Operational stabilization of fungal α-rhamnosyl-β-glucosidase by immobilization on chitosan composites

Article abstract of DOI:10.1016/j.procbio.2011.09.014

The diglycosidase α-rhamnosyl-β-glucosidase from Acremonium sp. DSM24697 was immobilized by adsorption and cross-linking. The supports screened included beads of chitosan composites (gelatin, arabic gum, silica gel), epoxy-activated agarose and chitosan, and macroporous polyvinyl-alcohol cryogel. The chitosan-silica gel beads were selected because of the highest immobilization efficiency obtained and their morphological properties (diameter 1.67 ± 0.99 mm, circularity 0.81 ± 0.05). The optimization of the immobilization - coating with polyethyleneimine, changes in the enzyme load - improved the immobilization efficiency up to 18%. The practical use of the enzyme deals with low water solubility substrates. The higher K_M for the immobilized enzyme - 8 mM vs. 1.8 mM hesperidin for the free enzyme - indicated that substrate diffusion limits the enzymatic reaction. The solvent dimethylsulfoxide (50%, v/v) was added in order to increase the substrate solubility, and 80% activity was retained (1 h, 60 °C) in contrast with the complete inactivation of the free form. The stability of the immobilized catalyst was extended to metal catalyzed oxidation where the enzyme was fully preserved in harsh conditions such as 1 mM CuSO₄ at 60 °C during 1 h.

Full text of DOI:10.1016/j.procbio.2011.09.014

Process Biochemistry 46 (2011) 2330–2335
Contents lists available at SciVerse ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Operational stabilization of fungal ␣-rhamnosyl-␤-glucosidase by
immobilization on chitosan composites
Lucrecia Pin˜uel, Laura S. Mazzaferro, Javier D. Breccia∗
INCITAP-CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas), Departamento de Química, Facultad de Ciencias Exactas y Naturales,
Universidad Nacional de La Pampa (UNLPam), Av. Uruguay 151, (6300) Santa Rosa, La Pampa, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 July 2011
Received in revised form
15 September 2011
Accepted 19 September 2011
Available online 28 September 2011
The diglycosidase ␣-rhamnosyl-␤-glucosidase from Acremonium sp. DSM24697 was immobilized by
adsorption and cross-linking. The supports screened included beads of chitosan composites (gelatin, ara-
bic gum, silica gel), epoxy-activated agarose and chitosan, and macroporous polyvinyl-alcohol cryogel.
The chitosan–silica gel beads were selected because of the highest immobilization efficiency obtained and
their morphological properties (diameter 1.67 0.99 mm, circularity 0.81 0.05). The optimization of the
immobilization—coating with polyethyleneimine, changes in the enzyme load—improved the immobi-
lization efficiency up to 18%. The practical use of the enzyme deals with low water solubility substrates.
The higher KM for the immobilized enzyme—8 mM vs. 1.8 mM hesperidin for the free enzyme—indicated
that substrate diffusion limits the enzymatic reaction. The solvent dimethylsulfoxide (50%, v/v) was added
in order to increase the substrate solubility, and 80% activity was retained (1 h, 60 ◦C) in contrast with the
complete inactivation of the free form. The stability of the immobilized catalyst was extended to metal
catalyzed oxidation where the enzyme was fully preserved in harsh conditions such as 1 mM CuSO4 at
60 ◦C during 1 h.
Keywords:
Dimethylsulfoxide
Glycoside hydrolase
Rutinose
Hesperidin
Hesperidin methylchalcone
Polyethyleneimine
Metal catalyzed oxidation
© 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Stable glycosidases with reutilization capacity is a key require-
ment for economically relevant biotransformations [4]. The low
Glycoside hydrolases (EC 3.2.1.-) are a widespread group
of enzymes, which hydrolyze the glycosidic bond, and from
a biotechnological point of view, they find extended applica-
tions in bulk biotransformations as well as in fine chemistry for
modification of biologically active compounds [1]. We recently
reported the enzyme ␣-rhamnosyl-␤-glucosidase (EC 3.2.1.168),
a distinctive glycosidase because of its substrate specificity for
7-O-rutinosides as well as its mode of action as a diglycosidase.
