Paper
RSC Advances
at the ultraviolet wavelength of 270 nm. The mobile phase ow and the reaction time does not change the enantioselectivity of
rate was 1 mL minꢀ1 and 20 mL of each sample was injected at CLEAs-YCJ01, indicating that the two congurations of
30 ꢁC. The retention times of all the substrates are presented in substrate with high ee values could be achieved by prolonging
the ESI.†
The ee values of R-monoacetate (ees) and S-diacetates (eep)
the reaction time or adding catalyst at every batch reaction.
Further, CLEAs-YCJ01 was evaluated for the resolution of
were calculated by HPLC. The yield of S-diacetates was deter- a series of 3-aryloxy-1,2-propanediols (six analogues of MPPD)
mined by HPLC. The conversion of 3-aryloxy-1,2-propanediols under the optimized reaction conditions for the resolution of
(c) was calculated as c (%) ¼ ees/(ees + eep) ꢂ 100 and the MPPD. The results showed that CLEAs-YCJ01 has relatively
enantioselectivity (E) was calculated as E ¼ ln[(1 ꢀ c)(1 ꢀ ees)/(1 strict enantioselectivity towards 3-aryloxy-1,2-propanediols with
ꢀ c)(1 + ees)].
a high yield ($49.3%), favorable ee values (94.8–99.4%) for S-
diacetates and high ee values (92.1–99.2%) for R-monoacetate.
Especially, towards 3-(4-methylphenoxy)-1,2-propanediol and 3-
phenoxy-1,2-propanediol, both of R- and S-type compounds
with >99% ee were obtained.
Water content
The substrates and solvents were separately pre-equilibrated at
different water contents (aw) prior to the resolution. All samples
were placed in a closed vessel above saturated salts, and equi-
librium was reached for 48 h at room temperature. The salts
used in this test included LiCl, aw ¼ 0.11; CH3COOK, aw ¼ 0.24;
MgCl2, aw ¼ 0.33; Mg(NO3)2, aw ¼ 0.56, and NaCl, aw ¼ 0.75.
Conflicts of interest
There are no conicts to declare.
Preparation of CLEAs of lipase YCJ01 (CLEAs-YCJ01)
Acknowledgements
The preparation of CLEAs-YCJ01 in this work was done
according to the procedures described by Schoevaart20 with
some modications. Crude lipase YCJ01 powder was prepared
in our previous work18 and dissolved in phosphate buffer (pH ¼
7.5 and 0.05 M) to a concentration of 1.2 mg protein per mL (53
U). Aggregates of lipase YCJ01 were prepared using precipitants
and glutaraldehyde was added to a total concentration of 0.25%
(v/v), then the resulting suspensions were stirred at 0 ꢁC for 4 h.
The CLEAs of lipase YCJ01 was separated by centrifugation and
obtained by volatilizing the residual solvent. All portions of the
ltrate were retained for the determination of protein concen-
tration and activity.
This work was supported by the National High Technology
Research and Development Key Program of China
(2012AA022205).
Notes and references
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Bioconversion and determination of operational stability
In a typical reaction, 20 mL isopropyl ether, CLEAs-YCJ01 (20
mg), MPPD (50 mM), and vinyl acetate (100 mM) were loaded
into 50 mL conical asks with stoppers. The mixture was kept at
35 ꢁC and rotated at 180 rpm. The samples were withdrawn
from the mixture and analyzed by HPLC, as described above.
When the determination of operational stability was performed,
the ltered CLEAs-YCJ01 was washed with fresh isopropyl ether
(3 ꢂ 5 mL) and loaded back into the conical asks for the next
cycle.
Conclusions
In this work, cross-linked enzyme aggregates of lipase YCJ01
(CLEAs-YCJ01) were prepared with t-butanol as the precipitant,
and 214.8% of activity recovery was achieved. CLEAs-YCJ01
showed
excellent
enantioselectivity
towards
3-(4-
methylphenoxy)-1,2-propanediol with ee values of 99.2% for S-
diacetates and 99.1% for monoacetate. For the rst time, R- and 10 Z. Sun, L. Wu, M. Bocola, H. C. S. Chan, R. Lonsdale,
S-type compounds with excellent ee values were simultaneously
X.-D. Kong, S. Yuan, J. Zhou and M. T. Reetz, J. Am. Chem.
obtained. CLEAs-YCJ01 also showed high operational stability
and maintained 91.2% residual activity aer ten batches. These 11 A. Janfalk Carlsson, P. Bauer, H. Ma and M. Widersten,
Soc., 2018, 140, 310–318.
˚
results showed that CLEAs-YCJ01 has good operational stability
Biochemistry, 2016, 55, 1940.
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RSC Adv., 2019, 9, 13757–13764 | 13763