β-xylosidase from Selenomonas ruminantium: Immobilization, stabilization, and application for xylooligosaccharide hydrolysis

Article abstract of DOI:10.1080/10242422.2016.1247817

The tetrameric β-xylosidase from Selenomonas ruminantium is very stable in alkaline pH allowing it to easily immobilize by multipoint covalent attachments on highly activated glyoxyl agarose gels. Initial immobilization resulted only in slight stabilization in relation to the free enzyme, since involvement of all subunits was not achieved. Coating the catalyst with aldehyde-dextran or polyethylenimine, fully stabilized the quaternary structure of the enzyme rendering much more stabilization to the biocatalyst. The catalyst coated with polyethylenimine of molecular weight 1300 is the most stable one exhibiting an interesting half-life of more than 10 days at pH 5.0 and 50 °C, being, therefore, 240-fold more stable than free enzyme. Optimum activity was observed in the pH range 4.0–6.0 and at 55 °C. The catalyst retained its side activity against p-nitrophenyl α-l-arabinofuranoside and it was inhibited by xylose and glucose. Kinetic parameters with p-nitrophenyl β-d-xylopyranoside as substrate were V_max 0.20 μmol.min^?1 mg prot.^?1, K_m 0.45 mM, K_cat 0.82 s^?1, and K_cat/K_m 1.82 s^?1 mM^?1. Xylose release was observed from the hydrolysis of xylooligosaccharides with a decrease in the rate of xylose release by increasing substrate chain-length. Due to the high thermostability and the complete stability after five reuse cycles, the applicability of this biocatalyst in biotechnological processes, such as for the degradation of lignocellulosic biomass, is highly increased.

Full text of DOI:10.1080/10242422.2016.1247817

Biocatalysis and Biotransformation
ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: http://www.tandfonline.com/loi/ibab20
β-xylosidase from Selenomonas ruminantium:
Immobilization, stabilization, and application for
xylooligosaccharide hydrolysis
César Rafael Fanchini Terrasan, Caio Casale Aragon, Douglas Chodi Masui,
Benevides Costa Pessela, Gloria Fernandez-Lorente, Eleonora Cano Carmona
& Jose Manuel Guisan
To cite this article: César Rafael Fanchini Terrasan, Caio Casale Aragon, Douglas Chodi Masui,
Benevides Costa Pessela, Gloria Fernandez-Lorente, Eleonora Cano Carmona & Jose Manuel
Guisan (2016): β-xylosidase from Selenomonas ruminantium: Immobilization, stabilization,
and application for xylooligosaccharide hydrolysis, Biocatalysis and Biotransformation, DOI:
10.1080/10242422.2016.1247817
To link to this article: http://dx.doi.org/10.1080/10242422.2016.1247817
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Date: 22 November 2016, At: 08:25

Biocatalysis and Biotransformation, 2016; Early Online: 1–11
RESEARCH ARTICLE
b-xylosidase from Selenomonas ruminantium: Immobilization,
stabilization, and application for xylooligosaccharide hydrolysis
1
CESAR RAFAEL FANCHINI TERRASAN , CAIO CASALE ARAGON1
,
´
DOUGLAS CHODI MASUI1 , BENEVIDES COSTA PESSELA2
GLORIA FERNANDEZ-LORENTE2 , ELEONORA CANO CARMONA3
& JOSE MANUEL GUISAN1
,
1
´
´
´
Departamento de Biocatalisis, Instituto de Catalisis y Petroleoquimica (ICP), Consejo Superior de Investigaciones
2
´
Cientıficas (CSIC), Campus Universidad Autonoma de Madrid (UAM), Madrid, Spain, Departamento de
´
´
´
Biotecnologıa y Microbiologıa de Alimentos, Instituto de Investigacion en Ciencias de los Alimentos (CIAL), Consejo
´
´
Superior de Investigaciones Cientıficas (CSIC), Campus Universidad Autonoma de Madrid (UAM), Madrid,
3
Spain, and Biochemistry and Microbiology Department, Biosciences Institute, Univ Estadual Paulista – UNESP,
˜
Rio Claro, Sao Paulo, Brazil
Abstract
The tetrameric b-xylosidase from Selenomonas ruminantium is very stable in alkaline pH allowing it to easily immobilize by
multipoint covalent attachments on highly activated glyoxyl agarose gels. Initial immobilization resulted only in slight sta-
bilization in relation to the free enzyme, since involvement of all subunits was not achieved. Coating the catalyst with aldehyde-
dextran or polyethylenimine, fully stabilized the quaternary structure of the enzyme rendering much more stabilization to the
biocatalyst. The catalyst coated with polyethylenimine of molecular weight 1300 is the most stable one exhibiting an interesting
half-life of more than 10 days at pH 5.0 and 50 ꢀC, being, therefore, 240-fold more stable than free enzyme. Optimum activity
was observed in the pH range 4.0–6.0 and at 55 ꢀC. The catalyst retained its side activity against p-nitrophenyl a-L-
arabinofuranoside and it was inhibited by xylose and glucose. Kinetic parameters with p-nitrophenyl b-D-xylopyranoside as
substrate were Vmax 0.20 mmol.minꢁ1 mg prot.ꢁ1, Km 0.45 mM, Kcat 0.82 sꢁ1, and Kcat/Km 1.82 sꢁ1 mMꢁ1. Xylose release was
observed from the hydrolysis of xylooligosaccharides with a decrease in the rate of xylose release by increasing substrate chain-
length. Due to the high thermostability and the complete stability after five reuse cycles, the applicability of this biocatalyst in
biotechnological processes, such as for the degradation of lignocellulosic biomass, is highly increased.
