Isolation and properties of β-xylosidase from Aspergillus niger GS1 using corn pericarp upon solid state fermentation

Article abstract of DOI:10.1016/j.procbio.2013.05.003

There is growing interest in developing high-yield and low-cost production of xylanolytic enzymes for industrial applications using agroindustrial byproducts. A native strain of Aspergillus niger GS1 was used to produce β-xylosidase (EC 3.2.1.37) on solid state fermentation using corn pericarp (CP) with innovative alkaline electrolyzed water (AEW) pretreatment at room temperature. β-xylosidase was purified by ammonium sulfate fractionation followed by anion exchange and hydrophobic interaction chromatographies. β-Xylosidase showed a molecular weight of 111 kDa, isoelectric point of 5.35 and specific activity of 386.7 U (mg protein)^-1, using p-nitrophenyl-β-d-xylopyranoside as substrate, at pH 5 and 60 C, and optimal activity at pH 4.5. Optimal temperature was 65 C, showing full activity after 1 h at 60 C. Activity was reduced by 1 mM β-mercaptoethanol (55.6 ± 0.1%), and enhanced by 1 mM SDS (11.0 ± 0.03%). K_m and V_max were 6.1 ± 0.9 mM and 1364 ± 105 U (mg protein)^-1, respectively, whereas k_cat was 5.1 s ^-1. A predominant α-helix (41%) was determined from circular dichroism on β-xylosidase, while thermal transition profiles produced a T_m of 54.1 ± 5.8 C, enthalpy change for unfolding of 67.4 ± 6.7 kJ/mol, and onset temperature of 37 C. Pre-treatment of CP using AEW is an ecologically friendly alternative to chemical and heat treatments for the production of relatively high levels of β-xylosidase.

Full text of DOI:10.1016/j.procbio.2013.05.003

Process Biochemistry 48 (2013) 1018–1024
Contents lists available at SciVerse ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Isolation and properties of ␤-xylosidase from Aspergillus niger GS1 using corn
pericarp upon solid state fermentation
a
a
b
F.I. Díaz-Malváez , B.E. García-Almendárez , A. Hernández-Arana ,
a
a,∗
A. Amaro-Reyes , C. Regalado-González
DIPA, PROPAC. Facultad de Química, Universidad Autónoma de Querétaro, C.U. Cerro de las Campanas s/n. Col. Las Campanas, C.P. 76010 Querétaro, Qro., Mexico
a
b
Departamento de Química, Universidad Autónoma Metropolitana-Iztapalapa, Av. San Rafael Atlixco No. 186, Col. Vicentina, Delegación Iztapalapa, C.P. 09340 México, D.F., Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
There is growing interest in developing high-yield and low-cost production of xylanolytic enzymes for
industrial applications using agroindustrial byproducts. A native strain of Aspergillus niger GS1 was used
to produce ␤-xylosidase (EC 3.2.1.37) on solid state fermentation using corn pericarp (CP) with innova-
tive alkaline electrolyzed water (AEW) pretreatment at room temperature. ␤-xylosidase was purified by
ammonium sulfate fractionation followed by anion exchange and hydrophobic interaction chromatogra-
phies. ␤-Xylosidase showed a molecular weight of 111 kDa, isoelectric point of 5.35 and specific activity
Received 13 November 2012
Received in revised form 7 April 2013
Accepted 2 May 2013
Available online 13 May 2013
Keywords:
−1
◦
of 386.7 U (mg protein) , using p-nitrophenyl-␤-d-xylopyranoside as substrate, at pH 5 and 60 C, and
␤
-xylosidase
◦
◦
optimal activity at pH 4.5. Optimal temperature was 65 C, showing full activity after 1 h at 60 C. Activity
Aspergillus niger
Purification
Solid state fermentation
Circular dichroism
was reduced by 1 mM ␤-mercaptoethanol (55.6 ± 0.1%), and enhanced by 1 mM SDS (11.0 ± 0.03%). Km
−
1
−1
and Vmax were 6.1 ± 0.9 mM and 1364 ± 105 U (mg protein) , respectively, whereas kcat was 5.1 s . A
predominant ␣-helix (41%) was determined from circular dichroism on ␤-xylosidase, while thermal tran-
◦
sition profiles produced a Tm of 54.1 ± 5.8 C, enthalpy change for unfolding of 67.4 ± 6.7 kJ/mol, and onset
◦
temperature of 37 C. Pre-treatment of CP using AEW is an ecologically friendly alternative to chemical
and heat treatments for the production of relatively high levels of ␤-xylosidase.
