Biochemical composite synthesized by stepwise crosslinking: An efficient platform for one-pot biomass conversion

Article abstract of DOI:10.1016/j.jcat.2015.04.021

This paper describes the development of a new bifunctional catalyst system that integrates enzyme and chemical material into a biochemical composite through a stepwise crosslinking approach. The as-prepared biochemical composite not only allows one-pot biomass conversion via sequential enzyme-catalyzed hydrolysis of biomass materials to glucose and metal-catalyzed hydrogenation of glucose to sorbitol, but also enables reusability of the catalyst. This design concept facilitates access to fuels and chemicals from the biomass-derived sorbitol and will attract more attention in the foreseeable future.

Full text of DOI:10.1016/j.jcat.2015.04.021

Journal of Catalysis 327 (2015) 78–85
Contents lists available at ScienceDirect
Journal of Catalysis
journal homepage: www.elsevier.com/locate/jcat
Biochemical composite synthesized by stepwise crosslinking: An
efficient platform for one-pot biomass conversion
a
a
a
a
a
a
a
Wei Wei , Cong Wang , Yu Zhao , Shichao Peng , Haoyang Zhang , Yipeng Bian , Hexing Li ,
b
a,
⇑
Xinggui Zhou , Hui Li
a
The Education Ministry Key Lab of Resource Chemistry and Shanghai Key Laboratory of Rare Earth Functional Materials, Shanghai Normal University, Shanghai 200234, PR China
State Key Laboratory of Chemical Engineering, East China University of Science and Technology, Shanghai 200237, PR China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
This paper describes the development of a new bifunctional catalyst system that integrates enzyme and
chemical material into a biochemical composite through a stepwise crosslinking approach. The
as-prepared biochemical composite not only allows ‘‘one-pot’’ biomass conversion via sequential
enzyme-catalyzed hydrolysis of biomass materials to glucose and metal-catalyzed hydrogenation of
glucose to sorbitol, but also enables reusability of the catalyst. This design concept facilitates access to
fuels and chemicals from the biomass-derived sorbitol and will attract more attention in the foreseeable
future.
Received 16 March 2015
Revised 18 April 2015
Accepted 20 April 2015
Keywords:
Biochemical composite
Yolk–shell nanostructure
Metallic catalysis
Ó 2015 Elsevier Inc. All rights reserved.
Enzymatic catalysis
Biomass conversion
1
. Introduction
configuration that provides protecting effect on the individual core
[
21], very recently, we designed yolk–shell nanoarchitectures con-
The production of fuels and chemicals from plentiful and
sisting of cores made of supported Ru encapsulated within porous
silica shells [22,23]. By combining such materials with amyloglu-
cosidase, one-pot hydrolysis–hydrogenation of dextrin has been
successfully conducted to produce sorbitol where the porous silica
shell separates the incompatible catalysts in different regions.
Specifically, the enzymatic hydrolysis of dextrin to glucose occurs
outside the yolk–shell nanoarchitectures owing to the blocking
effect of the silica shells on the large enzyme molecules.
Meanwhile, the permeation-selective porous silica shells offer a
convenient path for the produced small glucose molecules crossing
into the catalytically active cores for hydrogenation to sorbitol.
While promising, the present process still uses free enzyme which
decreases the economical attractiveness owing to the difficulty
associated with the reusability of enzyme and the protein contam-
ination of the final product. Therefore, immobilization of enzyme
directly onto the outer surface of shell is needed to ensure the
achievement of a real merging of such yolk–shell nanostructures
and enzyme.
renewable biomass resources has drawn immense attention in
recent years [1–5]. Sorbitol, a hexitol, is a valuable platform chem-
ical that can be converted by straightforward methods into a vari-
ety of useful products [6]. Nowadays, almost all extant sorbitol
production processes are usually based on the hydrogenation of
glucose catalyzed by metallic catalysts [7–13]. In fact, glucose
can be facilely obtained from biomass materials, primarily starch
[
14–16] and even cellulose, [17] through enzymatic hydrolysis bio-
processes. Apparently, a combined, one-pot hydrolysis–hydrogena
tion of biomass materials to sorbitol displays some advantages in
its step-saving and low costs mainly linked to both the separation
and the refining procedures [18–20]. Nevertheless, our previous
studies revealed the one-pot process contains incompatible param-
eters. More specifically, enzymes are easily poisoned when con-
tacting with metal catalysts, while metallic active sites would be
covered by enzymes and the colloidal substances originated from
hydrolysis of biomass materials, leading to a rapid deactivation
for the subsequent glucose hydrogenation. Noting that encaging
a functional material within another material can form a yolk–shell
With advances in material science, a number of techniques have
been developed for enzyme immobilization, such as support
binding (physical binding, ionic binding, or covalent binding),
entrapment, and crosslinking [24]. To enhance the operational
stability and reusability of amyloglucosidases for bioprocessing,
⇑
Corresponding author. Fax: +86 21 64322272.