Keywords: b-xylosidase, Selenomonas ruminantium, enzyme immobilization, enzyme stabilization, xylooligosaccharide
hydrolysis
Introduction
synergistic action of various enzymes, known as a
xylanolytic system (Biely 1985). Endo-b-1,4-xyla-
nase (E.C. 3.2.1.8) and b-D-xylosidase (E.C.
3.2.1.37) are the main enzymes in the degrad-
ation of the polymer. Xylanases cleave internal
b(1 ! 4) bonds in the xylan backbone liberating
xylooligosaccharides, and b-xylosidases are exogly-
cosidases that remove xylose from the non-
reducing ends of these xylooligosaccharides.
Xylan is the second most abundant biopolymer in
the plant cell walls and the main hemicellulosic
polysaccharide. It is composed of a b(1 ! 4)-D-
xylopyranose backbone substituted, at varying
degrees, by side chain residues as glucopyranosyl,
4-O-methyl-D-glucuronopyranosyl, a-L-arabinofura-
nosyl, acetyl, feruloyl, and p-coumaroyl (Saha 2003).
The complete hydrolysis of xylan requires the
Correspondence: Ce´sar Rafael Fanchini Terrasan, Departamento de Biocata´lisis, Instituto de Cata´lisis y Petroleoquimica (ICP), Consejo Superior de
´ ´
Investigaciones Cientıficas (CSIC), Campus Universidad Autonoma de Madrid (UAM), Cantoblanco, Calle de Marie Curie 28049, Madrid, Spain. E-mail:
cesarterrasan@hotmail.com
(Received 25 July 2015; revised 26 January 2016; accepted 19 August 2016)
ISSN 1024-2422 print/ISSN 1029-2446 online ß 2016 Informa UK Limited, trading as Taylor & Francis Group
DOI: 10.1080/10242422.2016.1247817

2
C. R. F. Terrasan et al.
Finally, a-L-arabinofuranosidases, acetyl xylan- and
ferulic or p-coumaric acid esterases are enzymes
responsible for removing side chains of the polymer
(Collins et al. 2005).
Materials and methods
Materials
Agarose 4BCL was purchased from Agarose Bead
Technologies (Madrid, Spain). p-nitrophenyl b-D-
xylopyranoside (pNPX), p-nitrophenyl a-L-arabino-
furanoside (pNPAra), xylose, glycidol, potassium
tetraborate tetrahydrate, sodium borohydride,
sodium periodate, ethanolamine, Leuconostocc spp
dextran (MW 1500–70,000), polyethylenimine (PEI,
MW 1300–1 ꢂ 106) and molecular weight standard
proteins were obtained from Sigma-Aldrich Co. (St.
Louis, MO). Cyanogen bromide (CNBr) Sepharose
4B and low molecular weight standard proteins were
purchased from GE Healthcare (Uppsala, Sweden).
The interest and application of xylan degrading
enzymes in different industries have increased over
the last decades. b-xylosidase is used on industrial
scale included in xylanases cocktails for the biocon-
version of lignocellulosic material to sugars and other
useful substances, clariEcation of juices and wines,
biobleaching in the pulp and paper industries and in
processing of fabrics. b-xylosidases are critical for the
systems since they carry the greatest work load in
terms of number of glycosidic bonds cleaved, as well
as in relieving product inhibition of xylanases (Jordan
and Wagschal 2010) and cellulases (Zhang and
Viikari 2012). Thus, its performance is of primary
importance especially when these enzymes are
applied together in processes aiming the complete
saccharification of lignocellulosic biomass.
Selenomonas
ruminantium
b-xylosidase
(Lot
100301a) and standards xylooligosaccharides (DP
2-6) were purchased from Megazyme (Wicklow,
Ireland). All reagents were of analytical grade.
Xylobiose and xylooligosaccharides (xylotriose and
larger than six residues xylooligosaccharides) from
beechwood xylan (Sigma-Aldrich Co., St. Louis,
MO) and from Cannabis spp xylan (obtained locally
from textile industry) were prepared in our labora-
tory by enzymatic hydrolysis with the purified
xylanase from Humicola brevis var. thermoidea
(Masui et al. 2012).
The b-xylosidase from the ruminal anaerobic
bacterium Selenomonas ruminantium is a very well
characterized enzyme, corresponding to a bifunc-
tional b-D-xylosidase/a-L-arabinofuranosidase glyco-
side hydrolase belonging to family 43 (GH 43) and
structural clan F in the CAZy database. The enzyme
is a homotetramer in solution, with MW of approxi-
mately 60 kDa of each subunit. It shows a pI of 4.4,
pH stability in the range from 4.3 to 10.0 and
stability up to 50 ꢀC in pH 5.3 for 1 h (Jordan et al.