It cleaves the flavonoid hesperidin and other 7-O-rutinosylated
flavonoids in the heterosidic bond to yield the disaccharide
rutinose (␣-rhamnopyranosyl-␤-glucopyranose) and the corre-
sponding aglycone [2]. On account of its hydrolytic activity,
␣-rhamnosyl-␤-glucosidase has potential use for industrial pro-
cessing of plant-based foods [3]. On the other hand, the ability
of the enzyme to transglycosylate rutinosyl units to OH-acceptor
compounds, using as starting material an abundant and inexpen-
sive by product of citric industry, such as hesperidin and hesperidin
methylchalcone, makes this enzyme a promissory system for the
development of new drugs [25].
stability of the proteins in water-soluble organic co-solvents con-
stitutes a problem for using them in synthetic reactions [5]. Many
applications demand the use of these solvents in the reaction mix-
tures to increase the kinetic constant and yield of the processes
by raising substrates solubility. In that way, several glycosidases
have been immobilized onto a number of supports in view of their
application in food processing and fine chemistry [6–8]. Moreover,
the immobilization of the biocatalyst allows the kinetic control
of a given process avoiding the final thermodynamic equilibrium
that could lead with the hydrolysis of the synthesized products.
In this work we have assessed different strategies to obtain an
immobilized preparation of ␣-rhamnosyl-␤-glucosidase stable in
operational conditions. The polymer chitosan was chosen as the
main constituent of the support based on its compatibility with
proteins, chemical stability and the non-toxic character [9].
2. Materials and methods
2.1. Chemicals
Hesperidin, hesperidin methylchalcone, rutinose, chitosan, glutaraldehyde 50%
(v/v), polyethyleneimine PEI (MW 60 kDa), dimethylsulfoxide (DMSO), were pur-
chased from Sigma Chemical (St. Louis). All other chemicals were from standard
sources.
∗
Corresponding author. Tel.: +54 2954 436787x26; fax: +54 2954 432535.
E-mail address: javierbreccia@exactas.unlpam.edu.ar (J.D. Breccia).
1359-5113/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2011.09.014

L. Pi˜nuel et al. / Process Biochemistry 46 (2011) 2330–2335
2331
2.2. Production and partial purification of ˛-rhamnosyl-ˇ-glucosidase
Abeads
EI =
× 100
Aloaded − Aunbound
The ␣-rhamnosyl-␤-glucosidase was purified from the culture supernatant of
Acremonium sp. DSM24697 as described before [2]. The procedure includes salt
precipitation and hydrophobic interaction chromatography (butyl-agarose) to
yield 30.4% of the initial activity and purified 45.7 fold. The product was desalted
and freeze-dried before storing at −18 ◦C.
where Aloaded is the enzyme activity loaded, Aunbound is the enzyme activity remaining
in the supernatant, and the activity bound to the support (Abeads) was calculated as
the units of enzyme activity per gram of wet beads
2.7. Stability in the presence of a co-solvent (dimethylsulfoxide) and Cu2+ ions
2.3. Enzyme assay
The immobilized enzyme (25 mg beads) was incubated with 0–50% (v/v) of
DMSO or 1 mM CuSO4 at 60 ◦C during 1 h in 50 mM citrate buffer (pH 5.0). Then,
the support was washed exhaustively with distilled water to remove rests of DMSO
or Cu2+ ions. Determination of the residual activity was carried out as described
above. The sample without additives was fixed as 100% activity.