E-mail address: lihui@shnu.edu.cn (H. Li).
http://dx.doi.org/10.1016/j.jcat.2015.04.021
021-9517/Ó 2015 Elsevier Inc. All rights reserved.
0

W. Wei et al. / Journal of Catalysis 327 (2015) 78–85
79
they have been immobilized via various methods by far. Using
2. Experimental section
polyethyleneimine-coated sepabeads as supports, Torres et al.
successfully immobilized glucoamylase via ionic adsorption [25].
In comparison with ionic binding, covalent binding of enzyme
to insoluble carriers is even stronger. In 2008, Kamal et al.
reported the covalently immobilization of glucoamylase on
polypropylene-grafted fibers by using carbodiimide as a coupling
agent [26]. By using glutaraldehyde [27] or polyglutaraldehyde
2.1. Catalyst preparation
The synthesis of biochemical composite involves the fabrication
of yolk–shell structured chemical catalyst (Scheme 1A) and the
integrating of the above material and enzyme (Scheme 1B).
Firstly, uniform dispersing of Ru–B amorphous alloys within the
porous channels of amino-functionalized mesoporous carbon
nanospheres (af-mCarbon) was achieved by ultrasound-assisted
incipient wetness infiltration of (NH ) RuCl onto af-mCarbon, fol-
lowed by reduction with borohydride (Ru–B/af-mCarbon) [31].
Afterward, the Ru–B/af-mCarbon was coated by co-condensation
of tetraethoxysilane (TEOS) and N-(amino-ethyl)-amino-propyl tri-
methoxy silane (APTES) in the presence of cetyltrimethylammo-
nium bromide (CTAB), generating
Ru–B/af-mCarbon@CTAB/af-SiO , where CTAB/af-SiO2 refers to a
mesostructured CTAB/silica composite coated on the surface of
the Ru–B/af-mCarbon core. Finally, the as-synthesized core–shell
structured Ru–B/af-mCarbon@CTAB/af-SiO2 was etched with hot
water to achieve a yolk–shell structured configuration (Ru–B/af-
[
28] as coupling agent, amyloglucosidase was attached to silanized
magnetic nanoparticles or gelatin. Compared to other coupling
agents, glutaraldehyde-based coupling reaction requires mild con-
ditions. In those reports, immobilized amyloglucosidases were
found to be more beneficial relative to the corresponding free
enzymes. Despite the well-known advantages for enzyme immobi-
lization, the immobilization of amyloglucosidase has not been per-
formed frequently in industrial because the macromolecular
enzyme loading is still a tough issue. Crosslinking technique has
proved to be a promising approach for carrier-free immobilization
of enzyme, which permits multipoint attachment through inter-
molecular crosslinking between enzyme molecules [29]. Based on
this technique, Talekar et al. recently developed a combi-CLEAs
strategy to prepare carrier-free co-immobilization of macromolec-
ular enzymes, glucoamylase, and pullulanase [30]. In our research,
we sought to address these concerns by both covalently attaching of
yolk–shell nanostructures onto amyloglucosidase and ensuring an
insoluble and robust biochemical catalyst.
4
2
6
a core–shell structured
2
mCarbon@air@af-mSiO ). The integrating of the yolk–shell struc-
2
tured Ru–B/af-mCarbon@air@af-mSiO2 and enzyme was con-
ducted through
a stepwise crosslinking method, including
covalent attachment of chemical catalyst onto enzyme through a
glutaraldehyde-based crosslinking technique (Ru–B/af-mCarbon@
Herein, we design a recyclable bifunctional biochemical com-
posite. The synthesis of such composite is achieved through a step-
wise crosslinking method that involves the covalent attachment of
yolk–shell structured chemical catalyst onto amyloglucoamylase
with glutaraldehyde and the subsequent coupling of the composite
in the presence of modified dextran. The biochemical composite
enables the efficient synthesis of sorbitol in one pot from dextrin,
cellobiose, and even cellulose. More importantly, the biochemical
composite could be used repetitively many times, showing a good
potential in industrial applications.
air@af-mSiO –A-I) and coupling of the obtained crosslinked com-
posite with modified dextran (Ru–B/af-mCarbon@air@af-mSiO2–
A-II). More details about the catalyst preparation can be found in
the Supporting Information.