2007b; Jordan 2008; Jordan and Wagschal 2010).
Currently, the main concern is the rational use of
b-xylosidases for the complete hydrolysis of xylooli-
gosaccharides to the final product, D-xylose. Enzyme
immobilization poses as a possibility to improve the
characteristics of an enzyme in terms of stability,
stabilization, and catalysis, as well as for processes
improvement since it allows the reuse of the
biocatalyst for many operational cycles (Mateo
et al. 2007). Many b-xylosidases have been produced
and characterized (Knob et al. 2010) and some
studies have evaluated covalent immobilization of
these enzymes as that from Talaromyces thermophilus
(Guerfali et al. 2009) immobilized on chitosan or
that from Trichoderma reesei QM 9414 immobilized
on nylon powder (Duen˜as and Estrada 1999).
Nevertheless, to our knowledge, there is no report
about the immobilization of b-xylosidases by multi-
point covalent immobilization using agarose gels
activated with glyoxyl groups as support.
Methods
All results represent the average of three experi-
ments. The experimental error was lower than 5%.
Enzyme assay and protein determination
Standard b-xylosidase activity assays were carried out
in a reaction mixture containing 5 mM pNPX in
0.05 M sodium acetate buffer pH 5.0 and appropri-
ately diluted enzyme solution or catalyst suspension
to 1 ml final volume. The reaction was stopped with
1 ml of a saturated potassium tetraborate solution
and the absorbance was read at 405 nm (Kersters-
Hilderson et al. 1982). One unit of activity was
deEned as the amount of enzyme required to release
1 mmol of product equivalent perꢁm1 in in the assay
conditions at 25 ꢀC (e ¼ 18,300 M cmꢁ1).
Protein concentration was determined with the
modified Bradford’s method, with bovine serum
albumin as standard (Sedmak and Grossberg 1977).
The aim of this study was to immobilize the
b-xylosidase from Selenomonas ruminantium by mul-
tipoint covalent immobilization, evaluate some phy-
sicochemical properties of the immobilized enzyme
and also verify its performance on the hydrolysis of
xylooligosaccharides.
Preparation of glyoxyl-agarose support
A mass of 105 g of 4% cross-linked agarose beads
was suspended in water to a final volume of 180 ml
(0.7 g of agarose is roughly equivalent to 1 ml).

ꢀ-xylosidase from Selenomonas ruminantium
3
After mild homogenization, 50 ml of 1.7 M NaOH
containing 1.425 g of NaBH4 was slowly added. In
an ice bath, 36 ml of glycidol was added drop-wise
and the mixture was then gently stirred at room
temperature for 18 h. After this period, the gel was
finally washed with an excess of distilled water.
Then, the wet glyceryl-activated gel was oxidized
with aqueous NaIO4 solution under very gentle
stirring. After 2 h, the gel was washed with distilled
water, vacuum dried (maintaining the pores of
agarose gels filled with water) and stored at 4 ꢀC
0.5 h, as described above) was added to 5 ml of 5%
(w/v) PEI solutions, pH adjusted to 7.0. PEI with
MW of 1300, 25,000, 75,000, and 600,000–
1 ꢂ 106 Da was evaluated. The suspensions were
gently agitated overnight, washed abundantly with
water and filtered under vacuum.
Immobilization on CNBr-activated Sepharose: it
was performed in very mild conditions by using a
100ꢂ diluted enzyme solution in 0.1 M sodium
phosphate buffer pH 7.0 at 4 ꢀC. After 15 min, the
catalyst was filtered and suspended in 10 ml of a 1 M
ethanolamine solution pH 8.0 for 2 h to block any
remaining reactive group, according to the supplier.
The immobilization yield was defined as the ratio
between the activities (or protein) in the supernatant
compared with the activity (or protein) in the
control. Expressed activity was defined as the ratio
of the activity in the final suspension after the
immobilization process and the initial activity of
offered enzyme.
´
(Guisan 1988).
Preparation of aldehyde-dextran
Leuconostoc spp dextran (MW 1500–70,000) was
fully oxidized by adding 4.36 g of sodium periodate
to 50 ml of an aqueous suspension containing 1.67 g
dextran. The suspension was gently agitated by 2 h
and then dialyzed (4ꢂ) against distilled water.
Stability of soluble enzyme at pH 10.0
Optimum pH and temperature
The enzyme was 400-fold diluted in 0.1 M sodium
bicarbonate buffer pH 10.0. The solution was
incubated at 25 ꢀC, and aliquots were withdrawn at
different intervals and the activity was assayed as
previously described.
Optimum pH was determined by assaying b-xylosi-
dase activity at 25 ꢀC at various pH in the range from
4.0 to 9.0. The following buffers were utilized:
0.05 M sodium acetate buffer pH 4.0 and 5.0,
0.05 M sodium phosphate buffer pH 6.0 and 7.0,
and 0.05 M Tris HCl buffer pH 8.0 and 9.0.
Optimum temperature was determined by assay-
ing b-xylosidase activity at temperatures ranging
from 35 to 60 ꢀC, with 5 ꢀC intervals, in 0.05 M
sodium acetate buffer pH 5.0.
