J. Cruz et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 7–14
13
Acknowledgments
This work was supported by Grant No. 1102-489-25428 from
COLCIENCIAS and Universidad Industrial de Santander (VIE-UIS
Research Program) and Grant CTQ2009-07568 from Spanish Min-
isterio de Ciencia e Innovación. The support of COLCIENCIAS (PhD
Program Fellowship-2008) is gratefully recognized. Authors also
gratefully recognize the kind supply of enzymes by Novozymes.
The authors would like to thank Mr. Ramiro Martínez (Novozymes,
Spain S.A.) and Ing. Sandra La-Rotta (Coldanzimas Ltda) for kindly
supplying the enzymes used in this research. The help and
comments from Dr. Ángel Berenguer (Instituto de Materiales, Uni-
versidad de Alicante) are gratefully recognized.
References
[1] V. Gotor-Fernández, R. Brieva, V. Gotor, J. Mol. Catal. B: Enzym. 40 (2006)
111–120.
[2] Y. Fan, J. Qian, J. Mol. Catal. B: Enzym. 66 (2010) 1–7.
[3] A. Bajaj, P. Lohan, P.N. Jha, R. Mehrotra, J. Mol. Catal. B: Enzym. 62 (2010) 9–14.
[4] M. Guncheva, D. Zhiryakova, J. Mol. Catal. B: Enzym. 68 (2011) 1–21.
[5] F.J. Contesini, D.B. Lopes, G.A. MacEdo, M.D.G. Nascimento, P.D.O. Carvalho, J.
Mol. Catal. B: Enzym. 67 (2010) 163–171.
[6] R.C. Rodrigues, R. Fernandez-Lafuente, J. Mol. Catal. B: Enzym. 66 (2010) 15–32.
[7] R.C. Rodrigues, R. Fernandez-Lafuente, J. Mol. Catal. B: Enzym. 64 (2010) 1–22.
[8] U. Derewenda, L. Swenson, R. Green, Y. Wei, S. Yamaguchi, R. Joerger, et al.,
Protein Eng. 7 (1994) 551–557.
Fig. 9. Confocal microscopy of CAL/BSA-CLEAs. The equipment was a Hirox KH-
7700, using a lent OL-700. Photo was taken at 280× at 15 kV. The samples were in
aqueous medium.
[9] N. Miled, F. Beisson, J. De Caro, A. De Caro, V. Arondel, R. Verger, J. Mol. Catal.
B: Enzym. 11 (2001) 165–171.
[10] S.S. Kumar, N. Arora, R. Bhatnagar, R. Gupta, J. Mol. Catal. B: Enzym. 59 (2009)
41–46.
[11] J.M. Palomo, G. Fernández-Lorente, J.M. Guisán, R. Fernández-Lafuente, Adv.
Synth. Catal. 349 (2007) 1119–1127.
[12] A. Chaubey, R. Parshad, S. Koul, S.C. Taneja, G.N. Qazi, J. Mol. Catal. B: Enzym.
42 (2006) 39–44.
(50% v/v). The crosslinking reagent concentration was fixed at the
higher level (1.5% w/v). Thus, after these two experimental designs,
the best conditions for preparation of CALB–BSA-CLEA were: pro-
tein concentration, 3 mg/mL; precipitant concentration, 50% v/v;
[13] Z. Cabrera, G. Fernandez-Lorente, R. Fernandez-Lafuente, J.M. Palomo, J.M.
Guisan, J. Mol. Catal. B: Enzym. 57 (2009) 171–176.
[14] G. Fernandez-Lorente, J.M. Palomo, Z. Cabrera, R. Fernandez-Lafuente, J.M.
Guisán, Biotechnol. Bioeng. 97 (2007) 242–250.
3.3. Stability of CALB–BSA-CLEA
[15] V. Gotor-Fernández, E. Busto, V. Gotor, Adv. Synth. Catal. 348 (2006) 797–812.
[16] E.M. Anderson, K.M. Larsson, O. Kirk, Biocatal. Biotransform. 16 (1998) 181–204.
[17] J. Uppenberg, S. Patkar, T. Bergfors, T.A. Jones, J. Mol. Biol. 235 (1994) 790–792.
[18] K. Hernandez, C. Garcia-Galan, R. Fernandez-Lafuente, Enzyme Microb. Tech-
nol. 49 (2011) 72–78.
[19] J.A. Laszlo, K.O. Evans, J. Mol. Catal. B: Enzym. 58 (2009) 169–174.
[20] A. Bastida, P. Sabuquillo, P. Armisen, R. Fernández-Lafuente, J. Huguet, J.M.J.M.
Guisán, Biotechnol. Bioeng. 58 (1998) 486–493.
