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TRYPSIN IMMOBILIZATION ON DERIVATIZED CELLULOSE BEADS BY BIOSPECIFIC AVIDIN-BIOTIN INTERACTION AND CHARACTERIZATION OF THE IMMOBILIZED ACTIVITY1

Authors :
Harold E. Swaisgood
Violeta G. Janolino
Source :
Journal of Food Biochemistry. 26:119-129
Publication Year :
2007
Publisher :
Hindawi Limited, 2007.

Abstract

Trypsin was immobilized on cellulose beads using the biospecific and high affinity avidin-biotin interaction. Trypsin and cellulose beads were biotinylated with sulfosuccinimidyl-6-(biotinamido) hexanoate (NHS-LC-biotin). Avidin and biotinylated trypsin were sequentially adsorbed to the biotinylated cellulose beads. A similar procedure was carried out using controlled-pore glass (CPG) beads. The properties of the two trypsin bioreactors were examined and compared. The substrate used for the assay of trypsin activity was p-tosyl-L-arginine methyl ester and the extent of biotinylation of biotinylated trypsin and of immobilized biotin on cellulose beads and on CPG beads were determined using the 2-[4'-hydroxyazobenzene]benzoic acid dye-binding method. Biotinylated trypsin in solution retained about 82% of the specific activity of native trypsin. Cellulose beads contained 0.184 μmol/mL (1.15 μmol/g) biotin and CPG beads, 0.329 μmol/mL (0.987 μmol/g). After regeneration, the biotin contents became slightly lower, namely, 0.159 μmol/mL for cellulose beads and 0.315 μmol/mL for CPG beads. The specific activities of trypsin immobilized on cellulose beads and CPG beads were 32 U/mL (202 U/g) and 43 U/mL (130 U/g), respectively. These studies indicate that cellulose beads can be biotinylated for use as bioselective support. Cette etude utilise l'interaction avidine-biotine comme modele pour l'adsorption selective et l'immobilisation de proteines de fusion enzyme-streptavidine recombinantes pour la preparation de bioreacteur. L'application de cette technologie permet de realiser la purification et l'immobilisation dans une seule etape. Cette etude caracterise enfin la trypsine immobilisee par cette methode en comparaison avec celle immobilisee sur des billes de verre.

Details

ISSN :
01458884
Volume :
26
Database :
OpenAIRE
Journal :
Journal of Food Biochemistry
Accession number :
edsair.doi...........8c0016ab89357c4e07f89c2c4755f127
Full Text :
https://doi.org/10.1111/j.1745-4514.2002.tb00869.x