Multipoint Immobilization of Invertase on Agarose: Stability and Kinetic Properties

Order of Publishing in Issue: 
12
Volume :2
Issue :3
July, 2008
Page No: 
462-470
Authors: 
Antonio José Goulart1, Ana Claudia Elias Pião Benedetti2, Olga Luisa Tavano1, Daniela Parreira Marques2, Jonas Contiero3, Eleonora Cano Carmona3 and Rubens Monti2*
Address: 
1Department of Biochemistry and Technological Chemistry, Institute of Chemistry – São Paulo State University –
Address: 
UNESP, Araraquara, SP, Brazil; 2Department of Food and Nutrition, School of Pharmaceuticals Sciences – São Paulo
Address: 
State University – UNESP, Araraquara, SP, Brazil; 3Department of Biochemistry and Microbiology – Institute of Biosciences – São Paulo State University – UNESP, Rio Claro, SP, Brazil

Enzyme immobilization is a specific method for restricting the enzyme freedom ofmovement. There are strategies for the proteins multipoint immobilization by amine-terminalresidues, lysine residues and carboxylic groups. In the present work, a commercial invertase ofSaccharomyces cerevisiae underwent multipoint immobilization on glyoxyl-agarose, amineagaroseand glutaraldehyde-agarose supports. Derivatives kinetic properties were determined and compared with the properties of the soluble enzyme. The copper influence on enzyme activityand its inhibition by fructose were also investigated. Amine-agarose exhibited activityclosest to the soluble enzyme (93.3%). This same derivative maintained approximately 50% ofinitial activity when 800mM of fructose were added to the reaction medium. However,glutaraldehyde-agarose exhibited the best stability to temperature and pH and none of thederivatives lost inhibition by copper. Glyoxyl derivative exhibited the lowest Km (0.023mM)and amine derivative achieved the highest maximum velocity (1666.7 U/mg prot.).

Keywords: 
enzyme immobilization and stabilization, invertase immobilization, multipoint immobilization
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