Formation and hydrolysis of amide bonds by lipase A from Candida antarctica; Exceptional features

Article abstract of DOI:10.1039/b920939p

Various commercial lyophilized and immobilized preparations of lipase A from Candida antarctica (CAL-A) were studied for their ability to catalyze the hydrolysis of amide bonds in N-acylated α-amino acids, 3-butanamidobutanoic acid (β-amino acid) and its ethyl ester. The activity toward amide bonds is highly untypical of lipases, despite the close mechanistic analogy to amidases which normally catalyze the corresponding reactions. Most CAL-A preparations cleaved amide bonds of various substrates with high enantioselectivity, although high variations in substrate selectivity and catalytic rates were detected. The possible role of contaminant protein species on the hydrolytic activity toward these bonds was studied by fractionation and analysis of the commercial lyophilized preparation of CAL-A (Cat#ICR-112, Codexis). In addition to minor impurities, two equally abundant proteins were detected, migrating on SDS-PAGE a few kDa apart around the calculated size of CAL-A. Based on peptide fragment analysis and sequence comparison both bands shared substantial sequence coverage with CAL-A. However, peptides at the C-terminal end constituting a motile domain described as an active-site flap were not identified in the smaller fragment. Separated gel filtration fractions of the two forms of CAL-A both catalyzed the amide bond hydrolysis of ethyl 3-butanamidobutanoate as well as the N-acylation of methyl pipecolinate. Hydrolytic activity towards N-acetylmethionine was, however, solely confined to the fractions containing the truncated form of CAL-A. These fractions were also found to contain a trace enzyme impurity identified in sequence analysis as a serine carboxypeptidase. The possible role of catalytic impurities versus the function of CAL-A in amide bond hydrolysis is further discussed in the paper. The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/b920939p

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Formation and hydrolysis of amide bonds by lipase A from Candida
antarctica; exceptional features†
Arto Liljeblad,*a Pauli Kallio,b Marita Vainio,a Jarmo Niemib and Liisa T. Kanervaa
Received 6th October 2009, Accepted 24th November 2009
First published as an Advance Article on the web 4th January 2010
DOI: 10.1039/b920939p
Various commercial lyophilized and immobilized preparations of lipase A from Candida antarctica
(CAL-A) were studied for their ability to catalyze the hydrolysis of amide bonds in N-acylated a-amino
acids, 3-butanamidobutanoic acid (b-amino acid) and its ethyl ester. The activity toward amide bonds is
highly untypical of lipases, despite the close mechanistic analogy to amidases which normally catalyze
the corresponding reactions. Most CAL-A preparations cleaved amide bonds of various substrates with
high enantioselectivity, although high variations in substrate selectivity and catalytic rates were
detected. The possible role of contaminant protein species on the hydrolytic activity toward these bonds
was studied by fractionation and analysis of the commercial lyophilized preparation of CAL-A
(Cat#ICR-112, Codexis). In addition to minor impurities, two equally abundant proteins were
detected, migrating on SDS-PAGE a few kDa apart around the calculated size of CAL-A. Based on
peptide fragment analysis and sequence comparison both bands shared substantial sequence coverage
with CAL-A. However, peptides at the C-terminal end constituting a motile domain described as an
active-site flap were not identified in the smaller fragment. Separated gel filtration fractions of the two
forms of CAL-A both catalyzed the amide bond hydrolysis of ethyl 3-butanamidobutanoate as well as
the N-acylation of methyl pipecolinate. Hydrolytic activity towards N-acetylmethionine was, however,
solely confined to the fractions containing the truncated form of CAL-A. These fractions were also
found to contain a trace enzyme impurity identified in sequence analysis as a serine carboxypeptidase.
The possible role of catalytic impurities versus the function of CAL-A in amide bond hydrolysis is
further discussed in the paper.
a tetrahedral intermediate between the substrate and a serine
Introduction
hydroxyl at the enzyme active site. This is coupled to the formation
of a covalent acyl-enzyme complex (acylation phase), which results
in the release of the first product, the nucleophilic part of the
acyl donor. In the subsequent step, the ester intermediate is
hydrolyzed (deacylationphase) generating the secondproduct, free
carboxylic acid. Interestingly, although many amidases are known
to readily hydrolyze both esters and amides, lipases generally do
not cleave amide bonds. This may be due to the higher activation
energy requiredfor the formation ofthe enzyme-boundtetrahedral
transition state of amides in comparison to the more polar ester
substrates. Thus, contrary to in amides, in esters the vicinity of the
relatively electronegative oxygen restricts electron delocalization
over the adjacent bonds, resulting in the reduction of resonance
stabilization at the ground state. A structural explanation for
the ester versus amide specificity may be the efficiency of the
protonation of the leaving group, which has been demonstrated to
influence the enzymatic cleavage of unnatural amide substrates.1
In addition, it has been shown that the presence of the transition-
state stabilizing hydrogen-bonded water network reaching to the
amide hydrogen is decisive for the amide hydrolyzing activity of
esterase from Bacillus subtilis.2
The physiological role of secreted lipases, such as lipase A from
the yeast Candida antarctica (CAL-A), is to decompose triacyl-
glycerols into free fatty acids and glycerol. Typically the lipase-
catalyzed hydrolytic reactions of the carboxyester bond are highly
regioselective and enantioselective with a substrate preference for
long-chain acylglycerols (>10 carbons). As a group, lipases are
known to be diverse catalysts which can hydrolyze a broad range
of different fatty acid esters and, in reverse, catalyze esterification
between carboxylic acids and alcohols. These features, combined
with characteristic stability in demanding conditions, such as in
organic solvents, render lipases useful for numerous applications
in organic synthesis and industry.
The active site topology and hydrolytic mechanism of lipases
is common to the members of the serine hydrolase family. The
hydrolytic reactions of both ester and amide bonds proceed via
a well-characterized double displacement mechanism involving
aInstitute of Biomedicine, Pharmacology, Drug Development and Therapeu-
tics/Laboratory of Synthetic Drug Chemistry and Department of Chemistry,
University of Turku, Lemminka¨isenkatu 5 C, FIN-20520, Turku, Finland.