Enzyme immobilization
Immobilization on glyoxyl agarose: the S. ruminan-
tium b-xylosidase was 400-fold diluted in 10 ml of
0.1 M sodium bicarbonate buffer pH 10.0 at 4 ꢀC.
The solution (0.31 g prot./l) was added to 1 g of
glyoxyl agarose support, and the suspension was
gently stirred at 25 ꢀC. The period of immobilization
was determined after carrying out this procedure by
0.5 and 14 h. Both supernatant and suspension
samples were withdrawn periodically and the
enzyme activity was measured. After these periods,
catalysts were reduced by adding 10 mg of sodium
borohydride for 30 min, then washed with water and
filtered under vacuum.
Thermal stability
A mass of 0.1 g of each catalyst was suspended in
1.0 ml of 0.05 M acetate buffer pH 5.0 and
incubated at 50 and 55 ꢀC. In all cases, samples of
the suspension were withdrawn at several intervals
and the activity was assayed as previously described.
Residual activity was calculated as the ratio between
activity at a given time and the activity in the
beginning of incubation.
Dextran-coated catalysts: a mass of 0.5 g of the
uncoated catalyst (immobilization carried out by
0.5 h, as described above) was added to 5 ml of
aldehyde-dextran suspensions, pH adjusted to 7.0.
Dextrans with MW of 1500, 6000, 25,000, and
75,000 Da were evaluated. The suspensions were
gently agitated overnight, then 5 ml of 0.1 M sodium
bicarbonate buffer pH 10.0 was added and the
catalysts were reduced by adding 10 mg of sodium
borohydride for 30 min, washed abundantly with
water and filtered under vacuum.
pH stability
A mass of 0.1 g of each catalyst was suspended in
1.0 ml of different buffers to compose the pH range
from 4.0 to 9.0. The following buffers were utilized:
0.05 M acetate buffer for pH 4.0 and 5.0, 0.05 M
phosphate buffer for pH 6.0 and 7.0, and 0.05 M
Tris HCl buffer pH and 8.0 and 9.0. The suspension
was incubated at 50 ꢀC. Samples were withdrawn at
several intervals and the activity was assayed as
PEI-coated catalysts: a mass of 0.5 g of the
uncoated catalyst (immobilization carried out by

4
C. R. F. Terrasan et al.
previously described. Residual activity was calculated
as the ratio between activity at a given time and the
activity in the beginning of incubation.
Analysis of hydrolysis products by high-performance
liquid chromatography
High-performance liquid chromatography (HPLC)
was coupled with low-temperature evaporative light-
scattering detection LT-ELSD using a detector
Sedex75 (Sedere, Orleans, France). Analyses were
carried out at 80 ꢀC on a Rezec R50-oligosaccharide
Ag+ 4% column (200 ꢂ 10 mm) (Phenomenex,
Torrance, CA). The elution was carried out with
Milli-Q ultrapure apyrogenic water at a 0.3 ml/min
flow rate using a TSP P100 pump and AS3000 auto
sampler Spectra Series (Thermo Electron Co., San
Jose, CA). Data acquisition and processing was
performed using the Chromquest 4.1 software
(Thermo Electron Co., San Jose, CA). The quanti-
fication of products was based on an external
calibration using standard xylose, xylobiose, and
XOS solutions (DP 3-6). Samples of each hydrolyzed
substrate were previously centrifuged (10,000ꢂg,
20 min), filtered (0.22 mm filters) and the adequately
diluted supernatant was analyzed. Analyses were
carried out in duplicate and data were expressed as
mean value.
Specificity and kinetics
The specificity of the catalyst was verified by assaying
the activity against 0.04 M pNPAra in 0.05 M
sodium acetate buffer pH 5.0 at 25 ꢀC. The results
were expressed in relation to the activity on pNPX.
Kinetics of the free and immobilized enzyme was
carried out by assaying b-xylosidase activity with
pNPX (0.1–3 mM) in 0.1 M succinate-NaOH buffer
pH 5.3 at 25 ꢀC. Maximum velocity (Vmax) and
Michaelis–Menten constant (Km) were calculated
with MMfit version 1.3.0 (2013, J.P.G. Malthouse).
Protein
concentration
of
31 g lꢁ1
and
0.95 mg g biocatalystꢁ1 for the free and immobilized
enzyme, respectively, was used to calculate Kcat
.
SDS-PAGE
SDS-PAGE was performed in 8% (w/v) gels
(Laemmli 1970). The resolved protein bands were
visualized after staining with 0.1% (w/v) Coomassie
brilliant blue R-250 dissolved in methanol, acetic
acid, and distilled water (4:1:5, v/v/v). Standard
proteins were phosphorylase b (97 kDa), bovine
serum albumin (66 kDa), ovalbumin (45 kDa), car-
bonic anhydrase (30 kDa), trypsin inhibitor
(20.1 kDa), and a-lactalbumin (14.4 kDa).
