Food Chemistry 214 (2017) 39–46

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Purification and characterization of a naringinase from a newly isolated

strain of Bacillus amyloliquefaciens 11568 suitable for the transformation
of flavonoids
Yunping Zhu a,c, Huiyong Jia d, Menglu Xi b, Liya Xu c, Shaoming Wu b, Xiuting Li a,b,⇑
a
Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology & Business University (BTBU), Beijing 100048, China
b
School of Food and Chemical Engineering, Beijing Technology and Business University, No. 33, Fucheng Road, Beijing 100048, China
c
Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology and Business University (BTBU), Beijing 100048, China
d
College of Food Science and Technology, Agricultural University of Hebei, Baoding 071000, China

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

Article history: An intracellular naringinase from Bacillus amyloliquefaciens 11568 isolated from soil was purified,
Received 31 March 2016 identified, and characterized. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Received in revised form 28 June 2016 (SDS-PAGE) analysis of the purified enzyme gave a single protein band corresponding to a molecular
Accepted 29 June 2016
mass of 32 kDa. The optimum pH and temperature for naringinase and its a-L-rhamnosidase and
Available online 30 June 2016
b-D-glucosidase activities were pH 7.5 and 45 °C, respectively. The enzymes were stable below 45 °C
between pH 3.5 and 8.5. The Km and the Vmax of the isolated naringinase were 0.95 mmol/L and
Keywords:
3847.3 mmol/(L
min), respectively. The isolated naringinase was capable of hydrolyzing naringin,
Naringinase
Bacillus amyloliquefaciens
neohesperidin, and other glycosides. Additionally, a concentration of 4 U/mL of the enzyme in citrus juice
Debittering was sufficient to remove the naringin and alleviate the bitterness of the juice. These results provide an
Naringin in-depth insight into the structure of the naringinase and the hydrolysis of naringin and other flavonoids.
Purification Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction wine and the bioavailability of polyphenols (Puri, Kaur, Singh,

Naringinase (EC 3.2.1.40), a hydrolytic enzyme possessing both
a-L-rhamnosidase and b-glucosidase activities, hydrolyzes narin-
gin (4,5,7-trihydroxy flavonone 7-rhamnoglucoside) into prunin
that can then be converted by the flavonoid b-D-glucosidase to
naringenin (4,5,7-trihydroxyflavone). The a-L-rhamnosidase activ-
ity of the enzyme is of particular significance to the structural
determination of polysaccharides, glycosides, and glycolipids
(Shintaro Kamiya, 1985). Naringin and its hydrolyzed product of
naringenin are useful in inhibiting the activity of acyl-coA-choles
terol-o-acyltransferase, which helps in preventing the accumula-
tion of the macrophage-lipid complex that causes hepatic diseases
(Myung-Sook, 2000). The a-L-rhamnosidase activity of naringinase
is useful not only for elucidation of the structures of bioactive
compounds containing a-L-rhamnose but also for its potential
application in the food and beverage industries, such as the
debittering of citrus fruit juices, the improvement of the flavor of
⇑ Corresponding author at: Beijing Advanced Innovation Center for Food
Nutrition and Human Health, Beijing Technology & Business University (BTBU),
Beijing 100048, China.
E-mail addresses: lixt@th.btbu.edu.cn, li_xiuting@163.com (X. Li).
Schwarz, & Kaur, 2010).
Naringinase has been found in several fungi including Aspergil-
lus niger (Puri & Kalra, 2005), Aspergillus nidulans (Manzanares,
Orejas, Ibanez, Valles, & Ramon, 2000), Aspergillus flavus (Yadav,
Yadav, Yadava, & Singh, 2011), Penicillium decumbens (Mamma
et al., 2004), and Penicillus ulaiense (Rajal, Cid, Ellenrieder, &
Cuevas, 2009), and the characteristics of naringinase from fungi
have been thoroughly documented. Comparatively, the production
of this enzyme from bacteria has, to date, received very little
attention, partially because its industrial application has led to its
protection as a trade secret. Only a few reports on naringinase
activity from bacteria have been found. These reports have docu-
mented the purification of naringinase from Sphingomonas pauci-
mobilis (Miyata et al., 2005), Lactobacillus plantarum (Avila et al.,
2009), Staphylococcus xylosus (Puri, Kaur, Barrow, & Singh, 2011)
and Bacillus methylotrophicus (Mukund, Belur, & Saidutta, 2014).
Commonly, bacterial naringinases have optimal activity in neutral
or alkaline environments, which is more suitable for the transfor-
mation of flavonoids including 6-O-a-L-rhamnopyranosyl-b-D-
glucopyranosides, naringin, hesperidin, and rutin.
In our previous study, a novel naringinase-producing strain,
Bacillus amyloliquefaciens 11568, was isolated. However, the

