Highly enantioselective recombinant thermoalkalophilic lipases from Geobacillus and Bacillus sp

Article abstract of DOI:10.1016/j.tetasy.2004.07.041

Thermoalkalophilic lipases from Geobacillus and Bacillus strains have been cloned and characterised, several of which show excellent activity and enantioselectivity towards various substrates. The hydrolysis of methyl 2-methyldecanoate proceeds to give (R)-2-methyldecanoic acid in good yield and >99% enantiomeric excess (E>200). Crown Copyright

Full text of DOI:10.1016/j.tetasy.2004.07.041

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 2975–2980
Highly enantioselective recombinant thermoalkalophilic
lipases from Geobacillus and Bacillus sp
a
Lauren M. Hutchins, Luke Hunter, Noosha Ehya, Moreland D. Gibbs,
a
b
b
b,c
Peter L. Bergquist and Craig A. Hutton
a,d,
*
a
School of Chemistry, The University of Sydney, Sydney NSW 2006, Australia
Biotechnology Research Institute, Macquarie University, North Ryde, Sydney NSW 2109, Australia
b
c
Department of Molecular Medicine and Pathology, University of Auckland Medical School, Private Bag 92019, Auckland,
New Zealand
d
School of Chemistry, The University of Melbourne, Melbourne VIC 3010, Australia
Received 1 June 2004; accepted 13 July 2004
Available online 11 September 2004
Abstract—Thermoalkalophilic lipases from Geobacillus and Bacillus strains have been cloned and characterised, several of which
show excellent activity and enantioselectivity towards various substrates. The hydrolysis of methyl 2-methyldecanoate proceeds
to give (R)-2-methyldecanoic acid in good yield and >99% enantiomeric excess (E >200).
Crown Copyright ꢀ 2004 Published by Elsevier Ltd. All rights reserved.
1
. Introduction
esterification reactions in organic solvents, such as tert-
butylmethylether, toluene and heptane. Other potential
industrial applications of lipases include the production
of fatty acids and glycerol via hydrolysis of oils and fats,
and there are opportunities for both environmental
enhancement and adding value to beef tallow by its
hydrolysis to fatty acids. Beef tallow and some other fats
are not in a liquid form at mesophilic enzyme tempera-
tures and clearly, efficient enzymatic processes for han-
dling these and related by-products require suitable
thermostable lipases capable of hydrolysing long-chain
fatty acids. Most lipases on the market are impure and
poorly characterized and importantly, operate under rel-
atively mild conditions at low temperatures. We have
described consensus primers for the isolation of lipases
Enzymes such as hydrolases play an important role in
biotechnology because of their extreme versatility with
respect to substrate specificity and stereoselectivity.
1
,2
The use of lipases as catalysts for enantioselective organic
reactions is often limited by unacceptably low selectivi-
ties. Lipases are versatile enzymes that can provide an
environmentally friendly means of breaking down fats
for environmental clean-up and the production of
value-added products. They can also be used as biocata-
lysts in sophisticated reactions in chemistry for the phar-
3
maceutical industry. Lipases catalyse three types of
reactions: hydrolysis, esterification and acyl transfer.
The hydrolytic reaction mode is frequently used for
the preparation of both carboxylic acids and alcohols
in, for example, the lipase-catalysed hydrolysis of a race-
mic carboxylic ester. One significant application of lip-
ases in organic chemistry is the production of optically
active compounds that may be used in sophisticated
procedures for the synthesis of pharmaceuticals. Lipases
are useful catalysts for the preparation of a broad range
of optically active compounds and labile organic com-
pounds. As well as regiospecific and stereoselective
hydrolysis, they can be used for esterification or trans-
4
by Genomic Walking PCR and from expression librar-
5
ies. The genes for thermophilic lipases from several
Geobacillus strains and from biomass from microbial
tallow hydrolysis at high temperature have been isolated
and sequenced. The enzymes have had a His-tag added
and they have been isolated and purified after expression
in Escherichia coli. We have previously described the
cloning and expression of a lipase from a thermophilic
Geobacillus strain, Tp10A.1, and demonstrated its high
6
enantioselectivity on selected racemic ester substrates.