Reuse of the catalyst and storage
Reuse was verified by suspending the PEI-coated
glyoxyl catalyst plus inert agarose in 3 mM pNPX (in
0.05 M sodium acetate buffer at pH 5.0) at the
proportion 1:49:100 (w/w/v). Successive batch
hydrolysis cycles were carried out for 1 h at 25 ꢀC
under magnetic stirring (300 rpm). After each cycle,
the catalyst was washed three times with 0.05 M
sodium acetate buffer pH 5.0, filtered under vacuum
and re-suspended in the same buffer (1:10, w/v) to
measure residual activity. Then it was filtered under
vacuum again and new substrate was added for the
following cycle. In order to verify the stability after
long-term storage, the catalyst was maintained in
sealed flasks at 4 ꢀC. After 6 months, the catalyst was
re-suspended in 0.05 M sodium acetate buffer pH
5.0 and the residual activity was assayed.
Hydrolysis of xylooligosaccharides and analysis of the
products
Solutions of xylotetraose, xylopentaose, xylotetrose,
xylopentose, and xylohexaose (0.5 g/l) were prepared
in 5 mM sodium acetate buffer pH 5.0. The PEI-
coated glyoxyl catalyst was added (50 mg to 5 ml
reaction medium) and hydrolysis of each substrate
was carried out at 50 ꢀC, under magnetic stirring
(300 rpm), and samples were taken at several interval
times.
The hydrolysis of beechwood and Cannabis spp
xylans was carried out by Humicola brevis var.
thermoidea xylanase immobilized on glyoxyl agarose.
After 3 days, the medium was filtered and the
supernatant containing xylobiose, xylotriose, xyloo-
ligosaccharides with DP 46, and non-hydrolyzed
xylan (unpublished data) was submitted to a second
enzymatic hydrolysis with S. ruminantium immobi-
lized b-xylosidase (PEI-coated glyoxyl catalyst). The
hydrolysis was carried out for 120 min in 0.05 M
sodium acetate buffer pH 5.0 (200 mg catalyst to
10 ml reaction) at 25 ꢀC.
Results
Immobilization
The soluble b-xylosidase from Selenomonas ruminan-
tium was very stable in pH 10.0, maintaining 100%
of its activity up to 3 h-incubation in this condition.
After 20 h, 57% of the activity was still observed (not
shown). The high stability allowed the enzyme to be
submitted to multipoint covalent immobilization at
this pH, an ideal condition for the immobilization on
glyoxyl. Initially, a catalyst was prepared by carrying

ꢀ-xylosidase from Selenomonas ruminantium
5
out immobilization for 0.5 h. No activity was
detected in the final supernatant and the process
presented 71.4% yield based on initial activity.
Despite the 100% protein immobilization observed
after this period, a second catalyst was prepared by
carrying out immobilization during 15 h.
and 60-fold stabilized in relation to the free enzyme
and the CNBr catalyst, respectively. The stabilization
of the catalyst coated with PEI MW 1300 was much
higher (more than 240-fold in relation to the free
enzyme) since 52% of the activity was observed even
after 10 days of incubation.
Comparing the stability at 55 ꢀC, the catalyst
coated with dextran MW 1500 was sparingly stable,
while the catalyst coated with PEI MW 1300
presented half-life of 40 min (not shown). At this
temperature and in different pH, the highest
In a third stage, the catalyst prepared for 0.5 h
immobilization was incubated with polymers, i.e.,
different MW dextran and PEI in order to coat the
enzyme surface. The coating procedure and/or the
second sodium borohydride reduction (dextran
coated catalysts) caused some loss in the activity
and the coated catalysts maintained 65% of the
activity observed in the non-coated catalyst.
100
80
60
40
20
0
Soluble enzyme was the first control and the
CNBr catalyst, corresponding to a one point
immobilized enzyme, was a second control.
Catalysts characterization
Despite the possibility to obtain higher stability, the
catalyst prepared with 15 h immobilization was less
stable than that prepared by 0.5 h immobilization
(Figure 1). In fact, the half-life of this catalyst (0.5h)
was even lower than those observed for the free
enzyme (1 h) and the CNBr catalyst (2 h) at 50 ꢀC.
At this temperature, the glyoxyl catalyst prepared by
0.5 h immobilization presented half-life of 7 h, and
thus it was 7- and 3.5-fold stabilized in relation to the
free enzyme and the CNBr catalyst, respectively.
Due to the considerable stabilization obtained, this
catalyst was chosen for the subsequent experiments.
The stability of the dextran-coated catalysts at
50 ꢀC (Figure 2) was indirectly related to the MW of
dextran, i.e., the lowest thermostability was observed
for the catalyst coated with MW 70,000 dextran,
presenting half-life lower than 30 min, while the
highest thermostability was observed with MW 1500
dextran. This catalyst retained more than 50% of the
activity after 5 days, indicating low MW dextrans to
be better in adequately coating the enzyme subunits.
Intermediate half-life values, between 1 and 2 h, were
observed for the catalysts coated with MW 6000 and
25,000 dextrans.
0
1
2
3
4
5
6
7
Time (h)
Figure 1. Thermostability of the free b-xylosidase from S.
ruminantium and its catalysts. The incubation was carried out in
0.05 M sodium acetate buffer pH 5.0 at 50 ꢀC and the activity was
assayed in the same buffer at 25 ꢀC. Dash lines: b-xylosidase
activity of glyoxyl catalysts with immobilization carried out by 0.5 h
(˙) and 15 h (m). Solid lines: b-xylosidase activity of free enzyme
(m) and CNBr catalyst (n).
