Purification and characterization of a naringinase from Aspergillus aculeatus JMUdb058

Article abstract of DOI:10.1021/jf303512q

A naringinase from Aspergillus aculeatus JMUdb058 was purified, identified, and characterized. This naringinase had a molecular mass (MW) of 348 kDa and contained four subunits with MWs of 100, 95, 84, and 69 kDa. Mass spectrometric analysis revealed that the three larger subunits were β-d-glucosidases and that the smallest subunit was an α-l-rhamnosidase. The naringinase and its α-l-rhamnosidase and β-d-glucosidase subunits all had optimal activities at approximately pH 4 and 50 C, and they were stable between pH 3 and 6 and below 50 C. This naringinase was able to hydrolyze naringin, aesculin, and some other glycosides. The enzyme complex had a K_m value of 0.11 mM and a k_cat/K_m ratio of 14 034 s^-1 mM ^-1 for total naringinase. Its α-l-rhamnosidase and β-d-glucosidase subunits had K_m values of 0.23 and 0.53 mM, respectively, and k_cat/K_m ratios of 14 146 and 7733 s ^-1 mM^-1, respectively. These results provide in-depth insight into the structure of the naringinase complex and the hydrolyses of naringin and other glycosides.

Full text of DOI:10.1021/jf303512q

Article
pubs.acs.org/JAFC
Purification and Characterization of a Naringinase from Aspergillus
aculeatus JMUdb058
YueLong Chen,† Hui Ni,*,†,‡,§ Feng Chen,‡,§ HuiNong Cai,†,‡,∥ LiJun Li,†,‡,∥ and WenJin Su†,‡,∥
‡
†College of Bioengineering and Research Center of Food Microbiology and Enzyme Engineering Technology, Jimei University,
Xiamen, Fujian Province 361021, P.R. China
§Department of Food, Nutrition and Packaging Sciences, Clemson University, Clemson, South Carolina 29634, United States
∥Research Center of Food Biotechnology of Xiamen City, Xiamen 361021, P.R. China
S
* Supporting Information
ABSTRACT: A naringinase from Aspergillus aculeatus JMUdb058 was purified, identified, and characterized. This naringinase
had a molecular mass (MW) of 348 kDa and contained four subunits with MWs of 100, 95, 84, and 69 kDa. Mass spectrometric
analysis revealed that the three larger subunits were β-D-glucosidases and that the smallest subunit was an α-L-rhamnosidase. The
naringinase and its α-L-rhamnosidase and β-D-glucosidase subunits all had optimal activities at approximately pH 4 and 50 °C,
and they were stable between pH 3 and 6 and below 50 °C. This naringinase was able to hydrolyze naringin, aesculin, and some
other glycosides. The enzyme complex had a Km value of 0.11 mM and a kcat/Km ratio of 14 034 s−1 mM−1 for total naringinase.
Its α-L-rhamnosidase and β-D-glucosidase subunits had Km values of 0.23 and 0.53 mM, respectively, and kcat/Km ratios of 14 146
and 7733 s−1 mM−1, respectively. These results provide in-depth insight into the structure of the naringinase complex and the
hydrolyses of naringin and other glycosides.
KEYWORDS: naringinase, Aspergillus aculeatus, identification, hydrolysis, kinetics
glucosidase using the Davis and p-nitrophenyl methods,4,10 the
hydrolysis kinetic study of naringin, which represents its most
INTRODUCTION
■
Naringinase is an enzyme complex that possesses the activities
important application, has been difficult because of the intrinsic
of both α-L-rhamnosidase (EC 3.2.1.40) and β-D-glucosidase
limitations of these methods. Naringinase is commonly assayed
(EC 3.2.1.21). This enzyme has many important applications in
by the Davis method, which is based on the colonization
the food and pharmaceutical industries because of its ability to
reaction between flavonones and alkaline diethylene glycol.26
hydrolyze many glycosides, e.g., 6-O-α-L-rhamnopyranosyl-β-D-
glucopyranosides, naringin, hesperidin, and rutin.1,2 Naringi-
However, naringin (substrate), prunin (intermediate), and
nase specifically hydrolyzes naringin to naringenin,1,3,4 resulting
naringenin (product) all generate colonization complexes with
in an improvement in the taste of citrus juice.5 In addition, it
similar absorption spectra, which makes it difficult to accurately
determine the kinetic parameters of naringinase.27 The artificial
substrates p-nitrophenyl-α-L-rhamnopyranoside (pNPR) and p-
nitrophenyl-α-L-glucopyranoside (pNPG) allow rapid analysis
of α-L-rhamnosidase and β-D-glucosidase,28,29 but they could
not monitor the reaction velocity in the hydrolysis of special
glycosides such as naringin. In comparison, a high-performance
liquid chromatography (HPLC) method based on distinguish-
ing naringin, prunin, and naringenin could determine the
activities of naringinase, α-L-rhamnosidase, and β-D-glucosidase
using naringin and prunin as substrates.19 This HPLC method
would overcome the limitations of the Davis and p-nitrophenyl
methods and enable the kinetic study of the hydrolyses of
naringin and other glycosides.
efficiently removes the hesperidin haze from orange products1
and enhances the aroma of wine and grape juices.6,7 Moreover,
it also plays an important role in modification of flavonoids to
yield compounds of high bioactivity.2,8
Naringinase has been found in some fungi, yeasts, and
bacteria, e.g., Aspergillus niger,9 Penicillium decumbens,10
Williopsis californica,11 and Staphylococcus xylosus.12 Among
these, the fungi Aspergillus has been regarded as the best source
because many strains of Aspergillus have been approved by the
FDA to produce enzymes for the food industry, and their
naringinases are active and stable at acidic pH values.1 Several
research groups have purified naringinase, α-L-rhamnosidase,
and β-D-glucosidase from Aspergillus and other organisms8,13−19
and cloned some of these genes.14,20−25 However, most
naringinases exhibited a single band upon sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) anal-
ysis,1,16−19 which made it difficult to separate the subunits and
study their structures. Thus far, the structure (how do the α-L-
rhamnosidase and β-D-glucosidase subunits form the naringi-
nase complex) has yet to be elucidated.
