Catalysis Communications 55 (2014) 6–10

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Catalysis Communications
journal homepage: www.elsevier.com/locate/catcom

Short Communication

Immobilization of Aspergillus niger xylanase A on Fe3O4-coated chitosan

magnetic nanoparticles for xylooligosaccharide preparation
Ming-qi Liu ⁎, Xian-jun Dai, Rong-fa Guan, Xin Xu
Zhejiang Provincial Engineering Laboratory of Quality Controlling Technology and Instrumentation for Marine Food, China JiLiang University, Hangzhou 310018, China

a r t i c l e i n f o a b s t r a c t

Article history: Aspergillus niger xylanase A (XylA) was immobilized onto Fe3O4-coated chitosan magnetic nanoparticles pre-
Received 20 April 2014 pared by the layer-by-layer self-assembly approach. The Fe3O4-coated chitosan magnetic nanoparticles showed
Received in revised form 5 June 2014 a high binding capacity of 162.2 mg∙g−1-particles and a recovery activity of 56.5% for XylA. The immobilized XylA
Accepted 7 June 2014
showed improved thermostability and storage stability compared with free XylA. The immobilized XylA retained
Available online 17 June 2014
87.5% activity after seven successive reactions by magnetic separation. Xylotriose and xylohexaose were the main
Keywords:
products released from birchwood xylan and wheat bran insoluble xylan by immobilized XylA, respectively.
Xylanase © 2014 Elsevier B.V. All rights reserved.
Immobilization
Magnetic nanoparticles
Xylooligosaccharides
Layer-by-layer (LBL)

1. Introduction polyelectrolyte, nontoxicity, and biocompatibility. Chitosan is also

Endo-β-1,4-xylanase (EC. 3.2.1.8) is one of the important enzymes
involved in the degradation of xylan [1]. Xylanase has attracted consid-
erable research interest because of its industrial application and poten-
tial application, such as production of xylooligosaccharides (XOs) in the
food industry [2]. XOs, as high value-added ingredients for functional
foods, can be produced by chemical and/or enzymatic (immobilized or
free xylanase) methods from xylan-containing raw materials. XOs
showed favorable technological features and can be utilized by benefi-
cial gastrointestinal microflora, namely, Bifidobacterium and Lactobacilli,
to suppress the activity of the entero putrefactive bacteria, and inhibit
Shiga-like toxins from Escherichia coli O157:H7 [3,4].
Magnetic nanoparticles have long been studied for the purpose of
enzyme immobilization because of their native magnetic properties.
The immobilized enzyme based on Fe3O4 magnetic nanoparticles can
be easily separated from the reaction system for reuse [5]. Fe3O4 mag-
netic nanoparticles are chemically inert; hence, cannot be directly com-
bined with enzymes by covalent bonding. Fe3O4 magnetic nanoparticles
should be pre-coated by natural macromolecules to obtain active func-
tional groups for direct reactions with enzymes or undergo chemical
modification before use. The layer-by-layer (LBL) self-assembly
approach has been applied to prepare nanometer- and submicrometer-
sized charged particles as core to produce supports for enzyme immobi-
lization. Chitosan can spontaneously combine with anionic polyelectro-
lyte by the LBL self-assembly approach because of its cationic
⁎ Corresponding author. Tel.: +86 571 87676372; fax: +86 571 86834449.
E-mail addresses: mqliu524@163.com, tony_cjlu@aliyun.com (M. Liu).
extensively used in biomedical and pabular applications [6,7].
Aspergillus niger xylanase A (XylA) has been purified and character-
ized in our previous study [8]. In this study, XylA was immobilized on
Fe3O4-coated chitosan magnetic nanoparticles prepared by the LBL
self-assembly approach and characterized in detail.
2. Experimental
2.1. Preparation of Fe3O4-coated chitosan support and immobilization
of XylA
Fe3O4 nanoparticles were synthesized using the co-precipitation
method [5]. Up to 320 mg of Fe3O4 nanoparticles was added to 80 ml
of sodium dodecanesulfonate solution (0.06%, w/v), and then subjected
to ultrasonication for 20 min. The supernatant was removed after incu-
bation at 20 °C for 12 h. The precipitate was added to 10 ml of 1.0%
chitosan in 1.0% acetic acid solution and stirred at 80 rpm for 12 h.
The mixture was then centrifuged and the non-adsorbed chitosan was
removed. The Fe3O4-coated chitosan particles were cross-linked by
5 ml of 2.5% glutaraldehyde at 20 °C for 4 h. The precipitate was then
washed by deionized water three times.
The Fe3O4-coated chitosan supports (200 mg) were added to partial-
ly purified XylA solution (sodium acetate–acetic acid buffer, 0.1 M,
pH 5.5, 40,000 U) and incubated at 30 °C for 4 h. The particles were
then separated by neodymium magnet treatment and washed with
sodium acetate buffer (0.1 M, pH 5.5) three times. The immobilized
xylanase was stored at 4 °C.