For quantification of ␣-rhamnosyl-␤-glucosidase activity, each reaction con-
tained 450 ␮l of substrate 0.11% (w/v) hesperidin in 50 mM sodium citrate buffer
pH 5.0 and 50 ␮l of suitably diluted enzyme solution. The reaction was performed
for 1 h at 60 ◦C and stopped by adding 500 ␮l of 3,5-dinitrosalicylic acid (DNS)
[10]. For immobilized enzyme activity, each reaction contained 50 mg of beads in
500 ␮l substrate. The immobilized enzyme was removed before adding DNS. The
tubes were placed in a boiling water bath for 10 min and cooled before measur-
ing the absorbance at 540 nm. One unit of ␣-rhamnosyl-␤-glucosidase activity was
defined as the amount of enzyme required to release 1 ␮mol of rutinose/min. The
kinetic parameters for the immobilized enzyme were determined using hesperidin
and hesperidin methylchalcone as substrates. The initial velocities of the reaction
were plotted against substrate concentration and the data were adjusted using the
Michaelis–Menten equation by the least squares method.
3. Results and discussion
3.1. Immobilization of Acremonium sp. DSM24697 ˛-rhamnosyl-
ˇ-glucosidase on chitosan and chitosan composites beads
The enzyme ␣-rhamnosyl-␤-glucosidase was immobilized onto
chitosan beads by adsorption and cross-linking. The covalent bind-
ing of adsorbed ␣-rhamnosyl-␤-glucosidase with the cross-linking
agent (glutaraldehyde) was essential to avoid the enzyme leakage,
although it was deleterious for the expressed activity on the sup-
port (Abeads) (data not shown), as it had been demonstrated for the
immobilization of other glycoside hydrolases [6,8,13].
2.4. Preparation of chitosan composites beads
Chitosan flakes (0.5 g) were dissolved in 1 M acetic acid (50 ml). The additive
50% (w/w) (silica gel, arabic gum and gelatin) was added to the chitosan solution. It
yielded a viscous solution that was dropped at a constant rate, into a neutralizing
solution containing 15% (w/v) NaOH and 96% ethanol in a volume ratio of 4:1 with
magnetic stirring at 300 rpm. The formed beads were washed with deionized water
until neutral pH was reached, and stored at 4 ◦C for further uses. The 32-bit color
images of the beads were split into red, green and blue (RGB) components using
the software ImageJ (National Institutes of Health, USA: http://rsb.info.nih.gov/ij/).
Images corresponding to the green component were chosen for morphological
characterization due to the highest signal to noise ratio. The morphological charac-
terization of the beads was performed with the free program ImageJ 1.42q (National
Institutes of Health). Size distribution was fitted with the Gauss curve:
Purification process steps of proteins are preferably avoided for
immobilization purposes. In this study, both the supernatant of
the submerged culture and the purified enzyme of Acremonium sp.
DSM24697 were used as biocatalyst source for the evaluation of
the process. The immobilization efficiency (EI) on chitosan beads
was low, with EI of 0.04 and 0.5% for the supernatant and the
pure enzyme, respectively (Fig. 1). Moreover, the chitosan beads are
not suitable for some operational conditions, i.e. agitated reactor,
because of the low mechanical stability in presence of shear forces.
In order to improve the immobilization efficiency and the mechan-
ical stability, the beads were blended with several additives that
were chosen in basis of their different chemical nature: silica gel,
gelatin and arabic gum. The immobilization showed a better per-
formance for chitosan-composites beads than for chitosan beads
(Fig. 1). The efficiency was increased 3–5 fold for pure protein and
25–75 fold for culture supernatant (Fig. 1). In this case, the higher
efficiency for the supernatant samples suggested that some com-
ponents of the supernatant might have a protective effect on the
structure of ␣-rhamnosyl-␤-glucosidase. The binding of a protein
in presence of other compounds such as brown colored compounds
and polysaccharides may impair the immobilization yield [14]. By
the other side, the co-immobilization of two or more proteins has
been previously demonstrated to lower the immobilization stress
by competing for the binding sites of the support, promoting a
non-distorting conformation of the immobilized enzyme [15,16].