©
2013 Elsevier Ltd. All rights reserved.
1
. Introduction
glycosidases have potential applications; food (fruit, vegetable
processing, brewing, wine production, baking), feed (animal feed),
technical (paper and pulp, textile, bioremediation/bioconversion)
and biofuel (second generation biofuels) industries. Important
applications have been attributed to thermophilic hemicellulases,
such as processes where high temperatures are required to increase
bioavailability and/or solubility of substrates, to reduce viscos-
ity and/or to reduce risk of contamination [3,6–8]. ␤-xylosidase
hydrolyses xylobiose and xylooligosaccharides to xylose from the
non-reducing end [9,10]. Filamentous fungi secrete ␤-xylosidases
into the medium and are thus particularly interesting producers
of this enzyme from an industrial point of view [10,11]. Several
fungi such as Trichoderma reesei, Aspergillus niger and Penicillium
sp. have been employed for hemicellulases production using solid-
state fermentation (SSF) in which basal mineral salts medium is
used for moistening the substrate [12,13]. SSF is defined as that
occurring in absence or near absence of free water [14]. How-
ever, the substrate must possess enough moisture to support
growth and metabolism of microorganisms and it has recently
gained importance for the production of microbial enzymes due
to several economic advantages over conventional submerged fer-
mentation [13]. Recently, acidic and alkaline electrolyzed water
have been used as pretreatment for partial removal of plant cell wall
complex structures followed by commercial enzymatic treatment
Hemicellulose is the second most common polysaccharide in
nature and represents about 20–35% of lignocellulosic biomass.
Xylans are the major portion of plant cell walls hemicellulose, and
are heteropolymers containing mainly xylose and arabinose [1,2].
Agroindustrial residues such as corn pericarp (CP), cobs and stub-
ble; wheat, and rice straw contain about 20–40% hemicellulose [3].
CP is a byproduct from the wet milling industry and represents
5
.3% of whole corn kernel, comprising up to 70% hemicellulose
−
1
[
4]. CP is highly available in Mexico (∼0.1 million tons year
)
and may provide a low cost feedstock. However, high value added
biological products such as enzymes, fuels and chemicals, may be
produced with environmental and strategic advantages [5]. Due to
heterogeneity and complex chemical nature of plant xylans, sev-
eral hemicellulolytic enzymes with diverse specificities and modes
of action are needed for their complete breakdown. ␤-xylosidase
(
EC 3.2.1.37) in synergistic action with endo-␤-xylanases and
debranching enzymes namely ␣-glucuronidases, esterases and
∗ Corresponding author. Tel.: +52 442 1921307.
E-mail addresses: carlosr@uaq.mx, regcarlos@gmail.com
(
C. Regalado-González).
1
359-5113/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.procbio.2013.05.003

F.I. Díaz-Malváez et al. / Process Biochemistry 48 (2013) 1018–1024
2.4. Scanning electron microscopy (SEM)
1019
for biofuels production [15–19]. In all studies substrate pretreat-
◦
ment was successful at temperatures >170 C suggesting expensive
SEM was used to observe superficial morphological changes of CP before and
pressure tight reactors and a high energy process involved. The
molecular weight of ␤-xylosidases ranges 37.5–360 kDa and can
be monomeric, dimeric, or trimeric which most from fungi show
molecular weights above 100 kDa, and those from A. niger exhibit
optimum pH in the range 5–6.5 [10,20]. Based on their primary
sequence, ␤-xylosidases have been grouped into different fami-
lies (CAZy database, http://www.cazy.org) the so-called glycoside
hydrolases GH 3, 30, 39, 43, 52 and 54 [13,15,21]. The majority of
the isolated and characterized ␤-xylosidases are optimally active
at temperatures ranging 25–40 C, hampering their application as
robust tools for the industry, bearing in mind that xylan degra-
dation products have the potential as energy source in the future
10].
The aim of this work was to isolate and evaluate the properties of
a new ␤-xylosidase produced from Aspergillus niger GS1, using solid
◦
after the alkaline pretreatment. Samples were dehydrated at 120 C for 1 h, mounted
on stubs and sputter-coated with gold/palladium for 300 s (EMS, model S550, Hat-
field, PA, USA), using high vacuum and a voltage acceleration of 20 kV. SEM was
performed in a Jeol JSM 5200 (Tokio, Japan) on previously coated CP.
2.5. Protein determination, ˇ-xylosidase and xylanase enzymatic assays
Protein concentration was determined using the method of Bradford [32] with
bovine serum albumin (BSA) as standard. Xylosidase activity was measured by the p-
nitrophenol method [33], using 10 mM p-nitrophenol-␤-d-xylopyranoside (PNPX)
in 50 mM sodium acetate buffer, 30 ␮L of enzyme solution at pH 5.0 in a total reac-
tion volume of 1.5 mL. After incubation at 60 C for 10 min, the reaction was stopped
by adding 1.0 M Na2CO3 (JT Baker, Phillipsburg, NJ, USA) to a final concentration of
◦
◦
0
4
.3 mM, and the p-nitrophenol released was measured spectrophotometrically at
10 nm. One unit of ␤-xylosidase was considered as the amount of enzyme produc-
[
ing 1 ␮mol equivalent of p-nitrophenol per min. Xylanase activity was determined
−
1
using birchwood xylan (5 g L ; Sigma), dissolved in 50 mM sodium acetate buffer,
pH 5.5. Enzyme extracts (0.4 mL) were added to the substrate (0.4 mL) and incubated
state fermentation on alkaline electrolyzed water (AEW) pretreated
CP.