http://dx.doi.org/10.1016/j.catcom.2014.06.002
1566-7367/© 2014 Elsevier B.V. All rights reserved.
 M. Liu et al. / Catalysis Communications 55 (2014) 6–10
The obtained Fe3O4 nanoparticles and immobilized enzyme were
characterized by transmission electron microscopy (TEM), Fourier
transform infrared spectroscopy (FTIR), and vibrating sample
magnetometery (VSM).
2.2. Xylanase activity assay
Xylanase (immobilized and free) activity and kinetic parameters
were assayed according to Dai et al. [8]. The activity recovery of
immobilized enzyme was defined as the ratio of immobilized enzyme
activity to the total enzyme activity added into the immobilization sys-
tem. Triplicate measurements were performed for each assay to obtain
the mean value and standard deviation.
2.3. Optima and stability of immobilized and free XylA
Optima of immobilized and free XylA were measured according to
Dai et al. [8]. Thermal stability of xylanase was determined by assaying
the residual activity after incubation at 40 °C to 90 °C and pH 6.0 for
4 min. To determine the pH stability, immobilized XylA was suspended
in various pH buffers at 25 °C for 1 h, and the residual activities were
measured at optimal conditions.
2.4. Storage stability of immobilized and free XylA
Both immobilized and free XylA were stored at 20 °C. The samples
were obtained at 5 day intervals over a 30 day period. The residual
activity of the enzymes was measured at optimal conditions.
2.5. Reuse stability of the immobilized XylA
Several consecutive hydrolysis operating cycles were performed
by birchwood xylan reactions under the optimum conditions of
immobilized XylA. At the end of each reaction, immobilized XylA was
recovered by magnetism and washed three times by deionized water.
The residual activity was measured using the original activity of 100%.
The recycling process was repeated seven times.
2.6. Hydrolysis of xylans and standard xylooligosaccharides by immobilized
XylA
The birchwood xylan and wheat bran insoluble xylan (1.0%, w/v,
deionized water, pH 7.0) were incubated with the immobilized XylA
A B
Fig. 1. Transmission electron microscopy (TEM) micrographs of Fe3O4 nanoparticles (A) and Fe3O4-coated chitosan XylA (B).
7
at 40 °C with constant shaking (200 rpm) for 24 h, respectively. The
samples were analyzed by high performance liquid chromatography
(HPLC) [8]. The standard xylooligosaccharides (XOs) (deionized water,
pH 7.0) were incubated with immobilized XylA at 40 °C, and the
samples at different time intervals were analyzed by HPLC.
3. Results and discussion
3.1. Characterization of supports and immobilized XylA
TEM results showed that the prepared Fe3O4 particles were nano-
spheres with an equal size of approximately 10 nm (Fig. 1A). Aggregat-
ed particles from the aggregation during separation were also observed.
The VSM analysis showed that the saturation magnetization (Ms), rem-
nant magnetization (Mr), and coercivity (Hc) of the Fe3O4 particles
were 55.625 emu/g, 23.543 emu/g, and 0.340 Oe, respectively. The
magnetization curves indicated the existence of superparamagnetism
in the nanoparticles. The average size of immobilized XylA particles
was similar to that of prepared Fe3O4 (Fig. 1B). The Ms, Mr, and Hc
of the immobilized XylA were 40.259 emu/g, 10.918 emu/g, and
0.414 Oe, respectively.
The FTIR spectrum of Fe3O4 particles and immobilized XylA revealed
a strong absorption at 585 and 567 cm− 1 (Supplementary material,
Fig. 1). The strong absorption can be attributed to the Fe\O the bond
of magnetic materials [5]. The two peaks at 1045 and 1179 cm− 1 of
immobilized XylA reflected the C\O\C bond of chitosan [9]. The peak
at 1557 cm−1 of the immobilized enzyme was referred to as the C_N
bond formed by the Schiff-base reaction and the amide of the XylA
[10]. The peaks at 2849 and 2917 cm− 1 of the immobilized enzyme
were attributed to the\CH group [11]. The peak at 3423 cm−1 of
Fe3O4, which was wide and blunt, was attributed to the\OH of water
[11].
3.2. Assay of immobilized XylA
XylA was immobilized on Fe3O4-coated chitosan particle by covalent
bonds that formed between the aldehyde group of glutaraldehyde and
the amino group of enzyme, representing a typical Schiff-base reac-
tion [12]. When the concentration of XylA was 0.15 mg ml−1, the
immobilized enzyme amount and recovery activity were 110.0 mg/g
and 56.5%, respectively. However, 0.25 mg/ml enzyme was deemed as
sufficient to saturate support (160 mg/g support), with a recovery activ-
ity of around 35%. The high initial enzyme concentration led to an
8 M. Liu et al. / Catalysis Communications 55 (2014) 6–10