The chitosan-composites beads were morphologically charac-
terized as is shown in Table 1. The chitosan–silica beads showed
the highest circularity and homogeneity—as evidenced by size dis-
persion around the media. Moreover, chitosan–silica gel beads
were previously reported as more resistant material in comparison
with the other chitosan-composites used, which is in agreement
with its inorganic nature [13]. Because of the higher immobiliza-
tion efficiency obtained and their physical properties we selected
chitosan–silica gel beads for further studies.
A
2
/w
2
)
e−2(x−x
c
y = y0
+
ꢀ
w
ꢀ/2
where y0 is the medium diameter of the beads and w represents the size dispersion.
Circularity was calculated by the following equation:
area
Circularity = 4ꢀ
perimeter2
A circularity value of 1.0 indicates a perfect circle. As the value approaches 0.0,
it indicates an increasingly elongated polygon.
2.5. Preparation of PEI–chitosan–silica gel beads
Polyethylenimine (PEI) (MW 60 kDa) was adsorbed to the support throughout
electrostatic interactions with the silica (SiO2) fraction [11]. PEI-coated beads were
prepared as described Pessela et al. with slight modifications [12]. Wet support
(2.2 g) was suspended in 135 ml of 10% (w/v) PEI. The suspension was maintained
under mild stirring at 25 ◦C for 3 h. Then, the beads were treated with 10 mg/ml
sodium borohydride (2 h at 25 ◦C). Finally, the suspension was filtered and washed
consecutively with 100 mM sodium acetate buffer pH 5.0, 100 mM sodium borate
buffer pH 7.8, and an excess of distilled water. The beads were stored at 4 ◦C in an
aqueous solution containing 50% (v/v) ethanol. The presence of the PEI-coating onto
the beads was confirmed by incubating them in 1 mM Cu2SO4 solution, visualizing
the blue complex formed with copper ions.
2.6. Optimization of the immobilization of ˛-rhamnosyl-ˇ-glucosidase
The ␣-rhamnosyl-␤-glucosidase from Acremonium sp. DSM24697 was immobi-
lized by adsorption-cross linking using freeze-dried pure enzyme or culture broth.
The enzyme (0.017–0.067 U/ml) was incubated (18 h at room temperature) with
400 mg of wet chitosan composites beads suspended in 3.4 ml of 50 mM sodium
phosphate buffer pH 7.0. The cross-linking was carried out incubating the mixture
with 0.5% (v/v) glutaraldehyde (GA) at room temperature for 30 min [6]. Afterwards,
the beads were washed thoroughly with distilled water.
Table 1
Morphological characterization of the chitosan composite beads.
The immobilization yield (YI) and the immobilization efficiency (EI) were
defined as:
Additive
Diameter (mm)
Circularity
Arabic gum
Gelatin
Silica-gel
2.24 1.03
3.25 1.20
1.67 0.99
0.74 0.09
0.60 0.09
0.81 0.05
Aloaded − Aunbound
YI =
× 100
Aloaded

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L. Pi˜nuel et al. / Process Biochemistry 46 (2011) 2330–2335
Fig. 1. Immobilization of ␣-rhamnosyl-␤-glucosidase on chitosan composite beads using ( ) culture supernatant and (ꢀ) pure ␣-rhamnosyl-␤-glucosidase of Acremonium
sp. DSM24697.
3.2. Chitosan–silica beads coated with polyethyleneimine
Interestingly, the Abeads was not modified in the subsequent cycles.
In addition, during long-term storage, the immobilized enzyme was
shown to retain 100% of the activity after 65 days at 8 ◦C.
In order to prevent the decrease of the enzyme activity dur-
ing the first cycle, the beads were coated with polyethyleneimine.
The recycling of the biocatalyst was evaluated during five uses of
1 h-length at 60 ◦C (Fig. 2). After the first use, the enzymatic activity
on the support, Abeads, was diminished to 15% of the initial activity.
Fig. 2. Operational stability of the immobilized ␣-rhamnosyl-␤-glucosidase from Acremonium sp. DSM24697 using crude extract as enzyme source and beads of (
)
chitosan–silica–PEI and (ꢀ) chitosan–silica. Hundred percent correspond to the activity on the support before the recycling: 0.17 U/g for chitosan–silica–PEI and 0.13 U/g for
chitosan–silica beads.

