Development and evaluation of an HPLC method for accurate determinations of enzyme activities of naringinase complex

Article abstract of DOI:10.1021/jf402711h

An HPLC method that can separate naringin, prunin, and naringenin was used to help accurately measure the activities of naringinase and its subunits (α-l-rhamnosidase and β-d-glucosidase). The activities of the naringinase and β-d-glucosidase were determined through an indirect calculation of the naringenin concentration to avoid interference from its poor solubility. The measured enzymatic activities of the naringinase complex, α-l-rhamnosidase, and β-d-glucosidase were the as same as their theoretical activities when the substrates' (i.e., naringin or prunin) concentrations were 200 μg/mL, and the enzyme concentrations were within the range of 0.06-0.43, 0.067-0.53, and 0.15-1.13 U/mL, respectively. The β-d-glucosidase had a much higher V_max than either naringinase or α-l-rhamnosidase, implying the hydrolysis of naringin to prunin was the limiting step of the enzyme reaction. The reliability of the method was finally validated through the repeatability test, indicating its feasibility for the determinations of the naringinase complex.

Full text of DOI:10.1021/jf402711h

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Article
Development and evaluation of an HPLC method for accurate
determinations of enzyme activities of naringinase complex
Hui Ni, An-Feng Xiao, Ya Qi Wang, Feng Chen, Hui-Nong Cai, and Wen-Jin Su
J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 26 Sep 2013
Downloaded from http://pubs.acs.org on October 2, 2013
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Page 1 of 32
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Development and evaluation of an HPLC method for accurate
determinations of enzyme activities of naringinase complex
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†,‡,§
†,§,‖
‡
*,†,‡,§
†,§,‖
†
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Hui Ni , AnꢀFeng Xiao
, Ya Qi Wang , Feng Chen
, HuiꢀNong Cai
, WenꢀJun Su
†
‡
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0
1
2
College of Bioengineering, Jimei University, Fujian Province, 361021, P.R. China
Department of Food, Nutrition and Packaging Sciences, Clemson University, Clemson, SC
29634, USA
§
Research Center of Food Microbiology and Enzyme Engineering Technology, Jimei
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University, Xiamen, 361021, P.R. China
‖
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1
Research Center of Food Biotechnology of Xiamen City, Xiamen 361021, P.R. China
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Short version title: Determination of naringinase by HPLC method
Corresponding author:
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Professor Feng Chen
College of Bioengineering
Jimei University
Fujian Province, 361021, P.R. China
and
Department of Food, Nutrition and Package Sciences
Clemson University
Clemson, South Carolina 29634
USA
Phone: 864ꢀ656ꢀ5702
Eꢀmail: fchen@clemson.edu
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ABSTRACT:
An HPLC method that can separate naringin, prunin and naringenin was used to help
accurately measuring the activities of naringinase and its subunits (αꢀLꢀrhamnosidase and
βꢀDꢀglucosidase). The activities of the naringinase and βꢀDꢀglucosidase were determined
through an indirect calculation of the naringenin concentration to avoid interference from its
poor solubility. The measured enzymatic activities of the naringinase complex,
αꢀLꢀrhamnosidase and βꢀDꢀglucosidase were as same as their theoretical activities when the
substrates’ (i.e., naringin or prunin) concentrations were 200 ꢁg/mL, and the enzyme
concentrations were within the range of 0.06ꢀ0.43 U/mL, 0.067ꢀ0.53 U/mL and 0.15ꢀ1.13
U/mL, respectively. The βꢀDꢀglucosidase had a much higher Vmax than both the naringinase
and the αꢀLꢀrhamnosidase, implying the hydrolysis of naringin to prunin was the limiting step
of the enzyme reaction. The reliability of the method was finally validated through the
repeatability test, indicating its feasibility for the determinations of the naringinase complex.
KEYWORDS: HPLC, naringinase, αꢀLꢀrhamnosidase, βꢀDꢀglucosidase, kinetics, Aspergillus
niger
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■ INTRODUCTION
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Naringinase is an enzyme complex that contains both an αꢀLꢀrhamnosidase (EC 3.2.1.40)
that hydrolyzes naringin to prunin, and a βꢀDꢀglucosidase (EC 3.2.1.21) that further degrades
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prunin to naringenin.
The naringinase activity refers to its capability to convert naringin to
naringenin and glucose (Figure1). The naringinase has many valuable applications, such as
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debittering citrus fruit juices,
producing rhamnose, enhancing wine aromas, removing
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hesperidin crystals from canned orange products, yielding clinical important products by (or
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from) modification of steroids, etc.