◦
at 50 C for 10 min followed by immersion in iced water. Reducing sugars released
were quantified using the DNS method [34], and a xylose standard curve. One activ-
ity unit (U) was defined as the amount of enzyme that releases 1 ␮mol of xylose per
◦
min, at 50 C.
2. Materials and methods
2
.6. Enzyme concentration and purification
2.1. Materials
The crude extract was concentrated by tangential flow ultrafiltration using a
All chemicals were of analytical grade and were purchased from Sigma (St. Louis,
Pellicon unit (Millipore, Billerica, Massachusetts, USA), employing a 0.5 m2 Biomax-
5 (Millipore) polyethersulfone membrane of 5 kDa molecular weight cut off, at
4
MO, USA), except as indicated. CP was obtained from Ingredion, San Juan del Río,
Querétaro, México. AEW containing NaOH = 0.55–0.60 g L , pH = 11.5–12.5 (Limy®
−1
)
◦
−2 −1
h , followed by dialysis against distilled water.
C. Tangential flux was 0.20 L m
was provided by Grupo EcoRus AEQ, Mexico.
Samples of the concentrated extract were partially purified by differential ammo-
◦
nium sulfate precipitation at 4 C, using the following gradient: 0–25%, 25–50%,
5
1
0–80% and 80–100% saturation. After every step, samples were gently stirred for
h at 4 C, centrifuged at 14,000 × g for 45 min, and supernatants were subjected
2
.2. Microorganism
◦
to next precipitation stage and treated similarly. Precipitates were redissolved in
50 mM sodium acetate buffer, pH 5.5, dialyzed at 4 C against the same buffer with
three changes every 8 h, filtered through 0.45 ␮m membrane (Durapore, Millipore,
Ireland) and tested for activity. The concentrated extract was partially purified using
the best activity recovery conditions and was freeze dried (LabConCo, Freezone 18,
Kansas City, MO, USA).
Aspergillus niger GS1 isolated from copra paste and molecularly identified (NCBI
◦
◦
No. GU 95669) [22] was used as a source of ␤-xylosidase. It was propagated at 30
for seven days in potato dextrose agar. Inoculum was prepared by suspending the
conidia from PDA by adding sterile 0.1% Tween-80 solution.
C
A 2.5 cm × 23 cm column filled with Macro-Prep High Q [binding capacity 40 mg
2
.3. Substrate and solid state fermentation
−
1
protein (mL resin) , Bio-Rad, Hercules, CA, USA] strong anion exchange, was pre-
equilibrated with 50 mM sodium acetate buffer (pH 5.5). Ten mL (100 mg) protein
sample was injected into the column. Elution was conducted using 80 mL of buffer at
CP was washed with distilled water and treated with AEW diluted in a 1:4
ratio with distilled water, achieving 150 ppm NaOH, pH 9.2, contacted for 5 h at
room temperature (25 ± 2 C) with gentle stirring followed by rinsing with distilled
−
1
◦
a flow rate of 1 mL min , followed by 200 mL of a linear NaCl gradient (0–0.6 M) of
same buffer, and a final elution using 200 mL of 0.6 M NaCl. Four mL fractions were
collected, using a R-200 fraction collector (Pharmacia, Uppsala, Denmark). Active
fractions were pooled, dialyzed and freeze dried, as previously mentioned.
Saturated ammonium sulfate was added to the anion exchange fraction con-
taining ␤-xylosidase activity to 1.5 M final concentration. A sample of 10 mL
water, dried and milled (Cemotec, Tecator, Hillerød, Denmark). The resulting powder
was separated by sieving and particle size in the range 0.40–1.40 mm was used. CP
was analyzed for moisture (gravimetrically) [23], crude protein (Nx6.25) [24], ether
extract [25], and ash [26]. Hemicellulose, cellulose and lignin (organic matter based)
were determined from neutral (NDF) [27] and acid (ADF) detergent fibers [28] using
an ANKOM 200 digestion equipment (ANKOM Technology, Macedon, NY, USA). Pre-
liminary experiments varying carbon and nitrogen sources concentration as well as
moisture content did not produce better results than those obtained from previously
optimized experimental conditions [29]. Thus, the nutritional supplement used con-
(
90 mg protein) was injected to a column (1.5 cm × 54 cm) filled with Macro-Prep
methyl hydrophobic interaction chromatography [HIC, binding capacity >25 mg (mL
−
1
resin) , Bio-Rad], equilibrated with 20 mM sodium acetate buffer (pH 5.5) con-
taining 1.5 M (NH4)2SO4. The column was washed with 2-bed volumes of same
buffer and eluted with a 7-bed volume decreasing (NH4)2SO4 gradient (1.5–0 M)
−
1
−1
−1
−1
tained yeast extract 16.6 g L , (NH4)2SO4 1.44 g L , glucose 8.43 g L , Na2HPO4
4
0
−
1
−
1
−1
−1
at flow rate of 1 mL min . Four mL fractions were collected and active fractions
were pooled, dialyzed and freeze dried.