Recently, the naringinase synthesizing Aspergillus aculeatus
strain JMUdb058 has been isolated in our lab. In this study, its
naringinase (an enzyme complex consisting of α-L-rhamnosi-
dase and β-D-glucosidase) was purified to determine its primary
Received: August 22, 2012
Revised: December 16, 2012
Accepted: January 5, 2013
Published: January 5, 2013
Although some studies have focused on determining the
kinetic parameters of naringinase, α-L-rhamnosidase, or β-D-
© 2013 American Chemical Society
931
dx.doi.org/10.1021/jf303512q | J. Agric. Food Chem. 2013, 61, 931−938

Journal of Agricultural and Food Chemistry
Article
Figure 1. (A) Chromatographs of the reaction mixtures before (solid line) and after (dashed line) enzymatic hydrolysis of naringin (4,5,7-trihydroxy-
flavanone-7-rhamnoglucoside). Activity of naringinase complex that releases naringenin (4,5,7-trihydroxyflavonone) from naringin is determined by
quantification of the liberated naringenin using naringin as the substrate, and the α-L-rhamnosidase activity that hydrolyzes naringin to release prunin
(4,5,7-trihydroxy-flavanone-7-glucoside) and rhamnose is determined by quantification of the consumed naringin. (B) Chromatographs of reaction
mixtures before (solid line) and after (dashed line) the enzymatic hydrolysis of prunin, from which either the prunin consumed or the naringenin
generated is quantified to determine the β-D-glucosidase activity.
was centrifuged (Avanti J-25, Beckman Coulter, USA) at 4 °C and 15
000g for 20 min. Then the supernatant (450 mL, 1.10 U/mL) was
fractionally precipitated by ammonium sulfate from 40% to 80%
saturation. The resultant pellet was dissolved in the minimum
necessary quantity of 20 mM citrate buffer (pH 6.0), followed by
dialysis against the same buffer for 24 h at 4 °C. The dialysate was
applied to a DEAE-Sepharose Fast Flow column (1.6 × 20 cm,
Amersham Biosciences Inc., Uppsala, Sweden) that was previously
equilibrated with five times the column volume of 20 mM citrate
buffer (pH 6.0). With a fraction size of 3 mL, elution (after washing
with 300 mL of 20 mM citrate buffer) was performed at a flow rate of
1 mL/min with a linear NaCl gradient from 0 to 0.8 M in a final
volume of 200 mL and an isocratic elution of 0.8 M NaCl. The
naringinase fractions were pooled and concentrated using an ultrafilter
membrane (cutoff of 10 kDa) and an Amicon 8050 Ultra filtration Cell
(Millipore Corp., MA). Subsequently, the concentrated fraction was
loaded onto a Sephacyl S 300 HR column (1.6 × 60 cm, Amersham
Biosciences Inc., Uppsala, Sweden) that had been equilibrated with 20
mM citrate buffer containing 0.15 M sodium chloride. This process
was followed by elution with a fraction size of 1 mL at a flow rate of
0.4 mL/min with 20 mM citrate buffer. The fractions were collected to
analyze the activity of naringinase and the concentration of protein.
Analysis of Enzyme Activities. Enzyme activities were
determined according to the reaction scheme proposed by Puri and
Banerjee1 and Yadav et al.4 As shown in Figure 1, naringin was used as
the substrate to determine the activities of α-L-rhamnosidase and
naringinase, and prunin was applied to determine the activity of β-D-
glucosidase. Units of naringinase and β-D-glucosidase were defined as
the amounts of the enzymes that hydrolyzed naringin and prunin,
respectively, to release 1 μM of naringenin per minute at 50 °C and
pH 4.0. Additionally, one unit of α-L-rhamnosidase was defined as the
amount of enzyme that consumed 1 μM of naringin per minute at 50
°C and pH 4.0.
structure, which would reveal the basic structure of the enzyme.
In addition, kinetic study of the hydrolysis of naringin was
performed using an HPLC method to monitor enzyme activity,
which provides insight into the kinetic characteristic of the
hydrolysis of this glycoside.
MATERIALS AND METHODS
■
Chemicals and Reagents. DEAE-Sepharose Fast Flow, Sephacryl
S 300HR, acrylamide, and sodium dodecyl sulfate (SDS) were
purchased from Amersham Biosciences Inc. (Uppsala, Sweden).
Protein markers for SDS-PAGE were products from Fermentas VAB
(Vilnius, Lithuania). Prunin was purchased from Extrasysthese (Genay
Cedex, France). Naringin, naringenin, dithiothreitol (DTT), bovine
serum albumin (BSA), Coomassie Brilliant Blue-R250, and ammo-
nium persulfate were purchased from Sigma-Aldrich (St. Louis, MO).
Hesperidin, arbutin, myricitrin, salicin, and aesculin were purchased
from Xian Xiaocao Botanical Development Co. Ltd. (Xian, China).
HPLC-grade methanol and acetonitrile were products from Tedia Co.
Inc. (Fairfield, OH). All other reagents were of analytical grade.
Strain and Cultivation. JMUdb058 was identified as a strain of A.
aculeatus. In brief, its mycelium and spores were identical to
characteristics of a typical A. aculeatus. In the NCBI database, the A.
aculeatus JMUdb058 26S rDNA sequence (accession number
JQ301899) was 100% homologous with the 26S rRNA gene
(accession number EU381181.1) of the A. aculeatus strain NRRL 360.