http://dx.doi.org/10.1016/j.foodchem.2016.06.108
0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.
40 Y. Zhu et al. / Food Chemistry 214 (2017) 39–46
characteristics of the purified naringinase were still unknown. In
this study, we show for the first time the purification and charac-
terization of a naringinase from a bacterial strain. In addition, the
primary structure of the naringinase was investigated to reveal
its basic structure. The kinetics of the hydrolysis of naringin and
its application in debittering fruit juices were also investigated.
2. Materials and methods
2.1. Chemicals
Naringin was obtained from Sigma (St. Louis, MO, USA).
Acrylamide, high molecular weight markers for gel filtration, Q
Sepharose Fast Flow and Sephacryl S-200 were purchased from
Amersham (Uppsala, Sweden). All other chemicals were of
analytical grade.
2.2. Microorganism and culture conditions
B. amyloliquefaciens 11568 was isolated from soil in a previous
study and identified by the Institute of Microbiology of Chinese
Academy of Sciences (Beijing, China) using 16S rDNA PCR-RFLP.
The strain was cultivated with medium containing 10 g/L tryptone,
5 g/L yeast extract, 10 g/L NaCl, 15 g/L maltose, 20 g/L (NH4)2HPO4
and 7 g/L naringin. The pH of the medium was adjusted to 7.5. The
seed culture was prepared by inoculating bacteria from the main-
tenance plate into the culture medium and incubating at 40 °C for
48 h. The inoculum (2%, v/v) was cultured for the production of the
enzyme in a 250 mL triangle bottle containing 50 mL of medium.
The culture was maintained at 40 °C and 180 r/min for 48 h. The
collected cells were lysed using ultrasonication at 25 W for 2.5 s
intervals on and off for a total of 10 min. The lysis solution was
then centrifuged at 10,000g at 4 °C for 10 min, and the supernatant
was assayed for enzyme activity.
2.3. Assay of naringinase activity
Naringinase activity was assayed with respect to naringin at
40 °C with minor modifications (Davis, 1947). A typical assay mix-
ture contained 2.0 ml of naringin (0.1%), the appropriately diluted
enzyme buffered with 0.2 M sodium acetate (pH4.0) and 0.1 mL
enzyme solution. The assay mixture was incubated at 40 °C for
30 min, after which a 0.1 mL aliquot of the assay mixture was
added to 5 mL of diethylene glycol (90%); then, 0.5 mL sodium
hydroxide (1 M) was added to the mixture. The samples were
maintained at ambient temperature for 15 min. The intensity of
the resultant absorbance was determined at 420 nm. One unit of
activity (IU) was defined as the amount of enzyme required to
hydrolyze 1 lmol of naringin under the described assay conditions.
The activity of a-L-rhamnosidase was determined using
p-nitrophenyl-a-L-rhamnose (pNPR) as a substrate (Manzanares,
van den Broeck, de Graaff, & Visser, 2001). The reaction mixture,
consisting of 10 mM pNPR and the appropriately diluted enzyme
in 25 mM citrate-phosphate buffer (pH 5.0) was incubated at
45 °C for 30 min; then, the reaction was terminated by the addition
of 0.25 M sodium hydroxide, and the absorption caused by the
released p-nitrophenyl (pNP) was measured at 405 nm. One unit
of a-L-rhamnosidase activity was defined as the amount of enzyme
required that releases 1 mmol of p-nitrophenol at 30 °C in 50 mM
McIlvaine buffer (citrate-phosphate buffer), pH 5.0.
The activity of b-D-glucosidase was measured by the
p-nitrophenyl-a-L-glucosidase (pNPG) method (Zhao et al., 2009).
The reaction mixture of 10 mM pNPG and the appropriately diluted
enzyme buffered with 25 mM acetate (pH 5.0) was incubated at
45 °C for 30 min. The reaction was then terminated by the addition
of 0.25 M sodium hydroxide, and the absorption caused by the
released p-nitrophenyl (pNP) was determined at 405 nm.
2.4. Analysis of the concentration of protein
The protein concentration was determined according to the
method of Lowry et al. (Randall, 1951) using bovine serum albumin
