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TRYPSIN IMMOBILIZATION ON DERIVATIZED CELLULOSE BEADS BY BIOSPECIFIC AVIDIN‐BIOTIN INTERACTION AND CHARACTERIZATION OF THE IMMOBILIZED ACTIVITY 1
Author(s) -
JANOLINO VIOLETA G.,
SWAISGOOD HAROLD E.
Publication year - 2002
Publication title -
journal of food biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.507
H-Index - 47
eISSN - 1745-4514
pISSN - 0145-8884
DOI - 10.1111/j.1745-4514.2002.tb00869.x
Subject(s) - biotinylation , trypsin , chemistry , biotin , chromatography , avidin , cellulose , biochemistry , enzyme
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.