2
2.2. Catalyst characterization
Fourier transform infrared (FTIR) spectra were obtained using a
Thermo Nicolet Magna 550 spectrometer. The bulk composition
2
Scheme 1. Illustration of the synthesis process of (A) yolk–shell structured Ru–B/af-mCarbon@air@af-mSiO and (B) biochemical composite through stepwise cross-linking
technique.

8
0
W. Wei et al. / Journal of Catalysis 327 (2015) 78–85
a
d
b
c
e
Fig. 1. TEM images of (a) af-mCarbon, (b) Ru–B/af-mCarbon, (c) Ru–B/af-mCarbon@CTAB/af-SiO
2 2
, and (d) Ru–B/af-mCarbon@air@af-mSiO . (e) FESEM image of Ru–B/af-
mCarbon@air@af-mSiO
2
. The inset in (e) is a partially crushed sphere.
a
b
2
0
30
40
50
60
70
80
20
30
40
50
60
70
80
2
θ (degree)
2θ(degree)
c
d
Ru 3d
B 1s
Ru 3d
B 1s
2
84.2
284.5
Ru 3d
Ru 3d
3/2
3
/2
1
88.1
188.1
1
90.3
190.5
2
80.0
280.0
Ru 3d
5/2
Ru 3d
5
/2
3
00
290
280
195
190
185
300
290
280
195
190
185
Binding energy (eV)
Binding energy (eV)
2
Fig. 2. Wide-angle XRD patterns of (a) Ru–B/af-mCarbon and (b) Ru–B/af-mCarbon@air@af-mSiO . XPS spectra of (c) Ru–B/af-mCarbon and (d) Ru–B/af-mCarbon@air@af-
mSiO . The insets in (a) and (b) are the SAED images of the Ru–B particles.
2
and Ru loading were analyzed by means of inductively coupled
plasma optical emission spectrometry (ICP-OES; Varian
VISTA-MPX). Enzyme loading was determined by bicinchoninic
acid (BCA) assay. The amorphous structure was investigated by
Halenda model. The surface electronic states were determined by
X-ray photoelectron spectroscopy (XPS; ULVAC-PHI PHI5000
VersaProbe system using Al Ka radiation).
both X-ray diffraction (XRD; Rigaku D/Max-RB with Cu K
a radia-
2.3. Catalytic performances test
tion) and selective-area electronic diffraction (SAED; JEOL
JEM2100). The material shapes and morphologies were observed
by both field emission scanning electron microscopy (FESEM;
HITACHI S-4800) and transmission electron microscopy (TEM,
In a typical experiment, the one-pot hydrolysis–hydrogenation
of dextrin to sorbitol was carried out in a Parr 4848 autoclave con-
taining Ru–B/af-mCarbon@air@af-mSiO –A-II (6.5 mg Ru and
2
JEOL JEM2100).
obtained at 77 K using a Micromeritics TriStar II apparatus. By N
adsorption, the Brunauer–Emmett–Teller (BET) surface area (SBET
was calculated by using the multiple-point BET method in the
relative pressure range of P/P = 0.05–0.2. The pore volume and
pore size distribution curve were obtained by the Barrett–Joyner–
N
2
adsorption–desorption isotherms were
25.9 mg amyloglucosidase), 0.25 g of dextrin, 25 mL of water, and
2
4.0 MPa of H at 333 K. The reaction system was stirred vigorously
2
)
(800 rpm) to eliminate the diffusion effect. The reaction mixture
was sampled at intervals for product analysis on a liquid-phase
chromatograph (Agilent 1200) equipped with a carbohydrate
column (Shodex, SC1011) and a refractive index detector at
0

W. Wei et al. / Journal of Catalysis 327 (2015) 78–85
81
a
b
c
d
amyloglucosidas
Fig. 3. FESEM images of (a) amyloglucosidase, (b) Ru–B/af-mCarbon@air@af-mSiO
mCarbon@air@af-mSiO –A-II.