(BSA) as a standard.
2.5. Purification of naringinase
The broth from the bioreactor, containing the released intracel-
lular naringinase, was precipitated with ammonium sulfate at
40–80% saturation. The precipitated protein obtained by centrifu-
gation (10,000g for 10 min) was collected and dialyzed against
Tris-HCl buffer (20 mM, pH 8.5) overnight at 4 °C. The dialyzed
precipitate was then lyophilized and dissolved in a minimal
quantity of sodium acetate (0.2 M, pH 5.5). The concentrated
sample was applied to an anion exchange column (Q Sepharose,
25  1 cm) equilibrated with 20 mM buffer, pH 8.5. Elution of the
column was performed with a linear gradient of NaCl (0–0.5 M)
in Tris-HCl buffer (20 mM, pH 8.5) with a flow rate of 1 mL/min,
and 15 mL fractions were collected. The fractions of the elution
with enzyme activity were pooled and loaded onto a Sephacryl
S-200 (1  20 cm) column that had been previously equilibrated
with 50 mM phosphate buffer (pH 7.2) containing 50 mM sodium
chloride. Elution was performed at a flow rate of 0.1 mL/min
(50 mM phosphate buffer). Fractions (1 mL) with naringinase
activity were collected, and the purity was checked on native
PAGE. The purified enzyme was used for the characterization assay.
2.6. Polyacrylamide gel electrophoresis
SDS-PAGE was carried out with an SE600 Ruby Complete Dual
Cooled Vertical Slab Unit (GE Healthcare Technologies, USA) using
12.5% (w/v) acrylamide gels according to the Laemmli method
(Laemmli, Molbert, Showe, & Kellenberger, 1970). The gels were
stained with Coomassie brilliant blue R-250. The following
unstained protein molecular weight markers (Fermentas SM031)
were used as standards: b-galactosidase, 116.0 kDa; Bovine serum
albumin, 66.2 kDa; Ovalbumin, 45.0 kDa; Lactate dehydrogenase,
35.0 kDa; REase Bsp98 l, 25.0 kDa; b-lactoglobulin, 18.4 kDa;
Lysozyme, 14.4 kDa. Here, the native-PAGE was similar to
SDS-PAGE but without the addition of denaturing reagents, such
as SDS and mercaptoethanol, to maintain the activity of proteins
during the process of polyacrylamide gel electrophoresis. The
concentration of the substrate was 1 mmol/L for both pNPR and
pNPG, with reaction times of 0.5 h and 1 h, respectively.
2.7. Peptide identification
The protein bands on the native-PAGE gel were excised and
placed in Eppendorf tubes. In each tube 200–400 lL of
100 mmol/L NH4HCO3/30% ACN was added for decolorization.
After lyophilization, 5 lL of 2.5–10 ng/lL sequencing grade trypsin
(Promega) solution was added and incubated at 37 °C for 20 h.
Then, the resulting hydrolyzate was transferred to a new
Eppendorf tube. In the original tube another 100 lL of 60%
ACN/0.1% TFA was added to form a mixture with the previous
solution, which was homogenized by ultrasonication for 15 min.
The hydrolyzate was then desalted with Ziptip (Millipore). A total
of 2 L of the hydrolyzate was taken after lyophilization and
reconstituted with 20% acetonitrile;1 L of the lysate was applied
to the 384 Opti-TOF 123 mm  81 mm ss ABI, allowing the solvent
to dry naturally. Then, 0.5 lL of the supersaturated CHCA matrix
solution (solvent 50% ACN, 0.1% TFA) was taken to be air-dried.
 Y. Zhu et al. / Food Chemistry 214 (2017) 39–46
Identification of the peptides was performed using a 5800 Plus
MALDI TOF/TOFTM Analyzer (ABI, Foster City) with a laser source
of Nd, wavelength of 355 nm and acceleration voltage of 2 kV.
Positive ion and automatic data acquisition modes were applied.
The PMF mass scan range was set to 800–4000 Da, and parent
ions SNR greater than 50 were selected for secondary mass
spectrometry (MS/MS) analysis. The number 8 on the parent ion
was selected for each sample point, and 2 MS/MS laser excitation
was set to 2500, collision energy was set to 2 kV, and CID was
set to close. Peptide identification was accepted with a significance
threshold of P < 0.05.