g L , MgSO4·7H2O 0.5 g L , FeSO4·7H2O 0.31 g L , KH2PO4 2 g L , ZnSO4·7H2O
−
1
−
1
.1 g L , CuSO4·5H2O 0.0138 g L . The inoculum of A. niger was added at a final
7
−1
−1
count of 1 × 10 conidia (g dry CP) . Moisture was adjusted to 80% (w v ) and
−1
packing density was 0.6 g mL , measured by adding a known weight of wet sub-
2.7. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and
isoelectric point (pI)
◦
strate to an identified volume of the column. Fermentation was kept at 30 C by
circulating water inside a jacketed stainless steel cylindrical bioreactor, containing
a helical tubing allowing for water circulation along the central axis of the cylin-
der [30]. A previous report from our group showed that fermentation time could
be limited to 24 h [29]. In addition, according to the supplier CP contains residual
starch, which might have accelerated fungal growth. Compressed air supply was
sterilized using a Millex-FG50 5 cm filtration disk, with 0.2 ␮m pore size (Millipore,
Bedford, MA, USA), bubbled through a 3 L flask containing sterile distilled water to
produce nearly saturated air. Air flow was set at 0.2 vvm [(volume of air) (volume of
substrate) (min )]. Air passed through a stainless steel mesh located 5 cm above
the bottom of the reactor and covered with 2 layers of cheese cloth. Aeration rate
required by A. niger ranges 0.2–0.5 vvm for high enzyme productivity [31]. Higher
air flow tended to dry the top section of the substrate in the reactor, while increas-
ing moisture content at the bottom. The fermented product (5.5 kg) was mixed with
Aliquots from pooled active fractions from HIC were subjected to SDS-PAGE
using 10 T (% acrylamide plus bis-acrylamide in gelling solution) according to
Laemmli [35] without ␤-mercaptoethanol, and heating at 60 C for 5 min. Protein
◦
bands were stained with Coomassie brilliant blue G-250 (Bio-Rad). ␤-xylosidase
activity was detected in the gel after electrophoresis by cutting the bands from non-
stained gels. Each band was washed three times with Triton X-100 solution (2.5%,
−
1
vv ), placed in a microtube containing 300 ␮L substrate solution (10 mM PNPX),
−
1
−1
◦
and incubated for 60 min at 60 C. The reaction was stopped by adding 300 ␮L of
1 M Na2CO3, and absorbance was measured at 400 nm, against a blank obtained by
using a gel fragment without protein.
High molecular weight markers (GE Healthcare, Fairfield, CT, USA) were used to
estimate the molecular weight of the purified enzyme. The isoelectric point of ␤-
xylosidase was determined by using ultrathin (0.2 mm) polyacrylamide gels (5 T) for
electrofocusing (100 mm × 125 mm) in a Bio-phoresis cell (Bio-Rad) system with IEF
ampholites (3–10, Serva, Heidelberg, Germany). Protein bands were detected using
silver staining [36].
5
0 mM sodium acetate buffer, pH 5.5, using an equal weight ratio, and the mixture
◦
was stirred in an orbital shaker at 150 rpm, 4 C, for 60 min. Subsequently, the liquid
extract was separated and centrifuged at 10,000 × g for 30 min. The supernatant was
filtered thought Whatman No. 1 filter paper to obtain a crude extract.

1
020
F.I. Díaz-Malváez et al. / Process Biochemistry 48 (2013) 1018–1024
Fig. 1. Scanning electron microscopy (SEM) of CP surface. (A) untreated CP; (B) CP pretreated with AEW. Pretreated CP clearly shows degradation of a layer covering the
hemicellulose fibers.
Table 1
2.8. Kinetic properties and effect of temperature and pH on ˇ-xylosidase activity
CP proximal analysis of pretreated and untreated CP.
