R. J. Kazlauskas et al.
FULL PAPER
with bovine serum albumin (BSA) as the standard. Solutions were stored in
a 96-well assay block mother plate equipped with aluminum sealing tape
Maarten Egmond (Unilever Research), and Biocatalysts (Pontypridd,
UK) for gifts of hydrolases, and the Swedish Chemical Society and the
Bengt Lundqvist Commemorative Foundation for fellowships to ACL.
(
�
2 mL maximum volume in each well, Corning Costar, Acton, MA) at
208C. This mother plate speeds up repeated screens that use the same
hydrolases, and is a convenient way to store large libraries of hydrolases.
Hydrolytic activity of the libraries is maintained over several months.
Received: April 22, 1998 [F1114]
Screening of commercial hydrolases with pH indicators: The assay
solutions were prepared by mixing solketal butyrate (420 mL of a 30.0mm
solution in acetonitrile), acetonitrile (470 mL), 4-nitrophenol (6000 mL of a
[
[
1] S. M. Roberts, Preparative Biotransformations, Wiley, Chichester,
992 ± 1997; K. Faber, Biotransformations in Organic Chemistry, 3rd
ed., Springer, Berlin, 1997.
2] For example, R. J. Kazlauskas, A. N. E. Weissfloch, A. T. Rappaport,
L. A. Cuccia, J. Org. Chem. 1991, 56, 2656 ± 2665; M. C. R. Franssen,
H. Jongejan, H. Kooijman, A. L. Spek, N. L. F. L. Camacho Mondril,
P. M. A. C. Boavida dos Santos, A. de Groot, Tetrahedron Asymmetry
0
5
.9115mm solution in 5.0mm BES, pH 7.2), and BES buffer (5110 mL of a
.0mm solution, pH 7.2). Hydrolase solutions (5 mL/well) were transferred
1
from the mother plate to a 96-well microtiter plate using an 8-channel
pipette. Assay solution (100 mL/well) was quickly added to each well using
a 1200 mL 8-channel pipette. The final concentrations in each well were
1.0mm substrate, 4.65mm BES, 0.434mm 4-nitrophenol, 7.1% acetonitrile.
The plate was quickly placed in the microplate reader and shaken for 10 s to
ensure complete mixing, and the decrease in absorbance at 404 nm was
monitored at 258C as often as permitted by the microplate software,
typically every 11 seconds. The starting absorbance was typically 1.2. Data
were collected for one hour to ensure we detected slow reactions and
reactions with a lag time. Each hydrolysis was carried out in quadruplicate
and was averaged. The first 10 s of data were sometimes erratic, possibly
due to dissipation of bubbles created during shaking. For this reason, we
typically excluded the first 10 s of data from the calculation of the initial
rate. Activities were calculated from slopes in the linear portion of the
curve, usually over the first two hundred seconds. The initial rates were
calculated from the average dA/dt by means of Equation (2), where De
1
4
996, 7, 497 ± 510; S. T. Chen, J. M. Fang, J. Org. Chem. 1997, 62, 4349 ±
357.
[
3] C. S. Chen, Y. Fujimoto, G. Girdaukas, C. J. Sih, J. Am. Chem. Soc.
1
982, 104, 7294 ± 7299.
[
[
4] M. J. Wajzer, C. R. Hebd. S e ances Acad. Sci. 1949, 229, 1270 ± 1272.
5] R. A. John in Enzyme Assays (Eds.: R. Eisenthal, M. J. Danson), IRL,
Oxford, 1992, pp. 81 ± 82.
6] R. M. Rosenberg, R. M. Herreid, G. J. Piazza, M. H. OꢀLeary, Anal.
Biochem. 1989, 181, 59 ± 65.
7] B. H. Gibbons, J. T. Edsall, J. Biol. Chem. 1963, 238, 3502 ± 3507.
8] O. H. Lowry, N. R. Roberts, M.-L. Wu, W. S. Hixon, E. J. Crawford, J.
Biol. Chem. 1954, 207, 19 ± 37.
[
[
[
�
1
� 1
1
0
7300m cm (experimentally determined for our conditions) and l
[
9] R. A. Darrow, S. P. Colowick, Methods Enzymol. 1962, Vol. V, 226 ±
� 1 � 1
.306 cm. To calculate specific activity (mmolmin mg protein), we took
2
35.
into account the total amount of protein in each well.
[
10] R. K. Crane, A. Sols, Methods Enzymol. 1960, Vol. I, 277 ± 286.
Screening of commercial hydrolases with pH indicators under interfacial
activation conditions: The procedure was the same as outlined above
except that the BES buffer (5mm, pH 7.2) contained Triton X-100
[11] R. G. Whittaker, M. K. Manthey, D. S. Le Brocque, P. J. Hayes, Anal.
Biochem. 1994, 220, 238 ± 243.
[12] R. G. Khalifah, J. Biol. Chem. 1971, 246, 2561 ± 2573; an appendix
includes a derivation of the pH dependence of the buffer factor, Q.
(
8.45mm). The final concentration of Triton X-100 in the wells was 2.8mm.