2.8. Effect of pH on enzyme activity and stability
The effect of pH on the activity of naringinase,
a-L-rhamnosidase and b-glucosidase was studied at 45 °C in seven
different buffers (100 mM) with a range of pH values (3.8–10.15):
acetate buffer (pH 3.80–5.80), MES buffer (pH 5.20–7.20) MOPS
buffer (pH 6.20–8.20), Tris-HCl buffer (pH 7.00–9.00), CHES buffer
(pH 8.15–10.15). The purified enzyme was incubated in these
buffers to determine its stability at 45 °C for 30 min, and the
residual enzyme activities were measured by the standard assay
procedure.
2.9. Effect of temperature on enzyme activity and stability
The optimum temperature for naringinase, a-L-rhamnosidase
and b-glucosidase activity was determined by incubating the
enzyme at different temperatures (30–60 °C) in 100 mM Tris-HCl
buffer (pH 7.5). To determine the temperature stability of naringi-
nase, the purified enzyme was incubated at different temperatures
(30–60 °C) at pH 7.5 for 5 h. The treated enzyme samples were
drawn at 0.5 h, 1 h, 2 h, 3 h, 4 h and 5 h and were cooled on ice
for 30 min. The residual enzyme activities were measured by
standard assay procedures as 2.3 described.
2.10. Effect of metals and EDTA on naringinase activity
The influence of metal ions and a chelating agent (EDTA) on
naringinase activity was investigated using the standard assay
conditions. Purified naringinase was incubated in the presence of
metal ions and other agents (0.1, 1, 5 and 10 mmol/L) with various
concentrations, and its activity was compared with that of the
control without metal ions or other agents.
2.11. Determination of kinetic parameters for the hydrolysis of
naringin
The naringinase activity at various concentrations of substrate
(200, 400, 600, 400, or 1000 lg/mL) was determined at 45 °C and
pH 7.5. Apparent Km and Vmax values for naringin were calculated
using a Lineweaver–Burk plot, and the turnover constant (Kcat)
and the catalytic coefficient (ratio of Kcat to km) were then
determined.
2.12. Investigation of substrate specificity
To study the hydrolysis of different substrates, a typical assay
mixture contained 2.0 mL of 50 lg/mL substrate (naringin, neohes-
peridin, ginsenosides, hesperidin, rutin, or quercetin) dissolved in
0.2 M sodium acetate buffer (pH 4.0), and 0.1 mL of enzyme solu-
tion (4 U/mL) was used. The assay mixture was incubated at
40 °C for 30 min, which was followed by measurement of the con-
centration changes of substrates and products using the HPLC
method according to the method of Ni et al. (2012) with a little
modification. An Agilent 1260 HPLC coupled with a zorbax eclipse
41
plus C18 reverse phase column (4.6  100 mm, 5 lm) and a UV
detector was used to determine the concentrations of the sub-
strates and products. Following an injection of 25 lL of reaction
mixture, the column was eluted using a gradient elution at 30 °C
and 0.5 mL/min. The mobile phase was composed of water (A),
methanol (B), and acetonitrile (C). The gradient procedure began
with A:B:C = 26:12:62 within 0–9 min. This procedure was fol-
lowed by a linear change to A:B:C = 15:35:50 within 9–11 min,
another linear change to A:B:C = 15:35:50 within 11–15 min, a lin-
ear 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 UV detector at 280 nm.
2.13. Efficient hydrolysis of naringin by naringinase in pomelo juice
Bitter components, such as naringin, limonin and nomilin, are
not acceptable in commercial products because of their bitterness.
An enzymatic debittering process has little negative impact on
nutrients due to its specificity toward bitter compounds. The fruit
named Guanxi honey pomelo picked from the Fujian province in
China was selected to evaluate the debittering efficiency of the
naringinase. The juice was homogenized in an Omni mixer homog-
enizer at speed 3 for 1 min and was divided into portions of
200 mL/cup. Hydrolysis was initiated by the addition of various