2
–A-I, and (c) Ru–B/af-mCarbon@air@af-mSiO
2
–A-II. (d) TEM image of Ru–B/af-
2
3
33 K with water as the movable phase at 0.6 mL/min. After the
mixture cooled to room temperature at the end of the reaction,
the biochemical composite catalyst was separated by centrifuga-
tion and washed with deionized water for further characterization
and applications. To test the catalyst durability, the used Ru–B/a
1235
f-mCarbon@air@af-mSiO
washed thoroughly with deionized water after each run of the
reaction. Then, the Ru–B/af-mCarbon@air@af-mSiO –A-II was
2
–A-II catalyst was centrifuged and
2
reused with a fresh charge of dextrin for subsequent recycle run
under the same reaction conditions. The supernatant was collected
for BCA assay.
1
557
407
1
1657
3
. Results and discussion
4
000
3500
3000
2500
2000
1500
1000
500
-
1
3.1. Catalyst characterization
Wavenumber (cm )
2
Fig. 4. FTIR spectrum of Ru–B/af-mCarbon@air@af-mSiO –A-II.
As shown in TEM image (Fig. 1a), the as-synthesized
af-mCarbon is present in the form of uniform spheres with an aver-
age diameter of ꢀ550 nm, which is similar to the pure mCarbon
reported recently [23]. This demonstrates that the resulting
amino-functionalized mCarbon preserves the characteristic spher-
ical morphology of mCarbon. Meanwhile, the TEM image reveals
that these nanospheres contain highly ordered mesoporous chan-
nels similar to the pure mCarbon [23]. The pore size is roughly esti-
80
60
40
20
0
Amyloglucosidase
Amyloglucosidase + af-mCarbon
Amyloglucosidase + Ru-B/af-mCarbon
mated to be ꢀ2.5 nm. N
2
physisorption experiment for the
as-synthesized af-mCarbon further confirms the ordered meso-
Amyloglucosidase + Ru-B/af-mCarbon@air@af-mSiO2
Ru-B/af-mCarbon@air@af-mSiO -A-II
porous structure centered approximate 2.6 nm with high SBET of
2
2
À1
9
31 m g (Fig. S1). The successful incorporation of amino groups
into the network of mCarbon was demonstrated by FTIR character-
ization. The FTIR spectra (Fig. S2) reveal that, besides those absor-
bance bands observed in the pure mCarbon, the acid-treated
mCarbon displays additional absorbance bands at 1728 and
0
1
2
3
4
5
À1
1
222 cm , corresponding to the stretching vibration of C@O bond
Reaction time (h)
and the bending vibration of O–H bond from the grafted –COOH
groups on mCarbon [32]. Grafting TETA on the acid-treated
mCarbon resulted in the remarkable decrease of O–H bending
Fig. 5. Dextrin hydrolysis in different catalyst systems. Reaction conditions: dextrin
0.25 g), amyloglucosidase (20 L), a catalyst (containing 6.5 mg Ru), water (25 mL),
T = 333 K, PH2 = 4.0 MPa, stirring rate = 800 rpm.
(
l
vibration and
a blueshift of C@O stretching vibration to

8
2
W. Wei et al. / Journal of Catalysis 327 (2015) 78–85
À1
1
662 cm , indicating the formation of the amide bond [32].
interaction between Ru and B in the Ru–B alloys makes Ru electron
enriched. The higher electron density on Ru active sites might facili-
tate the formation of H species, which would be anticipated to
Additionally, other features should be given attention are 3041,
930, 2849, 1453, and 1112 cm
À1
À
2
due to N–H stretching, C–H
asymmetric stretching, C–H symmetric stretching, N–H bending,
and C–N stretching, respectively [33]. CO temperature-program
med-desorption (CO -TPD) was also used to confirm the incorpora-
tion of amino group into the surface of mCarbon. In case of the pure
mCarbon, no CO uptake was found for the used adsorption condi-
tions (Fig. S3a); while the af-mCarbon shows pronounced CO des-
orption peaks (Fig. S3b). All of these features indicate that the TETA
have been grafted onto mCarbon successfully. Fig. 1b demonstrates
that the ordered mesostructure of af-mCarbon can be well main-
tained after depositing Ru–B nanoparticles (NPs). Meanwhile, it
can be observed that the Ru–B NPs are uniformly dispersed into
the pore channels. The Ru loading was determined as 2.2 wt% by
ICP analysis. From Fig. 1c, one can see that the Ru–B/af-mCarbon
core is completely coated by silica shell with a thickness around
increase glucose hydrogenation activity [33,38].