[
M
13] The enantioselectivity is the ratio of the specificity constants (kcat/K )
Small-scale reactions with 1mm (Æ)-solketal butyrate: These small-scale
reactions mimic the conditions in the microplate during pH indicator
activity screening except that no indicator is present. Hydrolase solutions
for each enantiomer. By measuring initial rates of the enantiomers
separately, we eliminate competitive binding between the two
enantiomers. At saturating substrate conditions, the relative initial
rates equal the relative kcat values; at partially saturating conditions,
(
1
50 mL) were added to solutions of (Æ)-solketal butyrate (3.50 mL of a
4.4mm solution in acetonitrile) and BES buffer (46.45 mL of a 5.0mm
solution, pH 7.2) for a final reaction volume of 50 mL (1.0mm substrate,
.65mm BES, 7% acetonitrile). After stirring at room temperature for a
the initial rates also depend on the K
separately measured initial rates ignores some or all of the effect of
on enantioselectivity. In spite of this inaccuracy, the relative initial
M
values. Thus, the ratio of
4
K
M
time estimated from the pH indicator screening, the mixture was extracted
with diethyl ether (3 Â 20 mL). The extracts, which contained both the ester
substrate and the alcohol product, were combined, washed with water and
dried with magnesium sulfate, filtered, and evaporated to dryness.
rate provides an estimated enantioselectivity.
[
[
14] J. Jurczak, S. Pikul, T. Bauer, Tetrahedron 1986, 42, 447 ± 488.
15] E. Vänttinen, L. T. Kanerva, Tetrahedron: Asymmetry 1997, 8, 923 ±
9
33 and references therein. The microorganism Comamonas testos-
Small-scale reactions with 50 mM (Æ)-solketal butyrate: Hydrolase
solutions (250 mL for CRL, ROL, HLE, AOP, E013; 50 mL for cutinase)
were added to solutions of (Æ)-solketal butyrate (352 mL of a 0.715m
solution in acetonitrile) and BES buffer (4,398 mL of a 5.0mm solution,
pH 7.2) for a final reaction volume of 5.0 mL (50mm substrate, 4.65mm
BES, 7% acetonitrile). Reactions were worked up as outlined above.
teroni also catalyzes the enantioselective oxidation of (R)-solketal
with an enantiomeric ratio of 49: A. Geerlof, J. Stoorvogel, J. A.
Jongejan, E. J. T. M. Leenen, T. J. G. M. van Dooren, W. J. J. van
den Tweel, J. A. Duine, Appl. Microbiol. Biotechnol. 1994, 42, 8 ± 15.
16] V. Partali, A. G. Melbye, T. Alvik, T. Anthonsen, Tetrahedron:
Asymmetry 1992, 3, 65 ± 72.
[
Determination of enantiomeric purity by GC: Gas chromatography
analysis was performed on a Hewlett Packard 5890 Series II Gas
Chromatograph equipped with a Chirasil-DEX CB chiral stationary phase
[17] The Merck Index, 10th ed., Merck, Rahway, NJ, 1983, p. 950. We also
measured the pK of 4-nitrophenol (10 mg in 10 mL of doubly distilled
a
water) by measuring the midpoint of the pH change as standardized
base was added. The experimental result agreed with the reported
value and did not change upon addition of 7% acetonitrile.
[18] Ref. [5], p. 80. The extinction coefficients change slightly upon
addition of cosolvent and should be determined experimentally.
[19] E. Banyai in Indicators (Ed.: E. Bishop), Pergamon, Oxford, 1972,
p. 75. With polyaromatic indicators, a control should be run to ensure
there is no enzyme inhibition by the indicator.
[20] The pathlength in a 96-well plate depends on the volume of the
solution in the well, since the light passes from the top of the plate
through the solution. Thus, the maximum indicator concentration
varies with the solution volumes. With other acid ± base indicators,
poor water solubility can also limit the maximal concentration. The 4-
nitrophenol concentration in our solutions (0.45mm or 0.006%) was
well below its solubility limit, 0.08%: see ref. [19], p. 92.
(
25 m  0.25 mm  0.25 mm Chrompack, Raritan, NJ). For analysis, solketal
was converted to the acetate by dissolving the mixture of solketal and
solketal butyrate in ethyl acetate (5 mL) containing acetic anhydride, 4-
pyrrolidinopyridine, and anhydrous potassium carbonate. The solution was
stirred for one hour at room temperature, then filtered, washed with brine,
then water, dried with magnesium sulfate and evaporated to dryness. Both
the starting material, solketal butyrate, and the acetate of the product were
simultaneously separated with baseline resolution by means of a temper-
ature gradient (1008C to 1308C, 28Cmin ). Solketal butyrate: k'
(
reported in the tables are the mean of three injections. We did not observe
any racemization of solketal or its esters during derivatization.
�
1
1
8.11
S), a 1.04; solketal acetate: k' 4.21 (S), a 1.10. The ee values
1
Acknowledgments: We thank NSERC (Canada) for financial support,
Alexandra N. Weissfloch for the gift of the pure enantiomers of solketal
butyrate, Fluka (Buchs, Switzerland), Thermogen (Chicago, IL), Dr.
[21] R. J. Beynon, J. S. Easterby, Buffer Solutions, The Basics, IRL, Oxford,
1996, p. 72.
2
330
ꢁ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1998
0947-6539/98/0411-2330 $ 17.50+.50/0
Chem. Eur. J. 1998, 4, No. 11