The traditional method to measure the enzymatic activities of the αꢀLꢀrhamnosidase and
βꢀDꢀglucosidase often uses the following two artificial substrates, i.e.,
pꢀnitrophenylꢀαꢀLꢀrhamnopyranoside (pNPR) and pꢀnitrophenylꢀβꢀDꢀglucopyranoside
12ꢀ15
(pNPG),
respectively. However, lack of specificity of the substrates has cast some doubts
on the method for the accurate determinations of the enzymatic activities of the naringinase
and its subunits, i.e., αꢀLꢀrhamnosidase and βꢀDꢀglucosidase. Another commonly used
method for determination of the naringinase activity is the Davis’s method, which is based on
the spectrophotometric determination of the reaction product between flavonones (e.g.,
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naringin) and diethyleneglycol under an alkaline condition. Likewise, this method can not
accurately determine the activities of naringinase and the αꢀLꢀrhamnosidase because naringin
(the substrate), prunin (an enzymatic reaction intermediate) and naringenin (the product) have
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similar absorptive spectra.
Later, a combined procedure consisting of an aldohexose
analysis and naringenin analysis was proposed to measure the activities of the
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αꢀLꢀrhamnosidase and naringinase, but its application was limited owed to its operation
complexity.
It has been reported that HPLC is able to conduct accurate and precise determination of
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naringin, prunin and naringenin.
Recently, an HPLC method has been used to analyze the
concentration changes of naringin, prunin and naringenin after the hydrolysis of naringin,
resulting in measurements of the activities of naringinase, αꢀLꢀrhamnosidase and
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βꢀDꢀglucosidase. However, there was a problem that was neglected in the previous report
that naringin is subject to precipitation or crystallization in aqueous solution at room
temperature, which might lead to an inhomogeneous reaction solution and affect the accuracy
and repeatability of the determinations. Moreover, the poor solubility of naringenin in water
might cause an inaccurate HPLC determination of the chemical, leading to inaccurate
determinations of the activities of naringinase and/or βꢀDꢀglucosidase.
Therefore, the general objective of the present study was to develop a reliable HPLC
analysis for simultaneous determination of the activities of naringinase, αꢀLꢀrhamnosidase
and βꢀDꢀglucosidase regardless of the limited solubility of naringin, prunin and naringenin in
the enzymatic solution. It included the following four specific aspects: (1) determining
appropriate ranges of concentrations of the substrates (i.e., naringin or prunin) in concern of
their solubilities in the enzymatic reaction solutions; (2) developing a reliable measurement of
naringenin to ensure accurate determinations of the enzymatic activities of naringinase and
βꢀDꢀglucosidase; (3) investigating the proper ranges of the enzyme concentrations and
enzymatic kinetics for the accurate determinations of these enzymes activities; (4) validating
the reliability of the aforementioned method based on the repeatability of the enzymatic
activities.
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■ MATERIALS AND METHODS
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Materials and Reagents. Naringin and naringenin were bought from SigmaꢀAldrich Inc.
(St. Louis, MO, USA). Prunin was bought from Extrasysthese (Genay Cedex, France). HPLC
grade methanol and acetonitrile were purchased from Tedia Company Inc. (Ohio, USA). All
other chemicals in analytical grade were obtained from various chemical companies in China.
HPLC Condition. Qualitative and quantitative analyses of naringin, prunin and
naringenin were conducted on a Waters 1525 HPLC instrument which was equipped with a
Symmetry C18 reversed phase column (4.6×150 mm, 3.5 ꢂm) (Waters Corporation, Milford,
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MA, USA).
Briefly, twenty microliters of the sample solution which was preꢀfiltered
through a 0.22 ꢁm PTFE filter were injected into the column, and immediately isocratically
eluted by a mobile phase in composition of 11.4% methanol, 26.6% acetonitrile and 62%
purified deionized water at a flow rate of 0.4 mL/min at 35ºC for 28 min. Signals were
detected at 280 nm by a 2487 UV detector.
Enzymatic Reaction Condition. The substrate naringin was used for analyses of the
naringinase and αꢀLꢀrhamnosidase, and the substrate prunin was used for analysis of the
βꢀDꢀglucosidase. The enzymatic reaction solutions and reaction conditions were set up
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, 12, 15, 20ꢀ25
according to the previous studies.
In brief, two milliliters of the stock solutions of
the substrates were mixed with 1.9 mL of 20 mM citric acid buffer (pH 5.0) and 0.1 mL of the
enzyme solution followed by incubation at 50ºC for 5 min, heated at 100ºC for 5 min, and
then cooled down to room temperature.