*
Corresponding author. Tel.: +61 3 8344 6482; fax: +61 3 9347
180; e-mail: chutton@unimelb.edu.au
Here, we present biochemical data on three other cloned
thermophilic lipases in hydrolytic reactions and describe
5
0
957-4166/$ - see front matter Crown Copyright ꢀ 2004 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.07.041

2976
L. M. Hutchins et al. / Tetrahedron: Asymmetry 15 (2004) 2975–2980
their outstanding enantioselectivity towards the hydrol-
ysis of (RS)-1-phenylethyl acetate and methyl (RS)-2-
methyldecanoate.
dues in 389. The TP10 A.1 enzyme, which exhibits
slightly different pH and temperature optima and sub-
strate selectivity has significant primary sequence differ-
ences, including a 29-residue N-terminal extension and
several residue substitutions.
2
. Results and discussion
2
.2. Enantioselectivities of lipases
2
.1. Characterisation of lipases
The enantioselectivities of the three new lipases and
Tp10 A.1 lipase were investigated for a number of sub-
strates. Tp10 A.1 lipase was previously used in assays of
the hydrolysis of (RS)-1-phenylethyl acetate 1 and
methyl (RS)-2-methyldecanoate 3 and showed excellent
enantioselectivity (E >200) for both substrates at
pH7.5, 50ꢁC. (In general, synthetically useful enzyme-
catalysed resolutions require an E value of 50 or greater,
The purified recombinant lipases were assayed for opti-
mal temperature and pH and chain-length specificity to-
wards fatty acid p-nitrophenyl esters (PNP esters). The
results are shown in Table 1.
Table 1. Optimal pH, temperature and substrate selectivity (fatty acid
PNP esters) of the lipases
7
with values higher than 100 considered excellent.)
Lipase
Optimal pH
Optimal
temperature (ꢁC)
Substrate
selectivity
In the current work, the Tp10 A.1 lipase and the three
new lipases were assayed for the hydrolysis of (RS)-1-
phenylethyl acetate 1 at 20 and 50ꢁC, and at pH7.5
and 9.0 (results shown in Table 3). Tp10 A.1 lipase again
showed excellent selectivity at 20 and 50ꢁC at pH7.5,
with only a slight drop in E value at pH9.0, presumably
due to an increase in the background rate of hydrolysis.
Of the new enzymes, both OK4 A.2 and B. therm lipases
are slightly more selective than Tp10 A.1, and also more
active, particularly under thermoalkalophilic conditions
B. therm.
Mk1 A.1
OK4 A.2
Tp10 A.1
9
9
9
8
50
50
50
60
C:8, 10
C:10
C:10
C:8, 10, 12
The optimal conditions of the enzymes correlate with
their sequence similarities, shown in Table 2. The B.
therm., Mk1 A.1 and OK4 A.2 lipases exhibit similar
pH and temperature optima, and fatty ester selectivities.
These enzymes are remarkably similar in terms of their
amino acids sequences, differing by at most three resi-
(pH9, 50ꢁC). The Mk1 A.1 lipase, however, is consider-
ably less selective, and does not exhibit thermoalkalo-
philic properties, with no increase in activity occurring
at high temperature and pH. Indeed, a dramatic de-
crease in selectivity is observed under thermoalkalophi-
lic conditions. All lipases were selective for hydrolysis
of the (R)-enantiomer of 1.
Table 2. Sequence similarities of the lipases (%)
B. therm.
Mk1 A.1
OK4 A.2
Tp10 A.1
B. therm.
Mk1 A.1
OK4 A.2
Tp10 A.1
100
99.2
100
99.7
99.5
100
93.4
93.2
93.7
100
99.2
99.7
93.4
Assays of the hydrolysis of methyl (RS)-2-methyldecano-
ate 3 (Table 4) indicated that the Tp10 A.1, OK4 A.2
and B. therm lipases all exhibit excellent enantioselectiv-
99.5
93.2
93.7
Table 3. Hydrolysis of (RS)-1-phenylethyl acetate
lipase
OAc
OH
1
2
Lipase
Temp (ꢁC)
pH
Time (h)
% Conv.