The integrating of the yolk–shell structured Ru–B/af-mCarbon@
2
air@af-mSiO and amyloglucosidase was conducted through a
stepwise crosslinking method. As shown in Fig. 3a, the free amy-
loglucosidase has tree-like appearance showing pinnatisect. The
width of the segments can be determined as 200–300 nm.
Glutaraldehyde-based crosslinking technique has proven one of
the most facile methods to immobilize an enzyme on functional-
ized support. Therefore, glutaraldehyde was first used as crosslin-
2
2
2
2
ker to covalently attach Ru–B/af-mCarbon@air@af-mSiO
amyloglucoamylase. The resulting Ru–B/af-mCarbon@air@af-mSi
–A-I preserves the characteristic tree-like shape of the free amy-
loglucoamylase and Ru–B/af-mCarbon@air@af-mSiO particles can
2
onto
O
2
2
be found to hang on the segments of amyloglucosidase, presenting
decorated tree-like structure (Fig. 3b). We presume that the
1
00 nm. To protect the amino functionality attached on the mate-
rial, hot water (363 K) was used as etching agent for the generating
of yolk–shell nanostructures in the present research. Fig. 1d reveals
that, after being etched with hot water, the thickness of silica shell
decreased about 20 nm, together with the formation of a space
around 20 nm between the silica shell and the Ru–B/af-mCarbon
core. From the FESEM image (Fig. 1e), the average diameter of
2
crosslinking process to form Ru–B/af-mCarbon@air@af-mSiO –A-I
was due to the interaction of glutaraldehyde with both the amino
functionalities on the surface of chemical catalyst and the amino
group residues in the enzyme. To further enhance the insolubility
and robustness of the biochemical composite, additional coupling
of Ru–B/af-mCarbon@air@af-mSiO
using the modified dextran as crosslinker. Under SEM, Ru–B/af-m
Carbon@air@af-mSiO –A-II appears as aggregates (Fig. 3c), sugges-
2
–A-I was implemented by
2
the as-prepared Ru–B/af-mCarbon@air@af-mSiO was estimated
to be ꢀ550 nm, which was in good line with the TEM observation.
2
The attached FESEM image of broken Ru–B/af-mCarbon@air@af-
tive of the tying of the decorated tree-like composite with the
modified dextran. From the TEM image of Ru–B/af-mCarbon@ai
2
mSiO further confirms the achievement of yolk–shell structured
configuration (inset in Fig. 1e). From the high-magnification TEM
image of the yolk–shell structures in Fig. 1d, continuous
mesochannels throughout the shell with openings at surface and
radially oriented to the sphere surface can be clearly observed
for the silica shell. Such a unique pore orientation is due to the per-
pendicular alignment of surfactant mesophases induced by the
equal attractivity to polar and nonpolar species of the interface
between the CTAB/silica phase and the water/ethanol solution
r@af-mSiO
rbon@air@af-mSiO
stepwise crosslinking was never associated with damage to the
structure of chemical catalyst. Because amyloglucoamylase is
extremely sensitive to the high-energy electron beam in TEM anal-
ysis, only a trail of devastation was left beside the yolk–shell struc-
tured Ru–B/af-mCarbon@air@af-mSiO
Furthermore, the formation of biochemical composite can be fur-
ther confirmed by the FTIR spectrum. As shown in Fig. 4, Ru–B/a
2
–A-II (Fig. 3d), the yolk–shell structured Ru–B/af-mCa
can be also observed, demonstrating that the
2
2
(marked with arrow).
[
34–36]. The perpendicular mesoporosity in the silica shell is antic-
ipated to increase the accessibility of the Ru–B/af-mCarbon core
and thus enhancing the efficiency of mass transport. The pore size
in the silica shell can be measured to be 2.6 nm by nitrogen
physisorption experiment (Fig. S4).
f-mCarbon@air@af-mSiO
bands at 1657, 1557, 1407, and 1235 cm , which are ascribed to
the functional groups of amino acid in amyloglucoamylase. It
should be noted that the C@N vibration peaks (1600–1650 cm
2
–A-II displayed additional absorbance
À1
À1
)
The wide-angle XRD patterns (Fig. 2a and b) reveal that the
Ru–B NPs in both the Ru–B/af-mCarbon and the Ru–B/af-mCarbon@
as the formation of Schiff’s base are covered by the characteristic
peaks from amino acid groups.
air@af-mSiO
2
are present in the typical amorphous structure, corre-
More importantly, the biochemical composite, denoted as
sponding to a broad peak at ꢀ2h = 45° [37,38], which is further con-
firmed by the consecutive diffraction halos in the attached SAED
pictures [39]. The XPS spectra (Fig. 2c and d) demonstrate that all
the Ru species in both the Ru–B/af-mCarbon and the yolk–shell struc-
Ru–B/af-mCarbon@air@af-mSiO
the yolk–shell structured Ru–B/af-mCarbon@air@af-mSiO
cellulase by the same method, demonstrating the generality of this
stepwise crosslinking strategy.