E-mail: artlilje@utu.fi; Fax: +358 2 333 7955; Tel: +358 2 333 7956
Despite the relative stability of amides, some lipases and
esterases have been reported to be capable of hydrolyzing
amide bonds. Electrophilic substituents facilitating polarization
of electrons (e.g. halogens) and structural features which prevent
resonance stabilization can activate amide substrates towards
bDepartment of Biochemistry, University of Turku, FIN-20014, Turku,
Finland
† Electronic supplementary information (ESI) available: 1H, COSY,
HMBC and HSQC NMR spectra of 3-butanamidobutanoic acid. See
DOI: 10.1039/b920939p
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activation at the lipid–water interface (interfacial activation),21
related classical property of lipases associated with the hydrolysis
of fatty acid glycerol esters, the 3D fold assigns CAL-A as the first
representative of a novel subclass of lipases.
hydrolytic cleavage by lipases. Thus, many lipases cleave the
strained four-membered b-lactam ring by water or alcohol3-6
and lipase from Candida rugosa acts on N-trifluoroethyl 2-
chloropropanamide.7 Lipases from Rhizomucor miehei and Pseu-
domonas stutzeri have recently been reported to hydrolyze
N-acetyl-DL-alanine, a natural amino acid amide, with low
enantioselectivity.8 In addition, a lipase from Pseudomonas aerug-
inosa has been shown to hydrolyze the amide bond of oleoyl 2-
naphthylamide, an activity which has further been enhanced by di-
rected evolution.9 Perhaps the most extensively studied member of
the esterase family with reported amide activity is the lipase B from
Candida antarctica (CAL-B) which has been shown to hydrolyze
substrates like trans-8-methyl-N-vanillyl-6-nonenamide, N-(1-(4-
chlorophenyl)ethyl)acetamide and N-methylacetamide.10-12 It is
worth pointing out that many of the experiments have been carried
out using semi-pure cell extracts or bulk-purified commercial
enzyme preparations which may be expected to contain enzyme
impurities. For instance, the amide bond hydrolytic activity
observed for the lipase extract from porcine pancreas appeared
to result from a contaminant enzyme in the preparation.13
Thus, the possible contribution of trace protein activities should
be considered and analyzed whenever unexpected activities are
encountered.
a
The interest towards CAL-A has largely evolved from its
exceptional tolerance to heat, pH and organic solvents, which
together with exceptionally broad substrate specificity and stere-
oselectivity make it a versatile biocatalyst for organic synthesis
and biotechnological applications.15,22 CAL-A has been shown
to exhibit many unique catalytic properties, such as substrate
preference for trans-fatty acids and the ability to catalyze highly
enantioselective acylation reactions of bulky sterically hindered
secondary and tertiary alcohols which are not usually accepted as
substrates by lipases.23-29 Of specific interest to this study, CAL-
A preparations have also been shown to catalyze secondary N-
acylation of a-amino esters,30-32 and a range of other a- and b-
amino esters in organic solvents.14-16 All these features make CAL-
A a potential candidate for protein engineering.33 CAL-A has been
heterologously expressed in Aspergillus oryzae,34 Pichia pastoris35
and Escherichia coli36 and at present is commercially available
from various companies as lyophilized preparations, cross-linked
enzyme aggregates (CLEA preparation) as well as in immobilized
form on different solid supports.
As the starting point for this work, the lipase CAL-A was
expected to be a potential candidate for hydrolyzing amides,
as it has been previously shown to catalyze N-acylation of
various amine precursors.14-16 CAL-A is a unique member of
the superfamily of ester hydrolases, and it has been extensively
characterized for catalytic and physical features in parallel to
recent studies on crystal structure.17,18 CAL-A is a secreted
enzyme composed of 431 amino acids in the mature form. In
the primary translation product the sequence is preceded by an
N-terminal signal peptide of 21 amino acids and a propeptide of
10 amino acids.19 In primary sequence comparison CAL-A shares
substantial amino acid similarity with a wide array of secreted
lipases and esterases. However, related lipase homolog crystal
structures have not been resolved, and the most closely related
folds found in a DALI search are annotated as peptidases. The
enzymes with the highest sequence identity in a BLAST search are
lipase from Kurtzmanomyces sp. I-11 (77%), protein from Ustilago
maydis 521 with a conserved esterase/lipase domain (68%) and
secreted lipase 1 precursor from Malassezia furfur (35%). There is
no apparent amino acid sequence homology between the Candida
antarctica lipases CAL-A and CAL-B (sequence identity 7.8%
in EBI EMBOSS alignment). Thus, they are not directly related
despite their similar nomenclature, function and the common
origin.
As expected by the comparisons, the structure of CAL-A
encloses the central structural features characteristic for the
mechanistically related superfamilies of hydrolases, involving the
a/b hydrolase fold and a catalytic triad of Ser, Asp and His
arranged in the order typical to lipases.18 The topology of the active
site pocket of CAL-A appears to be well suited to accommodate
long chain fatty acids and, in agreement with the putative function,
the enzyme has been reported to have catalytic preference for long-
chained carboxylic acids over smaller acyl groups.20 Importantly,
the structure also reveals a distinct lid domain characteristic of
lipases, but without apparent sequence homology to previously
known lid structures.18 Together with the reported catalytic
We have herein studied the behavior of CAL-A with amide sub-
strates in detail (Scheme 1). New marketed CAL-A preparations
were first screened for N-acylation of 1a, followed by the analysis
of selected candidates for the studies on amide bond hydrolysis
of various racemic a- and b-amino acid derivatives 1b–c, 2a–c,
3a and 4a. A parallel objective was to investigate the possible
effect of contaminating enzymes, because some commercial CAL-
A preparations are known to contain enzyme impurities.37 Based
on detected activities of interest, the commercial CAL-A prepa-
ration Cat#ICR-112 was selected for fractionation, followed by
comparison of the resolved catalytic activities and the different
protein species found.
Scheme 1 The model compounds for the CAL-A reactions.
Results and discussion
b-Amino acid derivatives as substrates for CAL-A
As the first experiment, commercial CAL-A preparations were
compared for their ability to catalyze the N-acylation of ethyl 3-
aminobutanoate rac-1a in neat butyl butanoate, a reaction earlier
reported for CAL-A (Scheme 2, Table 1).38 All the enzyme prepara-
tions were in immobilized form as provided by the manufacturers
or, in the case of the lyophilized Chirazyme L5 and Cat#ICR-112,
immobilized on Celite as described earlier (entries 1 and 2).39 The
preparations clearly catalyzed the reaction, enantioselectivities
(E around 40) being similar to those previously observed for
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Scheme 2 CAL-A-catalyzed N-acylation of ethyl 3-aminobutanoate rac-1a.
