H. Zhao et al. / Journal of Molecular Catalysis B: Enzymatic 126 (2016) 18–23
19
Scheme 1. The generation process of microspheres.
use. Another group of researchers were trying to address this prob-
lem by using surface-modified calcium alginate to adsorb GI to get
much more stable recycle use, however, the stability range of pH
and temperature was narrow [4]. High enzyme activity could be
obtained only under the pH of 7.5 and temperature of 60 ◦C. More-
over, chitosan–polyacrylic acid hybrid microspheres were prepared
as the GI carriers to improve the stability of pH, temperature and
operation [16]. Although it was found that the immobilized GI
had better operation stability due to crosslinking after GI adsorp-
tion, the process was complicated and dependent on the presence
of ions with low enzyme activity. Therefore, it is necessary to
develop a simple approach to prepare immobilized GI with higher
enzyme activity and wider stability range against the environmen-
tal changes.
Silica/chitosan core–shell hybrid microspheres with good
mechanical intensity and strong interaction with metal ions were
prepared using microfluidic technology in our previous work [17].
The silica/chitosan supported catalyst can decrease mass-transfer
resistance effectively while increasing reaction velocity of the
bined with chitosan was located on the shell of microspheres.
Moreover, chitosan has outstanding properties of biocompatibil-
ity, adsorption and being environment-friendly, it is beneficial
for the exposure of active sites of enzyme and interaction with
enzyme strongly [18]. Hence, silica/chitosan core–shell hybrid
microspheres can be an ideal carrier candidate to immobilize GI.
Therefore, the immobilized GI was prepared simply by in situ
encapsulation of GI in the silica/chitosan hybrid mic rospheres in
this work. High yield was obtained in 10 min using immobilized GI
as catalyst and the stability range of pH, temperature and storage
was expanded to a large extent without dependence on metal ions.
Furthermore, the immobilized GI can be recycled for many times
with high catalytic activity.
as the solidification bath in which glutaraldehyde was served as
the cross-linking reagent. Glucose isomerase from Streptomyces
rubiginosus (Zhengzhou Zhongxin Chemical Reagent Co., Ltd.) and
d-glucose (Beijing Chemical Works) were used as purchased and
all of the reagents were analytically or chemically pure.
2.2. Preparation of silica/chitosan hybrid microspheres supported
GI
The preparation procedure of silica/chitosan hybrid micro-
spheres was described in detail in our previous work [17]. Dual
co-axial microfluidic device was used, as shown in Scheme 1.
2.0 g of glucose isomerase powders were dissolved in 100 mL of
deionized water and then centrifuged to get the supernatant as
GI solution. 1.80 g of aqueous solution with 2.0 wt.% chitosan and
2.0 wt.% acetic acid was mixed with 0.20 g GI solution, which was
used as middle fluid. Silica sol, the inner fluid, was obtained by
stirring the aqueous solution with 2.0 wt.% acetic acid and 2.0 wt.%
TEOS for 12 h at room temperature. They were dispersed into
droplets by the shearing force of continuous fluid consisting of n-
octane at the intersection of the microchannel. The droplets were
collected out of the microchannel in the solidification bath con-
sisting of n-octanol with 0.5 wt.% glutaraldehyde and 2.0 wt.% Span
80. The droplets were pre-solidified into microspheres through
the Schiff’s base reaction between CHO from glutaraldehyde and
NH2 from chitosan and GI and the extraction of water out of
droplets by octanol. The microspheres were pre-solidified for a cer-
tain time (15–45 min) in the solidification bath and then washed
with n-octane followed by a submersion for 24 h to gelate the sil-
ica sol. The silica/chitosan supported GI was then obtained after
freeze-drying.
The immobilized GI prepared by adsorption of GI onto the micro-
spheres were also prepared for comparison in this work. First,
silica/chitosan hybrid microspheres were prepared according to the
procedure mentioned above, with the exception that no GI solution
was added in the middle fluid. Then silica/chitosan hybrid micro-
spheres were incubated in the GI solution placed in a water-bath
shaker under 25 ◦C, 130 rpm for 24 h for GI adsorption. The micro-
spheres were then separated and washed with deionized water to
remove the remnant GI followed by immersion in glutaraldehyde
solution (0.2 wt.%) under 25 ◦C for 30 min enabling crosslink-
ing between chitosan and GI for further immobilization. Lastly,
the microspheres were washed with buffer solution to remove
the residue glutaraldehyde and stored in fridge under 4 ◦C after
freeze-drying.
2. Experimental
2.1. Materials and chemicals
Aqueous solution (10.0 g) of chitosan(0.20 g) with degree of
deacelation below 95% (Sinopharm Chemical Reagent Co., Ltd., Bei-
jing, PR China) dissolving in acetic acid (0.20 g, VAS Chemical Co.,
Ltd., Tianjin, PR China) was served as the middle fluid. Polymer
aqueous solution (10.0 g) with tetraethoxysilane (TEOS) (0.20 g)
dissolving in acetic acid (0.20 g) was used as the inner fluid whereas
n-octanol (VAS Chemical Co., Ltd., Tianjin, PR China) as the contin-
uous phase. n-Octane (10.0 g) with glutaraldehyde (0.040 g) and
Span 80 (0.20 g, VAS Chemical Co., Ltd., Tianjin, PR China) was used