Y. K. Sim et al. / Tetrahedron Letters 52 (2011) 1041–1043
1043
creased by 5% at each run, but the value is still smaller than the
5a
activity loss (ꢀ15% at each run) in the previous report, although
Liu and coworkers reported lower losses (ꢀ10% loss after six runs)
in recycling experiments of hydrolysis at the oil/water interface by
Candida cylindracea lipase immobilized on oleic-acid–Pluronic-
8
a
coated magnetic particles.
In conclusion, our approach demonstrates that the introduction
of hydrophilic polymers provides advantages in retaining the
intrinsic activity of enzymes over direct coating of enzymes to
the magnetic nanoparticle surface in covalent immobilization of
enzymes. This study also showed that introducing a functional
group to be used for enzyme conjugation during the polymer coat-
ing step reduces the effort for overall manufacturing processes.
Figure 3. The AFM images of a magnetic nanobead: (A) A PMMA-coated magnetic
nanobead, (B) a CAL-B-conjugated nanobead.
Acknowledgments
This research was supported by Basic Science Research Program
through the National Research Foundation of Korea (NRF) funded
by the Ministry of Education, Science and Technology (NRF-2007-
3
31-C00170). The authors acknowledge Mr. Eunkyeom Kim (Univ.
of Seoul) and also the Korea Research Institute of Chemical
Technology.
Figure 4. Recycling of the lipase-conjugated nanobeads in aqueous media. The
lipase-conjugated beads were recovered by a magnet for consecutive runs.
Supplementary data
Supplementary data (experimental details, analysis of the PS-
coated magnetic beads by SEM, measuring diameters of the mag-
netic beads and hydrolysis of p-nitrophenyacetate by immobilized
In order to investigate the detailed morphologies of the surfaces
of a single magnetic nanobead, three dimensional atomic force
microscopy (AFM) images were obtained (Fig. 3). The surface of a
lipase-conjugated nanobead contains fewer valleys and the valleys
have longer peak-to-valley distance compared to that of a PMMA-
encapsulated magnetic bead, although the individual lipase mole-
cules could not be detected.
References and notes
For evaluation of the catalytic efficiency of the immobilized li-
pases on the magnetic nanobeads, we measured the reaction con-
version as well as enantioselectivity toward hydrolysis of rac-1-
phenylethyl butanoate in an aqueous solution as a model reaction
1. (a) Bornscheuer, U. T. Angew. Chem., Int. Ed. 2003, 42, 3336–3337; (b) Cao, L.
Curr. Opin. Chem. Biol. 2005, 9, 217–226.
2
3
.
.
Rotticci, D.; Norin, T.; Hult, K. Org. Lett. 2000, 2, 1373–1376.
Jung, S.; Park, S. Biotechnol. Lett. 2009, 31, 107–111.
4. (a) Lu, A.-H.; Salabas, E. L.; Schüth, F. Angew. Chem., Int. Ed. 2007, 46, 1222–
1244; (b) Zhao, M.; Josephson, L.; Tang, Y.; Weissleder, R. Angew. Chem., Int. Ed.
(Table 1). The kinetic resolution of rac-1-phenylethyl alcohol by li-
2003, 42, 1375–1378; (c) Weissleder, R.; Kelly, K.; Sun, E. Y.; Shtatland, T.;
pases has been extensively studied and well documented in the lit-
Josephson, L. Nat. Biotechnol. 2005, 23, 1418–1423; (d) Bulte, J. W.; Kraitchman,
D. L. NMR Biomed. 2004, 17, 484–499; (e) Gu, H.; Ho, P. L.; Tsang, K. W. T.;
Wang, L.; Xu, B. J. Am. Chem. Soc. 2003, 125, 15702–15703; (f) Xu, C.; Xu, K.; Gu,
H.; Zhong, X.; Guo, Z.; Zheng, R.; Zhang, X.; Xu, B. J. Am. Chem. Soc. 2004, 126,
1
0
erature. The immobilized lipases catalyzed the hydrolysis with
0–75% conversion (entries 1–10) but protease S showed no con-
1
version (entry 11). Protease S does not accept rac-1-phenylethyl
butanoate as a substrate rather than becoming inactive by immo-
bilization because even the hydrolysis catalyzed by the free form
of protease S did not occur (data not shown). In addition, the reac-
tion of protease-S-conjugated nanobeads with p-nitrophenylace-
tate as a substrate showed equivalent activity to that of the free
enzyme (Fig. S3 in the Supplementary data). Among seven lipase-
conjugated nanobeads, CAL-B-, BCL-, and CRL-conjugated ones
showed high conversion as well as high enantioselectivity (entries
3
392–3393; (g) Gu, H.; Xu, K.; Xu, C.; Xu, B. Chem. Commun. 2006, 941–949; (h)
Lee, I. S.; Lee, N.; Park, J.; Kim, B.-H.; Yi, Y.-W.; Kim, T.; Kim, T. K.; Lee, I. H.; Paik,
S. R.; Hyeon, T. J. Am. Chem. Soc. 2006, 128, 10658–10659; (i) Lee, J.-H.; Huh, Y.-
M.; Jun, Y.-W.; Seo, J.-W.; Jang, J.-T.; Song, H.-T.; Kim, S.; Cho, E.-J.; Yoon, H.-G.;
Suh, J.-S.; Cheon, J. Nat. Med. 2007, 13, 95–99; (j) Yi, D. K.; Selvan, S. T.; Lee, S. S.;
Papaefthymiou, G. C.; Kundaliya, D.; Ying, J. Y. J. Am. Chem. Soc. 2005, 127,
4
2
990–4991; (k) Stoeva, S. I.; Huo, F.; Lee, J.-S.; Mirkin, C. A. J. Am. Chem. Soc.