Table 1 N-Acylation of rac-1a (0.100 M) in butyl butanoate (1 cm3) with
CAL-A preparations (40 mg cm-3) at 3 h. The CAL-A preparations tested
later for hydrolysis of a- and b- amino acid derivatives are shown bold
(entries 1–5)
Entry CAL-A preparation
Conv. [%]
ees [%]
88
eeP [%]
83
E
1
2
Chirazyme L5 on
Celitea,b
51
48
32
39
1
1
Cat#ICR-112 on
82
88
Celiteb
3
4
5
CAL-A-CLEA
Cat#NZL-101-IMB
Immozyme
CAL-A-T2-150
VZ1030-2
VZ1030-3
VZ1030-4
VZ1030-10
VZ1030-11
VZ1030-12
VZ1031-2
VZ1031-3
VZ1031-5
33
55
46
45
98
76
92
80
88
34
42
39
2
3
1
6
7
8
9
10
11
12
13
14
9
20
48
25
47
52
37
53
37
9
24
82
31
78
94
54
95
55
95
95
91
94
89
85
93
85
93
48
51
50
41
41
41
46
46
46
2
1
1
1
1
1
1
1
1
Fig. 1 Cat#NZL-101-IMB-catalyzed (100 mg cm-3) hydrolysis of rac-1c
(ꢀ, 0.050 M) and formation of the products (S)-1b (᭹), (R)-1a (᭢) and
(R)-1d (ꢀ). Ee-values have been marked in parentheses.
(S or R)-1d (Scheme 3; Table 2; Fig. 1). Although the reactions
proceeded slowly, this confirmed that both the amide and the
ester groups of the substrate were hydrolyzed. The observed
amide hydrolysis was highly enantioselective with the preparations
Cat#ICR-112, Chirazyme L5 and CAL-A-CLEA, ee values being
>95 for (R)-1a (Table 2; entries 1–3). With Cat#NZL-101-IMB
and Immozyme CAL-A-T2-150 the corresponding reaction took
place with only moderate enantioselectivity (entries 4 and 5).
Configuration of the formed product 1d was either R (Cat#NZL-
101-IMB and CAL-A-CLEA) or S (CAL-A-T2-150, Cat#ICR-
112 and Chirazyme L5) depending on the relative proportions
and hydrolytic rates of (S)-1b and (R)-1a in each case.
a Ref. 38, t = 24 h. b Commercial lyophilized CAL-A (10%) on Celite (78%)
and sucrose (12%).
CAL-A on Celite (cf. Table 1; entry 1).38 The immobilized CAL-
As of ViaKit gave the highest E values of around 50 (entries 6–8
and 12–14).
Five of the CAL-A preparations analyzed above (Chirazyme L5,
Cat#ICR-112, CAL-A-CLEA, Cat#NZL-101-IMB and CAL-A-
T2-150) were selected for the trial to test the amide bond hydrolysis
of rac-1c, the resolution product of Scheme 2 (Scheme 3). In
contrast to the previous reaction series, the lyophilized prepa-
rations Chirazyme L5 and Cat#ICR-112 were analyzed without
immobilization on Celite, as the enzymes may be detached from the
solid support in aqueous reaction conditions. All the preparations
yielded three different conversion products (S)-1b, (R)-1a and
a-Amino acid derivatives as substrates for CAL-A
In the subsequent reaction series the same five CAL-A prepa-
rations shown in Table 2 were analyzed for their ability to hy-
drolyze the amide bonds of N-protected methionine rac-2a–c, N-
acetylphenylglycine rac-3a and N-acetylvaline rac-4a (Scheme 4,
Table 3). Both lyophilized CAL-A preparations (Chirazyme L5
and Cat#ICR-112) were found to catalyze the hydrolysis of
the amide substrates 2a, 2c, 3a and 4a in phosphate buffer
(0.100 M, pH 7.5) (Table 3; entries 1–2). The reactions were
highly enantioselective with E values of over 200, in contrast to
the corresponding N-acylation reactions of a-amino esters earlier
reported to proceed with low or moderate enantioselectivity.31 The
highest conversions were observed with N-acetylmethionine 2a,
whereas the hydrolysis of 3a with a sterically more constrained a-
phenyl substituent was less efficient. The only compound that was
not accepted as a substrate by the lyophilized CAL-A preparations
was N-Boc-methionine 2b with the bulky tert-butoxycarbonyl
substituent.
Immozyme CALA-T2-150 was also found to have activity
toward some of the substrates tested; it slowly catalyzed the amide
bond hydrolysis of 2a, 2c and 4a with high enantioselectivity
Scheme 3 CAL-A-catalyzed sequential resolution of rac-1c.
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Table 2 CAL-A-catalyzed hydrolysis of b-amino acid derivative rac-1c (0.050 M) in phosphate buffer (0.100 M, pH 7.5) at room temperature after 24 h
Entry
CAL-A preparation
ee1c (%)/Yield [%]
ee (R)-1a (%)/Yield [%]
ee(S)-1b (%)/Yield [%]
ee1d (%)/Yield [%]/Abs. config.
1
2
3
4
5
Chirazyme L5, lyo.a
Cat#ICR-112, lyo.a
CAL-A-CLEAc
53/35
77/33
10/4
22/41
4/39
98/44
71/18
64/47
29/73
49/8
-/3/S
-/3/S
-/1/R
82/8/R
8/5/S
>95/17
>95/22
89/43
Cat#NZL-101-IMBb
Immozyme CAL-A-T2-150c
50/19
67/37
a 5 mg cm-3. b 100 mg cm-3. c 75 mg cm-3.
Table 3 Enzyme-catalyzed (5 mg cm-3) hydrolysis of a-amino acid derivatives 2a–c, 3a and 4a (0.050 M) in phosphate buffer (pH 7.5; 0.100 M) at room
temperature (t = 24 h, except for 2c 2 h)
Entry
CAL-A preparation
2a Conv. [%]/E
2b Conv. [%]/E
2c Conv. [%]/E
3a Conv. [%]/E
4a Conv. [%]/E
1
2
3
4
5
Chirazyme L5, lyo.
Cat#ICR-112, lyo.
CAL-A-CLEAa
49/>200
50/>200
0/-
0/-
14/>200
0/-
0/-
0/-
0/-
0/-
17/>200
31/>200
0/-
6/>200
2/>200
0/-
37/>200
42/>200
0/-
Cat#NZL-101-IMBb
Immozyme CAL-A-T2-150
0/-
<1/>200c
0/-
0/-
1/>200c
1/>200c
a 20 mg cm-3. b 75 mg cm-3. c determined by taking samples after several days at later stage of the reaction.
Scheme 4 CAL-A-catalyzed hydrolysis of N-acyl-a-amino acids.
(E > 200), just as observed for the two lyophilized preparations
(Table 3; entry 5).
purposes, and may contain catalytically active enzyme impurities
which originate from the expression host strain.34,37 To analyze the
possible role of impurities in relation to the observed activities,
one of our target CAL-A preparations (lyophilized Cat#ICR-112,
Codexis) was selected as a representative for a detailed fractiona-
tion study. The other CAL-A preparations, except Chirazyme L5,
were covalently immobilized and thus not suitable for the purpose.