A. aculeatus JMUdb058 was cultured by a modified method of our
previous study.19 This strain was activated on media containing (g/L)
MgSO4·7H2O (1.0), KH2PO4 (1.0), (NH4)2SO4 (1.5), KCl (0.5),
KNO3 (1.5), CaCl2 (0.1), yeast extract (2.0), naringin (2.5), and agar
(20) at 28 °C for 96 h. Spores were then collected in water.
Subsequently, 50 mL of the spore suspension (OD600 = 0.2) were
inoculated into a NBS Bioflo-110 7-l fermenter with 5 L of medium
containing (g/L) MgSO4·H2O (0.5), KH2PO4 (1.5), (NH4)2SO4
(4.0), ZnSO4·7H2O (0.09), CaCl2 (0.1), yeast extract (1.0), naringin
(5.0), soybean powder (2.0), and peptone (2.0), pH 6.0. After 7 days
of cultivation at 28 °C (300 rpm and air flow of 0.8 L/min), a broth
with a naringinase activity of 1.16 U/mL was obtained.
The analysis procedure, which included performing the enzymatic
reactions and quantifying the substrates and products, was conducted
according to Ni et al.11 with some modifications. An assay mixture
composed of 1.9 mL of 50 mM citrate buffer (pH 4.0), 2 mL of 200
μg/mL substrate solution (naringin for the analysis of naringinase and
α-L-rhamnosidase activities or prunin for the analysis of β-D-
glucosidase activity), and 0.1 mL of enzyme solution was incubated
Purification of Naringinase. Naringinase was purified by a
modified method based on previous studies.16,19 The broth (460 mL)
932
dx.doi.org/10.1021/jf303512q | J. Agric. Food Chem. 2013, 61, 931−938

Journal of Agricultural and Food Chemistry
Article
at 50 °C for 5 min followed by heating at 100 °C for 30 min to
inactivate the enzyme. After filtration through a 0.22 μm filter
(Generay Biotech Co., Ltd., Shanghai, China), the reaction mixture
was subjected to HPLC for analysis. This process was repeated for the
control experiment using inactivated enzyme instead of fresh enzyme
solution. A Waters 2695 HPLC coupled with a Symmetry C18 reverse
phase column (bonded C18 ligands on a high-purity base-deactivated
silica) (4.6 × 150 mm, 3.5 μm) and a 2487 UV detector (Waters
Corp., Milford, MA) was used to determine the concentrations of the
substrates and products. Following an injection of 20 μL of reaction
mixture, the column was eluted using a gradient elution at 35 °C and
0.4 mL/min. The mobile phase was composed of water (A), methanol
(B), and acetonitrile (C). The gradient procedure began with A:B:C =
62:12:26 within 0−7 min. This procedure was followed by a linear
change to A:B:C = 15:35:50 within 7−9 min, another linear change to
A:B:C = 15:35:50 within 9−15 min, a linear return to A:B:C =
62:12:26 within 15−17 min, and maintaining at A:B:C = 62:12:26
within 17−20 min. The target compounds were captured in the 2487
UV detector at 280 nm.
Analysis of the Concentration of Protein. The protein content
in the purification was monitored by ultraviolet spectrophotometry
(A280), whereas the concentration of protein for the kinetic studies was
determined using the Bradford assay with bovine serum albumin
(BSA) standards.30
Determination of the Molecular Mass (MW) of Naringinase
and Its Subunits. The MW of naringinase was analyzed by
comparing the elution volume of the enzyme with the protein
standard (HMW, GE Health, USA) using a standard curve. The MWs
of the subunits were measured by SDS-PAGE using a Mini-protean III
dual-slab cell electrophoresis31 and a 10.0% gel, which was stained with
Coomassie Brilliant Blue R-250.
Identification of Subunits of Naringinase. Naringinase was
separated on a 10.0% native-PAGE gel, which was run at 8 mA and 4
°C for approximately 120 min. The resulting protein bands were
extracted to analyze the activities of α-L-rhamnosidase and β-D-
glucosidase. Furthermore, the protein bands on the SDS-PAGE gel
from the above section were extracted followed by washing with water
twice, destaining with 10% ethanol and 10% acetic acid, dehydrating
twice with acetonitrile for 20 min, and in-gel digesting overnight with a
sequencing grade modified porcine trypsin. Peptides were extracted
with a 50% acetonitrile and 0.1% formic acid solution, dried
thoroughly using a Savant SpeedVac concentrator (Thermo Electron
Corp., Pittsburgh, PA), and dissolved in a 5% acetonitrile and 0.1%
formic acid solution. Identification of the peptides was performed on a
Nano Aquity UPLC system (Waters Corp., Milford, USA) connected
to an LTQ Orbitrap XL mass spectrometer (Thermo Electron Corp.,
Bremen, Germany) equipped with an online nanoelectrospray ion
source (Michrom Bioresources, Auburn, USA). Peptides were
separated by a Cap trap column (0.5 × 2 mm, Michrom Bioresources
Inc.) and a 15 cm reverse-phase column (100 μm i.d., Michrom
Bioresources Inc.).
The peptide mixtures were injected onto the trap column at a flow
of 20 μL/min for 5 min and subsequently eluted with a 0.1% formic
acid solution (mobile phase A) and acetonitrile containing 0.1% formic
acid (mobile phase B) by a three-step linear gradient (0−35 min, B
increased from 5% to 45%; 35−40 min, B increased to 80%; 40−45
min, B held at 80%; 45−60 min, B reduced from 80% to 5%). Flow
rate was maintained at 500 nL/min, and column temperature was kept
at 35 °C. An electrospray voltage of 1.9 kV versus the inlet of the mass
spectrometer was used.
The MS/MS spectrum was identified by searching against the
Swissprot database using the Mascot algorithm. Search parameters
were set up as follows: taxonomy of fungi; partial trypsin (KR)
cleavage; two missed cleavage; fragment mass tolerance of 1 Da;
instrument ESI-TRAP. Peptide identification was accepted with
significance threshold P < 0.05.