005, 127, 15362–15363.
5
.
(a) Dyal, A.; Loos, K.; Noto, M.; Chang, S.; Spagnoli, C.; Shafi, K.; Ulman, A.;
Cowman, M.; Gross, R. J. Am. Chem. Soc. 2003, 125, 1684–1685; (b) Gardimalla,
H. M. R.; Mandal, D.; Stevens, P. D.; Yen, M.; Gao, Y. Chem. Commun. 2005,
4
432–4434; (c) Lee, J.; Lee, Y.; Youn, J.; Na, H.; Yu, T.; Kim, H.; Lee, S.; Koo, Y.;
1
, 3, and 5, respectively). The reactions catalyzed by the free form
Kwak, J.; Park, H. Small 2008, 4, 143–152; (d) Lee, D.; Ponvel, K.; Kim, M.;
Hwang, S.; Ahn, I.; Lee, C. J. Mol. Catal. B: Enzym. 2009, 57, 62–66; (e) Huang, S.;
Liao, M.; Chen, D. Biotechnol. Prog. 2003, 19, 1095–1100; (f) Lee, K. S.; Woo, M.
H.; Kim, H. S.; Lee, E. Y.; Lee, I. S. Chem. Commun. 2009, 3780–3782.
Rossi, L.; Quach, A.; Rosenzweig, Z. Anal. Bioanal. Chem. 2004, 380, 606–613.
Liao, M.; Chen, D. J. Mol. Catal. B: Enzym. 2002, 16, 283–291.
of those three lipases were also carried out to check the changes in
the enzyme activities caused by the current immobilization meth-
od (entries 2, 4, and 6). Interestingly, the reactions by the lipase-
conjugated nanobeads in this study showed comparable conver-
sions to the reactions by the free lipases, although it is generally re-
ported that the activity of covalently immobilized enzyme
6
7
.
.
8. (a) Mohammod, I.; Guo, C.; Xia, H.; Ma, J.; Jiang, Y.; Liu, H. Ind. Eng. Chem. Res.
2
2
008, 47, 6379–6385; (b) Wang, W.; Xu, Y.; Li, D. I. C.; Wang, Z. J. Am. Chem. Soc.
009, 131, 12892–12893.
5
,11
decreases.
9. Bradford, M. M. Anal. Biochem. 1976, 72, 248–254.
In addition, we conducted recycling experiments of the three li-
pase-conjugated magnetic nanobeads. The lipase-conjugated
beads were isolated by a magnet for consecutive runs after a reac-
tion had been completed. As shown in the Figure 4, the conversion
in the reactions by CAL-B- and BCL-conjugated magnetic nanobe-
ads was consistent during six-time recycling with maintaining
the same enantioselectivity (E P 200). For the CRL-conjugated
magnetic nanobeads, it was observed that the conversion de-
10. (a) Park, S.; Kazlauskas, R. J. J. Org. Chem. 2001, 66, 8395–8401; (b) Schofer, S.
H.; Kaftzik, N.; Wasserscheid, P.; Kragl, U. Chem. Commun. 2001, 425–426; (c)
Shah, S.; Gupta, M. N. Bioorg. Med. Chem. Lett. 2007, 17, 921–924; (d) Csajagi, C.;
Szatzker, G.; Toke, E. R.; Urge, L.; Darvas, F.; Poppe, L. Tetrahedron: Asymmetry
2008, 19, 237–246; (e) Kourist, R.; de Maria, P. D.; Bornscheuer, U. T.
ChemBioChem 2008, 9, 491–498; (f) Jung, S.; Park, S. Biotechnol. Lett. 2008, 30,
717–722; (g) Wang, Y. H.; Wang, R.; Liz, Q. S.; Zhang, M.; Feng, Y. J. Mol. Catal. B:
Enzym. 2009, 56, 142–145.
11. Moreno, J.-M.; Arroyo, M.; Hernáiz, M.-J.; Sinisterra, J.-V. Enzym. Microb.
Technol. 1997, 21, 552–558.