% Ee (P)
E
B. therm
B. therm
B. therm
B. therm
Mk1 A.1
Mk1 A.1
Mk1 A.1
Mk1 A.1
OK4 A.2
OK4 A.2
OK4 A.2
OK4 A.2
Tp10 A.1
Tp10 A.1
Tp10 A.1
Tp10 A.1
RT
50
7.5
7.5
9
187
23
22.0
37.2
47.1
49.7
41.9
9.3
99.0
98.9
98.6
96.1
97.1
95.5
97.0
71.3
98.9
98.4
94.4
97.1
98.8
98.5
97.3
96.5
>200
>200
>200
188
142
47
RT
50
69
19
9
RT
50
7.5
7.5
9
147
47
RT
50
121
70
30.9
17.2
24.9
37.2
48.4
49.6
17.0
20.7
44.4
43.4
99
9
6.9
RT
50
7.5
7.5
9
190
71
>200
>200
103
RT
50
193
23
9
>200
198
174
RT
50
7.5
7.5
9
168
46
RT
50
170
42
174
124
9

L. M. Hutchins et al. / Tetrahedron: Asymmetry 15 (2004) 2975–2980
2977
ity for this substrate. These three lipases also display dis-
tinct thermoalkalophilic properties, with high activity at
pH9 and 50ꢁC. The Mk1 A.1 lipase does show excellent
selectivity at pH7.5, but again appears to be unstable
under thermoalkalophilic conditions. All the lipases
show the same sense of enantioselectivity, being selective
for hydrolysis of the (R)-enantiomer of methyl 2-methyl-
decanoate. Confirmation of the absolute configuration
of the products was obtained by isolating the hydrolysed
product acid and determination of its specific rotations.
This enantioselectivity is the opposite sense to nearly all
Given the high selectivity of three of the four lipases to
these two substrates, 1 and 3, further assays were con-
ducted. The hydrolysis of methyl 2-phenylpropionate 5
was tested, but all lipases showed virtually no enantiose-
lectivity for this substrate (Table 5). The lipase-catalysed
acetylation of 1-phenylethanol 2 in organic solvent was
also investigated (Table 6). While the Tp10 A.1 and B.
therm lipases showed very poor activity in this transfor-
mation, Mk1 A.1 and OK4 A.2 lipases were reasonable
catalysts. The OK4 A.2 lipase was the most selective en-
zyme, giving E values of up to 68, though its activity was
still only moderate.
8
,9
other lipases towards this substrate.
Table 4. Hydrolysis of methyl (RS)-2-methyldecanoate
lipase
CO2Me
CO2H
7
7
3
4
Lipase
Temp (ꢁC)
pH
Time (h)
% Conv.
% Ee (P)
E
B. therm
B. therm
B. therm
B. therm
Mk1 A.1
Mk1 A.1
Mk1 A.1
Mk1 A.1
OK4 A.2
OK4 A.2
OK4 A.2
OK4 A.2
Tp10 A.1
Tp10 A.1
Tp10 A.1
Tp10 A.1
RT
50
7.5
7.5
9
261
42
34.1
20.3
45.7
37.8
17.8
42.3
25.4
0.77
27.5
23.2
49.1
45.9
45.0
32.6
36.7
48.7
99.5
99.3
99.7
99.4
99.7
99.1
99.2
99.1
99.1
99.4
99.3
99.6
99.6
99.6
99.7
99.7
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
RT
50
67
20
9
RT
50
7.5
7.5
9
234
141
187
48
RT
50
9
RT
50
7.5
7.5
9
145
95
RT
50
45
17
9
RT
50
7.5
7.5
9
264
69
RT
50
97
18
9
Table 5. Hydrolysis of (RS)-2-phenylpropionate
lipase
CO2Me
CO2H
5
6
Lipase
Temp (ꢁC)
pH
Time (h)
% Conv.