100
100
50
80
0
0
50
100 150
60
40
20
0
The PEI-coated catalysts were all highly stabi-
lized. The stabilization with PEI was similar to that
obtained with the catalyst coated with MW 1500
dextran, although the size of the polymer had low
influence on enzyme thermostability (Figure 3).
Catalysts coated with PEI MW 1300 and 25,000
were more stable maintaining around 60% of the
activity while the others maintained around 50%
after 5 days. If considering these two latter catalysts
maintained around 50% of the activity after 5 days of
incubation at 50 ꢀC, the enzyme was therefore 120-
3
0
1
2
Time (h)
Figure 2. Thermostability of the dextran-coated glyoxyl catalysts
of the b-xylosidase from S. ruminantium. The incubation was
carried out in 0.05 M sodium acetate buffer pH 5.0 at 50 ꢀC, and
the activity was assayed in the same buffer at 25 ꢀC. b-xylosidase
activity of catalysts coated with dextrans of MW 1500 (m, in
detail), 6000 (ꢃ), 25,000 (g) and 70,000 (˙).

6
C. R. F. Terrasan et al.
4
5
3
1
2
100
97 kDa
66 kDa
45 kDa
80
60
40
20
0
30 kDa
20 kDa
14.4 kDa
0
10
20
30
120
Time (h)
Figure 3. Thermostability of the PEI-coated glyoxyl catalyst of the
b-xylosidase from S. ruminantium. The incubation was carried out
in 0.05 M sodium acetate buffer pH 5.0 at 50 ꢀC, and the activity
was assayed in the same buffer at 25 ꢀC. b-xylosidase activity of
catalysts coated with PEI MW 1300 (ꢃ), 25,000 (g), 75,000 (˙),
and 600,000–1 ꢂ 106 (m).
Figure 5. SDS-PAGE of the free b-xylosidase from S. ruminan-
tium and its catalysts. Row 1: Standard proteins: phosphorylase b
(97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa),
carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa), and
a-lactalbumin (14.4 kDa), row 2: uncoated glyoxyl catalyst
(0.07 mg prot.), row 3: dextran-coated glyoxyl catalyst (0.07 mg
prot.), row 4: CNBr catalyst (0.05 mg prot.), row 5: free enzyme
(0.2 mg prot.).
100
80
60
40
20
0
and the higher stability indicate that low MW
aldehyde-dextran and, similarly, different MW PEI
are adequate to coat the immobilized enzyme,
promoting the full stabilization of its quaternary
structure.
Optimum activity of the catalysts was observed in
the pH range from 4.0 to 6.0, while the free enzyme
presented optimum activity in pH 4.0. Besides,
elevated activity of the immobilized enzyme, higher
than 80%, was observed in pH 7.0, while the activity
of the free enzyme was considerably lower (Figure
6a). Optimal temperature for the free enzyme and for
the catalysts was observed at 50–55 ꢀC (Figure 6b).
In addition, the side a-L-arabinofuranosidase activity
was preserved in the catalyst corresponding to 18%
of the b-xylosidase activity.
Table 1 shows the different values obtained for the
kinetic constants on pNPX as substrate of the free
and immobilized enzyme (PEI coated glyoxyl cata-
lyst). The immobilized enzyme presented lower
initial rates (Figure 7), resulting in Vmax that is half
of that observed for the free enzyme. Km of 0.45 mM
observed for the immobilized enzyme was lower than
the 0.72 mM observed for the free enzyme, indicat-
ing the immobilized enzyme with higher affinity for
the substrate.
0
1
2
Time (h)
Figure 4. Stability at different pH values of the PEI-coated glyoxyl
catalyst of the b-xylosidase from S. ruminantium. The incubation
was carried out at 55 ꢀC in 0.05 M sodium acetate buffer pH 4.0
(ꢃ) and 5.0 (m), and 0.05 M sodium phosphate buffer pH 6.0
(g). The activity was assayed in 0.05 M sodium acetate buffer pH
5.0 at 25 ꢀC.
thermostability of the PEI-coated glyoxyl catalyst was
verified in pH 4.0, i.e. after 2 h the remaining activity
still was around 90%. The lowest stability was
verified in pH 6.0, while intermediate half-life value
was observed in pH 5.0 (Figure 4).
When the catalysts were incubated under dena-
turing conditions, it can be observed that the
enzyme was partially covalently immobilized since
bands of the enzyme subunits were observed. Full
stabilization of quaternary structure was only
obtained after coating the surface of the biocatalyst
with dextran (Figure 5). Considering this results
The catalysts were also inhibited by xylose and
glucose, being inhibition by xylose stronger than that
by glucose. The PEI-coated catalyst and the free
enzyme presented similar inhibition pattern and
CNBr catalyst is a little more inhibited by xylose.