Table 1 attributed to the (a) repulsive interaction caused by the support;

Kinetic constants for immobilized and free XylA. Note. Xylanase activities were measured
under their optimum conditions described in Experimental. The concentration of
birchwood xylan was from 1.0 mg/ml to 10 mg/ml.
Km (mg ml−1) kcat (s−1)
(b) change in the structure of enzyme during the binding process of
the enzyme on Fe3O4-coated chitosan particle, and (c) low accessibility
of substrate to the active site of the immobilized enzyme [12,18].
kcat/Km ml (mg s)−1

Immobilized XylA 10.53 293.95
Free XylA 9.24 540.50
increased amount of immobilized enzyme, whereas the activity recov-
ery decreased. The immobilization performance of the carrier was af-
fected by the attached functional groups, such as the amino group,
hydroxyl group, aldehyde group, epoxy, and oxirane ring [13–15].
The Michaelis–Menten constant (Km) of immobilized XylA was
slightly higher than that of the free one, and the kcat of the immobilized
XylA was much lower than that of the free XylA (Table 1). The vast ma-
jority of the immobilized enzymes showed lower catalytic activity, but
increased Km compared with the free one [16]. The decreased catalytic
activity can be attributed to the distortion of the 3D structure of enzyme,
resulting from the high reactivity of glutaraldehyde [17]. Reduction in
the affinity of the immobilized enzyme to the substrate can also be
27.92 3.3. Optima and stability of immobilized and free XylA
58.50
The optimum temperature for immobilized and free XylA activity
was 60 °C and 55 °C, respectively (Fig. 2A). When treated at 70 °C and
pH 6.0 for 4 min, the residual activities of immobilized and free XylA
were 84.6% and 56.2%, respectively. The immobilized and free XylA
were stable at temperatures below 60 °C (Fig. 2B).
The immobilized and free XylA showed high activity in a pH range of
4.0–7.0, with optimal pH at 5.5 and 5.0, respectively (Fig. 2C). Over 80%
of xylanase activity was retained after treatment of the immobilized en-
zyme after incubation over a pH range of 4.0–9.0 for 1 h at 25 °C
(Fig. 2D). The polyionic matrices prompted partitioning of protons
between the bulk phase and enzyme microenvironment during the hy-
drolysis, which caused a shift in the optimum pH. The shift depends on
both enzyme reaction and charge in the structure of the carriers [10,19].
The optimum pH of immobilized XylA on Fe3O4-coated chitosan

A B
110
110
100
100
90
90
80
80
Relative activity (%)

Residual activity (%)

70
70
60
60
50
50
40 40
Free Immobilized
30 Free Immobilized 30
20 20

10 10

0 0
20 30 40 50 60 70 80 20 30 40 50 60 70 80
Temperature (°C) Temperature (°C)

C D
110 110

100 100

90 90

80 80
Relative activity (%)

Residual activity (%)

70 70

60 60

50 50

40 40

30 30 Free Immobilized

20 20

10 Free Immobilized 10

0 0
2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0
pH pH

Fig. 2. Optima (A and C), thermostability (B) and pH stability (D) of immobilized and free XylA. Note. 1% birchwood xylan was used as substrate. The highest activity was taken as 100% in
assay of optima (temperature optimum, A; pH optimum, C). The xylanase activity under optimum conditions was taken as 100% in thermostability (B) and pH stability (D) assays.
 M. Liu et al. / Catalysis Communications 55 (2014) 6–10 9