2
–C-II, can be also achieved from
and
2
2
tured Ru–B/af-mCarbon@air@af-mSiO are present in metallic state,
corresponding to the binding energy (BE) of 280.0 eV in Ru 3d5/2
,
3.2. Catalytic performances
while the B species are present in both the elemental state and the
oxidized B, with BE of 188.1 and 190.5 eV in B 1s level. The B 1s BE
of the elemental B exceeds that of pure B by 1.0 eV [40], suggesting
the formation of Ru–B alloy in which partial electrons transfer from
B to Ru. The failure in observing the BE shift of the metallic Ru can
be understood by considering its relatively greater atomic weight
compared with the B atom. As a result, the XRD, SAED data coupled
with that of XPS, confirmed the formation of Ru–B amorphous alloy.
Plenty of studies had demonstrated that Ru–B amorphous alloy has
enhanced catalytic activity relative to the monometallic Ru in many
reactions, including the hydrogenation of glucose to sorbitol [33].
On one hand, the unique amorphous alloy structure of Ru–B endows
them with a stronger synergistic effect between Ru active sites and
more highly unsaturated Ru active sites than the monometallic cata-
lyst, which may promote the adsorption of reactants and favor hydro-
genation activity [33,38]. On the other hand, the strong electronic
The as-prepared biochemical composites were subjected to
one-pot production of sorbitol via hydrolysis–hydrogenation of
biomass materials. We began by exploring the enzymatic efficiency
of amyloglucosidase for saccharification of dextrin in different cat-
alyst systems (Fig. 5). Note that blank run performed using amy-
loglucosidase accompanying with af-mCarbon delivers similar
enzymatic efficiency to that of the free amyloglucosidase.
Nonetheless, significant inhibiting effects on the dextrin hydrolysis
activity can be observed when using amyloglucosidase in the pres-
ence of Ru–B/af-mCarbon. This implies that amyloglucosidase is
easily poisoned once directly contacting with metallic Ru, in line
with the results reported in our recent studies [22,23]. Hardly,
any difference in the enzymatic efficiency can be found using amy-
loglucosidase accompanying with the yolk–shell structured Ru–B
2
/af-mCarbon@air@af-mSiO , apparently owing to the protective

W. Wei et al. / Journal of Catalysis 327 (2015) 78–85
83
Ru-B/af-mCarbon@air@af-mSiO -A-I
2
100
Dextrin
Glucose
Sorbitol
Ru-B/af-mCarbon@air@af-mSiO -A-II
2
8
6
4
2
0
0
0
0
0
8
6
0
0
40
2
0
0
1
2
3
4
5
6
0
1
2
3
4
5
Recycling number
Reaction time (h)
Fig. 6. Recycling test of the biochemical composites for dextrin hydrolysis. Reaction
conditions: dextrin (0.25 g), biochemical composite (containing 6.5 mg Ru and
Fig. 7. Reaction profile in one-pot hydrolysis–hydrogenation of dextrin by Ru–B/af-
mCarbon@air@af-mSiO –A-II. Reaction conditions: dextrin (0.25 g), 0.9 g of bio-
2
2
5.9 mg amyloglucosidase), water (25 mL), T = 333 K,
PH2 = 4.0 MPa, stirring
chemical composite (containing 6.5 mg Ru and 25.9 mg amyloglucosidase), water
rate = 800 rpm. Each run was conducted for 5 h in recycling test. Each run was
conducted for 6 h in recycling test.
(25 mL), T = 333 K, PH2 = 4.0 MPa, stirring rate = 800 rpm.
Conversion of dextrin
Yield of sorbitol
2
effect of the af-mSiO shell that prevents amyloglucosidase from
crossing over the shell to contact with the Ru-containing core.
Furthermore, almost entire retention of the enzymatic efficiency
for saccharification of dextrin can be observed on Ru–B/af-mCarb
80
2
on@air@af-mSiO –A-II, compared to the free amyloglucosidase.