As for the activities of interest, Cat#ICR-112 catalyzed the
amide bond hydrolysis of various a- and b-amino acid derivatives
(Tables 2 and 3), and the corresponding immobilized derivative
catalyzed the N-acylation of b-amino acid derivative ethyl 3-
aminobutanoate rac-1a in butyl butanoate (Table 1; entry 2).
To investigate whether the activities could be separated from
one another, and whether they overlap with the CAL-A elution
fraction instead of any possible impurity fractions, the preparation
was subjected to preparative size exclusion chromatography,
SDS-PAGE analysis with sequence-based identification and ac-
tivity characterization.
The remaining immobilized preparation (Cat#NZL-101-IMB)
and the cross-linked derivative (CAL-A-CLEA) were either
inactive or had only very low activity towards the substrates
tested (Table 3; entries 3–4). Similar results were obtained in an
additional reaction series, in which the 18 immobilized CAL-As of
ViaKit (not included in Table 3) gave only traces of the hydrolyzed
product (S)-2d (c < 3% in 24 h) from rac-2a.
Fractionation and analysis of the commercial CAL-A preparation
(Cat#ICR-112, Codexis)
Overview. Several CAL-A preparations in this study were
found to have unexpected amidase activity. Of the enzymes
which generally catalyze the hydrolysis of non-peptide carbon–
nitrogen bonds of linear amides (E.C. 3.5.1), hydrolysis of
2a–4a corresponds to that of acylase I-type enzymes (N-acyl-L-
amino-acid amidohydrolases, E.C. 3.5.1.14) which by definition
are specific for different N-acyl-L-amino acids.40 Members of this
functional class of amidohydrolases have been widely used as
biocatalysts in the preparation of enantiopure a-amino acids,41
which makes the activity discovered for the CAL-A preparations
of distinct scientific interest.
SDS-PAGE and LC-MS/MS characterization
SDS-PAGE analysis of the CAL-A preparation Cat#ICR-112
revealed multiple protein bands of varying intensities ranging from
less than 30 kDa to above 60 kDa in size (Fig. 2). Five distinct
bands of potential interest migrating between ~30–55 kDa were
selected as targets for in-gel digestion and LC-MS/MS analysis
Bulk-purified commercial enzymes such as the available CAL-A
preparations are not primarily prepared for analytical or research
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Da). In fragment LC-MS/MS analysis and MASCOT sequence
comparison both these bands shared over 40% coverage (with >10
distinct identified peptides for each) with the CAL-A sequence. To
avoid cross-contamination between the bands, Cat#ICR-112 was
subsequently fractionated using preparative gel filtration and re-
analyzed (see the next section).
The smallest analyzed band (band 5 in Fig. 2), migrating
above 30 kDa on SDS-PAGE gel, was identified as a degradation
product of CAL-A. The sequence coverage was 22% with 6 distinct
identified peptides exclusively at the N-terminal end of the target
protein sequence.
The two analyzed fragments migrating around 55 kDa (bands
3–4 in Fig. 2) were clearly larger in size than that expected for CAL-
A. In sequence-based identification these bands matched with a
good score value and sequence coverage with a-amylase (best hit
54.8 kDa from Aspergillus shirousami). Although some peptides
of lipases were identified in these two samples probably due to
sample smearing during electrophoresis, apparently these proteins
are not related to CAL-A, but they are contaminants originating
from the expression host organism.
Fig. 2 SDS-PAGE analysis (Bio-Rad Criterion 10% Bis-Tris/silver
stained) of the commercial CAL-A preparation (Cat#ICR-112 from
Codexis) against Bio-Rad standard #161-0318. The bands submitted
for in-gel digestion, LC-MS/MS analysis and sequence comparison are
labeled 1–7. The best sequence hits: bands 1–2) CAL-A; bands 3–4)
a-amylase (Aspergillus shirousami); band 5) CAL-A N-terminal half;
band 6) maltase glucoamylase (Aspergillus); band 7) maltase glucoamy-
lase/serine carboxypeptidase (Aspergillus). Bands marked with * were only
or additionally analyzed from fractionated samples.
Gel filtration fractionation and further analysis
Cat#ICR-112 was fractionated using preparative gel filtration
size-exclusion chromatography to study the distribution of dif-
ferent proteins in more detail. Consistent with the heterogeneity
visualized in SDS-PAGE, the runs resulted in multiple eluting
peaks; two distinct, partially overlapping main peaks (one with a
clear shoulder) together with a few minor peaks (Fig. 3). Despite
attempts to optimize the run conditions the resolution remained
incomplete.
and compared against the CAL-A sequence34 and Sprot Trembl
database sequences for possible identification.
The two most intense bands on SDS-PAGE (bands 1–2 in
Fig. 2) migrated a few kDa apart from one another, near
the calculated molecular weight of the mature CAL-A (46129
SDS-PAGE analysis of the gel filtration fractions (Fig. 4)
showed that the two proteins preliminarily identified as
Fig. 3 UV–vis chromatogram of FPLC gel filtration fractionation of the CAL-A Cat#ICR-112. The fractions collected for analysis are highlighted.
The smaller chromatograms show the peak fractions 19 and 23 after second round of gel filtration.
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apex peak fractions. In fraction 23 the most intense band (~55
kDa) appeared to be composed of the putative a-amylase contam-
inants identified earlier in protein fragment characterization. In
fraction 18, the concentration procedure revealed two new bands
around 60 kDa, which were only barely visible and ignored in
earlier SDS-PAGE runs (bands 6–7 in Fig. 2). In the subsequent
protein fragment LC-MS/MS analysis and comparison against
the Sprot Trembl database, the bands were best matched with
Aspergillus maltase glucoamylase (sequence coverage 25% and
number of distinct peptides 24/sequence coverage 19% and
number of distinct peptides 14). Some peptides in the larger band
also matched with Aspergillus serine carboxypeptidase sequence
(4th hit; sequence coverage 13% and number of distinct peptides
identified 7).
Fig. 4 SDS-PAGE analysis of concentrated gel filtration fractions 15–23
of CAL-A Cat#ICR-112 against Fermentas Pageruler standard #SM0661
(self-casted 8% gel/Coomassie Brilliant Blue stained). The two dotted
horizontal lines between 40 kDa and 50 kDa show the migration fronts
of the two CAL-A proteins. The collected fraction 24 is missing from the
image.