80.00
90.00

80.00 A 70.00 B
70.00 60.00

60.00 50.00

50.00

MV
40.00
MV

40.00
30.00
30.00
20.00
20.00

9.791
11.463

11.517

14.133
14.117
10.00 10.00

0.00 0.00

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00
Minutes Minutes

80.00

80.00
6.495

C 70.00
D
70.00
60.00
60.00
50.00
50.00
40.00
MV
MV

40.00
30.00
30.00
20.00
20.00
8.610

11.403

11.447

14.117
9.788
9.703

8.003
8.633
10.00 10.00

0.00 0.00

5.00 10.00 15.00 20.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00
Minutes Minutes
1

80.00
6.573

E
60.00
MV

40.00

20.00
11.500
9.821
8.668
7.881
7.433

0.00

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00

Minutes

654 3 2

Fig. 3. HPLC analysis of hydrolytic products from XOs by immobilized XylA. Note. Panels A–E were HPLC analyses of hydrolytic products from X2–X6 by immobilized XylA after 120, 60, 30,
15 and 5 min of reaction, respectively. The positions of xylose (X), xylobiose (X2), xylotriose (X3), xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6) are shown.

nanoparticles slightly shifted toward the basic range compared to the
free XylA. The optimum pH of the immobilized XylA was higher
(0.5 units) than that of the free XylA. The relative activity of the
immobilized XylA above the pH 5.5 was higher than that of the free
XylA, indicating that the microenvironments of the immobilized XylA
on the Fe3O4-coated chitosan nanoparticles may have been buffered.
The activity of immobilized enzyme was less affected by the acidic–
neutral conditions.
3.4. Storage and reuse stabilities of immobilized XylA
Significant differences were observed between the residual activity
of immobilized and free XylA after five days of storage at 20 °C. The
free XylA lost its entire xylanase activity after 30 days of storage at
20 °C, whereas the immobilized XylA retained 83.7% of its activity
after 30 days. Therefore, the immobilization of XylA on Fe3O4 nanopar-
ticles reduced the inactivation rate of the enzyme. The immobilized
10 M. Liu et al. / Catalysis Communications 55 (2014) 6–10
XylA showed improved stability and longer shelf life compared to the
free XylA.
The immobilized XylA can be easily separated by magnetic decanta-
tion after the hydrolytic reaction and reused several times. The residual
activity of immobilized XylA was 87.5%, even after seven recycling
processes.
3.5. XOs released from xylans by immobilized XylA
The hydrolysis products from birchwood xylan by immobilized XylA
were X–X6, with X3 as the major product (Supplementary material,
Fig. 2B). After 24 h of incubation, approximately 26.9% and 30.6% of
the total hydrolysis products were X2 and X3, with a concentration of
1.782 and 2.023 mg ml−1, respectively. Hydrolysis products released
by immobilized XylA from wheat bran insoluble xylan were X2–X6
(Supplementary material, Fig. 2C). After 24 h of incubation, approxi-
mately 22.9% and 17.3% of the total reaction products were X2 and X3,
with concentrations of 0.667 and 0.504 mg ml− 1, respectively. The
immobilized and free XylA showed low hydrolytic activity on wheat
bran insoluble xylan because of its insolubility [8]. The kinds of hydro-
lytic products released from birchwood xylan by immobilized XylA
were similar to those released when free XylA was used [8]. However,
the kinds of hydrolytic products and main products released from
wheat bran insoluble xylan by immobilized XylA and the free XylA
were different from each other. Our previous study revealed that the
hydrolysis products released by XylA from wheat bran insoluble xylan
were X–X6 with X3 as the major product [8].
Wheat bran is a hemicellulose-rich by-product. Xylan represents
about 40% of the dry matter in wheat bran [20]. The purity of food-
grade XOs is usually within the range of 75% to 95%. The enzyme residue
in the hydrolysate makes purification of XOs difficult [21]. Immobilized
XylA hydrolyzed wheat bran insoluble xylan to a mixture of XOs. The
