24
L. Qian et al. / Journal of Molecular Catalysis B: Enzymatic 73 (2011) 22–26
acetate, 20/1, v/v); 1H NMR (500 MHz, CDCl3): ı/ppm 8.27 (d,
2H, J = 9.1 Hz), 7.28 (d, 2H, J = 9.3 Hz), 2.62–2.59 (t, 2H, J = 7.48 Hz),
1.68–1.66 (m, 3H), 0.97 (d, 6H, J = 6.05 Hz).
2.2.15. 4-Nitrophenyl 3-phenylpropanoate (15)
The reaction was carried out following the general proce-
dure starting with 16 mmol of 3-phenylpropanoic acid, yielding
3.5 g (80.7%) of 15. Pale yellow solid; TLC: Rf = 0.54 (petroleum
ether/ethyl acetate, 20/1, v/v); 1H NMR (500 MHz, CDCl3): ı/ppm
8.25 (d, 2H, J = 9.1 Hz), 7.35–7.32 (m, 2H), 7.27–7.25 (m, 3H), 7.19
(d, 2H, J = 9.1 Hz), 3.10–3.07 (t, 2H, J = 7.57 Hz), 2.96–2.92 (m, 2H,
J = 7.57 Hz).
2.2.8. 4-Nitrophenyl 3-methylpentanoate (8)
The reaction was carried out following the general procedure
starting with 16 mmol of 3-methyl pentanoic acid, yielding 3.4 g
(89.7%) of 8. Pale yellow oil; TLC: Rf = 0.73 (petroleum ether/ethyl
acetate, 20/1, v/v); 1H NMR (500 MHz, CDCl3): ı/ppm 8.27 (d, 2H,
J = 7.12 Hz), 7.27 (d, 2H, J = 9.1 Hz), 2.63–2.58 (m, 1H), 2.42–2.38 (m,
1H), 2.05–2.01 (m, 1H), 1.50–1.44 (m, 1H), 1.37–1.32 (m, 1H), 1.05
(d, 3H, J = 6.69 Hz), 0.98–0.95 (t, 3H, J = 7.43 Hz).
2.2.16. 4-Nitrophenyl 2-phenylpropanoate (16)
The reaction was carried out following the general proce-
dure starting with 16 mmol of 2-phenylpropanoic acid, yielding
3.8 g (87.6%) of 16. Pale yellow solid; TLC: Rf = 0.49 (petroleum
ether/ethyl acetate, 20/1, v/v); 1H NMR (500 MHz, CDCl3): ı/ppm
8.22 (d, 2H, J = 9.0 Hz), 7.39 (d, 4H, J = 4.3 Hz), 7.34–7.31 (m, 1H), 7.18
(d, 2H, J = 9.0 Hz), 4.02–3.97 (m, 1H), 1.63 (d, 3H, J = 7.1 Hz).
2.2.9. 4-Nitrophenyl 2-methylpentanoate (9)
The reaction was carried out following the general procedure
starting with 16 mmol of 2-methyl pentanoic acid, yielding 3.4 g
(89.7%) of 9. Pale yellow oil; TLC: Rf = 0.63 (petroleum ether/ethyl
acetate, 20/1, v/v); 1H NMR (500 MHz, CDCl3): ı/ppm 8.27 (d, 2H,
J = 9.0 Hz), 7.27 (d, 2H, J = 8.9 Hz), 2.75–2.71 (m, 1H), 1.83–1.77 (m,
1H), 1.60–1.53 (m, 1H), 1.49–1.41 (m, 2H) 1.31 (d, 3H, J = 7.0 Hz),
0.99–0.96 (t, 3H, J = 7.30 Hz); 13C NMR (500 MHz, CDCl3): ı/ppm
174.99, 156.29, 145.81, 125.75, 123.05, 40.04, 36.31, 20.99, 17.40,
14.53.
2.2.17. 4-Nitrophenyl 2-phenylbutanoate (17)
The reaction was carried out following the general procedure
starting with 16 mmol of 2-phenylbutanoic acid, yielding 3.5 g
(76.8%) of 17. Pale yellow oil; TLC: Rf = 0.59 (petroleum ether/ethyl
acetate, 20/1, v/v); 1H NMR (500 MHz, CDCl3): ı/ppm 8.24 (d,
2H, J = 9.1 Hz), 7.41–7.38 (m, 4H), 7.34–7.32 (m, 1H), 6.88 (d,
2H, J = 9.1 Hz), 3.74–3.71 (t, 1H, J = 7.68 Hz), 2.26–2.20 (m, 1H),
1.96–1.90 (m, 1H), 1.01–0.98 (t, 3H, J = 7.36 Hz).