% Ee (P)
E
B. therm
B. therm
B. therm
B. therm
Mk1 A.1
Mk1 A.1
Mk1 A.1
Mk1 A.1
OK4 A.2
OK4 A.2
OK4 A.2
OK4 A.2
Tp10 A.1
Tp10 A.1
Tp10 A.1
Tp10 A.1
RT
50
7.5
7.5
9
238
142
241
143
239
142
244
122
19
0.17
0.71
1.9
38.5
9.3
2.3
1.2
1.0
1.0
1.2
1.2
1.1
1.1
1.8
1.2
1.1
1.1
1.1
1.3
1.1
1.0
RT
50
2.0
1.7
9
6.2
RT
50
7.5
7.5
9
0.12
0.34
2.1
8.0
8.2
RT
50
2.7
2.5
9
9.0
RT
50
7.5
7.5
9
0.65
1.5
28.6
10.4
2.8
119
166
118
139
143
142
147
RT
50
1.2
9
10.1
0.05
0.21
0.96
3.5
2.5
6.3
RT
50
7.5
7.5
9
13.3
3.0
RT
50
9
0.56

2978
L. M. Hutchins et al. / Tetrahedron: Asymmetry 15 (2004) 2975–2980
Table 6. Acylation of (RS)-1-phenylethanol
lipase
OH
OAc
vinyl acetate
2
1
Lipase
Temp (ꢁC)
Time (h)
% Conv.
% Ee (P)
E
B. therm
B. therm
Mk1 A.1
Mk1 A.1
OK4 A.2
OK4 A.2
Tp10 A.1
Tp10 A.1
RT
50
148
44
4.6
3.2
92.6
90.6
93.3
89.0
96.4
93.2
71.5
43.7
27
21
34
18
68
34
RT
50
146
44
15.5
6.0
RT
50
142
43
19.0
17.1
0.7
RT
50
195
44
6.1
2.6
0.2
Given that the sequence similarities of these enzymes are
very high the differences in their selectivities at high tem-
perature and pH is surprising. The Mk1 A.1 lipase
exhibits a dramatic decrease in selectivity under these
conditions, particularly for the hydrolysis of (RS)-1-phen-
ylethyl acetate 1 and methyl (RS)-2-methyldecanoate 3
selectivity at high pH, attributed to a single Pro-Arg
substitution. The OK4 A.2 lipase is the most active
and selective of those isolated, considerably more so
than the previously isolated Tp10 A.1 lipase, retaining
high activity and enantioselectivity towards various
substrates at high pH and temperature.
(
Tables 3 and 4). The Mk1 A.1 lipase differs from the
OK4 A.2 lipase by just two amino acid substitutions––
P228R and G393S––and the B. therm. lipase differs by
just an additional V156M substitution. In order to
define the role of these substitutions in modifying
the Mk1 A.1 activity at high temperature and pH,
the thermostabilities of the enzymes were calculated
3. Experimental
3.1. Bacteria and genes
All genes were expressed in Escherichia coli BL21AP
(Invitrogen, Carlsbad, CA). This strain allows a tight
regulation of lipase expression, which is induced on
induction with arabinose and IPTG. The lipase from
Geobacillus sp. Tp10A.1 was prepared as previously
(
Table 7).
Table 7. Lipase thermostabilities
6
described. The genes for lipases from Geobacillus
Lipase
Temp. (ꢁC)
67
t
1/2 (min)