L. Pi˜nuel et al. / Process Biochemistry 46 (2011) 2330–2335
2333
Fig. 3. Influence of enzyme load on the inmobilization of purified ␣-rhamnosyl-␤-glucosidase from Acremonium sp. DSM24697. (ꢀ) Beads bound activity (U/g), (ꢁ) Immo-
bilization efficiency (%) and ( ) Immobilization yield (%).
Table 2
The binding capacity of chitosan–silica–PEI beads was
assessed loading different amounts of partially purified
␣-rhamnosyl-␤-glucosidase. The Abeads increased as the amount of
Effect of temperature on the leakage of ␣-rhamnosyl-␤-glucosidase immobilized on
chitosan–silica–PEI beads.
Temperature (◦C)
Enzyme leakage (U/g)
loaded enzyme was augmented, but the immobilization efficiency
was diminished (Fig. 3). The immobilization yield (YI) was found
to be 100% when 0.1 U/g were loaded; and the immobilization
efficiency was 18%, e.g. 0.03 U/g support. This behavior brought
about a tradeoff regarding the optimization of the conditions for
the immobilization process. The obtainment of 5 times higher
activity on the support required ∼400 times higher enzyme load-
ing, and the immobilization efficiency was as low as 2%. The profile
of the immobilization efficiency observed as the enzyme loading
was augmented agrees with the results previously reported for
glycoside hydrolases by several authors [19,20]. The immobiliza-
tion efficiency was low in comparison with a ␤-glucosidase from
Issatchenkia terricola immobilized in Eupergit C250L [19]. Eupergit
C250L is a commercial support composed of epoxy-activated
polyacrylate-based beads. The replacement of the support by
beads of macroporous polyvinyl-alcohol cryogel and epoxy-
activated supports (agarose and chitosan beads) did not improve
the efficiency of ␣-rhamnosyl-␤-glucosidase immobilization in
comparison with chitosan–silica–PEI beads (data not shown).
30
40
50
60
ND
0.63 0.005
1.44 0.095
1.16 0.080
The activity was measured in the supernatant after incubating the beads at different
temperatures for 1 h.
This polymer gets adsorbed on silica dispersions producing floc-
culated slurry in a wide range of pH values [11]. In this case it
produced a stable layer on the chitosan–silica beads most likely
by ionic interaction with the silica fraction. PEI is a polycationic
polymer (pKa ∼ 9) that forms complexes with acidic proteins due
to electrostatic forces [17,18]. Since the isoelectric point of ␣-
rhamnosyl-␤-glucosidase is acidic (pI 5.7), it is expected to interact
with PEI in the conditions of the immobilization procedure (pH 7).
The adsorption of enzymes onto composites with flexible poly-
mers containing a very high density of ion exchange moieties would
promote a very strong adsorption on a flexible coating bed [7].
Remarkably, the residual activity after the first use was increased
from 15% to 35% for the PEI-coated support (Fig. 2). The 65% loss
of activity during the first cycle was attributed to a temperature-
dependent leakage found between 40 and 60 ◦C but not below 30 ◦C
(Table 2). Although the immobilization efficiency was not modified
in the presence of PEI, the importance of the PEI coating resides in
the maintenance of the enzyme bound to the support at high tem-
peratures. Previously, Pessela et al. [7] reported that the adsorption
strength was much higher using PEI supports, where desorption of
a ␤-galactosidase could be prevented even at high ionic strength
(0.5 M NaCl at pH 7).
3.3. Kinetic parameters of immobilized
˛-rhamnosyl-ˇ-glucosidase
The enzyme ␣-rhamnosyl-␤-glucosidase was shown to be
active against flavonoid 7-O-rutinosides, with the highest affinity
for hesperidin (KM 1.7 mM), a compound scarcely soluble in water
(0.324 mM) [2]. The diffusion hindrance of the substrate is one of
the phenomena that would explain the low activity expressed on
chitosan–silica–PEI beads. The kinetic parameters of the immobi-
lized enzyme were determined using hesperidin and hesperidin
Table 3
Influence of immobilization process on kinetic parameters.