6
4
0
0
This result indicates that the present stepwise crosslinking method
is promising and the resulting biochemical composite is antici-
pated to catalyze the one-pot biomass conversion.
Industrial application of an enzyme in an immobilized form
greatly relies on its stability and handling convenience [24]. Next,
we first investigated the durability of the biochemical composites
during the hydrolysis of dextrin. Although Ru–B/af-mCarbon@
air@af-mSiO –A-I retained complete activity of the free enzyme,
2
its stability was rather undesirable since apparent deactivation
occurred during the recycling test (Fig. 6). During the hydrolysis
20
0
1
2
3
4
5
6
Recycling number
2
of dextrin catalyzed by Ru–B/af-mCarbon@air@af-mSiO –A-I, we
2
Fig. 8. Recycling test of Ru–B/af-mCarbon@air@af-mSiO –A-II for one-pot hydrol-
also determined the leaching amounts of amyloglucosidase by the
BCA assay in the supernatants. The analysis results revealed that
more than 11% and 75% of the applied enzyme leached out after
the first cycle and the six cycle, respectively, suggesting that Ru–B
ysis–hydrogenation of dextrin. Reaction conditions: dextrin (0.25 g), 0.9 g of
biochemical composite (containing 6.5 mg Ru and 25.9 mg amyloglucosidase),
water (25 mL), T = 333 K, PH2 = 4.0 MPa, and stirring rate = 800 rpm. Each run was
conducted for 5 h in recycling test.
/
af-mCarbon@air@af-mSiO
present conditions. In an effort to favor the stability of biochemical
composite, Ru–B/af-mCarbon@air@af-mSiO –A-I was further
coupled with modified dextran to afford Ru–B/af-mCarbon@air@
af-mSiO –A-II. As shown in Fig. 6, the further coupled biochemical
2
–A-I was soluble in water under the
saccharification of dextrin to release glucose molecules rapidly.
2
The produced glucose would diffuse through the af-mSiO
2
shell
in Ru–B/af-mCarbon@air@af-mSiO , followed by the hydrogena-
2
2
tion to the final product, sorbitol, over the catalytically active cores.
Our observations suggest that the mesoporous silica shell in
composite exhibited substantially enhanced stability under the
present reaction conditions. On the basis of these observations,
we can deduce that additional crosslinking of the biochemical
composites would tie up them together and thus rendering them
permanently insoluble and effectively preventing the leaching of
enzyme while maintaining the enzymatic efficiency.
The as-prepared insoluble and robust Ru–B/af-mCarbon@air@
2
af-mSiO –A-II was then subjected to one-pot conversion of dextrin
to sorbitol (Scheme 2). As shown in Fig. 7, the enzyme catalyzed
Ru–B/af-mCarbon@air@af-mSiO
the one-pot dextrin conversion. On one hand, af-mSiO
the big dextrin molecules and other colloidal substances
100–1000 nm) stemmed from the dextrin hydrolysis out of the
yolk–shell nanostructures, efficiently protecting the Ru active sites
from being covered. On the other hand, af-mSiO shell allows the
2
plays a key role in conducting
2
shell blocks
(
2
produced glucose (ꢀ1 nm) from dextrin hydrolysis diffuse across
OH
OH
OH
OH
H O
O
O H
O
O
Enzyme
OH
O
HO
H2
Ru-B
HO
HO
O
HO
OH
OH
OH
OH
OH
O H
Dextrin
Glucose
Sorbitol
Scheme 2. One-pot hydrolysis–hydrogenation of dextrin to sorbitol by merger of enzymatic and metallic catalysis.

8
4
W. Wei et al. / Journal of Catalysis 327 (2015) 78–85
a
b
1
00 nm
2
Fig. 9. (a) FESEM and (b) TEM images of the Ru–B/af-mCarbon@air@af-mSiO –A-II after being reused for 6 times.