strains OK4 A.2 and Mk1 A.1 and Bacillus thermoleovo-
rans were cloned by PCR amplification from genomic
DNA. The following primers were used: Mk1 A.1 and
B. thermoleovorans, BthFpet. AAAAAACGGGATCC-
AGCGGC AGTTTCACGCGCCA; and BthR-
B. therm.
21
87
50
51
67
Mk1 A.1
OK4 A.2
Tp10 A.1
51
67
101
17
51
67
26
15
pet,
GATAATCCCTAAGCTTAAGGCCGCAAA-
CTCGCC. The OK4 A.2 lipase gene was amplified by
nested PCR, with the following degenerate primers:
BLPF2, GAG/TAA/GC/GATGATGAAAT/GGCTG
and BLPR2, CC/TC/T G/TC/TA/TTTAAGGCCGC-
AAACTC. The PCR product from this amplification
was used as template with the BthFpet and BthRpet
primers above for the second amplification. The PCR
products were ligated into pET26B(+) and induced as
These results show that all the lipases show significant
thermophilicity (mesophilic lipases typically have a
half-life <10min at 67ꢁC). Interestingly, the Mk1 A.1
lipase is the most thermostable of the lipases, suggesting
that its different reactivity under thermoalkalophilic
conditions is due to high pH rather than high tempera-
ture. This is consistent with the single substitution of a
proline residue for an arginine in Mk1 A.1 lipase. It is
feasible that at high pH the arginine is no longer in
the protonated form, thereby dramatically changing
the electrostatic environment around this residue. Fur-
ther investigations of this effect are currently underway.
6
previously described. In addition, the pET-26B(+) vec-
tor carries an N-terminal pelB signal sequence for poten-
tial periplasmic localization, plus a C-terminal His-Tag
sequence for easy purification.
3.2. Enzyme purification
A small-scale (50mL) culture was performed to check
expression levels and to establish the optimal induction
period. The lipase was purified from the cell fraction
using Ni-NTA Magnetic Agarose Beads and samples
were analysed by SDS PAGE. Then a larger scale pro-
tein expression (700mL) was performed to produce
In conclusion, several new thermoalkalophilic lipases
have been cloned and characterised, which show excel-
lent activity and enantioselectivity towards various sub-
strates, particularly methyl 2-methyldecanoate. The
Mk1 A.1 lipase shows a substantial loss of activity and

L. M. Hutchins et al. / Tetrahedron: Asymmetry 15 (2004) 2975–2980
2979
enough enzyme for biochemical characterisation. Cells
were induced with arabinose (final concentration 2%)
and IPTG (final concentration 1mM) and lysed by pas-
sage through a French pressure cell. After centrifuga-
tion, the resulting cleared cell lysate was used for
further purification. The recombinant His-tagged lipase
was purified from the cell fraction using a Ni-column on
a BIOLOGIC LP chromatographic system (Amersham/
Pharmacia). The lipases appeared as a single band when
analysed by SDS-PAGE and had a molecular weight of
approximately 45kDa.
3.6. Assay 1: Hydrolysis of (RS)-1-phenylethyl acetate 1
To a stirred suspension of (RS)-1-phenylethyl acetate
1 (115lL, 0.72mmol) and Triton X-100 (1 drop,
ꢁ16mg) in aqueous Tris buffer (100mM, pH7.5 or
9, 2.5mL) at room temperature was added purified
lipase solution (50lL). A constant pH was maintained
by dropwise addition of 1M NaOH. The progress of
the reaction was monitored periodically by withdraw-
ing an aliquot (200lL) of the reaction mixture. The
aliquot was extracted with ethyl acetate (200lL), the
organic layer dried (Na SO ) and subsequently ana-
2
4
3
.3. Substrate preparation
lysed by gas chromatography, using a temperature
profile of 80ꢁC for 1min, then increasing by 2ꢁC/
min to 130ꢁC for 5min. The observed retention times
were as follows: (S)-1, 23.9min; (R)-1, 24.8min; (R)-2,
25.3min; (S)-2, 26min. The extent of conversion
and the enantiomeric excess of 2 were calculated
from the relative integrals of the peaks, which were
corrected using experimentally determined molar
Fatty acid p-nitrophenyl esters were purchased from
Sigma. Lipase substrates (RS)-1-phenylethyl acetate 1
and methyl (RS)-2-phenylpropionate 5 were prepared
from sec-phenethyl alcohol 2 and 2-phenylpropionic
acid 6, respectively, according to standard procedures.
2
-Methyldecanoic acid 4 was prepared according
1
0
4
À1
to the procedure of Berglund et al. Methyl (RS)-2-
methyldecanoate 3 was prepared from 4 as described
below.
response factors: (RS)-1, 1 · 10 counts nmol
;
3
À1
(RS)-2, 8.18 · 10 counts nmol . Results are dis-
played in Table 3.