Investigation of the Effects of pH and Temperature on
Naringinase. The optimal pH was investigated by analysis of the
enzymatic activities at different pH values using 50 mM of citrate
buffer (pH 3.0−6.0), phosphate buffer (pH 6.0−7.0), and Tris-HCl
buffer (pH 7.0−8.0) at 50 °C. The pH stability was assessed by
measuring the residual activities after preincubation of the enzyme by
the method described in Analysis of Enzyme Activities.
To study the optimum temperature, enzymatic activities were
determined at pH 4.0 in 50 mM of citrate buffer and between 30 and
80 °C. The thermal stability was investigated by incubating the enzyme
in the citrate buffer (50 mM, pH 6.0) at 30−80 °C for 60 min prior to
analysis of the residual enzyme activities by the method mentioned in
Analysis of Enzyme Activities.
Investigation of Substrate Specificity. Incubation of 0.1 mL of
naringinase (1.08 U/mL) with 1.9 mL of citrate buffer (50 mM, pH
4.0) and 2 mL of 200 μg/mL substrate (naringin, hesperidin,
myricitrin, prunin, arbutin, salicin, or aesculin) at 50 °C for 5 min was
followed by measurement of the concentration changes of substrates
and products using the HPLC method described in Analysis of
Enzyme Activities.
Determination of Kinetic Parameters for Hydrolysis of
Naringin. The enzyme activities of naringinase, α-L-rhamnosidase,
and β-D-glucosidase were determined at various concentrations of
substrate (6.25, 12.5, 25, 50, 75, 100, 150, and 200 μg/mL) at 50 °C
and pH 4.0. From the data, kinetic constants such as the Michaelis
constant (Km) and maximum velocity (Vmax) were computed from
Lineweaver−Burk plots, and the turnover constant (Kcat) and the
catalytic coefficient (ratio of Kcat to Km) were then estimated.
Statistical Analysis. Every experiment was performed in triplicate,
and the results represent the mean values that were calculated. SPSS
17.0 (SPSS Inc. H, Chicago, IL) was used to analyze the significant
difference (p < 0.05) of the various samples through Duncan’s multiple
range test.
RESULTS AND DISCUSSION
■
Purification of Naringinase. Many studies referred to
purification of naringinase, α-L-rhamnosidases, and β-D-
glucosidases.8 In general, a combined procedure is always
needed to purify naringinase,1,8,32 whereas affinity chromatog-
raphy is effective for purification of α-L-rhamnosidases and β-D-
glucosidases using one-step chromatography.15 A combined
procedure consisting of ammonium sulfate precipitation and
chromatography on a DEAE-Sepharose Fast Flow and a
Sephacyl S 300 HR column was used to purify naringinase in
this study. As shown in Figure 2A, naringinase activity was
eluted from the DEAE-Sepharose Fast flow column in 0.45−
0.55 M NaCl. The naringinase fraction was then separated from
other proteins on a Sephacyl S 300HR column (Figure 2B).
Table 1 showed that after the final purification step (size
exclusion chromatography with the Sephacyl S 300HR
column), the naringinase was purified by 17.2-fold with a
yield of 7.1%.
Molecular Mass and Structure of the Naringinase
Complex. Previous studies showed that the MW of
naringinase varied depending on its origin and fermentation
conditions.1,8,32 When analyzed by SDS-PAGE, most naringi-
nases behaved homogenously, appearing as a solo band.16−19
However, some microorganisms have the ability to synthesize
several α-L-rhamnosidases and β-D-glucosidases that result in
different MWs on an SDS-PAGE.14,15 Ni et al.19 purified a
naringinase that exhibited a MW of 132 kDa by gel filtration
An LTQ Orbitrap XL mass spectrometer was operated in the data-
dependent mode to allow an automatic switch between MS and MS/
MS acquisition. Full-scan MS survey spectra with two microscans (m/z
400−2000) were acquired using the Orbitrap, with a mass resolution
of 60 000 at m/z 400, followed by eight sequential LTQ-MS/MS
scans. Dynamic exclusion was used with two repeat counts: a 10 s
repeat duration and a 60 s exclusion duration. For MS/MS, the
precursor ions were activated using the 25% normalized collision
energy at the default activation q of 0.25.
933
dx.doi.org/10.1021/jf303512q | J. Agric. Food Chem. 2013, 61, 931−938

Journal of Agricultural and Food Chemistry
Article
activity and the remaining one was an α-L-rhamnosidase
(Figure 3A), which indicates that the naringinase contains at
Figure 3. (A) Native-PAGE analysis of the naringinase from Sephacyl
TM S 300 HR chromatography (at an elution volume of 56 mL). (B)
SDS-PAGE analysis of the purifications. Lane M was the protein
marker (#SM0661, Fermentas); lanes 1, 2, 3, and 4 (from left to right)
were crude broth, fractions pooled in (NH4)2SO4 precipitation,
DEAE-Sepharose Fast Flow chromatography, and Sephacyl S 300HR
chromatography (at 56 mL of elution volume), respectively.
Figure 2. In all, 90 mL (399−489 mL) of the fractions from the
DEAE-Sepharose FF column (A) was pooled and subsequently
concentrated to 2.5 mL prior to separation on a Sephacyl S 300HR
column (B). Naringinase exhibited the highest activity at an elution
volume of 56 mL. (B, insert) Linear relationship of the log [MW]
versus the elution volume (Ve). Ferritin (440 kDa), aldolase (158
kDa), conalbumin (75 kDa), and ovalbumin (44 kDa) were used as
standards and had Ves values of 53.66, 63.25, 69.74, and 73.94 mL,
respectively.
least of one α-L-rhamnosidase and two β-D-glucosidase
subunits. Furthermore, SDS-PAGE, which was performed for
150 min with a 10% gel, showed four protein components with
MWs of approximately 100, 95, 84, and 69 kDa (Figure 3B).