Activity in the gel filtration fractions
CAL-A eluted throughout the two main peaks. The smaller protein
(band 1 in Fig. 2) was distributed over the first main peak in
consecutive fractions 15–21 (Fig. 3), whereas the larger protein
(band 2 in Fig. 2) was mainly observed in the second main peak
fractions 23–24. In repeated rounds of preparative gel filtration
the two proteins were sufficiently separated from one another
(Fig. 3), digested and characterized by LC-MS/MS as before. The
total sequence coverage of the larger band was 47% in relation to
the mature CAL-A sequence (the number of different identified
peptides 12), and peptides corresponding to both the C-terminal
end and N-terminal end were identified. The sequence coverage
of the smaller band was 35% (the number of different identified
peptides 10) and notably without any identified peptides spanning
the C-terminal region of the target sequence. This suggests that
the smaller band (>45 kDa) may be a degradation product of
CAL-A which lacks a fragment of up to several kDa from the
C-terminus. The number of the missing amino acids could not,
however, be determined by protein mass spectrometry due to
remaining protein contaminants in the sample. Inspection of the
CAL-A crystal structure18 reveals that the C-terminal residues
425–435 form an antiparallel b-sheet secondary structure loop
(B10/B11) which resides on the surface of the protein, near the
active site cavity. The loop functions as an “active-site flap” which
restricts access to the active site in the inactive form, and it is turned
away upon activation to allow substrate binding. To exclude any
possible effect of non-uniform post-translational glycosylation on
the separation of the two CAL-A bands, the preparation was also
subjected to enzymatic N-glycosidase treatment. This, however,
had no apparent effect on the SDS-PAGE migration profile of the
bands, further supporting protein cleavage as the primary cause for
the observed size difference. The native N-terminal signal peptide
(21aa) or the propeptide (10aa) specific to the immature form of
CAL-A34 were not recognized in any of the runs for the protein
fragment characterization.
Each gel filtration fraction was individually analyzed for three
activities corresponding to those earlier measured for the different
CAL-A preparations: N-acylation and two amide bond hydrolytic
reactions. The first monitored reaction was N-acylation of methyl
pipecolinate rac-5a in tert-butyl methyl ether (TBME) (Scheme 5).
CAL-A has been shown to catalyze this reaction with high
enantioselectivity,30,31 whereas acylase I-type enzymes and most
lipases do not generally accept the bulky 5a as their substrate
for the N-acylation. This is contrary to the less-specific substrate
ethyl 3-aminobutanoate rac-1a (Scheme 2) which is known to be
converted by many different enzymes.42 Hence it was rejected
for this screening. The second test reaction was the hydrolysis
of ethyl 3-butanamidobutanoate rac-1c (Scheme 3). The activity
of interest, amide hydrolysis of b-amino acid-derivatives, is not
common to known amidases, although the corresponding ester
hydrolysis could be expected to be catalyzed by many related
enzymes. The third studied reaction was the hydrolysis of N-
acetylmethionine rac-2a, a catalytic activity characteristic to
acylase I-type enzymes and less typical to lipases (Scheme 4).
N-Acylation of methyl pipecolinate rac-5a. This activity was
found to be distributed over the entire elution front and, as seen in
quantitative comparison of relative activities (Table 4; Fig. 5a), the
highest values centered around the two main peaks, fractions 18–
19 and 23. In comparison to the corresponding band intensities on
the SDS-PAGE gel (Fig. 4), the relative activities appeared directly
proportional to the quantity of CAL-A in each fraction.
Hydrolysis of ethyl 3-butanamidobutanoate rac-1c. The main
reaction was clearly the ester hydrolysis from rac-1c to yield (S)-
1b (Table 4). However, formation of the alternate product (R)-1a
confirmed that also the amide bond of the b-amino acid derivative
rac-1c was hydrolyzed (Table 4; Fig. 5b). The amide hydrolytic
activity was observed in both main gel filtration elution peaks and
centered in fractions 18–19 and 23, overlapping almost precisely
The two CAL-A species eluted in rather broad fronts in gel
filtration and were no longer the most abundant proteins in the
Scheme 5 CAL-A-catalyzed N-acylation of methyl pipecolinate rac-5a.
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Fig. 5 Distribution of the relative activities (a) N-acylation of methyl pipecolinate rac-5a, (b) hydrolysis of ethyl 3-butanamidobutanoate rac-1c, and (c)
hydrolysis of N-acetylmethionine rac-2a in the CAL-A Cat#ICR-112 gel filtration fractions. Activities shown as % in relation to the most active fraction.
Table 4 Analysis of the gel filtration peak fractions 15–24 (Fig. 3) by
N-acylation of methyl pipecolinate rac-5a with trifluoroethyl butanoate
(A), hydrolysis of ethyl 3-butanamidobutanoate rac-1c (B) and by hydro-
lysis of N-acetylmethionine rac-2a (C)
of intracellular protein turnover and specific protein maturation
reactions. Although the protein could potentially have activity
towards the provided amide substrates, the effect of this (or any
other) impurity on the hydrolysis of N-acetylmethionine remains
unclear.
A
B
C
Unlike observed for the N-acylation of methyl pipecolinate
rac-5a and hydrolysis of ethyl 3-butanamidobutanoate rac-1c,
hydrolysis of N-acetylmethionine rac-2a was not detected in the
second gel filtration elution peak. This demonstrated that this
source of activity is absent in fractions 22–24 and that the reaction
is not catalyzed by the native form of CAL-A. In analogy to this
observation, the preparations Cat#NZL-101-IMB and CAL-A-
CLEA were shown to catalyze the N-acylation of rac-1a (Table 1),
but they did not hydrolyze N-acetyl-a-amino acids (Table 3).
Gel filtration fraction number
c [%]
[1b]/%
[1a]/%
c [%]
15
16
17
18
19
20
21
22
23
24
0.8
1.1
1.0
1.7
2.5
0.9
0.3
0.5
6.9
2.2
10
44
43
49
49
54
57
42
12
5
—
1
3
4
4
1
traces
traces
14
3
10
7
20
35
6
1
< 1
< 1
< 1
1
Conclusion
with the relative methyl pipecolinate N-acylation activity profile
described above (Fig. 5a,b).
In this paper we demonstrate that commercial lyophilized and
immobilized preparations of lipase A from Candida antarctica
(CAL-A) have distinct hydrolytic activity toward the amide bond
of various N-acyl-a- and b-amino acid derivatives. This property
is generally uncommon to carboxyester hydrolases, especially
classical lipases which naturally hydrolyze fatty acid glycerol
esters. The observed reactions catalyzed by the selected CAL-A
preparations (Chirazyme L5, lyo.; Cat#ICR-112, lyo.; Immozyme
CALA-T2-150, Cat#NZL-101-IMB; CAL-A-CLEA) were rather
slow but moderately or highly enantioselective depending on the
substrate structure. Although most of the eight amide substrates
were hydrolyzed, the results revealed clear differences in substrate
preference between the CAL-A preparations tested.
