Tuesday, April 20, 2010
10-29

Immobilization of β-Glucosidase: Reversible adsorption versus multipoint covalent attachment

Marcelo F. Vieira1, Angélica M. S. Vieira1, Gisella M. Zanin1, Paulo W. Tardioli2, Raquel L. C. Giordano2, Cesar Mateo3, and Jose M. Guisán3. (1) Chemical Engineering Department, State University of Maringá, Maringá, PR, Brazil, (2) Chemical Engineering Department, Federal University of São Carlos, Via Washington Luiz, Km 235, Monjolinho, São Carlos, SP, Brazil, (3) Department of Biocatalysis, Institute of Catalysis, CSIC, Madrid, Spain

The bioconversion of lignocellulosic biomass to fermentable sugars for production of ethanol requires a multienzyme system named cellulase. This system contains enzymes that act synergistically in the hydrolysis of cellulose: endoglucanase, cellobiohydrolase and β-glucosidase. The first two enzymes act directly on cellulose, yielding mainly glucose and cellobiose, which is hydrolyzed into glucose by β-glucosidase. An industrial process would be more economical by using immobilized systems that allow the reuse of the enzyme and improve the enzyme stability against different inactivation agents. Particularly, the hydrolysis of cellobiose would be performed using immobilized enzyme because cellobiose molecules are soluble in the reaction medium. In this work, β-glucosidase was immobilized onto an agarose matrix derivatized with different reactive groups, e.g. polyethylenimine (PEI), glyoxyl (linear aliphatic aldehydes) and amine-epoxy, trying to optimize the stability and activity of the immobilized enzyme. Using reversible attachment (immobilization by anion exchange), the derivatives were active, but with poor thermal stability; e.g. PEI agarose derivative was 6 times more stable than the soluble β-glucosidase. However, these derivatives have important characteristics for an industrial process: the reuse of the enzyme and/or the application of continuous systems. Among the activated supports with irreversible attachment (covalent immobilization), glyoxyl agarose did not reach a good thermal stability; it seems that the enzyme surface is very poor in amino groups from lysine residues. Better results were obtained with amine-epoxy agarose supports. β-glucosidase immobilized on that support kept 80% of its activity and was 200-fold more stable than the soluble enzyme.

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