The sum of the MWs of the four bands was equal to the
counterpart estimated by gel filtration chromatography (100 +
95 + 84 + 69 = 348 kDa), which indicate that naringinase
contains four subunits. Identification based on mass spectra
analysis and Mascot searching against the Swissport database
revealed that three of the subunits (bands 1 (100 kDa), 2 (95
kDa), and 3 (84 kDa) in Figure 3B) were β-D-glucosidase 1
precursors24 and the other (band 4 (69 kDa) in Figure 3B) was
an α-L-rhamnosidase A precursor14 from A. aculeatus (Table 2,
Supporting Information Tables 1−4). Although the native-
PAGE analysis produced different results from SDS-PAGE
analysis because more uncertain factors affect the native-PAGE
results, the SDS-PAGE and mass spectra analyses indicated that
this naringinase was a tetramer consisting of one α-L-
rhamnosidase subunit and three β-D-glucosidase subunits.
These results confirmed the assumption that naringinase is
composed of α-L-rhamnosidase and β-D-glucosidase subu-
nits.32,33
A. aculeatus synthesizes two α-L-rhamnosidases (RhaA and
RhaB) that are encoded by different genes (rhaA and rhaB);14
however, only α-L-rhamnosidase A (RhaA) was present in the
naringinase of this study. Thus far, α-L-rhamnosidase has been
associated with the glycoside hydrolysis family (GH) of 28, 78,
and 106,33 but only the α-L-rhamnosidase of GH 78 is found in
naringinase,14,35,36 which further indicates the sequence
selectivity of naringinase. A crystal structure of α-L-
rhamnosidase of GH 78 was resolved by Cui et al. (2007).33
This structure showed that the catalytic domain was an (α/α)
6-barrel fold structure with several highly conserved residues,
such as Asp 567, Glu 572, Asp 579, and Glu 841, thus providing
a potential interpretation for sequence selectivity.
Table 1. Purification Summary of Naringinase from A.
aculeatus JMUdb058
total
protein
(mg)
total
activity
(U)
specific
purification
activity (U/ purification yield
step
mg)
fold
(%)
crude extract
385.0
268.0
532.2
411.3
1.38
1.53
1
100
(NH4)2SO4
precipitation
1.1
77.3
DEAE-
Sepharose
FF
23.5
1.6
146.6
38.0
6.24
4.5
27.5
7.1
Sephacyl S
300HR
23.75
17.2
chromatography and 66 kDa by SDS-PAGE. Another
naringinase from A. niger was estimated to have a MW of
approximately 168 kDa by gel filtration chromatography and 85
kDa by SDS-PAGE analysis.16 A naringinase from A. sojae was
purified and determined to have a MW of 70 kDa by SDS-
PAGE.18 Two α-L-rhamnosidases with different protein
sequences, MWs, and properties were purified from A.
aculeatus.14 Two β-D-glucosidases and one α-L-rhamnosidases
were purified from the naringinase broth of A. terreus CECT
2663.15 Recently, Puri32 reviewed the latest research advance-
ment on the structure of naringinase, in which he assumed that
naringinase was a complex with different subunits. These
studies raise questions regarding the makeup of the naringinase
complex and whether all of the different α-L-rhamnosidases and
β-D-glucosidases are components of naringinase.
Naringinase from A. aculeatus JMUdb058 was estimated to
have a MW of 348 kDa based on the elution volume and
standard curve (Figure 2B). Native-PAGE analysis showed
three protein bands. Among these, two had β-D-glucosidase
Most α-L-rhamnosidases and β-D-glucosidases from the
Aspergillus family are glycoproteins with variable MWs due to
post-translational processing.14,24,25,29 The α-L-rhamnosidase
subunit had a MW (69 kDa) similar to that of the
934
dx.doi.org/10.1021/jf303512q | J. Agric. Food Chem. 2013, 61, 931−938

Journal of Agricultural and Food Chemistry
Article
a
Table 2. Identification of the Subunits by MS Analysis and Mascot Searching
band no.
MW (kDa)
matched peptide
score
accession no.
protein name
pI
1
2
3
4
100
95
50(36)
44(31)
36(24)
5(3)
3796
2188
1685
100
BGL1_ASPAC
BGL1_ASPAC
BGL1_ASPAC
gi|13241312
β-D-glucosidase
β-D-glucosidase
β-D-glucosidase
α-L-rhamnosidase A
5.0
5.0
5.0
5.8
84
69
a
Peptide identification was accepted with a significance threshold of P < 0.05. Band number corresponds to the number of bands mentioned in
Figure 3B. pI (isoelectric point) represents the theoretical results predicted based on the protein sequence. Matched peptide was the number of
parings between the experimental fragmentation spectra and the theoretical segments of the protein. Accession no. is the unique number given when
a protein sequence is entered into a primary or secondary database (i.e., GenBank).
deglycosylated α-L-rhamnosidase A (Rha A) from A.
aculeatus,14 indicating that the post-translational glycosylation
that is found in almost all liberated α-L-rhamnosidases did not
occur in formation of naringinase. On the other hand, the three
β-D-glucosidase subunits had different MWs, which is
consistent with an earlier study in which three different β-D-
glucosidases were purified from A. aculeatus.34 Because only
one gene encoding β-D-glucosidase was found in this fungus,24
it is reasonable to believe that the three β-D-glucosidases occur
from different post-translational processing. These results
indicate that the post-translational processing of β-D-glucosi-
dase plays an important role in forming the structure of the
naringinase complex.
Rhamnosidase was very stable between pH 3 and 8 (Figure
4B). However, the stability of the β-D-glucosidase ranged from
pH 3 to 6, which suggests that naringinase is stable from
approximately pH 3 to 6 (Figure 4B). These characteristics
allow the naringinase to hydrolyze naringin and hesperidin in
acidic pH conditions, which is found in wine and citrus juices.