To study the distribution of the observed activities of CAL-A, a
representative of the commercial lyophilized products (Cat#ICR-
112) was subjected to fractionation, sequence-based identification
and functional comparison. In addition to several proteins present
at low relative concentrations, SDS-PAGE and preparative gel
filtration revealed two main protein species of about equal
abundance. The two proteins migrated a few kDa apart, around
the ~46 kDa size range expected for CAL-A. The larger protein
Although these two monitored activities are not characteristic
to lipases, the results suggest that the two forms of CAL-A
identified in the commercial preparation (bands 1–2 in Fig. 2) may
both be correctly folded functional polypeptides: the lack of the
C-terminal end, as expected for the smaller CAL-A fragment, does
not necessarily abolish the catalytic activity of the protein.
Hydrolysis of N-acetylmethionine rac-2a; acylase I-type activity.
The activity was shown to be confined to the first gel filtration
elution peak, with the highest relative activities in fractions 18–
19 (Table 4, Fig. 5c). Within the elution peak, the distribution
of the relative hydrolytic activity and the amount of the smaller
CAL-A fragment as seen in SDS-PAGE seemed to perfectly
coincide (Fig. 4). The CAL-A fragment could not, however, be
further isolated from the co-eluting protein impurities to directly
demonstrate the function of the lipase fragment towards the
substrate. One of the trace impurities present in the fraction was
identified as a member of the serine carboxypeptidase family (EC
3.4.16).43 These enzymes catalyze the cleavage of the C-terminal
residues from polypeptides and participate in various reactions
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was identified by digestion/peptide LC-MS/MS analysis as the
mature CAL-A, whereas the smaller fragment appeared to be
a truncated form of the enzyme, lacking the C-terminal region.
Isolated gel filtration fractions of the two forms of CAL-A were
shown to have distinct catalytic properties: both peak fractions
catalyzed the N-acylation of methyl pipecolinate rac-5a with
trifluoroethyl butanoate and hydrolysis of the ester and amide
bonds of ethyl 3-butanamidobutanoate rac-1c. However, only
the fractions containing the truncated CAL-A catalyzed the
highly enantioselective hydrolysis of N-acetylmethionine rac-2a.
Based on the crystal structure of CAL-A, the C-terminal region
constitutes a motile antiparallel b-sheet structure which may
function as an “active-site flap”. This structure has been proposed
to affect the substrate binding properties of the enzyme in different
conformational states. Accordingly, lack of the C-terminal end
would leave the binding pocket more exposed, which could in turn
allow the enzyme to accept substrate molecules which cannot be
efficiently accommodated in the active site of the wild type enzyme.
Analysis of the commercial preparation also uncovered many
minor protein contaminants originating from the expression host
strain. One of these was identified as a serine carboxypeptidase
which co-eluted in the main fraction together with the smaller
CAL-A protein. This contaminant was present in trace quantities
in the original preparation and remained undetected until frac-
tionation and sample concentration. Although any direct effect of
the carboxypeptidase on the amide bond hydrolysis could not be
verified, the confirmed presence of protein impurities cannot be
ignored as a potential source for the activity.
As a conclusion, preparations of CAL-A show preliminary
potential to be used as an alternative for specific amidase-catalyzed
reactions. The amidase activity of lipases studied here is of
particular interest, because the reaction could be used for instance
as an alternative mild deprotection method for amides in organic
synthesis or for resolving racemic amides. CAL-A has several
desirable features for commercial use: exceptional stability in low
pHs, high temperature and organic solvents, together with an
unusually broad tolerance for various unnatural substrates. This
makes CAL-A a promising candidate for protein engineering to
alter its catalytic properties or substrate preference, for instance
toward different amide precursors.
calculation of conversion was based on the equation c = eeS/
(eeS + eeP).
N-Acylation of 1a (0.100 M) in butyl butanoate (1 cm3) was
started by adding the enzyme (40 mg cm-3). The amounts of the
enzyme preparations were kept constant, because the real protein
content in the preparations was unknown. Five samples (0.1 cm3)
were taken at intervals and derivatized with acetic anhydride.
Hydrolysis of 1b, 1c, 2a–c, 3a and 4a (0.050 M) was carried out
in phosphate buffer (pH 7.5; 0.100 M) at room temperature, the
amount of the enzyme being 5 mg cm-3 (except for enzymes of
ViaKit 20 mg cm-3 and other exceptions described in Tables 2 and
3). In all hydrolyses, the progress of the reactions was followed
by taking samples (400–1000 ml) and filtering off the enzyme with
10 K Nanosep centrifugal tubes. Only with 2c did we observe a
background chemical reaction which stayed negligible in 2 h, when
the reaction samples were analyzed.
The enantiomers of 2a–d, 3a, 3d, 4a and 4d were analyzed by
HPLC using Phenomenex D-penicillamine-column. Hydrolyses of
1b into 1d, and 1c into 1a/1b/1d, were followed by GC using
Chirasil-DEX-CB (Varian) as a column. Before analysis water
was evaporated from the samples (400–1000 mL), and the starting
material and the product were derivatized into amido esters by
adding acetic or butanoic anhydride (100 mL) and after five
minutes MeOH (200 mL). The relative proportions of 1a, 1b, 1c and
1d were determined from the peak areas by taking into account
the peak responses which were determined by making a solution
containing all the four compounds in equal amounts.
Absolute configurations were determined based on our previous
works.38,42,45
Preparation of the racemic starting materials
1d, 2a–d, 3a, 3d, 4a and 4d were commercially available. Prepara-
tion of 1a and 1c has been previously described.38 1b as a sodium
salt (611 mg, yield 90%, mp 120 ◦C) was prepared by hydrolyzing
the corresponding ethyl ester 1c (700 mg, 3.48 mmol) with sodium
hydroxide (139.1 mg; 3.48 mmol) dissolved in water (40 cm3).
rac-1b (sodium salt): 1H NMR (400 MHz, DMSO-d6) d (ppm):
8.05 (d, 1 H, J 8.0, NH), 3.95 (m, 1 H, J 6.8 and 14.4, CH), 2.06
(br m, 1 H, J 4.4 and 14.4, CH2CO2), 1.97 (m, 2 H, J 7.2 and 14.4,
CH2CONH), 1.96 (br m, 1 H, J 7.6, CH2CO2), 1.45 (m, 2 H, J
7.6, CH2CH3), 1.01 (d, 3H, J 6.8, CHCH3), 0.83 (m, 3H, J 7.6,
CH2CH3).
Experimental section
HRMS: MH+ = 196.0950, C8H15NNaO3 requires MH+
=
Materials and methods
196.0950.
Various forms of lipase A from Candida antarctica were the prod-
ucts of Roche (Chirazyme L-5, lyo.), Fluka (CAL-A-CLEA), Chi-
ralVision (Immozyme CALA-T2-150), ViaZym [ViaKit contain-
ing 18 immobilized CAL-As on macroporous supports (VZ1030-
and VZ1031-codes), silica gel (VZ101-codes and VZ1011-1) and
Celite (VZ102-codes)] and Biocatalytics (now Codexis; both free
(Cat#ICR-112) and immobilized (Cat#NZL-101-IMB) forms).
