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Candida rugosa lipase was covalently immobilized on silanized controlled pore silica
previously activated with glutaraldehyde in the presence of nonenzymatic proteins.
This strategy is suggested to protect the enzyme from aggregation effects or
denaturation that occurs as a result of the presence of silane precursors used in the
formation of the silica matrix. The immobilization yield was evaluated as a function
of the lipase loading and the additive type (albumin and lecithin) using statistical
concepts. In agreement with the mathematical model, the maximum coupling yield
(32.2%) can be achieved working at high lipase loading (450 units‚g-1 support) using
albumin as an additive. In these conditions, the resulting immobilized lipase exhibits
high hydrolytic (153.2 U‚mg-1) and esterification (337.6 mmol‚g-1‚min) activities. The
enhanced activity of the final lipase derivative is the sum of the benefits of the
immobilization (that prevents enzyme aggregation) and the lipase coating by additives
that increases the accessibility of active sites to the substrate.
addition of nonenzymatic proteins during the immobili- Gonçalves, A. M.; Schucht, E.; Matthijs, G.; Aires Barros, M.
zation procedure was studied. The immobilized biocata- R.; Cabral, J. M. S.; Gil, M. H. Stability Studies of a
lysts were used in the hydrolysis of olive oil and synthesis Recombinant Cutinase Immobilized to Dextran and Deriva-
of butyl butyrate. The data were compared with those tized Silica Supports. Enzyme Microb, Technol. 1999, 24, 60-
obtained with the immobilized biocatalyst in the absence 66.
of additive under the same operational conditions. Reetz, M. T.; Zonta, A.; Simpelkamp, J. Efficient Immobilization
In aqueous media the catalytic activity of immobilized of Lipases by Entrapment in Hydrophobic Sol-Gel Materials.
CPS-albumin-lipase is greater than that observed for Biotechnol. Bioeng. 1996, 49, 527.
CPS-lecithin-lipase, probably as a result of the limitations Rocha, J. M. S.; Gil, M. H.; Garcia, F. A. P. Effects of Additives
imposed by the physical properties of lecithin. However, on the Activity of a Covalently Immobilised Lipase in Organic
Media. J. Biotechnol. 1998, 66, 61.
in nonaqueous media both additives enhanced the activ-
ity of the final lipase derivative. The improved lipase Rocha, J. M. S. Aplicações de Agentes Tensioactivos em Biotec-
activity was considered to be the sum of the benefits of nologia. Bol. Biotecnol. 2000, 5, 25.
the immobilization (which prevents enzyme aggregation) Soares, C. M. F.; de Castro, H. F.; Moraes, F. F.; Zanin, G. M.
and the enzyme coating with the additive, which in- Characterization and Utilization of Candida rugosa Lipase
Immobilized on Controlled Pore Silica. Appl. Biochem. Bio-
creases the accessibility of active sites to the substrate.
technol. 1990, 77, 745.
References and Notes Triantafyllou, A. O.; Wehtje, E.; Adlercreutz, P.; Mattiasson, B.
Effects of Sorbitol Addition on the Action of Free and
Bosley, J. A. Supported Enzymes. Patent Application EQP Immobilized Hydrolytic Enzymes in Organic Media. Biotech-
424130 A1, 1991. nol. Bioeng. 1995, 45, 406.
Bosley, J. A.; Moore, S. R. Immobilized Lipases. Patent Applica-
Wehtje, E.; Adlercreutz, P.; Mattiasson, B. Improved Activity
tion WO 94/28118, 1994.
Box, G. E. P.; Hunter, W. G.; Hunter, J. S. Statistics for Retention of Enzymes Deposited on Solid Supports. Biotech-
Experimenters: An Introduction to Design, Data Analysis and nol. Bioeng. 1993, 41, 171.
Model Building; Wiley & Sons Inc.: New York, 1978; p 653.
Bradford, M. M. A. Rapid and Sensitive Method for the Accepted for publication March 3, 2003.
Quantification of Microgram Quantities of Protein Utilizing
the Principle of Protein-Dye Binding. Anal. Biochem. 1976,
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