amounts of naringinase (1–4 U/mL) into each portion of the juice,
kept at 45 °C for 60 min and followed by immediate heating to
100 °C for 5 min to thermally denature the enzyme. After cooling
to 45 °C, the concentrations of naringin and naringenin in the
hydrolysates were determined using HPLC method as 2.12
described. The hydrolysis rate (%) was calculated using the follow-
ing formula: (S1 S2)  100/S1; where S1 was the initial concentra-
tion of substrates before enzymatic reaction, and S2 was the
residual concentration of substrates after enzymatic reaction.
3. Results and discussion
3.1. Purification of the enzyme
A typical purification of naringinase from B. amyloliquefaciens
11568 is summarized in Table S1. The crude extract was subjected
to fractional precipitation with ammonium sulfate (40–80%), yield-
ing a 2.41-fold purification with a specific activity of 982 U/mg. The
precipitated sample was applied to a Q Sepharose anion exchange
column. The Q Sepharose step yielded a purification factor of 15
with a specific activity of 11,114 U/mg, whereas the percentage
recovery was 46% (Fig. S1A and Table S1). Further purification
was performed using a Sephacryl S-200column. A major peak
appeared when eluting with phosphate buffer containing 0.05 M
NaCl (pH 7.2) (Fig. S1B). Using this procedure, the naringinase
was purified approximately by 38-fold from the culture filtrate
with a specific activity of 15,609 U/mg and a recovery of 21%
(Table S1).
3.2. Molecular mass and its subunits
The relative molecular weight of the purified enzyme was
estimated to be 32.1 kDa using gel filtration chromatography on
a Sephacryl S-200 column. Native-PAGE analysis showed a solo
protein band exhibiting both a-L-rhamnosidase and b-D-glucosidase
activity (Fig. 1B). Furthermore, when analyzed by SDS-PAGE, the
current naringinases behaved homogenously, appearing as a solo
band with a molecular weight (MW) of 32 kDa (Fig. 1A). These
results indicated that naringinase is a single protein with both
a-L-rhamnosidase and b-D-glucosidase activity, which is signifi-
cantly different from previously reported naringinases.
42 Y. Zhu et al. / Food Chemistry 214 (2017) 39–46
Fig. 1. (A) SDS-PAGE analysis of the purified enzyme. Lane M is the protein marker; lanes 1, 2, 3, and 4 (from left to right) are crude broth, fractions pooled from the
(NH4)2SO4 precipitation, Q Sepharose Fast Flow chromatography, and Sephacryl S-200 samples, respectively. (B) Native-PAGE analysis of naringinase from Sephacryl S-200
chromatography reacted with pNPR and pNPG for 1 h.
The previously reported MW of naringinase varied from 60 to
200 kDa depending on its origin and fermentation conditions
(Ribeiro, 2011). Most naringinases from microorganisms showed
different MWs on SDS-PAGE with several a-L-rhamnosidase or
b-D-glucosidase subunits. Puri, Banerjee, and Banerjee (2005) puri-
fied a naringinase that exhibited a MW of 168 kDa by gel filtration
chromatography; however, on SDS-PAGE, it yielded two bands
with apparent molecular masses of 85 and 80 kDa. A naringinase
from Aspergillus aculeatus JMUdb058 was estimated to have a
MW of 348 kDa by gel filtration chromatography and had four pro-
tein components with MWs of approximately 100, 95, 84, and
69 kDa by SDS-PAGE (Chen et al., 2013). Identification of the
enzyme based on mass spectra analysis and Mascot searching
against the Swissport database revealed that three of the subunits
(100 kDa, 95 kDa, and 84 kDa) were b-D-glucosidase 1 precursors
and that the other subunit (69 kDa) was an a-L-rhamnosidase A
precursor. Naringinase from A. niger has a MW of 131 kDa; its sub-
unit was measured by SDS-PAGE to be approximately 65.5 kDa (Ni
et al., 2012). Until now, no naringinase with a single band on SDS-
PAGE without subunits has been reported. The present naringinase
from B. amyloliquefaciens 11568 is a novel naringinase.