Substrate
Free [2]
Immobilized
KM(i)/KM(f)
Hesperidin (H)
Hesperidin methylchalcone (HMC)
KM
KM
1.8
8.7
2
8.0
38.7
13
4.4
4.4
Vmax(HMC)/Vmax(H)
(i) Immobilized; (f) free enzyme.

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L. Pi˜nuel et al. / Process Biochemistry 46 (2011) 2330–2335
Fig. 4. Stability of ␣-rhamnosyl-␤-glucosidase in presence of dimethylsulfoxide at high temperature. The biocatalyst was incubated for 1 h at 60 ◦C with different concentration
of DMSO. (ꢀ) Free ␣-rhamnosyl-␤-glucosidase, ( ) immobilized ␣-rhamnosyl-␤-glucosidase. Hundred percent corresponds to the activity without additives 0.89 U/ml for
free enzyme and 0.08 U/g for the immobilized catalyst.
methylchalcone—a flavonoid 7-O-rutinoside that is water-soluble
(>300 mM) (Table 3). The apparent Michaelis constants for the
immobilized catalyst were found to be 4.4 times higher for both
substrates, in comparison with the free enzyme. The change in
the affinity of the enzyme for its substrate is probably caused by
structural changes in the enzyme introduced by the immobiliza-
tion procedure and by the lower accessibility of the substrate to
the active site of the immobilized enzyme [20]. By the other hand,
the Vmax for hesperidin was significantly reduced in comparison
with hesperidin methylchalcone for the immobilized catalyst. This
result indicated that the diffusion rate of the substrate limits the
enzymatic reaction. As a consequence, the diffusion limitation is
greater for the less soluble substrate.
and it was reported as a stabilizer against MCO for soluble pro-
teins [17,18,21]. Interestingly, both the polymer and the enzyme
are located in a restricted layer around the beads and the former
promotes a higher concentration of the metal at the surroundings
of the enzyme environment without affecting the activity. These
results indicate that immobilization process promotes stabilization
of ␣-rhamnosyl-␤-glucosidase against MCO in harsh conditions
like 60 ◦C and 1 mM CuSO4.
3.5. Organic co-solvent in the reaction mixture increases
substrate solubility and affects enzyme stability
The practical use of the immobilized ␣-rhamnosyl-␤-
glucosidase deals with the low water solubility of its main
substrate, hesperidin. The inclusion of an organic co-solvent in
the reaction medium has been frequently used to increase the
synthetic efficiency of glycosidase-catalyzed processes; the two
most commonly employed organic co-solvents in this respect
being dimethylsulfoxide (DMSO) and acetone. However, enzymes
are usually unstable under such conditions [22]. The stability of
immobilized ␣-rhamnosyl-␤-glucosidase in presence of the apro-
tic solvent DMSO is particularly interesting. The transglycosylation
as well as hydrolysis reactions are favored in presence of this
solvent, due to the increment in the solubility of the substrate
[23]. The residual activity was studied after 1 h incubation at
60 ◦C in presence of DMSO (Fig. 4). The residual activity of the
free-enzyme was strongly dependent on the DMSO concentration.
A reduction of 40% of the initial activity was found after incubation
in 20% (v/v) DMSO and the activity was practically undetectable
after incubation with 50% (v/v) DMSO. Previous reports had been
demonstrated that immobilization of a protein on a support with
a polymeric bed (dextran) would change the enzyme from a
hydrophobic environment to a hydrophilic one promoting the
enzyme stability in presence of organic solvents [24]. In this
case, the immobilized enzyme maintained 82% of the activity
independently of DMSO concentration after the exposure at high
temperature.