The optimal temperature of the α-L-rhamnosidase and β-D-
glucosidase subunits was 50 °C and within the range from 50 to
60 °C, which is consistent with some other α-L-rhamnosi-
dases32,35,36 and β-D-glucosidases29,37 and thus presents a
reasonable explanation for the occurrence of the optimal
temperature of naringinase at 50 °C (Figure 5A). In addition,
Effects of pH and Temperature on Naringinase. Most
of the fungal naringinases, α-L-rhamnosidases, and β-D-
glucosidases have an optimal activity at an acidic pH value
and are stable in a broad pH range.1,8,32 Naringinases from A.
niger 1344,16 P. decumbens PTCC 5248,10 and A. sojae18 have
optimum pH values of 4.0, 4.5, and 6.0, respectively.
Both α-L-rhamnosidase and β-D-glucosidase described in this
study exhibited optimal activity at pH 4.0, which is similar to
the optimal pH of some previously characterized α-L-
rhamnosidases8,14,33,34 and β-D-glucosidases1,8,29 and explains
why the naringinase had an optimal pH of 4.0 (Figure 4A). α-L-
Figure 5. Optimal temperature (A) was estimated by analysis of the
enzymatic activities at different temperatures (30−70 °C). pH stability
(B) was measured by analysis of the activity that remained after the
enzyme was incubated at different temperatures for 24 h in citrate
buffer.
the naringinase complex and its α-L-rhamnosidase and β-D-
glucosidase subunits exhibited similar temperature stability
ranges below 50 °C (Figure 5B), which is consistent with most
naringinases from fungi.8,10,16,18,32
Substrate Specificity. Naringinase hydrolyzed hesperidin,
myricitrin, arbutin, salicin, and aesculin (Figure 6) in addition
to naringin and prunin (Figure 1), which was supported by the
ability of α-L-rhamnosidase to cleave both α-1,2 and α-1,6
linkages to release the terminal rhamnose4 and of β-D-
glucosidase to cleave several types of beta glycosidic bonds.35
Among the substrates tested, naringin was the best substrate for
the α-L-rhamnosidase and aesculin and prunin were the best
Figure 4. Optimal pH (Figure 4A) was estimated by analysis of the
enzymatic activities in different buffers (pH 3−8). pH stability (Figure
4B) was measured by analysis of the activity that remained after the
enzyme was incubated at 4 °C for 24 h in buffers with different pH
values from 3 to 8. Relative activity was the percentage of a detected
activity compared with the activity at the peaks.
935
dx.doi.org/10.1021/jf303512q | J. Agric. Food Chem. 2013, 61, 931−938

Journal of Agricultural and Food Chemistry
Article
Figure 6. Chromatographs A−E show the reaction mixtures before (solid line) and after (dashed line) enzymatic hydrolysis of hesperidin, myricitrin,
arbutin, salicin, and aesculin, respectively. Hesperidin was hydrolyzed by α-L-rhamnosidase first, releasing rhamnose and hesperetin-7-glucoside,
which was further catalyzed to hesperetin and glucose by the β-D-glucosidase activity of naringinase. Myricitrin (myricetin-3-rhamnoside) was
hydrolyzed to myricetin and rhamnose by α-L-rhamnosidase. β-D-Glucosidase split arbutin (C), salicin (D), and aesculin (E) to generate
hydroquinone, salicyl alcohol, and aesculetin, respectively.
substrates for β-D-glucosidase (Table 3). These results suggest
the effectiveness of naringinase in modification of glycosides.
Kinetic Parameter for Hydrolysis of Naringin. Although
some studies have characterized naringinase using p-nitro-
phenol methods,8,14,29,32 the kinetic parameters of the
hydrolysis of naringin were difficult to accurately determine.
The HPLC method that distinguished naringin, prunin, and
naringenin was used to determine the reaction velocities of the
α-L-rhamnosidase (transforming naringin to prunin), β-D-
glucosidase (cleaving prunin to naringenin), and naringinase
complex (transforming naringin to naringenin via prunin;
Figure 1).19 This HPLC method can determine the kinetics of
the hydrolysis of naringin by the Lineweaver−Burk plot
method. As shown in Table 4, the α-L-rhamnosidase was
936
dx.doi.org/10.1021/jf303512q | J. Agric. Food Chem. 2013, 61, 931−938

Journal of Agricultural and Food Chemistry
Article
a
Table 3. Substrate Specificity of the Naringinase
AUTHOR INFORMATION
■
Corresponding Author
active components
substrate
hydrolysis rate (%)
α-L-rhamnosidase
naringin
myricitrin
hesperidin
aesculin
prunin
22.68 0.22a
5.54 0.76b
4.04 0.33c
97.28 0.04a
95.33 0.16b
55.06 0.71c
33.88 0.31d
*Phone: 0592-6181764. E-mail: nihui1973@yahoo.com.cn;
HUIN@clemson.edu.
Notes
β-D-glucosidase
The authors declare no competing financial interest.
arbutin
REFERENCES
■
salicin
a
(1) Puri, M.; Banerjee, U. C. Production, purification, and
characterization of the debittering enzyme naringinase. Biotechnol
Adv. 2000, 18, 207−217.
The substrate specificity of naringinase was studied by measuring the
hydrolysis rates of α-L-rhamnosidase and β-D-glucosidase. Naringin,
myricitrin, and hesperidin, three glycosides containing a terminal
rhamnose, were used to investigate the substrate specificity of the α-L-
rhamnosidase. Aesculin, prunin, arbutin, and salicin, four substrates
containing a terminal glucose, were used to analyze the substrate
specificity of β-D-glucosidase. The hydrolysis rate (%) was calculated
by the formula: (CI − CR) ×100/CI, where CI was the initial
concentration of the substrates before the enzymatic reaction and CR
was the residual concentration of the substrates after the enzymatic
reaction. The numbers followed by different lowercase superscript
letters indicate a statistical difference (P < 0.5).