To further identify the peptides of the naringinase, mass spectra
analysis and Mascot searching against the Swissport database were
performed. The band contained a peptide belonging to glycoside
hydrolase (gi | 746279102); its credibility exceeds the credible
threshold; the peptides somehow divided into 67; the molecular
weight of the enzyme was 36 kDa; and the peptide sequence of
the band from the SDS-PAGE of purified naringinase was
RAESVRADLQAKW.
3.3. Effect of pH and temperature on enzyme activity
Both the a-L-rhamnosidase and b-glucosidase activities exhib-
ited optimal activity at pH 7.5, which explains why the naringinase
had an optimal pH of 7.5 (Fig. 2A). a-L-rhamnosidase was stable
between pH 4.3 and 8.5 with an activity of over 90% (Fig. 2B).
The retained activity of b-glucosidase was approximately 80% in
a pH range of 3.8–8.5. This suggests that naringinase is stable from
pH 3.8–8.5. These characteristics allow the naringinase not only to
hydrolyze naringin and hesperidin in acidic pH conditions but also
to convert natural flavonoids to bioactive components in alkaline
conditions. Generally, naringinases from Bacillus showed optimal
activity at neutral or alkaline pH, whereas the optimal pH
values of most fungal naringinases were acidic. For example, the
a-L-rhamnosidase from Corynebacterium (1985) and Pseudomonas
paucimobilis FP 2001 (Miake et al., 2000a) have optimal pH values
of 8.5 and 7.8, respectively. The naringinase from Aspergillus
aculeatus JMUdbo58 (Chen et al., 2013), A. niger 1344 (Puri &
Kalra, 2005), and Penicillium corylopholum MTCC-2011 (Yadav,
Yadava, & Yadav, 2013) have optimal pH values of 4.0,4.0, and
6.0, respectively.
Both the a-L-rhamnosidase and b-glucosidase exhibited optimal
activity at 45 °C and were active over a broad temperature range of
30–60 °C, retaining over 50% of their original activity. This provides
a reasonable explanation for the optimal temperature of naringi-
nase of 45 °C (Fig. 2C). Manzanares, de Graaff, and Visser (1997)
reported that the optimum temperature of a-L-rhamnosidase from
A. niger was 65 °C. Munish Puri et al. and Norouzian et al. reported
that the optimum temperatures of naringinase from A. niger and
Penicillium were 50 °C and 55 °C, respectively (Norouzian,
Hosseinzadeh, Inanlou, & Moazami, 2000; Puri et al., 2005).
The temperature stability of the purified enzyme was deter-
mined according to the naringinase activity. The naringinase was
stable at temperatures up to 45 °C with a remaining enzyme activ-
ity over 90% after 5 h of incubation, but the enzyme was then
rapidly inactivated above 50 °C (Fig. 2D). At 60 °C, the enzyme
activity significantly decreased to less than 10% of its original activ-
ity after 5 h of incubation. Most naringinases from microorganisms
are stable below 50 °C. For example, naringinase from Aspergillus
sojae (Chang et al., 2011), P. paucimobilis FP2001 (Miake et al.,
2000b) and Aspergillus aculeatus JUMdb058 (Chen et al., 2013)were
stable at 37 °C, 40 °C and 50 °C, respectively.
3.4. Kinetic parameters
The purified naringinase was evaluated for its kinetic parame-
ters including maximum reaction velocity (Vmax) and apparent
Michaelis constant (Km) toward the natural substrate naringin
using a Lineweaver-Burk plot. Under optimal conditions (45 °C,
pH7.5), naringinase activity exhibited Michaelis-Menten type
kinetics. The Michaelis constant (Km) measured for naringin was
0.95 mM, and the Vmax was determined to be 3847.3 mM/(L
min)
(Table 1). The Km value was significantly lower than that of
a-L-rhamnosidase of A. niger (2.32 and 2.65 mM) (Caldini, Bonomi,
Pifferi, Lanzarini, & Galante, 1994; Kurosawa, Ikeda, & Egami,
1973), whereas a relatively low Km value of 1.52 mM was found
for a-L-rhamnosidase of Penicillum sp. (Manjón, Bastida, Romero,
Jimeno, & Iborra, 1985). As shown in Table 2, a-L-rhamnosidase
 Y. Zhu et al. / Food Chemistry 214 (2017) 39–46 43

