasic system where the polarity is controlled by the
addition of water-miscible organic solvents. Ideally, such
solvents should also function as a hydrogen source for
cofactor regeneration, preferably employing the same
enzyme that is used for the enantioselective reduction.
In fact, biocatalysts that function in such systems have
recently become available [24]. Whole cells of Rhodo-
coccus ruber DSM 44541 reduce aliphatic and aromatic
Current Opinion in Biotechnology 2004, 15:272–279
ketones in excellent enantioselectvity [25], and the cells
retain activity in the presence of 20% acetone or 50% 2-
propanol. An additional benefit is that the excess of
reducing agent drives the reduction to completion.
The responsible alcohol dehydrogenase has recently
been isolated and tolerates even larger amounts of organic
solvent in the water phase than lyophilized R. ruber cells
[26]. Employing the latter, enantioselective ketone
reductions with only one enzyme and substrate concen-
trations of up to 400 mM could be achieved [27].
Carbohydrates
N-Acetyl-D-neuraminic acid (NeuAc) and related com-
pounds are potential pharmaceuticals and have been
launched as neuraminidase inhibitors (e.g. Relenza from
GSK; http://www.gsk.com/) for the treatment of influ-
enza. Access to NeuAc is difficult and several biocatalytic
approaches have been developed to produce this com-
pound. All start from the easily available N-acetyl-D-
glucosamine that is epimerized to N-actyl-D-mannosa-
mine (ManNAc). There are two possible routes from
ManNAc: an aldol condensation with pyruvate, catalyzed
by NeuAc aldolase, or a NeuAc-synthetase-catalyzed
reaction with phosphoenolpyruvate (PEP). The first route
has been implemented [28], but the equilibrium of the
epimerization as well as the aldol-condensation is unfa-
vourable. The equilibrium problem can be solved by the
degradation of pyruvate after synthesis to facilitate work-
up [29], making use of a novel process for cheap access to
pyruvate by engineered E. coli cells [30]. By substituting
the alkaline epimerization with an enzymatic step, using
an epimerase from hog kidney, the reaction sequence
could be carried out with high productivity [31].
Researchers from Kyowa Hakko (http://www.kyowa.-
co.jp/eng/)_have established a very elegant alternative
approach by functionally expressing the epimerase gene
in E. coli. In addition, they substituted the pyruvate-
requiring aldolase for a PEP-requiring synthase. The
latter reaction is thermodynamically more favourable
[32]. The provision of PEP has been addressed by the
use of perforated Corynebacterium ammoniagenes that con-
verts glucose into PEP.
Given the importance of complex carbohydrate structures
for biological function [33], oligosaccharides have become
desirable drug ingredients. An attractive route for their
production is the assembly of oligosaccharides from their
monosaccharide constituents, in analogy to the Leloir-
pathway. This method depends on the availability of both
the bacterial glycosyltransferases and the activated forms
of the monosaccharides that are used by the glycosyl-
transferases to sequentially build up the oligosaccharide
structure. For example, Galb1-3GlcNAcb1-3Galb1-4Glc
(lacto-N-tetraose; LNT) is of use for fighting Pseudomonas
aeruginosa infections [34]. A suitable starting material for
LNT is lactose to which GlcNAc can be added to obtain
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 Innovations in industrial biocatalysis Panke, Held and Wubbolts 277
Figure 4
O Improved
OH
Prunus amygdalus
hydroxynitrile lyase
Cl Cl
(R)-2-Chloromandelonitrile
ee 96.5%
Hydroxynitrile lyase is used for the production of (R)-2-chloromandelonitrile. The enzyme has been engineered for several improvements: the
inclusion of a secretion signal, active-site modification and recombinant expression of only one of the almond tree (Prunus amygdalus) isoenzymes
[45]. The recombinant enzyme is also stable at acidic pH.
GlcNAcb1-3Galb1-4Glc (lacto-N-triose II; LNTII) using
b1-3GlcNAc transferase. In a second step, galactose (Gal)
is added with b1-3Gal transferase to obtain LNT. The
latter enzyme has recently been cloned from Streptococcus
agalactiae [35], while the former is available from Pasteur-
ella multocida [36]. Combined with Kyowa Hakko’s tech-
nology, which provides access to UDP-Gal [37] and UDP-
GlcNAc [38], a route from lactose to LNT and then to
Galb1-3GlcNAc is now available.
Manipulations of complex natural products
Biocatalysis is widely used for the manipulation of com-
plex natural products, such as antibiotics and steroids.