3.4. Immobilization promotes protein stability against
metal-catalyzed oxidation
Metal catalyzed oxidation (MCO) is associated to protein
modifications promoting aggregation, irreversible amino acid
modifications, increased susceptibility to proteolysis and/or frag-
mentation of the protein backbone [17,21]. As a consequence,
traces of metals are involved in activity losses during storage condi-
tions. ␣-Rhamnosyl-␤-glucosidase was incubated for 1 h at 60 ◦C in
presence of 1 mM CuSO4. The soluble form of the enzyme losts 97%
of the initial activity while no activity losses were detected for the
immobilized enzyme, regardless the coating with PEI (Table 4). PEI
is a chelating polymer for several metal ions like iron and copper
Table 4
Effect of metal catalyzed oxidation on the activity of ␣-rhamnosyl-␤-glucosidase.
State
Residual activity (%)
Free enzyme
Immobilized coated support
Immobilized uncoated support
2.9 0.3
105.1 8.5
92.4 4.0
The residual activity was measured after incubating the biocatalyst with 1 mM
CuSO4 at 60 ◦C for 1 h.

L. Pi˜nuel et al. / Process Biochemistry 46 (2011) 2330–2335
2335
4. Concluding remarks
[9] Krajewska B. Application of chitin- and chitosan-based materials for enzyme
immobilizations: a review. Enzyme Microb Technol 2004;35:126–39.
[10] Miller GL. Use of dinitrosalisylic acid (DNS) for determination of reducing sug-
ars. Anal Chem 1959;31:426–8.
[11] Lavanya A, Yee-Kwong L. Interactions of PEI (polyethylenimine)–silica particles
with citric acid in dispersions. Colloid Polym Sci 2011;289:237–45.
[12] Pessela B, Betancor L, López-Gallego F, Torres R, Dellamora-Ortiz GM, Alonso-
Morales N, Fuentes M, Fernández-Lafuenta R, Guisán JM, Mateo C. Increasing
the binding strength of proteins to PEI coated supports by immobilizing at high
ionic strength. Enzyme Microb Technol 2005;37:295–9.
[13] Spagna G, Barbagallo R, Greco E, Manenti I, Pifferi PG. A mixture of purified
glycosidases from Aspergillus niger for oenological application immobilized by
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natural supports. Process Biochem 1995;31:281–5.
The enzyme ␣-rhamnosyl-␤-glucosidase was reported recently
by our group for the hydrolysis of the flavonoid hesperidin into the
aglycone hesperetin and the disaccharide rutinose [2]. The prone
oxidation character of the enzyme and its low stability in presence
of organic solvent would limit the use of the enzyme. The immo-
bilization process onto chitosan–silica–PEI beads showed a good
performance to overcome the mentioned drawbacks for biotrans-
formation reactions.
Acknowledgements
[15] Morana A, Mangione A, Maurelli L, Fiume I, Paris O, Cannio R, Rossi M. Immo-
bilization and characterization of a thermostable ␤-xylosidase to generate a
reusable biocatalyst. Enzyme Microb Technol 2006;39:1205–13.
[16] Minakshi, Pundir CS. Co-immobilization of lipase, glycerol kinase, glycerol-3-
phophate oxidase and peroxidase onto aryl amine glass beads affixed on plastic
strip for determination of triglycerides in serum. Indian J Biochem Biophys
2008;45:111–5.
This work was supported by Consejo Nacional de Investiga-
ciones Científicas y Técnicas (CONICET), Universidad Nacional de La
Pampa (UNLPam), and Agencia Nacional de Promoción Científica y
Técnica (ANPCyT) of Argentina.
[17] Andersson MM, Breccia JD, Hatti-Kaul R. Stabilizing effect of chemical addi-
tives against oxidation of lactate dehydrogenase. Biotechnol Appl Biochem
2000;32:145–53.
[18] Mazzaferro LS, Breccia JD, Andersson MM, Hitzmann B, Hatti-Kaul R.
Polyethyleneimine–protein interactions and implications on protein stability.
Int J Biol Macromol 2010;47:15–20.
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