A 100 Table 1
Kinetic parameters of the purified naringinase.
90
1
Enzyme Km (mM) Vmax (U/mg) Kcat (s ) Kcat/km
80
Relative activity (%)

(s 1/mM)
70
Naringinase 1.44 ± 0.14 59.31 ± 3.21 22.62 15.27
60 a-L-rhamnosidase 0.74 ± 0.06 380.35 ± 16.84 145.67 195.94
50 Naringinase b-D-glucosidase 0.76 ± 0.07 314.67 ± 10.90 120.37 157.89
40 α-L-rhamnosidase
30 β-D-glucosidase
20
10 Table 2
0 Substrate Specificity of the naringinase.
3.5 4.5 5.5 6.5 7.5 8.5 9.5 10.5
Substrate Hydrolyzable Hydrolyzate Hydrolysis
pH bonds (%)

B 100 Naringin a-1,2 Naringenin, rhamnose, glucose 90

Neohesperidin a-1,2 Hesperetin, rhamnose, glucose 86
90
Hesperidin a-1,2 Hesperetin, rhamnose, glucose 83
Relative activity (%)

80
Rutin a-1,6 Quercetin, rhamnose, glucose 5
70 Quercetin Connected Quercetin, rhamnose 8
60 Naringinase
directly to it
50 α-L-rhamnosidase
40 β-D-glucosidase
30
20
10 was estimated to have a Km value of 0.74 mM, which is lower than
0 those from A. Aculeatus and A. niger, indicating that the present a-L-
3.5 4.5 5.5 6.5 7.5 8.5 9.5 10.5 rhamnosidase has a relatively high substrate affinity, whereas a
pH lower Km value of 0.62 mM was found for a-L-rhamnosidase of
C Penicillum sp. (Yadav et al., 2013). The b-D-glucosidase was esti-
100
mated to have a Km value of 0.76 mM, which is lower than those
Relative enzyme activity %

90
80 of the two b-D-glucosidases from A.terreus with Km values of
70 1.9 mM and 2.1 mM, indicating that our b-D-glucosidase had a
60 naringinase
higher affinity for pNPG than those from A.terreus (Soria,
50 α-L-rhamnosidase
Ellenrieder, Grasselli, Navarro, & Cascone, 2004).
40 β-D-glucosidase
30
20
10
3.5. Effect of metal ions and EDTA on enzyme activity
0
25 35 45 55 65 Table S2 shows the effect of metal ions and organic compounds
Temperature %
on naringinase activity. Zn2+, Ag+, Ba2+, Fe2+, Hg2+, Cu2+ and EDTA
D 100 were found to have completely inhibited the enzyme activity at a
90 concentration range of 0.1–10 mM, and Na+, K+, Li+, Pb2+, Al3+,
80 Ba2+, and Co2+ showed very little inhibition at 0.1–10 mM. EDTA
%