Concentrations of p-nitrophenyl-␤-d-xylopyranoside were varied from 0.3 to
Chemical composition
Pretreated CP %
Untreated CP %
(dry basis)
1
0 mM. Substrate dilutions were contacted with 0.2 ␮g ␤-xylosidase in sodium
(
dry basis)
◦
acetate buffer pH 5.0, at 60 C, in a total volume of 300 ␮L. Kinetic parameters were
estimated by fitting initial velocities (Vo) versus substrate concentrations to the
Michaelis–Menten equation using a non-linear correlation curve-fitting software
*
*
Moisture
8.75 ± 0.07
5.54 ± 0.15
0.36 ± 0.13
0.90 ± 0.02
8.15 ± 0.06
Protein (Nx6.25)
Ether extract
Ash
Neutral detergent fiber (NDF)
Acid detergent fiber (ADF)
Hemicellulose
Cellulose
5.47 ± 0.03
0.25 ± 0.03
0.8 ± 0.03
(
Graph-Pad Prism 5.00; San Diego, CA, USA).
◦
The effect of temperature on ␤-xylosidase activity, was evaluated between 40 C
◦
and 75 C, at optimal pH. The effect of pH was determined from 4.0 to 5.5 using
83.27 ± 0.47
18.42 ± 0.21
64.22 ± 0.64
17.14 ± 0.21
1.28 ± 0.06
84.95 ± 0.77
*
*
5
0 mM sodium acetate buffer, 50 mM sodium citrate buffer for pH 6.0, while 50 mM
20.70 ± 0.23
sodium phosphate buffer was used for pH between 6.5 and 7.5, at optimal tempera-
ture. Thermal stability was determined by incubating enzyme solutions in absence
64.25 ± 0.96
*
*
*
18.12 ± 0.23
◦
◦
◦
of substrate at 60 C, 65 C and 70 C, taking samples every 10 min up to 1 h and
immediately ice cooled. Residual activities were assayed under standard conditions.
Lignin
2.58 ± 0.40*
Mean of three replicates ± standard deviation.
*
Significant difference p < 0.05.
2
.8.1. Effect of chemicals on ˇ-xylosidase activity
To evaluate the effect of various metal ions on ␤-xylosidase activity, 1 mM solu-
tions of Fe2 , Ca , Cu , Hg , SDS, EDTA and 2-mercaptoethanol were added to the
reaction mixtures containing the enzyme in 50 mM sodium acetate buffer (pH 5.0),
at room temperature. A reaction mixture without any additive was used as control
and its xylosidase activity was designated as 100%.
+
2+
2+
2+
However, CP was treated with AEW in an attempt to enhance
xylanolytic enzymes production.
2.9. Far-UV circular dichroism (CD) spectra
3.2. Effect of AEW on CP
The CD spectrum of purified ␤-xylosidase in 20 mM sodium acetate buffer
pH 5.0, was recorded over the range 200–250 nm using a Jasco J-715 spectropo-
larimeter (Easton, MD, USA) fitted with a PTC-348WI Peltier-type cell holder. The
From SEM it was clear that AEW pretreatment modified the
superficial structure of CP (Fig. 1). Initially CP showed an exter-
nal layer probably comprising cutin and suberin (Fig. 1A), whereas
after AEW pretreatment this layer almost disappeared and hemicel-
lulose fibers were visible with thickness of about 5–6 ␮m (Fig. 1B).
The evident surface exposure of these fibers represented a more
available target for hemicellulolytic activity production during SSF.
The width of hemicellulose fibers of Gingko (Gingko biloba) was
about 3 ± 0.5 nm in a perpendicular plane orientation [39], while
the diameter of raw CP fiber was reported as about 5 ␮m [40].
A study using distiller’s dried grains with solubles (mainly corn
fiber) pretreated with AEW reported disrupted crystalline struc-
tures and structural alterations, leading to increased amounts of
fermentable sugars for biofuel production by microbial fermenta-
tion [41]. The amount of NaOH present in AEW confers surface
active properties, enabling partial removal of waxes (long chain
primary and secondary alcohols, aldehydes and ketones, and ester-
ified fatty acids) associated to cutin and suberin of CP [39,42,43].
However, the difference between lipid content (ether extract) of
AEW pretreated and untreated CP was not significant (Table 1),
suggesting another more significant effect of AEW pretreatment.
0
.10 cm wide quartz cell was under constant nitrogen flush. Protein concentration
−
1
(
0.10 mg mL ) was determined by absorbance at 280 nm using an extinction coeffi-
−1
−1
cient of 217,165 M cm , calculated from a recombinant A. niger ␤-xylosidase [22],
using the ProtParam tool of the ExPASy portal (http://web.expasy.org/protparam/).