Over the past couple of years, this approach has been
extended to a broader variety of natural products. The
most prominent example is the manufacturing process for
the anticancer drug Taxol (Paclitaxel), which in nature is
a low yield secondary plant metabolite (<0.1% in T.
brevifolia bark) [39]). To improve the supply of the key
intermediate 10-deacylbaccatin, different taxanes were
used as precursors together with a set of hydrolytic
enzymes [40]. A similar approach has also been used with
epothilones, complex macrolide structures that were iso-
lated from the myxobacterium Sorangium cellulosum and
which also have a role as anti-tumor agents [41]. Micro-
organisms have been isolated that selectively hydroxylate
particular positions of the complex molecule, which is
particularly important as unselective modification at car-
bon atoms C1 to C8 would lead to rapid inactivation [42].
The hydroxylation of epothilone at C21 has attracted
commercial interest and a novel epothilone B C21-amino
derivative has been synthesized [43]. This compound has
entered phase I clinical trials for the treatment of
advanced solid malignancies. Other selective biohydrox-
ylations involve the methylenes at C9 and C14 [43] as
well as the terminal C26 [44].
Cyanohydrins
Hydroxynitrile lyases are versatile enzymes for the synth-
esis of both (R)- and (S)-cyanohydrins, which are impor-
tant starting compounds for the production of pyrethroid
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OH
H+, ∆T
CN CO2H
Cl
(R)-2-Chloromandelic acid
Current Opinion in Biotechnology
insecticides and 2-hydroxy acids such as mandelic acid
derivatives. The production of ortho-substituted aromatic
cyanohydrins, such as (R)-2-chloromandelic acid, is typi-
cally hampered by low turnover frequencies and/or low ee
values. Furthermore, cyanohydrin formation from alde-
hydes typically requires a low pH to address the limited
stability of cyanohydrins at physiological pH. These
problems were approached in a step-by-step manner
(Figure 4). First, a promising (R)-hydroxynitrile lyase
isoenzyme from Prunus amygdalus (PaHNL) was effi-
ciently overexpressed in Pichia pastoris and was engi-
neered to contain a secretion signal. The limited
activity of the enzyme was addressed by rational muta-
genesis, based on the three-dimensional structure, result-
ing in a variant that was sixfold improved for the reaction
in question relative to the wild-type enzyme. In this way,
the production of (R)-2-chloromandelonitrile could be
carried out in excellent yield and with significantly
improved ee values (96.5%) [45].
Conclusions
Biocatalysis and other forms of biotransformation provide
access to (predominantly chiral) fine chemicals and are
increasingly being applied in industry. Trends that can be
observed are the continued use of hydrolases, where
novel enzymes are now accessible as a result of high-
throughput screening methods and genomics. In addition,
there is an increase in the industrial application of oxi-
doreductases, especially for ketone reductions, amino
transferases and carbon–carbon bond forming enzymes.
References and recommended reading
Papers of particular interest, published within the annual period of
review, have been highlighted as:
 of special interest
 of outstanding interest
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 Zelinski T: Industrial methods for the production of optically
active intermediates. Angew Chem Int Ed Engl 2004, 43:788-824.
Excellent overview of current methods — both chemical and biocatalysis
— applied in industry for the synthesis of enantiomerically pure com-
pounds.
Current Opinion in Biotechnology 2004, 15:272–279
278 Protein technologies and commercial enzymes
2. Li T, Kootstra AB, Fotheringham IG: Nonproteinogenic a-amino
 acid preparation using equilibrium shifted transamination.
Org Process Res Dev 2002, 6:533-538.
Elegant combination of an a- and d-aminotransferase to produce
L-a-amino acids in high yield.
3. May O, Verseck S, Bommarius A, Drauz K: Development of
dynamic kinetic resolution processes for biocatalytic
production of natural and nonnatural L-amino acids.
Org Process Res Dev 2002, 6:452-457.
4. Trauthwein H, May O, Dingerdissen U, Buchholz S, Drauz K: A new
biocatalytic route to enantiopure N-carbamoyl amino acids by
fast enzyme screening. Tetrahedron Lett 2003, 44:3737-3739.
5. Maier THP: Semisynthetic production of unnatural L-a-amino
 acids by metabolic engineering of the cysteine-biosynthetic
pathway. Nat Biotechnol 2003, 21:422-427.
Breakthrough methodology for the manufacture of a variety of unnatural
(S)-a-amino acids by fermentation/biotransformation based on the
cysteine biosynthesis pathway.
6. Sonke T, Kaptein B, Wagner AFV, Quaedflieg PJLM, Schultz S,
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amines via lipase-catalyzed methoxyacetylation.
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Good illustration of the potential of DERA and aldolases in general for
industrial asymmetric synthesis.
15. Kierkels JGT, Mink D, Panke S, Lommen FAM, Heemskerk D:
Process for the preparation of 2,4-dideoxyhexoses and 2,4,6-
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19. Burk M, Desantis G, Morgan B, Zhu Z: Manufacture of (R)-ethyl
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