immobilized XylA can be easily recycled from hydrolysis by magnetism.
Therefore, the purification of XOs from hydrolysate by the immobilized
XylA is easier than the purification from free XylA. The immobilized
XylA can be a potential candidate for the production of XOs from
wheat bran.
3.6. Degradation of standard XOs by immobilized XylA
The mode of action of immobilized XylA was determined using stan-
dard XOs as substrate. X2–X6 can be hydrolyzed by immobilized XylA.
Immobilized XylA showed very low activity on X2 and X3 (Fig. 3A and
B; Supplementary material, Fig. 3A and B). X2 and X3 were the main
products released separately from X4 and X5 (Fig. 3C and D; Supple-
mentary material, Fig. 3C and D). X6 was rapidly hydrolyzed and gener-
ated in XO mixture (X2–X5), with X3 as major product (Fig. 3E;
Supplementary material, Fig. 3E). X5 was among the hydrolysis prod-
ucts of X6, but no X was detected, suggesting that the formation of X5
may be the result of transglycosylation reaction. The presence of trace
amounts of X from XOs by immobilized XylA revealed that the enzyme
preferentially cleaved the internal glycosidic bonds of XOs, similar to the
free XylA [8]. However, no XOs with the degree of polymerization
higher than two in the hydrolysis product of X2 by immobilized XylA
were present. Therefore, X2 was directly hydrolyzed by the enzyme,
not by transglycosylation.
4. Conclusion
The carrier, Fe3O4-coated chitosan magnetic nanoparticles, were
successfully prepared by the LBL self-assembly approach. Fe3O4-coated
chitosan magnetic nanoparticles showed high binding capacity to
XylA (up to 162.2 mg/g). The immobilized XylA can be recycled easily
and exhibited improved stability during storage and thermal and pH
treatments. The immobilized XylA released XOs from wheat bran insol-
uble xylan and birchwood xylan and demonstrated a promising poten-
tial for the industrial production of XOs.
Acknowledgments
Our study was supported by the National Natural Science Founda-
tion of China (No. 31201831).
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.catcom.2014.06.002.
References
[1] T. Collins, C. Gerday, G. Feller, FEMS Microbiol. Rev. 29 (2005) 3–23.
[2] G.W. Tannock, Am. J. Clin. Nutr. 73 (2001) 410s–414s.
[3] K.M. Tuohy, G.C.M. Rouzaud, W.M. Brueck, G.R. Gibson, Curr. Pharm. Design 11
(2005) 75–90.
[4] M.J. Vazquez, J.L. Alonso, H. Dominguez, J.C. Parajo, Trends Food Sci. Technol. 11
(2000) 387–393.
[5] R.H. Hong, J.H. Li, H.Z. Li, J. Ding, Y. Zheng, D.G. Wei, J. Magn. Magn. Mater. 320
(2008) 1605–1614.
[6] M.N.V. Kumar, React. Funct. Polym. 46 (2000) 1–27.
[7] B. Krajewska, Enzyme Microb. Technol. 35 (2004) 126–139.
[8] X.J. Dai, M.Q. Liu, H.X. Jin, M.Y. Jing, Czech J. Food Sci. 29 (2011) 557–567.
[9] A. Bahrami, P. Hejazi, J. Mol. Catal. B Enzym. 93 (2013) 1–7.
[10] J.N. Talbert, J.M. Goddard, Colloid Surf. B 93 (2012) 8–19.
[11] J. Sun, S. Zhou, P. Hou, Y. Yang, J. Weng, X. Li, M. Li, J. Biomed. Mater. Res. A 80A
(2007) 333–341.
[12] W. Tischer, F. Wedekind, Top. Curr. Chem. 200 (1999) 95–126.
[13] A. Rekuc, J. Bryjak, K. Szymanska, A.B. Jarzebski, Process Biochem. 44 (2009)
191–198.
[14] C. Mateo, O. Abian, G. Fernández-Lorente, J. Pedroche, R. Fernández-Lafuente, J.M.
Guisan, A. Tam, M. Daminati, Biotechnol. Progr. 18 (2002) 629–634.
[15] C. Mateo, V. Grazu, J.M. Palomo, F. Lopez-Gallego, R. Fernandez-Lafuente, J.M.
Guisan, Nat. Protoc. 2 (2007) 1022–1033.
[16] C.W. Wu, J.G. Lee, W.C. Lee, Biotechnol. Appl. Biochem. 27 (1998) 225–230.
[17] A.A. Mendes, R.C. Giordano, R.L.C. Giordano, H.F. de Castro, J. Mol. Catal. B Enzym. 68
(2011) 109–115.
[18] W.H. Scouten, J.H.T. Luong, R.S. Brown, Trends Biotechnol. 13 (1995) 178–185.
[19] Z.L. Lei, S.X. Bi, Enzyme Microb. Technol. 40 (2007) 1422–1447.
[20] J. Beaugrand, D. CrÔnier, P. Debeire, B. Chabbert, J. Cereal Sci. 40 (2004) 223–230.
[21] R.G. Crittenden, M.J. Playne, Trends Food Sci. Technol. 7 (1996) 353–361.