Native ␤-xylosidase spectra were obtained at 25 C from freshly prepared samples,
which were then heated to 90 C and kept at this temperature until no further
changes in ellipticity were noticed. CD spectra were then recorded for denatured
␤
spectra of these samples once cooled to 25 C. Temperature-induced denaturation
transitions were monitored by recording the ellipticity at 210 nm from 25 C to
◦
◦
-xylosidase. Reversibility of thermal denaturation was tested by recording the
◦
◦
◦
◦
−1
9
0
C heating at a constant rate of 1.0 C min , using 1.0 cm cells. Renaturation
profiles were recorded, once thermal denaturation was completed, by cooling
◦
◦
−1
to 25 C at a rate of 1.0 C min . Ellipticities are reported as molar ellipticity
[
(
(
ꢀ] (degrees cm2 dmol ), based on an assumed mean amino acid residue weight
−1
−
1
MRW) of 112 Da. Thus [ꢀ] = ꢀ (100 MRW) (c l) , where c is protein concentration
mg mL 1), l is light path length (cm), and ꢀ is the measured ellipticity (degrees) [37].
−
CD spectra were reported as the mean of three scans corrected by subtracting the
adequate blank runs on xylosidase-free buffer. CD data were employed to calculate
secondary structure from the Dichroweb server (http://dichroweb.cryst.bbk.ac.uk)
using the K2D method [38]. The thermal denaturation profile was analyzed by means
of a two-state model, where the fraction of unfolded protein (fU) is expressed as fU = K
−
1
−1
−1
(
1 + K) , where the equilibrium constant is given by K = exp[−ꢁH(RT) + ꢁS(R) ].
All data are expressed as mean ± SD of 3–5 experiments.
−
1
Alkali pretreatment [2%, w v , Ca(OH) ] was reported to remove
2
3
. Results and discussion
the waxy cutin layer of CP leading to exposure of hemicelluloses
fibers [40].
3
.1. Proximal analysis of CP
Partial delignification of CP cell wall was observed after AEW
pretreatment, and this was confirmed by ADF determination, that
evaluates the sum of cellulose plus lignin which resulted in sig-
nificantly lower value for AEW pretreated than untreated CP
(Table 1). This effect probably allowed an easier fungal accessi-
bility to the substrate upon SSF. AEW pretreatment represents an
Hemicellulose was the major component of both CP pretreated
with AEW and untreated CP, followed by cellulose, while lignin
was relatively low (Table 1). The low lignin content allowed fungal
growth on CP and thus SSF was used without previous treatment.

F.I. Díaz-Malváez et al. / Process Biochemistry 48 (2013) 1018–1024
1021
5
4
3
2
1
0000
0000
0000
0000
0000
0
0.35
0.30
0.8
AEW treated CP
Untreated CP
Protein (A280
)
Xylosidase (A510
)
0
.6
NaCl
0.6
0
.25
0.20
.15
0.10
.05
0.00
0.4
0.4
0
0.2
0.2
0
1
0
15
20
25
30
35
40
45
0.0
0.0
Fermentation time (h)
0
20
40
60
80
100
120
Fraction No. (4 mL)
Fig. 2. Comparison of the total xylanase activity produced after fermentation of pre-
◦
treated CP with AEW diluted 1:4, contact time of 5 h at room temperature (25 ± 2 C).
Fig. 3. Macro-Prep high Q anionic exchange chromatography of ␤-xylosidase from
Aspergillus niger GS1. The active fractions were eluted with a linear gradient of NaCl
from 0 to 0.6 M.
Error bars indicate standard deviation of the mean of three replicates.
environmentally friendly alternative to the use of hazardous chem-
ical pretreatments, which then require appropriate disposal.
The basic structure of CP arabinoxylans consists of (1–4)-␤-d-
xylopyranosyl (Xylp) backbone, with ␣-l-arabinofuranosyl (Araf)
side units attached to O-2 and/or O-3 of backbone Xylp units. Addi-
tionally, ferulic acid can be esterified to the arabinose side-chains
and create cross-links with other arabinoxylan chains [44,45].
AEW pretreatment may have produced partial diferulates hydrol-
ysis leading to better fungal accessibility during SSF in relation to
higher xylanolytic enzymes production, with essentially no change
in hemicellulose content of AEW pretreated and untreated CP
this temperature. From anion exchange chromatography xylanase
activity was separated in fractions 15–24, giving an activity of
−
1
21 U mg . ␤-xylosidase activity was detected in fractions 70–82
eluted at 0.58–0.6 M NaCl gradient (Fig. 3). These fractions con-
tained 14% of total injected protein and showed a specific activity of
−
1
9.8 U (mg protein) , which were pooled, dialyzed and freeze dried.
After HIC ␤-xylosidase activity was detected in 10 fractions (results
not shown), which were pooled and dialyzed giving a specific activ-
−
1
ity of 386.7 U (mg protein) . A summary of the purification steps
required to achieve ␤-xylosidase is shown in Table 2.
(Table 1).