2.2.10. 4-Nitrophenyl cyclopropanecarboxylate (10)
The reaction was carried out following the general procedure
starting with 16 mmol of cyclopropanecarboxylic acid, yielding
2.4 g (72.7%) of 10. Pale yellow solid; TLC: Rf = 0.51 (petroleum
ether/ethyl acetate, 10/1, v/v); 1H NMR (500 MHz, CDCl3): ı/ppm
8.27 (d, 2H, J = 9.1 Hz), 7.29 (d, 2H, J = 9.1 Hz), 1.90–1.85 (m, 1H),
1.23–1.19 (m, 2H), 1.11–1.08 (m, 2H).
2.3. Enzyme assay
All enzyme samples were prepared by weighing the fresh solid
sample and diluting into a solution of 100 mg mL−1 in KPB (aq.
100 mM potassium phosphate, pH 7.0). The protein concentra-
tion was measured by Coomassie Brilliant Blue method. Based on
the protein concentration, the solution of enzyme samples was
diluted to a stock solution of 1 mg protein mL−1. All substrates
were diluted with DMSO from 100 mM stock solutions. The solu-
tions could be stored at 4 ◦C for several months without notable
decomposition.
2.2.11. 4-Nitrophenyl cyclobutanecarboxylate (11)
The reaction was carried out following the general proce-
dure starting with 16 mmol of cyclobutanecarboxylic acid, yielding
3.0 g (84.8%) of 11. Pale yellow solid; TLC: Rf = 0.54 (petroleum
ether/ethyl acetate, 40/1, v/v); 1H NMR (500 MHz, CDCl3): ı/ppm
8.28 (d, 2H, J = 9.1 Hz), 7.28 (d, 2H, J = 9.1 Hz), 3.46–3.39 (m, 1H),
2.49–2.33 (m, 4H), 2.14–1.98 (m, 2H).
2.2.12. 4-Nitrophenyl cyclopentanecarboxylate (12)
Assay was carried out in 96-well clear-bottom polystyrene
microtiter plates. A KPB solution of 180 L containing the enzyme
at an appropriate concentration was first placed in the wells.
The appropriate concentration of enzyme was obtained by pre-
measurements, in which the initial reactivity velocity of enzyme
samples could be tested accurately. The microtiter plate was incu-
bated at 30 ◦C for 2 min. Then the substrate solutions (20 L) were
added simultaneously into each well. The microtiter plate was
immediately placed in a Microplate Spectrophotometer (BioTek®
PowerWave XS2) and the reaction was monitored at 405 nm for
over 3 min. The spontaneous hydrolysis of each substrate was mea-
sured in absence of enzyme under the same condition.
The concentration of p-nitrophenol is linear under
the absorption at 405 nm. The calibration equation is
A405 = 8.00 × [pNP] + 0.055, in which A405 represents the absorp-
tion at 405 nm and the [pNP] represents the concentration of
p-nitrophenol with unit of mmol L−1. Therefore, the primary Abs-
versus-time data were first converted into product-versus-time
using the calibration equation. The steepest linear portion (<10%
conversion) of each curve was then used to calculate the apparent
initial reaction rate in each well. Given the spontaneous hydrolysis
of substrates, the real initial reaction rates of enzymes were
obtained by subtraction of the spontaneous hydrolysis rate from
the apparent rate.
The reaction was carried out following the general procedure
starting with 16 mmol of cyclopentanecarboxylic acid, yielding
3.2 g (85.1%) of 12. Pale yellow oil; TLC: Rf = 0.63 (petroleum
ether/ethyl acetate, 20/1, v/v); 1H NMR (500 MHz, CDCl3): ı/ppm
8.27 (d, 2H, J = 9.1 Hz), 7.27 (d, 2H, J = 9.0 Hz), 3.05–2.99 (m, 1H),
2.07–1.92 (m, 4H), 1.81–1.66 (m, 4H).
2.2.13. 4-Nitrophenyl cyclohexanecarboxylate (13)
The reaction was carried out following the general procedure
starting with 16 mmol of cyclohexanecarboxylic acid, yielding 3.6 g
(90.4%) of 13. Pale yellow oil; TLC: Rf = 0.76 (petroleum ether/ethyl
acetate, 20/1, v/v); 1H NMR (500 MHz, CDCl3): ı/ppm 8.27 (d, 2H,
J = 9.1 Hz), 7.26 (d, 2H, J = 9.0 Hz), 2.63–2.57 (m, 1H), 2.09–2.06
(m, 2H), 1.86–1.82 (m, 2H), 1.72–1.7 (m, 1H), 1.64–1.58 (m, 2H),
1.42–1.28 (m, 3H).
2.2.14. 4-Nitrophenyl benzoate (14)
The reaction was carried out following the general procedure
starting with 16 mmol of benzoic acid, yielding 3.3 g (84.9%) of
14. Pale yellow solid; TLC: Rf = 0.50 (petroleum ether/ethyl acetate,
20/1, v/v); 1H NMR (500 MHz, CDCl3): ı/ppm 8.34 (d, 2H, J = 9.1 Hz),
8.21 (d, 2H, J = 7.3 Hz), 7.71–7.68 (m, 1H), 7.57–7.54 (m, 2H), 7.43
(d, 2H, J = 9.1 Hz).