3
.4. Methyl (RS)-2-methyldecanoate 3
3.7. Assay 2: Hydrolysis of methyl (RS)-2-methyl-
decanoate 3
Thionyl chloride (2.04mL, 28.0mmol) was added to ice
cold methanol (60mL) and the solution treated with
To a stirred suspension of methyl (RS)-2-methylde-
canoate 3 (80lL, 0.40mmol) and Triton X-100 (1
drop, ꢁ16mg) in aqueous Tris buffer (100mM,
pH7.5 or 9, 1.4mL) at room temperature was added
purified lipase solution (50lL). A constant pH was
maintained by dropwise addition of 1M NaOH. The
reaction was periodically monitored by withdrawing
an aliquot (200lL) of the reaction mixture. The ali-
quot was extracted with ethyl acetate (200lL), the or-
ganic layer dried (Na SO ) and subsequently analysed
1
0
methyl (RS)-2-methyldecanoic acid
2
4
1.0mmol). The solution was stirred at room tempera-
(2.78mL,
ture under nitrogen for 1h. The reaction mixture was
concentrated in vacuo and the crude product purified
by flash chromatography, eluting with 5% ethyl ace-
tate/hexane, yielding a colourless oil (3.64g, 85%). H
NMR (300MHz) d 3.66 (s, 3H), 2.43 (m, 1H), 1.63
(m, 1H), 1.40 (m, 1H), 1.26 (s, 12H), 1.13 (d,
J = 6.9Hz, 3H), 0.87 (t, J = 6.4Hz, 3H); data in accord-
ance with literature values.
1
1
1
2
4
by gas chromatography, using a temperature profile of
80ꢁC for 1min, then increasing by 15ꢁC/min to 170ꢁC
for 20min, the observed retention times were as fol-
3
.5. Enantioselectivity assays
lows: (RS)-3, 10.02min; (S)-4, 18.22min and (R)-4,
1
Assays were analysed by gas chromatography using a
Hewlett Packard 5890 Series II chromatograph with a
Supelco Beta Dex 120 fused silica capillary column
8.75min. The extent of conversion and the enantio-
meric excess of 4 were calculated from the relative
integrals of the peaks, which were corrected by the
following experimentally determined molar response
(
length 30m, diameter 0.25mm, film thickness 0.25lm,
carrier gas He at 110kPa, Injector temperature 270ꢁC,
flame ionisation detector temperature 270ꢁC). The ex-
tent of conversion and the enantiomeric excess (e.e.) of
the products were calculated from the relative integrals
of the peaks, which were corrected by experimentally
determined molar response factors. E values (defined
as the ratio of the specificity constants of the
enzyme for the fast-reacting and slow-reacting substrate
enantiomers, respectively) were calculated according
4
À1
factors: (RS)-3, 1.57 · 10 counts nmol ; (RS)-4,
4
À1
1
Table 4.
.29 · 10 counts nmol . Results are displayed in
3.8. Isolation of (R)-2-methyldecanoic acid (R)-4 and
methyl (S)-2-methyldecanoate (S)-3
Methyl (RS)-2-methyldecanoate 3 (50lL, 0.25mmol)
was treated with Tp10 A.1 lipase for 69h as described
above. The assay mixture was extracted with ethyl ace-
tate (3 · 10mL) and the combined extracts were dried
1
2
to the method of Sih and co-workers using the
equation
ln½1 À cð1 þ eeðPÞÞꢀ
E ¼
(
MgSO ), concentrated and the residue was purified by
4
ln½1 À cð1 À eeðPÞÞꢀ
chromatography on silica, eluting with ethyl acetate/
hexanes (1:9). The ester (S)-3 was isolated as a clear
oil (11.3mg, 23%). The acid (R)-4 was also isolated as
where ÔcÕ denotes the extent of conversion and
Ôee(P)Õ denotes the enantiomeric excess of the reaction
product.
a clear oil (17.4mg, 40%), [a] = À15.4 (c 0.84, MeOH)
D
1
3
{lit. [a] = À15.6 (neat)}.