3
.4.1. Biochemical and kinetic properties of ˇ-xylosidase
3
.3. SSF of AEW pretreated CP
The optimal temperature of ␤-xylosidase under assay condi-
◦
tions was 65 C (Fig. 4A), which was lower than that of native and
recombinant ␤-xylosidase from Aspergillus niger [22,47]. In addi-
tion, the enzyme showed activity at acidic pH only with an optimum
pH of 4.5, and ≥70% activity was observed from pH 3.5 to 6.0
Total xylanase activity production by A. niger on AEW pretreated
CP after 24 h of fermentation was about 3.4 fold over the untreated
substrate. Adequate nutrients supplementation combined with
available carbon sources allowed a rapid fungal growth whereas
xylosidase activity could not be detected (Fig. 2). In a similar study
(Fig. 4B). These values were similar to other ␤-xylosidases isolated
from Aspergillus carbonarius [2]. Good thermal stability results were
found for ␤-xylosidase because it was fully stable at 60 C for 60 min
␤
-xylosidase could not be detected in the crude extract [46]. The
◦
−
1
protein solution (0.09 mg mL ) was concentrated by ultrafiltration
from 5.5 L to 200 mL. Activity recoveries from concentrated crude
extract using differential ammonium sulfate precipitation were:
◦
(Fig. 4C). Incubation at 65 C resulted in 70% activity retention after
◦
3
0 min, whereas at 70 C activity retained was 10% at same incuba-
tion time. Furthermore, 20% relative activity was retained after only
1
niger USP-67 was purified and immobilized in polyethyleneimine-
sepharose; it was more thermal stable than the soluble enzyme
and on other supports, and showed 50% activity loss after 50 min,
at 65 C [48]. Thus, thermal stability was slightly lower than our
purified ␤-xylosidase (62% activity retention after 50 min at 65 C,
Fig. 4C). On the other hand, A. niger IBT-3250 was more thermally
stable, showing 44% activity retention after 1 h at 75 C [33].
5
.7% for 0–25% fraction, 6.4% for 25–50% fraction, 64% for 50–80%
◦
0 min of incubation at 70 C (Fig. 4C). ␤-xylosidase produced by A.
fraction and 2.6% for 80–100% fraction. After using the 50–80% frac-
tion, the final extract was dialyzed achieving a volume of 80 mL
showing a protein concentration of 1.4 mg mL , and ␤-xylosidase
−
1
−
1
activity of 0.30 U (mg protein)
.4. Purification of ˇ-xylosidase
Xylanolytic activity was stable at room temperature for sev-
.
◦
◦
3
◦
From SDS-PAGE purified ␤-xylosidase showed a molecular
eral hours and therefore all purification steps were performed at
weight (MW) of 111 kDa (Fig. 5A) under reducing and non
Table 2
Summary of the purification procedure to obtain ␤-xylosidase from A. niger GS1.
Purification steps
Crude extract
Protein (mg)
Total activity (U)
Specific activity [U (mg protein)−1]
Yield (%)
Purification (fold)
4987
114.1
15.7
1514
156.4
153.9
7.28
0.30
1.37
9.80
386.7
100
1.00
4.57
32.67
1289
(
NH4)2SO4 precipitation and concentration
10.3
10.2
0.48
a
AEC
b
HIC
0.019
a
Macro-Prep High Q anion exchange chromatography (Bio-Rad).
Macro-Prep methyl hydrophobic interaction chromatography (Bio-Rad).
b

1
022
F.I. Díaz-Malváez et al. / Process Biochemistry 48 (2013) 1018–1024
(A)
100
80
60
40
20
0
40
45
50
55
60
65
70
75
80
o
Temperature ( C)
(B)
120
100
Fig. 5. (A) SDS-PAGE of purified ␤-xylosidase from Aspergillus niger GS1. Lane
, molecular weight markers: myosin rabbit muscle (220 kDa), 2-macroglobulin
bovine plasma (170 kDa), galactosidase from E.coli (116 kDa), human transferrin
80
60
40
20
0
1
(
(
76 kDa), bovine liver glutamic dehydrogenase (53 kDa). Lane 2, purified protein.
B) Isoelectric focusing. Lanes: 1 IEF ampholites (range 3-10); 2, purified protein.
Bands were detected by silver staining.
reducing conditions, suggesting a monomeric protein (results not
shown). Most fungal ␤-xylosidases exhibit MW above 100 kDa [20],
whereas those produced by Aspergillus sp. range 60–190 kDa [10],
suggesting that our enzyme size is expected for this type of fungi.
From isoelectric focusing purified ␤-xylosidase showed a pI of 5.35,
indicating acidic nature of the enzyme (Fig. 5B). We could only find
one report on the pI of ␤-xylosidase from Aspergillus and corre-
sponds to A. versicolor with a value of 5.6 [49], all other reported
3
4
5
6
7
8
pH
(C)
␤
-xylosidases show pI values above and below that found in this
1
20
00
study, but remain within the values reported for other filamentous
fungi (3.4–7.8) [10].