Fig. 1 shows that the optimized CALB–BSA-CLEA is much more
stable than CALB at 70 ◦C, in fact after 1 h the first remained fully
active, while the half live of CALB is under 10 min. The SDS-PAGE of
this new CLEA (results not shown) shows that this biocatalyst did
not release any enzyme molecule even after being boiled in SDS,
suggesting an effective crosslinking of the CALB and BSA molecules.
[21] K. Hernandez, R. Fernandez-Lafuente, Enzyme Microb. Technol. 48 (2011)
107–122.
[22] P.V. Iyer, L. Ananthanarayan, Process Biochem. 43 (2008) 1019–1032.
[23] L. Betancor, H.R. Luckarift, Trends Biotechnol. 26 (2008) 566–572.
[24] C. Mateo, J.M. Palomo, G. Fernandez-Lorente, J.M. Guisan, R. Fernandez-
Lafuente, Enzyme Microb. Technol. 40 (2007) 1451–1463.
[25] C. Oˇı’Fagain, Enzyme Microb. Technol. 33 (2003) 137–149.
[26] C. Garcia-Galan, A. Berenguer-Murcia, R. Fernandez-Lafuente, R.C. Rodrigues,
Adv. Synth. Catal. 353 (2011) 2885–2904.
3.4. Confocal microscopy of the CALB–BSA-CLEA
Fig. 9 shows the image of CALB–BSA-CLEA observed by confocal
microscopy. The CALB–BSA CLEA is formed by different individual
aggregates, forming ramified clusters of even 100 m. Each particle
may contain 105 individual aggregates.
[27] R.A. Sheldon, Appl. Microbiol. Biotechnol. 92 (2011) 467–477.
ˇ
[28] F. Sulek, D.P. Fernández, Z. Knez, M. Habulin, R.A. Sheldon, Process Biochem. 46
(2011) 765–769.
[29] L. Cao, L. van Langen, R.A. Sheldon, Curr. Opin. Biotechnol. 14 (2003) 387–394.
[30] D. Özdemirhan, S. Sezer, Y. Sönmez, Tetrahedron: Asymmetry 19 (2008)
2717–2720.
[31] B.L.A. Prabhavathi Devi, Z. Guo, X. Xu, J. Am. Oil Chem. Soc. 86 (2009) 637–642.
[32] Z.J. Dijkstra, R. Merchant, J.T.F. Keurentjes, J. Supercrit. Fluids 41 (2007)
102–108.
4. Conclusions
The use of a protein feeder seems to be necessary to obtain an
actual crosslinking on enzyme molecules having a low density of
Lys in its surface. BSA may be a good option as a feeder; it is a
molecule with many amino groups in the surface and that permits
and easy crosslinking using glutaraldehyde. In the case of CALB,
the use of some feeder has been clearly shown, even after using
RSM for the optimization enzyme molecules could still be released
from the solid, due to a low degree of crosslinking (visualized by
the high percentage of monomers of enzyme molecules in the SDS-
PAGE). The use of BSA as a feeder and RSM for the optimization of
the CLEA preparation has permitted an efficient preparation of the
CALB-CLEA, exhibiting a very high stability. RSM has revealed itself
as an interesting tool also in the design of CLEAs.
[33] H.R. Hobbs, B. Kondor, P. Stephenson, R.A. Sheldon, N.R. Thomas, M. Poliakoff,
Green Chem. 8 (2006) 816–821.
[34] F. López-Gallego, L. Betancor, A. Hidalgo, N. Alonso, R. Fernández-Lafuente, J.M.
Guisán, Biomacromolecules 6 (2005) 1839–1842.
[35] L. Wilson, G. Fernández-Lorente, R. Fernández-Lafuente, A. Illanes, J.M. Guisán,
J.M. Palomo, Enzyme Microb. Technol. 39 (2006) 750–755.
[36] B.K. Vaidya, S.S. Kuwar, S.B. Golegaonkar, S.N. Nene, J. Mol. Catal. B: Enzym. 74
(2012) 184–191.
[37] J. Pan, X.D. Kong, C.X. Li, Q. Ye, J.H. Xu, T. Imanaka, J. Mol. Catal. B: Enzym. 68
(2011) 256–261.
[38] J. Yan, X. Gui, G. Wang, Y. Yan, Appl. Biochem. Biotechnol. 166 (2012) 925–932.
[39] L. Wilson, A. Illanes, O. Abián, B.C.C. Pessela, R. Fernández-Lafuente, J.M. Guisán,
Biomacromolecules 5 (2004) 852–857.
[40] C. Mateo, B. Fernandes, F. Van Rantwijk, A. Stolz, R.A. Sheldon, J. Mol. Catal. B:
Enzym. 38 (2006) 154–157.
[41] S. Shah, A. Sharma, M.N. Gupta, Anal. Biochem. 351 (2006) 207–213.