(2) Vila-Reala, H.; Alfaia, A. J.; Rosa, M. E.; Calado, A. R.; Ribeiro, M.
H. L. An innovative sol-gel naringinase bioencapsulation process for
glycosides hydrolysis. Process Biochem. 2010, 45, 841−850.
(3) Busto, M. D.; Meza, V.; Ortega, N.; Perez-Mateos, M.
Immobilization of naringinase from Aspergillus niger CECT 2088 in
poly (vinyl alcohol) cryogels for the debittering of juices. Food Chem.
2007, 104, 1177−1182.
(4) Yadav, V.; Yadav, P. K.; Yadav, S.; Yadav, K. D. S. α-l-
Rhamnosidase: A review. Process Biochem. 2010, 45, 1226−1235.
(5) Ono, M.; Tosa, T.; Chibata, I. Preparation and properties of
immobilized naringinase using tannin-aminohexyl cellulose. Agric. Biol.
Chem. 1978, 42, 1847−1853.
a
Table 4. Kinetic Parameters of the Purified Naringinase
(6) Gunata, Z.; Bitteur, S.; Brillouet, J. M.; Bayonove, C.;
Cordonnier, R. Sequential enzymic hydrolysis of potentially aromatic
glycosides from grape. Carbohydr. Res. 1988, 184, 139−149.
(7) Caldini, C.; Bonomi, F.; Pifferi, P. G.; Lanzarini, G.; Galante, Y.
M. Kinetic and immobilization studies on fungal glycosidases for
aroma enhancement in wine. Enzyme Microbiol. Technol. 1994, 16,
286−291.
(8) Ribeiro, M. H. Naringinase: occurrence, characteristics, and
applications. Appl. Microbiol. Biotechnol. 2011, 90, 1883−1895.
(9) Bram, B.; Solomons, G. L. Production of the enzyme naringinase
by Aspergillus niger. Appl. Microbiol. 1965, 13, 842−845.
(10) Norouzian, D.; Hosseinzadeh, A.; Inanlou, D. N.; Moazami, N.
Production and partial purification of naringinase by Penicillium
decumbens PTCC 5248. World J. Microbiol. Biotechnol. 2000, 16, 471−
473.
(11) Ni, H.; Li, L.; Xiao, A.; Cao, Y.; Chen, Y.; Cai, H. Identification
and characterization of a new naringinase-producing strain, Williopsis
californica Jmudeb007. World J. Microbiol. Biotechnol. 2011, 27, 2857−
2862.
(12) Puri, M.; Kaur, A. Molecular identification of Staphylococcus
xylosus MAK2, a new α-L-rhamnosidase producer. World J. Microbiol.
Biotechnol. 2010, 26, 963−968.
kcat/Km
(s−1
enzyme
Km (mM)
Vmax (U/mg)
kcat (s−1
)
mM−1
)
naringinase
0.11 0.003
0.23 0.005
258.6 3.5
1500 20
3278 65
14 034
14 146
α-L-
565.1 11.2
rhamnosidase
β-D-glucosidase 0.53 0.002
707.6 2.9
4104 17
7733
a
The kinetic parameters were calculated at pH 4.0 and 50 °C.
estimated to have a Km value of 0.23 mM, which is less than
that of the pNPR (2.8 mM)14 and indicates that α-L-
rhamnosidase A has a higher affinity for naringin than pNPR.
Furthermore, the Km value of the α-L-rhamnosidase A is less
than that of the β-D-glucosidase (0.53 mM), which indicates α-
L-rhamnosidase has a stronger affinity for naringin than the β-D-
glucosidase has for prunin. However, the α-L-rhamnosidase
showed lower values of both Vmax (565.1 vs 707.6 U/mg) and
kcat (3278 vs 4104 s−1) than β-D-glucosidase, which suggests
that α-L-rhamnosidase is the main factor affecting the velocity
of naringin hydrolysis. Although the β-D-glucosidase had lower
(13) Gallego, V. M.; Pinaga, F.; Ramon, D.; Valles, S. Purification and
characterization of a α-L-rhamnosidase from Aspergillus terreus of
interest in wine making. J. Food Sci. 2001, 66, 204−209.
(14) Manzanares, P.; van den Broeck, H. C.; de Graaff, L. H.; Visser,
J. Purification and characterization of two different a-L-Rhamnosidases,
RhaA and RhaB, from Aspergillus aculeatus. Appl. Environ. Microbiol.
2001, 67, 2230−2234.
kcat/Km (7733 s−1 mM−1) than α-L-rhamnosidase (14 146 s−1
×
mM−1), the kcat/Km (14 034 s−1 × mM−1) of the naringinase
was similar to that of the α-L-rhamnosidase because the β-D-
glucosidase had a higher Vmax. This result further suggests that
α-L-rhamnosidase is the rate-limiting step of the enzymatic
hydrolysis of naringin.
(15) Soria, F.; Ellenrieder, G.; Grasselli, M.; Navarro delCanizo, A.
̃
A.; Cascone, O. Fractionation of the naringinase complex from
Aspergillus terreus by dye affinity chromatography. Biotechnol. Lett.
2004, 26, 1265−1268.
(16) Puri, M.; Kalra, S. Purification and characterization of
naringinase from a newly isolated strain of Aspergillus niger 1344 for
the transformation of flavonoids. World J. Microbiol. Biotechnol. 2005,
21, 753−758.
(17) Gabor, F.; Pittner, F. Characterization of naringinase from
Penicillium sp. Hoppe-Seyler's Z. Physiol. Chem. 1984, 365, 914−916.