Definition of Enzymatic Activity Unit. One unit of naringinase was defined as the
amount of enzyme that releases 1 ꢁM of naringenin from naringin; one unit of
αꢀLꢀrhamnosidase was defined as the amount of enzyme that hydrolyzes 1 ꢁM of naringin to
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prunin; one unit of βꢀDꢀglucosidase was the enzyme that hydrolyzes 1 ꢁM of prunin to
naringenin.
Construction of Calibration Curves of Naringin, Prunin and Naringenin. The
standard solutions of naringin and prunin were prepared at 10, 50, 100, 150, 200, 300, 400
and 500 ꢁg/mL in 10 mM citric acid buffer (pH 5.0) followed by heating at 100°C for 5 min
(imitating inactivation of the enzyme) in a water bath prior to the HPLC analysis. The
naringenin solutions were prepared in methanol regarding its poor aqueous solubility. After
filtration through 0.22 ꢁm syringe filters (Thermo Fisher Scientific Inc., Pittsburgh, USA), all
the standard solutions were subject to the HPLC analysis as mentioned in HPLC Condition.
The calibration curves were plotted by the peak areas against concentrations in a linear
regression.
Effect of the Enzymatic Reaction Condition on the Enzyme Substrates and Products.
To investigate the reaction condition on the substrates and products (i.e., naringin, prunin and
naringenin), the standard solutions were prepared at 50, 150, 250, 350 and 450 ꢂg/mL in the
reaction solution, which were incubated at 50ºC for 5 min, then heated at 100°C for 5 min and
kept in a water bath at 50°C. Thereafter, the naringin and prunin solutions were filtered
through 0.22 ꢁm filters prior to the HPLC analysis; the naringenin solutions were dried in a
rotary evaporator, and subsequently dissolved in the same volume of methanol prior to the
HPLC analysis. Recovery (the percentage of a detected concentration to the actually prepared
concentration) and relative standard deviation (RSD) of the detected concentration of the
three chemicals were calculated.
Effect of Temperature on Solubility of Naringin, Prunin and Naringenin in the
Reaction Solutions. Solutions of naringin (50, 100, 150, 200, 300, 400 and 500 ꢂg/mL),
prunin (25, 50, 100, 150 and 200 ꢂg/mL) and naringenin (25, 50, 75, 100 and 150 ꢂg/mL)
were prepared in the reaction buffer. After incubation at 50ºC for 5 min, the solutions were
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subsequently heated at 100°C for 5 min, and respectively kept at 50°C, 20°C (room
temperature) and 4°C (store temperature) for 12 h followed by filtration through 0.22 ꢁm
filters prior to the HPLC analysis.
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Investigation of Desirable Ranges of Enzyme Concentrations for Enzymatic
Reactions
To investigate the desirable ranges of the enzyme concentrations for accurate
determinations of the enzymatic activities, the naringinase that was extracted from the A.
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niger fermentation broth and purified to have an activity about 7.24 U/mL was serially
diluted by 2, 4, 8, 16, 32, 64 and 128 times. One hundred microliters of the respectively
prepared enzyme solutions were mixed with 2 mL of a substrate (i.e., naringin or prunin)
stock solution in a concentration of 400 ꢁg/mL and 1.9 mL of 20 mM citric acid buffer
(pH=5.0) at 50°C for 5 min, and subsequently heated at 100°C for 5 min and cooled down to
room temperature. After filtration through 0.22 ꢁm filters, the solutions were subject to the
HPLC analysis. Thereafter, the enzymatic activities were obtained by multiplying the activity
of each dilutee with its corresponding diluted factor.
Repeatability of Enzymatic Activities
To test the repeatability of the measured enzymatic activities, the enzymatic reactions
were repeated 5 times, from which the RSD of each sample was calculated. Two samples, a
broth of A. niger which was preꢀdiluted by 8 times and a purified naringinase which was
preꢀdiluted by 32 times, were used to conduct the reaction.