70 did not inhibit enzyme activity, even at 1 mM. However, at

5 mM, 50% of the enzyme activity was inhibited. Many types of
Relative activity

60
50 these metal ions were present in the media as either trace ele-
40 ments or mineral ions, but would not have reached the concentra-
30 tions necessary to inhibit naringinase activity.
20 30 35 40
45 50 55
10 60 3.6. Substrate specificity
0
0 1 2 3 4 5 Naringinase hydrolyzed naringin, neohesperidin, hesperidin,
Incubation time h rutin and quercetin (Fig. 3) via the activity of a-L-rhamnosidase,
Fig. 2. (A) Effect of pH on the activity of the purified enzyme from B. amylolique-
which cleaves L-rhamnose residues and b-glycoside residues linked
faciens 11568.The influence of pH on naringinase, a-L-rhamnosidase and b-D- by the covalent bonds of a-1,2 and a-1,6. Naringinase was also
glucosidase activity was determined at 40 °C using 50 mM of different buffers capable of hydrolyzing substrates to which L-rhamnose was
(pH3.8–10.15). Relative activity was calculated as the percentage of a detected directly connected. Table 2 shows that the best substrate for
activity compared with the activity at the peaks. (B) Effect of pH on the stability of
hydrolysis by naringinase is naringin.
the purified enzyme produced by B. amyloliquefaciens 11568. The remaining activity
was measured after incubation for 30 min at 40 °C in buffers with different pH Fig. 3 showed the reaction mixtures before (solid line) and after
values from 3.8–10.15. Relative activity was calculated as the percentage of the (dashed line) enzymatic hydrolysis of naringin, neohesperidin,
detected activity compared with the initial activity. (C) Optimal temperature of the hesperidin, rutin and quercetin glycosides. Neohesperidin was
purified enzyme produced by B. amyloliquefaciens 11568. The temperature profile hydrolyzed by a-L-rhamnosidase activity, releasing rhamnose
was measured at different temperatures using the standard assay as described in
the Material and Methods section at an optimal pH of 7.5 in 50 mM Tris-HCl buffer.
and hesperetin-7-glucoside, which was further hydrolyzed to
(D) Thermal stability of the purified enzyme from B. amyloliquefaciens 11568. The hesperetin and glucose by the b-D-glucosidase activity of naringi-
temperature profile was measured at different temperatures using the standard nase. Hesperidin was also hydrolyzed to hesperetin, glucose and
assay as described in the Material and Methods section at an optimal pH of 7.5 in a-L-rhamnose. Rutin and quercetin glycosides were hydrolyzed
50 mM Tris-HCl buffer.
by naringinase to quercetin and a-L-rhamnose, respectively.
44 Y. Zhu et al. / Food Chemistry 214 (2017) 39–46

Fig. 3. Hydrolysis of different substrates detected by HPLC. (A–E): Chromatographs of the reaction mixtures before (solid line) and after (dashed line) the enzymatic
hydrolysis of naringin, neohesperidin, ginsenosides, hesperidin, rutin and quercetin.
 Y. Zhu et al. / Food Chemistry 214 (2017) 39–46
Fig. 4. Efficient hydrolysis of naringin by the naringinase in pomelo juice. (Insert A) Standard curve of naringin; (Insert B) concentration changes of naringin with different
amounts of enzyme.
3.7. High efficiency of the naringinase in debittering citrus juice
The existence of bitter substances in citrus juice is the main
problem in the production of sweet pomelos (Maier & Beverly,
1968). Naringinase played an important role in reducing juice
bitterness by hydrolyzing naringin (Busto, Meza, Ortega, & Perez-
Mateos, 2007; Johnson & Chandler, 1981; Puri & Banerjee, 2000;
Yadav, Yadav, Yadav, & Yadav, 2010). Debittering citrus juice with
naringinase has many advantages, such as high specificity of the
enzyme for the bitter substances, good debittering effects, simple
operation and mild reaction conditions without any damage to
the nutritional ingredients or flavor of the citrus juice. Considering
these advantages, this application is expected to become the most
promising method for debittering citrus juice.
In this study, the naringin in citrus juice was efficiently hydro-
lyzed to naringenin by naringinase (Fig. 4). HPLC analysis of citrus
juice treated with naringinase showed that the concentration of
naringin was less than 30 lg/mL when the naringinase was main-
tained at or greater than 4 U/mL, indicating that naringinase was
able to efficiently remove the bitter taste of citrus juice according
to the taste threshold of naringin. A similar result was reported
for the naringinase from A. niger (Ni et al., 2012). However, the
enzyme purified from Penicillium (Puri, Seth, Marwaha, & Kothari,
2007) cannot sufficiently debitter citrus juice. The broad pH
stability and substrate specificity of the present naringinase from
Bacillus amyloliquefaciens suggests its potential application in
biotechnological processes such as debittering citrus fruit juice
and improving the bioavailability of polyphenols.
Acknowledgements
This research was financially supported by the National Natural
Science Foundation of China (Nos. 31371723, 31571872), and the
Development of Innovative Teams and Teachers’ Occupation
Advancement Project of Beijing Municipal Universities and
Colleges (No. IDHT20130506).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2016.
06.108.
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