96
M. Fujiwara et al. / Journal of Molecular Catalysis B: Enzymatic 109 (2014) 94–100
2.6. Catalytic tests of enzymes by hydrolysis of 4-nitorophenyl
acetate
keeping the mass ratio of catalyst/substrate in the first run. The
esterification of ( )-1 using lipase PS-IM (40 U, 20 mg) was carried
out by means of the same procedure.
Hydrolytic activities of enzymes were performed by measur-
ing the increase of 4-nitrophenol produced by the hydrolysis of
4-nitrophenyl acetate. To a 50 mL-Erlenmyer flask containing 0.1 M
Tris–HCl buffer (pH 7.0, 10 mL) were added a 2 M acetone solution
of 4-nitrophenyl acetate (100 L). In the case of commercially avail-
able lipase PS, a 100 L-portion of the enzyme solution in 0.1 M
Tris–HCl buffer (pH 7.0, 5.0 mg particle in 1 mL buffer) was added
to the mixture, and the solution was shaken with 100 min−1 at
25 ◦C. On the other hand, in the case of the immobilized lipase PS
Cap, 5.0 mg of the enzyme and 100 L of 0.1 M Tris–HCl buffer
(pH 7.0) were added, and the reaction was carried out in the
same way as mentioned above. After 4, 8, and 12 min, a 50 L-
portion of the mixture was added to a cuvette containing 0.1 M
Tris–HCl buffer (pH 7.0, 950 L), and the absorbance at 405 nm
was immediately measured by a Shimadzu PharmaSpec UV-1700
(UV/Vis spectrophotometer). One unit of hydrolytic activity was
defined as the amount of enzyme necessary to release 1 mmol of
4-nitrophenol per minute under the reaction conditions.
2.10. Typical procedure for the enantioselective hydrolysis of
(
)-2-acetoxyhexyl tosylate (2) with lipase PS in aqueous media
To a 200-mL Erlenmeyer flask containing 126 mg (0.400 mmol;
sub. conc., 10 mM) of ( )-2 was added 4 mL of diisopropyl ether and
36 mL of 0.1 M Tris–HCl buffer (pH 7.6). To the mixture was added
600 mg (24 U) of lipase PS Cap treated with zinc chloride solution,
and the solution was incubated for 24 h at 30 ◦C. Centrifugation of
the mixture with 8500 rpm for 5 min gave a supernatant and pre-
cipitate. After re-suspension of the precipitate in 0.1 M Tris–HCl
buffer (pH 7.6), the mixture was subjected to the next centrifuga-
tion. The products were extracted from the combined supernatant
with ethyl acetate (×3), washed with brine, and dried over Na2SO4.
Evaporation and purification by column chromatography on sil-
ica gel (n-hexane/ethyl acetate = 3/1) afforded (S)-2 (80 mg, 64%,
46% ee) and (R)-2-hydroxyhexyl tosylate (4, 31 mg, 28%, 99.7% ee).
The spectral data were in full agreement with those reported [1,3].
The ees of the resulting (S)-2 and (R)-4 were determined by HPLC
analysis, and the absolute configurations were also confirmed by
comparing the retention times with those reported [1,3]. HPLC
conditions: column, CHIRALCEL AD-H (Daicel Chemical Industries,
Ltd.); eluent, n-hexane/2-propanol = 90/10; flow rate, 0.5 mL/min;
254 nm; temperature, 25 ◦C; retention time, 2: 18 (S) and 19.5 (R)
min, 4: 32 (R) and 43 (S) min.
2.7. Determination of protein content
tracting the amount of protein in supernatant of the immobilization
suspension from the total amount of protein offered for immobi-
lization. Protein assay was performed by the Bradford method using
On the other hand, the recovered lipase PS Cap (540 mg)
as the precipitate was dried under reduced pressure, and was
examined for the next run. The reuse of the recovered enzyme
was performed on the basis of keeping the mass ratio of cata-
lyst/substrate in the first run. The hydrolyses of ( )-2 using lipase
2.8. Immobilization efficiency
Immobilization yield (%) and expressed yield (%) were calculated
using the following equations [32]:
Total protein offered − Protein in the supernatant of the immobilization
Immobilization yield (%) =
Expressed yield (%) =
× 100
Total protein offered
Actual activity of the derivative
× 100
Expected activity considering the immobilized enzyme
PS-IM (60 U, 30 mg) and lipase PS Cap (50 U, 300 mg) were carried
out by means of the same procedure, while in the case of lipase
PS-IM the enzyme was recovered by filtration with a filter paper.
2.9. Typical procedure for the enantioselective esterification of
(
)-1-phenylethanol (1) with lipase PS in organic media
To a 20-mL recovery flask were added 122 mg (1.00 mmol, sub.
conc. 250 mM) of ( )-1 and 4.0 mL of vinyl acetate. After the addi-
tion of lipase PS Cap (200 mg, 34 U), the mixture was stirred for
24 h at 30 ◦C. The mixture was filtered through a filter paper, and
the recovered enzyme was washed with ethyl acetate. Evaporation
under reduced pressure and purification by column chromatog-
raphy on silica gel (n-hexane/ethyl acetate = 4/1) afforded (S)-1
36%, 99.0% ee). The spectral data were in full agreement with
those of commercial sources. The ees of the resulting (S)-1 and
(R)-3 were determined by GC analysis, and the absolute con-
figuration of 1 was confirmed by comparing the retention time
140 ◦C; detection, 140 ◦C; oven, 120 ◦C; carrier gas, He; head pres-
sure, 2.4 kg/cm2; retention time, 1: 14.5 (R) and 14.7 (S) min, 3:
14.7 (S) and 15.2 (R) min. The reaction conversions and E val-
ues in Tables 2–4 were calculated using ees/(ees + eep) and using
ln[(1 − conv.)(1 − ees)]/ln[(1 − conv.)(1 + ees)], respectively [34].
On the other hand, the recovered lipase PS Cap (156 mg) was
dried under reduced pressure, and was examined for the next run.
The reuse of the recovered enzyme was performed on the basis of
3. Results and discussion
3.1. Encapsulation of lipase into calcium carbonate microcapsule
(ꢀCap) and its catalytic activity
The immobilization of lipase into calcium carbonate was carried
out by the analogous process to the phase transformation method
recovered solid was washed thoroughly with deionized water and
dried. The encapsulation of lipase in this sample (lipase PS Cap)
As shown in Fig. 1, a typical UV absorption of lipase at about 280 nm
tion around 280 nm was found in the Cap before the encapsulation
treatment (not shown here) [29]. Since the lipase PS Cap sample
was thoroughly washed with water, the immobilized lipase in Cap
must be strongly incorporated into the calcium carbonate matrix.
Fig. 2 shows the X-ray diffraction patterns of two calcium carbon-
ate samples, the original Cap and the lipase PS Cap. The XRD
pattern of the original Cap shows a typical vaterite one, and the