In the presence of glucose, CNBr catalyst is a little
more inhibited than the free enzyme and this effect is

ꢀ-xylosidase from Selenomonas ruminantium
7
(b)
(a)
100
80
60
40
20
0
100
80
60
40
20
0
35
40
45
50
55
60
4
5
6
7
8
9
Temperature (°C)
pH
Figure 6. Activity profile at different pH values (a) and temperatures (b) of the free b-xylosidase from S. ruminantium and its catalysts. (a)
The activity was assayed at 25 ꢀC in the following buffers: 0.05 M sodium acetate buffer pH 4.0 and 5.0; sodium phosphate buffer pH 6.0
and 7.0; and Tris HCl buffer pH 8.0 and 9.0. (b) The activity was assayed in 0.05 M sodium acetate buffer pH 5.0 at 35, 40, 45, 50, 55, and
60 ꢀC. PEI-coated glyoxyl catalyst (*), CNBr catalyst (m), and free enzyme (g).
Table 1. Kinetic parameters for the free and immobilized b-xylosidase from S. ruminantium.
Enzyme
Vmax (mmol minꢁ1 mg prot.ꢁ1
)
Km (mM)
Kcat (sꢁ1
)
K )
cat/Km (sꢁ1 mMꢁ1
Free
PEI-coated glyoxyl catalyst
0.43
0.20
0.72
0.45
1.83
0.82
2.47
1.82
The activity was assayed at different pNPX concentrations in 0.1 M succinate-NaOH buffer pH 5.3 at 25 ꢀC.
Table 2. Influence of xylose and glucose on the free
b-xylosidase from S. ruminantium and its catalysts.
0,3
0,2
0,1
0
Relative activity (%)
Monosaccharide
(mM)
PEI
catalyst
CNBr
catalyst
Free
enzyme
Xylose
0
25
100
100
100
59.7
46.5
26.1
21.6
14.2
45.6
35.8
22.3
16.0
14.3
60.7
42.6
31.5
20.7
13.6
50
100
150
200
Glucose
0
25
50
100
150
200
100
100
93.2
88.5
81.9
43.5
100
100
100
100
98.9
89.7
76.1
98.1
92.5
77.5
67.8
49.2
0
1
2
3
pNPX (mM)
Figure 7. Saturation curves of initial velocity (V0) with p-nitro-
phenyl b-D-xylopyranoside for the free and immobilized b-xylosi-
dase from S. ruminantium. The activity was assayed at different
concentrations of pNPX in 0.1 M succinate-NaOH buffer pH 5.3
at 25 ꢀC. PEI-coated glyoxyl catalyst (*) and free enzyme (g).
The activity was assayed in 0.05 M sodium acetate
buffer pH 5.0 at 25 ꢀC.
Hydrolysis of xylooligosaccharides
The hydrolysis of various XOS by the PEI-coated
glyoxyl catalyst was carried out in pH 5.0 at 50 ꢀC,
and the reaction products were analyzed by HPLC.
Xylose release was observed from all substrates over
time, including from natural XOS. Productions of
0.19 and 0.07 g/l were obtained from XOS derived
little more pronounced in the PEI-coated catalyst
(Table 2). At 200 mM glucose, the enzyme in the
PEI-coated catalyst was more than 50% inhibited
while the free enzyme was only a little more than
20% inhibited when using pNPX at 5 mM.

8
C. R. F. Terrasan et al.
from beechwood and Cannabis xylans, respectively.
Product profile at different periods of the hydrolyzed
substrates is presented in Table 3. As observed, the
enzyme presented an exo-type action as the main
product formed after a short period was always one-
xylosyl residue shorter than the incubated substrate.
The rate of xylose release decreased with the
substrate chain-length and, consequently, increased
the period to obtain complete conversion of substrate
to xylose. The conversion of each substrate to xylose
decreased with time what can be attributed to
inhibition caused by xylose accumulation.
Catalyst reuse and storage
The application of a biocatalyst in industry requires
the stability of immobilized enzymes through many
operational cycles. Long-term storage is also an
important characteristic, since increased shelf life is a
desired characteristic of bioproducts. The PEI-
coated glyoxyl catalyst fully retained its activity
after five re-use cycles; once again indicating the
enzyme did not suffer subunit dissociation (Figure
8a). During these several hydrolysis–washing–dilu-
tion cycles, the curves of product were similar during
five-hydrolysis cycles (Figure 8b). From the sixth
cycle, the activity gradually decreased and conse-
quently lower product release was observed specially
in the beginning of the cycle. The biocatalyst also
demonstrated to be very stable during long-term
storage since after 6 months under refrigeration it
fully retained the hydrolytic activity.
Discussion
The b-xylosidase from the ruminal anaerobic bac-
terium Selenomonas ruminantium is a very well
characterized enzyme, and owing to its interesting
properties, the enzyme presents high potential for
application in industrial processes (Jordan et al.
2007a, 2007b; Jordan 2008; Jordan and Wagschal
2010). Due to its stability at alkaline pH, the enzyme
could be easily immobilized on agarose highly
activated with glyoxyl groups by carrying out immo-
bilization for 0.5 h at pH 10 and 25 ꢀC.
Immobilization on glyoxyl occurs through the
enzyme surface region having the highest density of
lysine residues and has been the chosen protocol
to obtain very intense enzyme-support multipoint
covalent attachment for many enzymes. Besides,
prolonged incubation period can increase protein-
support attachments that could positively influence
the catalyst properties in relation to its stability
under different conditions (Mateo et al. 2006).