D

2980
L. M. Hutchins et al. / Tetrahedron: Asymmetry 15 (2004) 2975–2980
3.9. Assay 3: Hydrolysis of methyl (RS)-2-phenylprop-
ionate 5
analysed by gas chromatography, as described for assay
1. Results are displayed in Table 6.
To a stirred suspension of methyl (RS)-2-phenylprop-
ionate 5 (115lL, 0.72mmol) and Triton X-100 (1 drop,
Acknowledgements
ꢁ
16mg) in aqueous Tris buffer (100mM, pH7.5 or 9,
.5mL) at room temperature was added purified lipase
This work was funded by Australian Research Council
Linkage grant LP0215935.
2
solution (50lL). A constant pH was maintained by
dropwise addition of 1M NaOH. The progress of the
reaction was monitored periodically by withdrawing
an aliquot (200lL) of the reaction mixture. The aliquot
was acidified with 3M HCl (1 drop), extracted with
dichloromethane (200lL), the organic layer dried
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. Jaeger, K.-E.; Dijkstra, B. W.; Reetz, M. T. Annu. Rev.
Microbiol. 1999, 53, 315.
2. Jaeger, K.-E.; Reetz, M. T. Trends Biotechnol. 1998, 16,
(
Na SO ) and subsequently analysed by gas chroma-
2
4
tography, using the temperature profile of 85ꢁC for
396.
3
4
. Zhu, B.; Panek, J. S. Org. Lett. 2000, 2, 2575–2578.
. Bell, P. J. L.; Sunna, A.; Gibbs, M. D.; Curach, N. C.;
Nevalainen, H.; Bergquist, P. L. Microbiology 2002, 148,
6
1
5min, then increasing by 10ꢁC/min to 200ꢁC for
0min, the observed retention times were as follows:
(
7
S)-5, 67.7min; (R)-5, 68.0min; (S)-6, 79.0min; (R)-6,
9.1min. The extent of conversion and the enantiomeric
2
283.
5
. Bell, P. J. L.; Nevalainen, H.; Morgan, H. W.; Bergquist,
P. L. Biotechnol. Lett. 1999, 21, 1003.
. Sunna, A.; Hunter, L.; Hutton, C. A.; Bergquist, P. L.
Enz. Microb. Technol. 2002, 31, 472.
7. Kazlauskas, R. J. Trends Biotechnol. 1994, 12, 464.
8. Reetz, M. T.; Zonta, A.; Schimossek, K.; Liebeton, K.;
Jaeger, K.-E. Angew. Chem., Int. Ed. 1997, 36, 2830.
excess of 6 were calculated from the relative integrals of
the peaks, which were corrected using the experimen-
tally determined molar response factors: (RS)-5,
6
3
À1
3
9
.36 · 10 counts nmol ; (RS)-6, 7.49 · 10 counts
nmol . Results are displayed in Table 5.
À1
9
. Berglund, P. Biomol. Eng. 2001, 18, 13.
3
.10. Assay 4: Acylation of (RS)-1-phenylethanol 2
1
1
1
1
0. Berglund, P.; Holmquist, M.; Hedenstr o¨ m, E.; Hult, K.;
H o¨ gberg, H. Tetrahedron: Asymmetry 1993, 4, 1869–1878.
1. Toda, S.; Miyamoto, M.; Kinoshita, H.; Inomata, K. Bull.
Chem. Soc. Jpn. 1991, 64, 3600.
2. Chen, C.-S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J.
J. Am. Chem. Soc. 1982, 104, 7294.
3. H o¨ gberg, H.-E.; Hedenstr o¨ m, E.; Wassgren, A.-B.; Hjal-
marrson, M.; Bergstr o¨ m, G.; L o¨ fqvist, J.; Norin, T.
Tetrahedron 1990, 46, 3007.
An aliquot of purified lipase (50lL) was evaporated
under reduced pressure. To the dry residue was added
vinyl acetate (2.5mL) and (RS)-1-phenylethanol 2
(
87lL, 0.72mmol) and the resulting mixture was stirred
at room temperature. The reaction was monitored
periodically by withdrawing an aliquot (200lL) of the
reaction mixture. The aliquot was filtered and the filtrate