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Using the versatility of glutaraldehyde chemistry to immobilize and stabilize different β-glucosidases: protocol optimization depends only on the enzyme

Authors:Roberto Fernandez-Lafuente
Groups of research:Optimization of biocatalysts and bioprocesses
Diandra de Andrades,1 Natália G. Graebin,1 Marina Kimiko Kadowaki2 Marco A. Z.
Ayub,1 Roberto Fernandez-Lafuente,3 Rafael C. Rodrigues1,*
1Biotechnology, Bioprocess, and Biocatalysis Group, Food Science and Technology
Institute, Federal University of Rio Grande do Sul, Av. Bento Gonçalves 9500, PO Box
15090, ZC 91501-970, Porto Alegre, RS, Brazil;
2Center of Medical Sciences and Pharmaceutical, Western Paraná State University, Rua
Universitária 2069, ZC 85819-110, Cascavel, PR, Brazil.
3Department of Biocatalysis, ICP-CSIC, Campus UAM-CSIC, Cantoblanco, ZC 28049,
Madrid, Spain
The β-glucosidases from Aspergillus niger and contained in Pectinex Ultra SP-L and Pectinex Ultra Clear, were
immobilized using different strategies: ionic adsorption on aminated (MANAE)-agarose beads at pH 5, 7, and 9,
followed by biocatalysts modification with glutaraldehyde, or on glutaraldehyde pre-activated supports. The pH of the
immobilization was altered to allow different enzyme molecule orientations on the support surface. The biocatalysts
from Pectinex Ultra SP-L showed the highest thermal and operational stabilities when immobilized on MANAEagarose-
glutaraldehyde at pH 7. The β-glucosidase from Pectinex Ultra Clear and from A. niger produced best results
when immobilized on MANAE-agarose beads at pH 5 and 7, respectively, which was later treated with glutaraldehyde.
The best immobilization results using pre-activated supports were observed for the enzyme present in Pectinex Ultra
SP-L, to which the highest thermal stabilities were obtained. Remarkably, the enzyme from A. niger, immobilized on
MANAE-agarose at pH 9 and subsequently treated with glutaraldehyde, produced the highest stabilization
(approximately 560 times more stable than soluble enzyme at 60 ºC). Results showed that optimal protocol for β-
glucosidases immobilizations using the glutaraldehyde chemistry must be individually tested and tailored to each type
of enzyme.
Keywords:MANAE-agarose; Glutaraldehyde; Enzyme stabilization; Ionic adsorption; Covalent attachment.
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