Sains Malaysiana 40(9)(2011): 965–972
Properties of Immobilized Candida
antarctica Lipase B on Highly
Macroporous Copolymer
(Sifat
Pegun Candida antarctica Lipase B ke atas Kopolimer yang Sangat
Mikroporos)
Nurrahmi
Handayani*, Sadijah Achmad & Deana Wahyuningrum
Department of
Chemistry, Bandung Institute of Technology, Indonesia
Nemanja Miletic
& Katja Loos
Zernike
Institute for Advanced Materials, University of Groningen, Netherland
Diserahkan: 16
Jun 2010 / Diterima: 3 Januari 2011
ABSTRACT
In
spite of their excellent catalytic properties, enzymes should be improved
before their implementation both in industrial and laboratorium scales.
Immobilization of enzyme is one of the ways to improve their properties. Candida
antarctica lipase B (Cal-B) has been reported in numerous publications to be
a particularly useful enzyme catalizing in many type of reaction including
regio- and enantio- synthesis. For this case, cross-linking of immobilized
Cal-B with 1,2,7,8 diepoxy octane is one of methods that proved significantly
more stable from denaturation by heat, organic solvents, and proteolysis than
lyophilized powder or soluble enzymes. More over, the aim of this procedure is
to improve the activity and reusability of lipase. Enzyme kinetics test was carried
out by transesterification reaction between 4-nitrophenyl acetate (pNPA) and
methanol by varying substrate concentrations, and the result is immobilized
enzymes follows the Michaelis-Menten models and their activity is match with
previous experiment. Based on the Vmax values, the immobilized
enzymes showed higher activity than the free enzyme. Cross-linking of
immobilized lipase indicate that cross-linking by lower concentration of
cross-linker, FIC (immobilized lipase that was incubated for 24 h) gave
the highest activity and cross-linking by higher concentration of cross-linker, PIC (immobilized lipase that was incubated for 2 h) gives the highest
activity. However, pore size and saturation level influenced their activity.
Keywords: Candida antarctica lipase B (Cal-B); cross-linking; enzyme
immobilization
ABSTRAK
Walaupun
sifat katalitisis yang sangat baik, enzim perlu ditingkatkan keupayaannya
sebelum diimplementasikan dalam skala industri maupun makmal. Imobilisasi enzim
merupakan salah satu cara untuk memperbaiki sifat enzim. Candida
antarctica lipase B (Cal-B) telah dilaporkan dalam sejumlah penerbitan
sebagai enzim yang dapat digunakan dalam mengkatalisis enzim dalam berbagai
tindak balas termasuk sintesis yang bersifat regio- dan enantio-selektif. Dalam
kajian ini didapati taut-silang dalam Cal-B terimobilisasi oleh 1,2,7,8
diepoksi oktana merupakan salah satu kaedah yang terbukti secara signifikan
lebih stabil terhadap penyahaslian oleh pemanasan, pelarut organik, dan
proteolisis dibandingkan dengan enzim dalam bentuk larutan maupun serbuk
terliofilik. Selain itu, tujuan kajian ini adalah meningkatkan keaktifan dan
kebolehulangan pemakaian lipase. Ujian kinetik enzim dilakukan melalui tindak
balas transesterifikasi antara 4-nitrofenil asetat (pNPA) dan metanol dengan
memvariasikan kepekatan substrat, dan hasilnya adalah bahawa enzim
terimobilisasi mengikuti model Michaelis-Menten serta keaktifannya sesuai
dengan hasil kajian sebelumnya. Berdasarkan nilai Vmax,
enzim yang terimobilisasi menunjukkan keaktifan yang lebih tinggi daripada
enzim bebas. Taut-silang pada lipase yang terimobilisasi menunjukkan bahawa
taut-silang oleh adanya rangkaian silang FIC (lipase terimobilisasi yang
diinkubasi selama 24 jam) berkepekatan rendah menunjukkan keaktifan tertinggi,
sedangkan taut-silang oleh adanya rangkaian silang PIC (lipase
terimobilisasi yang diinkubasi selama 2 jam) berkepekatan lebih tinggi
menunjukkan keaktifan tertinggi. Di samping itu, saiz liang dan aras ketepuan
memberikan pengaruh terhadap keaktfian enzim tersebut.
Kata kunci: Candida antarctica lipase
B (Cal-B); imobilisasi enzim; taut-silang
RUJUKAN
Cao, L., van
Rantwijk, F. & Sheldon, R.A. 2000. Cross-linked enzymes aggregates: a
simple and effective method for the immobilization of Penicillin acylase. Organic
Letter 2(10): 1361-1364.
Govardhan, C.P.
1999. Crosslinking of enzymes for improved stability and performance. Current
Opinion in Biotechnology 10(4): 331-335.
Jaeger, K.E.
& Eggert, T. 2002. Lipase for biotechnology. Current Opinion on
Biotechnology 13: 390-397.
Kim, M.I., Kim,
J., Lee, J., Jia, H., Na, H.B., Youn, J.K., Kwak, J.H., Dohnalkova, A., Grate,
J.W., Wang, P., Hyeon, T., Park, H.G. & Chang, H.N. 2006. Crosslinked
enzymes aggregates in hierarchically-ordered mesoporous silica: a simple and
effective method for enzyme stabilization. Biotechnology and Bioengineering 96(2):
210-218.
Kobayashi, J.,
Mori, Y. & Kobayashi, S. 2006. Novel immobilization method of enzymes using
a hydrophilic polymer support. Chem. Commun. 4227-4229.
Mc Kee-Mc Kee.
2004. The Molecular Basis of Life. 3rh edition. New York: McGraw Hill. pp. 125-126.
Miletic, N.,
Rohandi, R., Vucovic, Z., Nastasovic, A. & Loos, K. 2009. Surface
modification of macroporous poly(glycidyl methacrylate-co- ethylene glycol
dimethacrylate) resuns for improved Candida antarctica lipase B immobilization. Reactive & Functional Polymers 69: 68-75.
Rohandi, R.
2007. Modified epoxy functionalized
macroporous resins for Candida antarctica lipase B immobilization. Thesis (Unpublished), Institut Teknologi Bandung 43-55.
Schoevaart, R.,
Wolbers, M.W., Golubovic, M., Ottens, M., Kieboom, A.P.G., van Rantwijk, F.,
van der Wielen, L.A.M. & Sheldon, R. 2004. Preparation, optimization, and
structures of cross-linked enzyme aggregates (CLEAs). Biotechnology and
Bioengineering 87(6): 755-762.
Sheldon, R.A.,
Schoevaart, R. & Van Langen, L.M. 2005. Cross-linked enzyme aggregates
(CLEAs): A novel and versatile method for enzyme immobilization (a review). Biocatalysis
and Biotransformation 23(3/4): 141-147.
Smith, P.K., Krohn,
R.I., Hermanson, T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto,
E.K., Goeke, N.M., Olson, B.J. & Klenk, C. 1985. Measurement of Protein
Using Bicinchoninic Acid. Analytical Biochemistry 150: 76-85.
Wilson, L.,
Illanes, A., Abia, O., Pessela, B.C.C., Ferna’ndez-Lafuente, R. & Guisa,
J.M. 2004. Co-aggregation of Penicillin G acylase and polyionic polymers: an
easy methodology to prepare enzyme biocatalysts in organic media. Biomacromolecule 5: 852-857.
*Pengarang untuk
surat-menyurat; email: ami_chemie2003@yahoo.com
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