On The Immobilization of Candida antarctica Lipase B onto Surface Modified Porous Silica Gel Particles - Bio-Inspired Silicon-Based Materials - page 64

5.2.2 Enzyme Immobilization Protocol

The surface modification of the silica particles were performed by two different
methods—namely a wet method (aqueous solution) and a dry method (toluene)
[ 11 ]. In the dry method, about 1 g of porous silica gel particles were taken into
a glass vial and then kept in an oven at 150 °C under vacuum (500 mmHg) for
24 h. In the wet method, the silica gel particles were transferred into a glass vial
containing 10 ml of NaOH solution (pH ~ 10.6) and 100 µ l of 3-APS and kept for
15 min. While, in the dry method, the silica gel particles were transferred into a
glass vial containing 10 ml of toluene and 100 µ l of 3-APS and kept for 15 min.
Then the solutions from these glass vials were removed carefully with a micro-
pipette and the particles were washed with 5 ml of ethanol (twice) correspond-
ing to each of the vials. These two silica samples were labeled as wet silica and
dry silica, respectively and the subsequent treatment and enzyme immobilization
procedure followed was identical for both the methods. The surface coated silica
gel particles with 3-APS were then cured at 150 °C for 15 min. These particles
were dried in a vacuum chamber (400 mmHg) overnight. The surface modified
silica gel particles were then used for the immobilization of CALB as described
below.
The enzyme solution was prepared by dissolving 10 mg of CALB in 1 ml of
Tris buffer solution (pH = 7.5) taken in a glass vial. About 15.6 µl of glutaraldehyde
(conc. 24 %) was added to the glass vial and mixed for 2 min [ 19 ]. The solution
was transferred to a glass vial containing 100 g of 3-APS coated silica gel par-
ticles (wet-silica or dry-silica) and then the glass vial was placed on a shaker at
room temperature overnight. The remaining solution was carefully removed with
a micropipette. The silica gel particles were washed (five times) with 1 ml of Tris
buffer solution (pH = 7.5) and the washed solution was collected in five different
glass vials for the analysis of protein concentration using the Bradford assay [ 27 ].
The CALB immobilized on the surface modified silica gel particles were then dried
at room temperature in a vacuum chamber (400 mmHg) for 48 h and then stored
in a refrigerator at 5 °C (± 1 °C) for future use. For bioinspired silica immobilised
CALB, a literature method was adopted [ 23 ] except the amines used (PEI and PAH)
and their concentrations were altered.

5.2.3 Enzyme Activity Assay

The enzyme activity was determined by the esterification of 20 mg of 1-octanol and
lauric acid (1:1 mol ratio) catalyzed by 10 mg of the fresh or recovered lipase in
1 ml of isooctane at 37 °C for 1 h. The amount of product octyl laurate formed was
analyzed by gas chromatography (GC).