Nevertheless, the catalyst prepared by carrying out

ꢀ-xylosidase from Selenomonas ruminantium
9
(a)
(b)
100
80
60
40
20
0
0
10
20
30
40
50
60
Time (min.)
Figure 8. Operational stability (a) and product release (b) from p-nitrophenyl b-D-xylopyranoside hydrolysis by the PEI-coated glyoxyl
catalyst of the b-xylosidase from S. ruminantium. The substrate was prepared in 0.05 M sodium acetate buffer pH 5.0 and reaction was
carried out at 25 ꢀC. Enzyme activity was assayed in the same buffer at 25 ꢀC. (a) Residual b-xylosidase activity after consecutive cycles (g).
(b) Full-lines: product release from the first to the fifth cycle; dashed lines: product release from the sixth (m), seventh (J ), and eighth
cycle (I ).
immobilization for 15 h had lower stability than
previously observed, that may be attributed to the
distortion of the enzyme structure resulting from
excessive attachments (Rodrigues et al. 2013). The
marginal stability of biocatalysts has often prevented
or delayed their implementation in industry.
Consequently, there is great interest in understand-
ing effects of reaction conditions on protein stability,
as well as in developing strategies to improve protein
stability (Polizzi et al. 2007). In the case of multi-
meric enzymes, as the tetrameric S. ruminantium
b-xylosidase, the stabilization can be considered a
special and complex problem in which the subunits
dissociation can result in inactivation and even
contamination of the product. In this sense, post-
immobilization strategies such as coating the catalyst
with polymers can stabilize the quaternary structure
of immobilized enzymes rendering much more
b-xylosidases can be associated to the substrate
polymerization degree; for example, those from
Aspergillus phoenicis (Rizzatti et al. 2001) and
Aspergillus versicolor (Andrade et al. 2004) hydrolyze
only up to xylotriose, while that from Scytalidium
thermophilum (Zanoelo et al. 2004) hydrolyzed up to
xylotetraose. Similarly to free enzyme (Jordan et al.
2007a), the immobilized b-xylosidase from S.
ruminantium is able to hydrolyze up to xylohexaose,
and increasing chain-length reduces hydrolysis rate
of the substrate. After immobilization, kinetic par-
ameters of the b-xylosidase from S. ruminantium
were affected, i.e., Km and Vmax were 1.6- and 2-fold
decreased, respectively. Vmax values for other
glyoxyl-immobilized enzymes are also reduced such
as lactase from Kluyveromyces lactis (Mateo et al.
2004), invertase Saccharomyces cerevisiae (Goulart
et al. 2008) and penicillin G acylase from Escherichia
coli (Illanes et al. 2010). Despite the fact that
immobilization on glyoxyl agarose is considered a
very mild system due to the open pore structure and
geometric congruence between enzyme and support
(Mateo et al. 2006), mass transfer limitations are
relevant in the case of immobilized enzymes; besides,
factors other than diffusional restrictions, possibly
conformational change as a consequence of immo-
bilization, may also be playing a role in determining
the catalytic properties of immobilized enzymes
(Illanes et al. 2010). Changes in kinetic parameters
after immobilization may also lead to changes in
´
thermostability to the biocatalyst (Fernandez-
Lafuente et al. 1999; Bolivar et al. 2009). Thus,
covalent immobilization of the b-xylosidase from S.
ruminantium initially involves intense but controlled
multipoint attachments and full stabilization is
achieved by coating its surface with polyethylenimine
and aldehyde dextran, resulting in a catalyst that is
more than 240-fold more stable than the free
enzyme. Free and immobilized enzyme presented
similar characteristics as optimum temperature and
pH. The catalyst also retained its side activity against
pNPAra, a very important characteristic special
considering hydrolysis of arabinoxylans, once the
presence of side chains may hinder the action of
other xylanolytic enzymes involved in xylan degrad-
ation (Rahman et al. 2003) The activity of different
´
inhibition of enzyme (Garcia-Galan et al. 2001), as
verified for the lactase from Kluyveromyces lactis,
which inhibition by glucose was strongly decreased
(Mateo et al. 2004). The immobilized b-xylosidase

10 C. R. F. Terrasan et al.
from S. ruminantium is inhibited by xylose, similar to
previous observations for the free enzyme (Jordan
and Wagschal 2010). The free and immobilized
enzyme is also inhibited by glucose, an important
consideration particularly in the application of this
catalyst for biomass saccharification (Kristensen
et al. 2009).
Jose Manuel Guisan
1627-6522
http://orcid.org/0000-0003-
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The authors declare not having any conflict of
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Ministry of Science and Innovation (Project BIO-
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Cesar Rafael Fanchini Terrasan
0000-0002-7988-4755
http://orcid.org/
Caio Casale Aragon
1138-6290
http://orcid.org/0000-0002-
http://orcid.org/0000-
http://orcid.org/0000-
Douglas Chodi Masui
0002-3858-1467
Benevides Costa Pessela
0003-1747-0124
Gloria Fernandez-Lorente
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