Acylase I from Aspergillus genus was obtained from Tokyo Kasei
Chemicals (TCI). Reagents were purchased from Sigma, Aldrich,
Fluka, Bachem and Acros. Solvents of the highest analytical grade
were obtained from Aldrich, J. T. Baker and Lab-Scan.
Fractionation and analysis of the commercial CAL-A preparation
SDS-PAGE analysis. Samples of CAL-A were analyzed on
various SDS-PAGE systems, including PhastSystem (Amersham
Pharmacia Biotech) gradient 8-25 gels and 8% self-casted SDS-
PAGE gels stained with Coomassie Brilliant Blue dye, and
Criterion 10% Bis-Tris gels (Biorad) stained with Silver dye.
The bands were compared against commercial protein standard
ladders (Biorad pre-stained broad range SDS ladder 161-0318 and
Fermentas PageRulerTM ladders).
The determination of E was based on the equation E = ln[(1 -
c)(1 - eeS)]/ln[(1 - c)(1 + eeS)].44 Using linear regression E was
achieved as the slope of a line. Unless otherwise stated, the
Preparative gel filtration. 5–75 mg of the solubilized samples
of the CAL-A preparation (lyophilized Cat#ICR-112, Codexis)
¨
were fractionated using AKTA FLPC chromatography system
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(Amersham Biosciences) and Superdex 200 Hiload 26/60 gel
filtration columns (Amersham Biosciences). The samples were
injected in 5 cm3 volume of 100 mM Tris-HCl buffer pH 7.5 used
for the run, and 10 cm3 fractions were collected for subsequent
steps. CentriPrep YM-10 centrifugal filter devices (Biorad) were
used for fraction concentration.
and trifluoroethyl butanoate (0.100 M) in TBME. The reactions
were analyzed by GC equipped with a chiral a column (Varian
permethylated b-cyclodextrin).
Acknowledgements
Removal of glycosyl groups. Removal of potential N-linked
sugars was accomplished using commercial glycosidase PNGase
F (New England Biolabs) which cleaves between the innermost
GlNAc and asparagine residues of high mannose, hybrid and com-
plex oligosaccharides from N-linked glycoproteins. The procedure
was carried out as instructed by the manufacturer.
The authors thank the Technology Development Center of
Finland (TEKES) for financial support and ViaZym for ViaKit
containing immobilized CAL-A preparations.
References
Protein LC-MS/MS analysis for identification. The analy-
sis and sequence comparison was carried out at Proteomics
Facility (Turku Centre for Biotechnology/University of Turku
1 Y. Nakagawa, A. Hasegawa A, J. Hiratake and K. Sakata, Protein Eng.,
Des. Sel., 2007, 20, 339–346.
2 R. Kourist, S. Bartsch, L. Fransson, K. Hult and U. T. Bornscheuer,
ChemBioChem, 2008, 9, 67–69.
3 R. Brieva, J. Z. Crich and C. J. Sih, J. Org. Chem., 1993, 58, 1068–
1075.
˚
and Abo Akademi University). Proteins in the provided CAL-
A samples were separated on BioRad’s precast Criterion XT
gel (10% Bis-Tris with MOPS buffer), cut from the sil-
ver stained gel and in-gel digested46 for the LC-MS/MS
analysis.
Tryptic peptides were analyzed using a nanoflow LC system
(Famos, Switchos, and UltimatePlus; LC Packings - Dionex
Corporation, Sunnyvale, CA, USA) coupled to a QSTAR Pulsar
i ESI-hybrid Q-TOF tandem mass spectrometer (Applied Biosys-
tems/MDS Sciex, Toronto, Canada). Peptides were concentrated
and desalted on a precolumn (0.3 ¥ 5 mm C18 PepMap100,
LC Packings), and eluted at 200 ndm3 min-1 by increasing the
concentration of acetonitrile onto a C18 reverse phase column
4 W. Adam, P. Groer, H.-U. Humpf and C. R. Saha-Mo¨ller, J. Org.
Chem., 2000, 65, 4919–4922.
5 E. Forro´ and F. Fu¨lo¨p, Org. Lett., 2003, 5, 1209–1212.
6 X.-G. Li, M. La¨hitie and L. T. Kanerva, Tetrahedron: Asymmetry, 2008,
19, 1857–1861.
7 V. Gotor, R. Brieva, C. Gonzalez and F. Rebolledo, Tetrahedron, 1991,
47, 9207–9214.
8 C. Simons, J. G. E. van Leeuwen, R. Stemmer, I. W. C. E. Arends, T.
Maschmeyer, R. A. Sheldon and U. Hanefeld, J. Mol. Catal. B: Enzym.,
2008, 54, 67–71.
9 R. Fujii, Y. Nakagawa, J. Hiratake, A. Sogabe and K. Sakata, Protein
Eng., Des. Sel., 2005, 18, 93–101.
10 D. R. Duarte, E. L. Castillo, E. Ba´rzana and A. Lo´pez-Mungu´ıa,
Biotechnol. Lett., 2000, 22, 1811–1814.
11 H. Smidt, A. Fischer, P. Fischer, R. D. Schmidt and U. Stelzer, EP
1995, 812363.
12 M. V. Sergeeva, V. V. Mozhaev, J. O. Rich and Y. L. Khmelnitsky,
Biotechnol. Lett., 2000, 22, 1419–1422.
˚
(75 mm i.d. ¥ 15 cm, Magic 5 mm 100 A C18, Michrom
BioResources Inc.). A linear 25 min gradient from 98% solvent
A (97.9% water, 2% acetonitrile, and 0.1% formic acid) to 65%
solvent B (95% acetonitrile, 4.9% water, and 0.1% formic acid)
was used.
13 T. Maruyama, M. Nakajima, H. Kondo, K. Kawasaki, M. Seki and M.
Goto, Enzyme Microb. Technol., 2003, 32, 655–657.
14 L. T. Kanerva, P. Csomo´s, O. Sundholm, G. Berna´th and F. Fu¨lo¨p,
Tetrahedron: Asymmetry, 1996, 7, 1705–1716.
15 A. Liljeblad and L. T. Kanerva, Tetrahedron, 2006, 62, 5831–
5854.
Sequence comparison and protein identification. Data depen-
dent acquisition was performed in positive ion mode. Two of
the most intense doubly or triply charged precursor ions were
selected from m/z 250 to m/z 1500. Data from LC-MS/MS runs
were converted to peak list files with the Analyst QS software
(version 1.1) and searched using an in-house Mascot search
engine (version 2.2, Matrix Sciences, Boston, MA, USA). The
following parameters were used: (1) database Swiss-Prot/Tremb
(versions 51.5/34.5), taxonomy all, enzyme trypsin with up to
one missed cleavages, variable modifications carbamidomethyl
and methionine oxidation, and a mass accuracy 0.6 Da for
both precursor ions and MS/MS data or (2) database con-
taining only lipase A sequence,34 but other parameters same as
above.