1
o
6
0 C
From non-linear correlation curve using the Graph-Pad Prism
software, the Km of ␤-xylosidase was 6.1 ± 0.9 mM, Vmax was
−
1
−1
.
8
6
4
2
0
0
0
0
0
1364 ± 105 U (mg protein) , while the kcat value was 5.1 ± 0.1 s
−
1
The apparent second order rate constant [kcat (KM) ] was
−1 −1
0
.84 s mM . The KM value obtained here was higher than other
reported for recombinant (0.48 mM) [50] and native ␤-xylosidases
(0.25 ± 0.07 mM) [51], (0.17–0.22 mM) [2] and 0.66 mM [52], sug-
gesting lower substrate affinity. In this study Vmax was 35 times
higher than that reported for ␤-xylosidase from A. ochraceus [52]
implicating higher hydrolysis rate for same substrate concentra-
tion. The kcat value was smaller than that reported for ␤-xylosidase
from Aspergillus awamori X-100 (17.5 ± 2.0 s ) [51], indicating one
third catalytic efficiency. More studies are necessary to fully eval-
uate hydrolytic specificity and analysis of hydrolysis products.
o
6
5 C
o
70 C
−1
0
10
20
30
Time (min)
40
50
60
◦
3.4.2. Influence of chemicals on activity of ˇ-xylosidase
Fig. 4. Effect of pH and temperature on ␤-xylosidase activity. (A) Effect of pH at 25 C.
◦
The activity of ␤-xylosidase was measured in the presence of
metal ions and other agents (Table 3). The enzyme was not affected
Activity was measured at 60 C for 15 min at the indicated pH. (B) Effect of temper-
ature. Activity was measured at pH 5.0 for 15 min at the indicated temperature. (C)
Effect of temperature on ␤-xylosidase stability. Ordinate values are expressed as
relative activity, i.e. ratio of actual activity divided by maximum activity expressed
as percentage. Results represent the mean of three experiments, and bars represent
standard deviation.
2+
2+
2+
by 1.0 mM Fe , Ca , Cu and EDTA. The fact that EDTA did not
affect activity, suggests that no metal cofactors are needed for
the enzymatic reaction, whereas Hg2+ showed a partial inhibi-
tion. ␤-xylosidase was markedly inactivated by ␤-mercaptoethanol
indicating that disulfide bridges have a significant effect on protein

F.I. Díaz-Malváez et al. / Process Biochemistry 48 (2013) 1018–1024
1023
Table 3
which is slightly higher than that reported for a Kluyveromicesmarx-
ianus xylosidase [56]. Onset temperature (To, where the loss of
Influence of various chemicals on activity of ␤-xylosidase from A. niger GS1.
Chemical (1 mM)
% Relative activity
ellipticity signal reaches 5% of the original) for ␤-xylosidase was
◦
3
7 C, which indicates a relatively good initial stability.
None
SDS
EDTA
100
111
103
54
4. Conclusion
␤
-Mercaptoethanol
FeSO4
CaCl2
CuSO4
HgCl2
98
101
105
89
Pretreatment of CP with AEW increased total xylanase activ-
ity production by 3.4 fold after solid state fermentation and a new
-xylosidase from Aspergillus niger GS1 was identified and puri-
␤
fied. Biochemical, thermodynamic and structural properties of the
enzyme are different to those reported previously. Further studies
are needed to assess its applications in industrial processes such as
fruit juice clarification, enzymatic conversion of xylooligosaccha-
rides into monosaccharides and baking industry.
active conformation. Slight enzyme inhibition was found in stud-
ies where SDS was tested [22,53]. However, this detergent caused a
slight activity increase to our xylosidase, perhaps because the active
site was more exposed to the substrate than in native conformation.
Acknowledgments
3.5. CD studies on ˇ-xylosidase
The authors thank to Dr. Menandro Camarillo Cadena (UAM-
Iztapalapa, Mexico) for kindly support in DC studies. Finantial
support from the Consejo Nacional de Ciencia y Tecnología, Mexico,
is appreciated.
The far-UV CD spectrum of native ␤-xylosidase (Fig. 6) is char-
acteristic of proteins showing ␣/␤ structure [7], where the broad
minimum around 210 nm and the shoulder close to 220 nm suggest
-helix together with ␤-sheet conformations [54]. Deconvolution
of CD spectra showed 41% ␣-helix, 16% ␤-sheet and 43% random
root mean square deviation = 0.125). Secondary structure of Tri-
choderma reesei ␤-xylosidase showed little similarity to our results
23% ␣-helix, 27% ␤-sheet) [7], indicating that our enzyme prob-
␣
(
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