(18) Chang, H. Y.; Lee, Y. B.; Bae, H. A.; Huh, J. Y.; Nam, S. H.;
Sohn, H. S.; Lee, H. J.; Lee, S. B. Purification and characterization of
Aspergillus sojae naringinase: the production of prunin exhibiting
markedly enhanced solubility with in vitro inhibition of HMG-CoA
reductase. Food Chem. 2011, 124, 234−241.
ASSOCIATED CONTENT
■
S
* Supporting Information
This research was supported by grants from the National
Natural Science Foundation of China (No. 31271914), Science
Foundation for Distinguished Young Scholars of Xiamen City
(No. 3502Z20126008), and Foundation for Innovative
Research Team of Jimei University (2010A006).
This material is available free of charge via the Internet at
http://pubs.acs.org.
937
dx.doi.org/10.1021/jf303512q | J. Agric. Food Chem. 2013, 61, 931−938

Journal of Agricultural and Food Chemistry
Article
(19) Ni, H.; Chen, F.; Cai, H.; Xiao, A.; You, Q.; Lu, Y.
Characterization and preparation of Aspergillus niger naringinase for
debittering citrus juice. J. Food Sci. 2012, 77, C1−7.
(20) Machida, M.; Asai, K.; Sano, M.; Tanaka, T.; Kumagai, T.; Terai,
G.; et al. Genome sequencing and analysis of Aspergillus oryzae. Nature
2005, 438, 1157−1161.
(21) Pel, H. J.; de Winde, J. H.; Archer, D. B.; Dyer, P. S.; Hofmann,
G.; Schaap, P. J.; et al. Genome sequencing and analysis of the versatile
cell factory Aspergillus niger CBS 513.88. Nat. Biotechnol. 2007, 25,
221−231.
(22) van den Berg, M. A.; Albang, R.; Albermann, Kaj.; Badger, J. H.;
Daran, J. M.; Driessen, A. J. M.; et al. Genome sequencing and analysis
of the filamentous fungus Penicillium chrysogenum. Nat. Biotechnol.
2008, 26, 1161−1168.
(23) Arnaud, M. B.; Chibucos, M. C.; Costanzo, M. C.; Crabtree, J.;
Inglis, D. O.; Lotia, A.; Orvis, J.; Shah, P.; Skrzypek, M. S.; Binkley, G.;
Miyasato, S. R.; Wortman, J. R.; Sherlock, G. The Aspergillus genome
database, a curated comparative genomics resource for gene, protein
and sequence information for the Aspergillus research community.
Nucleic Acids Res. 2010, 38, D420−D427.
(24) Kawaguchi, T.; Enoki, T.; Tsurumaki, S.; Sumitani, J.; Ueda, M.;
Ooi, T.; Arai, M. Cloning and sequencing of the cDNA encoding beta-
glucosidase 1 from Aspergillus aculeatus. Gene 1996, 173, 287−288.
(25) Takada, G.; Kawaguchi, T.; Sumitani, J.; Arai, M. Expression of
Aspergillus aculeatus No. F-50 Cellobiohydrolase I (cbhI) and β-
glucosidase 1 (bgl1) genes by Saccharomyces cerevisiae. Biosci.
Biotechnol.Biochem. 1998, 62, 1615−1618.
(26) Davis, D. W. Determination of flavonones in citrus juice. Anal.
Chem. 1947, 19, 46−48.
(27) Habelt, K.; Pittner, F. A rapid method for the determination of
naringin, prunin, and naringenin applied to the assay of naringinase.
Anal. Biochem. 1983, 134, 393−397.
(28) Romero, C.; Manjon, A.; Bastida, J.; Iborra, J. L. A method for
assaying rhamnosidase activity of naringinase. Anal. Biochem. 1985,
149, 566−571.
(29) Decker, C. H.; Visser, J. Schreier, Peter.β-Glucosidases from five
black Aspergillus species: study of their physico-chemical and
biocatalytic properties. J. Agric. Food. Chem. 2000, 48, 4929−4936.
(30) Bradford, M. M. A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the principle
of protein-dye binding. Anal. Biochem. 1976, 72, 248−254.
(31) Laemmli, U. K. Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 1970, 227, 680−
685.
(32) Puri, M. Updates on naringinase: structural and biotechnological
aspects. Appl. Microbiol. Biotechnol. 2012, 93, 49−60.
(33) Cui, Z.; Maruyama, Y.; Hashimoto, W.; Murata, K. Crystal
structure of glycoside hydrolase family 78 α-L-Rhamnosidase from
Bacillus sp. GL1. J. Mol. Biol. 2007, 374, 384−398.
(34) Sakamoto, R.; Kanamoto, J.; Arai, M.; Murao, S. Purification and
physicochemical properties of three beta-glucosidases from Aspergillus
aculeatus No.F-50. Agric. Biol. Chem. 1985, 49, 1275−1281.
(35) Manzanares, P.; deGraaff, L. H.; Visser, J. Purification and
characterization of an alpha-L-rhamnosidase from Aspergillus niger.
FEMS. Microbiol. Lett. 1997, 157, 279−283.
(36) Koseki, T.; Mese, Y.; Nishibori, N.; Masaki, K.; Fujii, T.; Handa,
T.; Yamane, Y.; Shiono, Y.; Murayama, T.; Iefuji, H. Characterization
of an α-L-rhamnosidase from Aspergillus kawachii and its gene. Appl.
Microbiol. Biotechnol. 2008, 80, 1007−1013.
(37) Lin, J.; Pillay, B.; Singh, S. Purification and biochemical
characteristics of β-D-glucosidase from a thermophilic fungus,
Thermomyces lanuginosus-SSBP. Biotechnol. Appl. Biochem. 1999, 30,
81−87.
938
dx.doi.org/10.1021/jf303512q | J. Agric. Food Chem. 2013, 61, 931−938