16 M. Fitz, E. Forro´, E. Vigo´czki, L. La´za´r and F. Fu¨lo¨p, Tetrahedron:
Asymmetry, 2008, 19, 1114–1119.
17 A. Kasrayan, M. Bocola, A. G. Sandstro¨m, G. Lave´n and J.-E. Ba¨ckvall,
ChemBioChem, 2007, 8, 1409–1415.
18 D. J. Ericsson, A. Kasrayan, P. Johansson, T. Bergfors, A. G. Sand-
stro¨m, J.-E. Ba¨ckvall and S. L. Mowbray, J. Mol. Biol., 2008, 376,
109–119.
19 T. B. Nielsen, M. Ishii and O. Kirk, in Biotechnological Applications
of Cold-Adapted Organisms, ed. R. Margesin and S. Schinner, Lipases
A and B from Candida antarctica, Landes Bioscience, Austin, Texas,
1999, pp. 49–61.
20 O. Kirk and M. W. Christensen, Org. Process Res. Dev., 2002, 6, 446–
451.
21 M. Martinelle, M. Holmquist and K. Hult, Biochim. Biophys. Acta,
Lipids Lipid Metab., 1995, 1258, 272–276.
22 P. Dom´ınguez de Mar´ıa, C. Carboni-Oerlemans, B. Tuin, G. Bargeman,
A. Van Der Meer and R. van Gemert, J. Mol. Catal. B: Enzym., 2005,
37, 36–46.
Analysis of the test reactions. Hydrolysis of N-
acetylmethionine 2a was detected by adding 1 cm3 of each
fraction to N-acetylmethionine (1 cm3, 0.100 M). Hydrolysis of
ethyl 3-butanamidobutanoate 1c was started by adding 1 cm3
of each fraction to 1c (10.1 mg; 0.050 M). The reactions were
analyzed as described in Materials and Methods. For N-acylation
of methyl pipecolinate rac-5a (Scheme 5), Celite (15.6 mg) and
sucrose (2.4 mg) were dissolved in each fraction (2 cm3) and the
water was left to evaporate in a fume hood. Adsorped enzyme
preparation was then added into methyl pipecolinate (0.025 M)
23 R. Borgdorf and S. Warwel, Appl. Microbiol. Biotechnol., 1999, 51,
480–485.
24 S. H. Krishna, M. Persson and U. T. Bornscheuer, Tetrahedron:
Asymmetry, 2002, 13, 2693–2696.
25 J. A. Bosley, J. Casey, A. R. Macrae and G. Mycock, WO 95/01450,
1995.
26 E. Henke, J. Pleiss and U. T. Bornscheuer, Angew. Chem., Int. Ed., 2002,
41, 3211–3213.
27 A. J. Pihko, K. Lundell, L. Kanerva and A. M. P. Koskinen,
Tetrahedron: Asymmetry, 2004, 15, 1637–1643.
894 | Org. Biomol. Chem., 2010, 8, 886–895
This journal is
The Royal Society of Chemistry 2010
©

View Article Online
28 F. Tjosa˚s, T. Anthonsen and E. E. Jacobsen, ARKIVOC, 2008, vi, 81–
37 J. M. Palomo, G. Ferna´ndez-Lorente, C. Mateo, M. Fuentes, J. M.
Guisan and R. Ferna´ndez-Lafuente, Tetrahedron: Asymmetry, 2002,
13, 2653–2659.
38 S. Gedey, A. Liljeblad, F. Fu¨lo¨p and L. T. Kanerva, Tetrahedron:
Asymmetry, 2001, 12, 105–110.
90.
¨
29 D. Ozdemirhan, S. Sezer and Y. So¨nmez, Tetrahedron: Asymmetry,
2008, 19, 2717–2720.
30 A. Liljeblad, J. Lindborg, A. Kanerva, J. Katajisto and L. T. Kanerva,
Tetrahedron Lett., 2002, 43, 2471–2474.
39 L. T. Kanerva and O. Sundholm, J. Chem. Soc., Perkin Trans. 1, 1993,
2407–2410.
31 A. Liljeblad, A. Kiviniemi and L. T. Kanerva, Tetrahedron, 2004, 60,
671–677.
40 H. K. Chenault, J. Dahmer and G. M. Whitesides, J. Am. Chem. Soc.,
32 S. Alatorre-Santamar´ıa, M. Rodriguez-Mata, V. Gotor-Ferna´ndez,
M. C. de Mattos, F. J. Sayago, A. I. Jime´nez, C. Cativiela and V. Gotor,
Tetrahedron: Asymmetry, 2008, 19, 1714–1719.
33 A. G. Sandstro¨m, K. Engstro¨m, J. Nyhle´n, A. Kasrayan and J.-E.
Ba¨ckvall, Protein Eng., Des. Sel., 2009, 22, 413–420.
34 I. Hoegh, S. Patkar, T. Halkier and M. T. Hansen, Can. J. Bot., 1995,
73(s1), S869–S875.
35 J. Pfeffer, S. Richter, J. Nieveler, C.-E. Hansen, R. B. Rhlid, R. D.
Schmid and M. Rusnak, Appl. Microbiol. Biotechnol., 2006, 72, 931–
938.
1989, 111, 6354–6364.
41 A. S. Bommarius, K. Drauz and U. Groeger, in Chirality in Industry,
ed. A. N. Collins, G. N. Sheldrake and J. Crosby, Wiley, Chichester,
UK, 1995, pp 376–378.
42 S. Gedey, A. Liljeblad, F. Fu¨lo¨p and L. T. Kanerva, Tetrahedron:
Asymmetry, 1999, 10, 2573–2581.
43 K. Breddam, Biomed. Life Sci., 1986, 51, 83–128.
44 C.-S. Chen, Y. Fujimoto, G. Girdaukas and C. J. Sih, J. Am. Chem.
Soc., 1982, 104, 7294–7299.
45 A. Liljeblad, R. Aksela and L. T. Kanerva, Tetrahedron: Asymmetry,
2001, 12, 2059–2066.
46 A. Shevchenko, M. Wilm, O. Vorm and M. Mann, Anal. Chem., 1996,
68, 850–858.
36 J. Pfeffer, M. Rusnak, C.-E. Hansen, R. B. Rhlid, R. D. Schmid
and S. C. Maurer, J. Mol. Catal. B: Enzym., 2007, 45, 62–
67.
This journal is
The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 886–895 | 895
©