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Fe3O4-MSNs for the first and second cycles, the fructose yield
decreased from 47 to 6% after the fifth cycle. Such a gradual
decrease in the fructose yield in the case of enzyme-immobi-
lized Fe3O4-SSNs is indicative of a gradual loss of the enzyme
as the catalyst is recycled, and this could be due to the loose
adsorption of the enzyme onto the external surfaces of the
SSNs. Because the SSNs lack mesopores, a shielding effect for
enzyme immobilization would not be provided.
Synthesis of Fe3O4-loaded MSNs and Fe3O4-loaded nonpo-
rous silica nanoparticles
The Fe3O4-loaded MSNs were synthesized by a co-condensation
method as follows: Brij-97 (6.92 mL) was added to an aqueous so-
lution (180 mL) of magnetite with stirring at room temperature.
After complete dissolution of Brij-97, APTMS (0.3 mL) and DOP
(0.8 mL) were added to the mixture with stirring. After stirring for
30 min, TEOS (6.7 mL) was introduced, and the mixture was stirred
at room temperature for 1 d followed by heating at reflux at
1008C for another 24 h. Finally, the precipitate was collected by fil-
tration, washed with methanol several times to remove the surfac-
tant, and dried in a lyophilizer. The resulting sample was Fe3O4-
loaded MSNs.
In conclusion, we disclose a sequential enzymatic concept
for the direct conversion of cellulose into fructose in aqueous
solution. First, the reaction conditions of glucose isomerase
were optimized as 708C, 24 h, 3.3 mg (per 15 mg of glucose),
and phosphate buffer with pH 7.5 for the maximum produc-
tion of fructose. The enzyme was then immobilized successful-
ly into Fe3O4-loaded MSNs without losing its activity. We also
demonstrated that such enzyme-immobilized Fe3O4-loaded
MSNs could catalyze a continuous cellulose-to-glucose and
glucose-to-fructose conversion sequence and could achieve
a high fructose yield up to 50%, which is the same yield ob-
tained when using the free enzyme. In addition, the utilization
of Fe3O4-loaded MSNs as enzyme hosts provides both excellent
recyclability and stability. The results obtained in this study in-
dicate that enzyme-immobilized Fe3O4-loaded MSNs would be
effective, green, recyclable, and stable biocatalysts for various
enzymatic applications.
Fe3O4-loaded nonporous silica nanoparticles were synthesized by
using the same procedure but without the addition of the
surfactant.
Enzyme immobilization
For immobilization of cellulase, Fe3O4-loaded MSNs (50 mg) were
suspended in a citric buffer (10 mm, 2 mL, pH 4.8). The cellulase so-
lution (1 mL) was added to the citric buffer mixture, and the result-
ing mixture was stirred at 48C for 1 d.
For immobilization of isomerase, the same amount of Fe3O4-loaded
MSNs was suspended in a phosphate buffer (20 mm sodium phos-
phate/0.15m sodium chloride/5 mm magnesium sulfate, 2.5 mL,
pH 7.5). Then, the isomerase solution (0.5 mL) was added to the
phosphate buffer, and the mixture was stirred at 48C for 1 d.
Finally, the enzyme-immobilized Fe3O4-loaded MSNs were collected
with a magnet. The enzyme remaining in the supernatant was con-
sidered the nonadsorbed enzyme, and its amount was measured
by UV/Vis spectrometry at a wavelength of 280 nm. Therefore, the
amount of immobilized enzyme could be calculated from the initial
amount of the enzyme minus the amount remaining in the super-
natant. The final catalysts were washed with citric or phosphate
buffer several times and redispersed into citric or phosphate buffer
(1mL) for further use.
Experimental Section
Chemicals
Poly(oxyethylene) oleyl ether (Brij-97, C18H35EO10), ammonia hy-
droxide (37%), hydrochloride acid (37%), iron(II) chloride tetrahy-
drate, 3-aminopropyltrimethoxysilane (APTMS, 97%), dimethyl
phthalate (DOP, >99%), tetraethoxysilane (TEOS), ethanol (99.8%),
cellulase (Trichoderma reesei ATCC 26921), 1-butyl-3-methylimidazo-
lium chloride (BMIM), cellulose (powder, ca.20 micron), d-(+)-glu-
cose (>99.5%), d-(À)-fructose (>99%), sodium phosphate tribasic,
magnesium sulfate, and sodium chloride were purchased from
Sigma–Aldrich. Citric acid (anhydrous, powder), sodium hydroxide
(NaOH), and acetonitrile were purchased from J. T. Baker. Iron(III)
chloride hexahydrate (FeCl3·6H2O) was purchased from Alfa Aesar.
Methyl alcohol was purchased from Mallinckrodt Chemical. Glucose
isomerase (purified from Streptomyces rubiginosus) was purchased
from Hampton Research.
Characterization
The morphology of the Fe3O4-loaded MSNs was observed with
TEM. The porous properties were analyzed with nitrogen adsorp-
tion–desorption isotherms with a Micromeritics ASAP 2000 instru-
ment. The specific surface area and pore size were calculated by
using BET and BJH methods, respectively.
Pretreatment of cellulose with ionic liquids
Cellulose (50 mg) was added to [BMIM]Cl (0.95 mL), and the mix-
ture was stirred at 1208C for 1 h. Methanol (3 mL) was added to
the mixture to quench the reaction. Then, the resulting oligomer
cellulose was separated from the ionic liquid by centrifugation,
washed with methanol and water several times, and dried in a
lyophilizer.
Synthesis of magnetite (Fe3O4)
FeCl3 (hexahydrate, 1.349 g) and FeCl2 (tetrahydrate, 0.781 g) were
dissolved in deionized water (600 mL) with stirring. Then, ammonia
hydroxide (1.5m) was added to the iron-containing aqueous solu-
tion until the pH value of the solution increased to 9. The iron
oxides (i.e., Fe3O4) were then collected by magnetic force and
washed with deionized water and ethanol several times. The result
sample was redispersed to deionized water (600 mL) for further
use.
Enzymatic reactions
For cellulose-to-glucose conversion, pretreated cellulose (0.015 g)
was added to citric buffer (1 mL) containing free cellulase or cellu-
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ChemCatChem 2013, 5, 2153 – 2157 2156