OH
OH
(333 K vs. 348 K [22] and 343 K [23]). Additionally, only a shorter
time (5 h) in the present system is needed to obtain the similar sor-
bitol yield to the recent results (7 h [22] and 6 h [23]). As demon-
strated above, the dextrin hydrolysis efficiency can be retained
entirely in Ru–B/af-mCarbon@air@af-mSiO –A-II, the enhanced
2
catalytic efficiency of the present biochemical composite should
be thanks to the superior glucose hydrogenation activity of the R
OH
OH
H O
O
O H
O
O
OH
a
O H
Enzyme
HO
H2
HO
OH
HO
O
OH
HO
OH
Ru-B
OH
OH
OH
O H
Cellobiose
Glucose
Sorbitol
Conversion of cellobiose
Yield of sorbitol
1
00
u–B/af-mCarbon@air@af-mSiO
hand, the amino groups grafted in mCarbon serve as anchor points
33], favoring the uniform dispersion of Ru–B. On the other hand,
the amino groups enhance the concentration of ionized glucose
species via abstracting the proton from the anomeric hydroxyl
group in glucose [41]. Beenackers et al. [42] showed that, upon
generation of the glucose anion, it was susceptible to attack by
hydrogen adsorbed dissociatively on the neighboring metal sites.
We also discovered another desirable attribute of our process:
handling convenience and the stability. Ru–B/af-mCarbon@air@a
2
to the previous systems. On one
8
6
4
2
0
[
0
0
0
0
1
2
3
4
5
6
2
f-mSiO –A-II could be easily separated from the reaction solution
Recycling number
via centrifugation and used repetitively at least for 6 times with
only 9% decrease of sorbitol yield during the one-pot hydrolysis–
hydrogenation of dextrin (Fig. 8), showing its superiority over the
simple combination of the free amyloglucosidase and the yolk–
shell structured chemical catalysts [22,23]. During the recycling
test, the dextrin conversion decreased by 7% after being used 6
times. Accordingly, the slight decrease in sorbitol yield after 6
cycles should be attributed to the partial lost in enzymatic effi-
ciency for saccharification of dextrin. The FESEM and TEM images
OH
OH
OH
OH
OH
OH
O
HO
HO
OH
O
O
HO
HO
OH
O
O
O
HO
O
O
HO
O
O
O
OH
O
OH
O
Enzyme
H2
Ru-B
OH
OH
OH
OH
OH
OH
HO
HO
b
O
OH
O
OH
HO
OH
O
HO
O
HO
OH
OH
OH
OH
O
OH
O
Cellulose
Glucose
Sorbitol
Conversion of cellulose
Yield of sorbitol
1
00
(
Fig. 9) showed that both the decorated tree-like structure of the
biochemical composite and the yolk–shell structure morphology
of the chemical catalyst were still present after cycles.
8
6
4
2
0
6
0
However, the leaching amount of amyloglucosidase was deter-
mined as 5% by the BCA assay in the supernatant, demonstrating
that leaching of enzyme might be the main factor responsible for
the decrease in efficiency.
Finally, further investigations of the generality of the present
strategy revealed that one-pot synthesis of sorbitol can be also
achieved via hydrolysis–hydrogenation of cellobiose and even cel-
0
0
0
2
lulose by Ru–B/af-mCarbon@air@af-mSiO –C-II with 6 cycles of
1
2
3
4
5
6
successive use (Fig. 10). From the viewpoint of practical applica-
tions, transforming the inedible biomass materials into valuable
platform chemicals is more economical issue. Therefore, further
optimization may eventually make the approach industrially
viable for the conversion of non-food biomass into sorbitol as a
renewable platform chemical.
Recycling number
2
Fig. 10. Recycling test of Ru–B/af-mCarbon@air@af-mSiO –C-II for one-pot hydrol-
ysis–hydrogenation of (a) cellobiose and (b) cellulose. Reaction conditions:
cellobiose or cellulose (0.1 g), 0.68 g of biochemical composite (containing 2.6 mg
Ru and 0.33 g cellulase), water (20 mL), T = 333 K,
rate = 800 rpm. Each run was conducted for 6 h in recycling test.
PH2 = 4.0 MPa, and stirring
4
. Conclusions
the shell and then be hydrogenated to sorbitol over the
Ru–B/af-mCarbon core. Notably, the catalytic efficiency of the pre-
sent biochemical composite exceeds those we reported recently.
For example, the present biochemical composite enables the
one-pot dextrin conversion to proceed at moderate temperature
In summary, our research provides a paradigm for the utility of
crosslinking technique for the integrating of chemical catalyst and
the macromolecular enzyme into biochemical composite. The recy-
clable biochemical composite can be used to convert biomass

W. Wei et al. / Journal of Catalysis 327 (2015) 78–85
85
materials into sorbitol in high efficiency. This one-pot process may
find important applications for the efficient production of renew-
able platform chemicals from biomass materials without interme-
diate purification. Moreover, this strategy can potentially be
extended to other biochemical composites with different composi-
tion and thus versatile functions.
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