Determination of the Enzymatic Kinetic Parameters. The naringinase, which was
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purified from Aspergillus niger according to our previous study
and diluted to a final
concentration at about 0.43 U/mL, was applied to reactions with 200, 150, 100, 75, 50, 25,
12.5 and 6.25 ꢂg/mL of the substrate at 50ºC for 5 min. After the activities were measured,
their Vmax and K were estimated based on the Lineweaver–Burk plot.
m
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Investigation of the Enzyme Production in Submerged Cultivation of A. niger. The
cultivation of A. niger DB056 for the production of naringinase was conducted according to
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the previous studies
with minor modifications. Briefly, 50 mL of spore suspension
(OD600 at 0.2) of A. niger was inoculated into a NBS Biofloꢀ110 7 L fermentor (New
Brunswick Scientific, Connecticut, USA) which contained 5 L of fermental medium with
composition (g/L) of: naringin 10, MgSO
4 2 2 4 4 2 4
ꢃ7H O 0.5, KH PO 1.5, (NH ) SO 4.0,
ZnSO ꢃ7H O 0.09, CaCl 0.1, yeast extract 1.0, soybean powder 2.0, and peptone 2.0,
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followed by cultivation at 28ºC, pH 6.0 and 300 rpm for 8 days, during which samples were
fetched in every 24 h to measure the activities of naringinase, αꢀLꢀrhamnosidase and
βꢀDꢀglucosidase.
Statistical Analysis. Every experiment was done in triplicate except of the repeatability
that was conducted for 5 times, and means were calculated to represent the results. The SPSS
17.0 software (SPSS Inc. Chicago, IL) was used to analyze the significant difference (p<0.05)
of different samples through the Duncan’s multiple range method.
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Qualification and Quantitation of Naringin, Prunin and Naringenin. Naringin, prunin
and naringenin had their HPLC chromatographic retention times at 5.83, 6.79, and 21.64 min
(Figure 1), respectively. Obviously they were completely separated from each other under the
current HPLC condition. The chromatographic resolutions between the chemicals were more
than 2.0 (Figure1B, Figure 1C). The enzymatic hydrolysis of naringin resulted in a decrease
of concentration of naringin from a peak height of 1.2 AU before the hydrolysis reaction
(Figure 1A) to 0.6 AU after the reaction (Figure 1B), accompanying with appearance of
prunin and naringenin, which was in accordance with the results of the previous studies that
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the naringinase hydrolyzes naringin to naringenin through intermediate prunin.
This
result indicates the HPLC method is able to distinguish the substrate (naringin), intermediate
(prunin) and product (naringenin) after the naringinase hydrolysis, and able to be used for the
determination of the enzyme activity.
The established calibration curves of naringin, prunin and naringenin were expressed as:
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y (peak area in unit of 10 mAu*s)=0.0103x+0.0035 (x refers to the analyzed chemical
concentration in unit of ꢁg/mL), y= 0.0148x+0.0024 and y=0.017x+0.007 (Figure 2),
2
respectively. The R were 0.9997 for naringin, 0.9999 for prunin and 0.9991 for naringenin,
which indicated a good linearity of the three calibration curves within the concentration range
of 10ꢀ500 ꢁg/mL (Figure 2). After incubation and heating within the enzymatic reactions,
naringin showed a recovery of 98.0ꢀ100.2% and RSD of 1.4ꢀ2.8%; prunin had a recovery of
98.9ꢀ102.8 and RSD of 1.8ꢀ3.3%; naringenin displayed a recovery of 98.8ꢀ103.6% and RSD
of 1.7ꢀ2.9% (Table 1). This result suggests the naringin, prunin and naringenin are stable and
not significantly affected by the reaction condition
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Solubility of Naringin and Prunin in the Reaction Solution and Desirable Substrate
Concentration. The solubility of naringin in water was reported to be dependent on the
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temperature and acidity. A higher temperature favors a higher solubility,
while a higher
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acidity (lower pH) results in a lower solubility. Although naringin can be dissolved up to
500 ꢁg/mL at 50°C and showed an acceptable solubility (e.g., 200 ꢁg/mL) at 20°C and 4°C, it
cannot be sufficiently dissolved (e.g., 300 ꢁg/mL) at 20°C and 4°C (Figure3A). By contrast,
all the prunin solutions were detected to have their concentrations identical to the theoretical
(or the actually prepared) concentrations in the range of 25ꢀ200 ꢁg/mL at 50°C, 20°C and 4°C
(Figure 3B). Therefore, it was evident that choosing 200 ꢁg/mL of the substrates (naringin or
prunin) was desirable for this experiment in an effort to ensure the complete solubility of the
substrates (i.e., naringin and prunin) in the processes of the enzymatic reaction (50°C), sample
keeping (4°C) and room temperature (20°C).
Solubility of Naringenin in the Reaction Solution and the Approach for Its
Determination. Theoretically, there are three kinds of enzyme activities resulted from the
naringinase complex, i.e., naringinase, αꢀLꢀrhamnosidase and βꢀDꢀglucosidase. The activity
of naringinase (the combination of the activities of both αꢀLꢀrhamnosidase and
βꢀDꢀglucosidase) can be measured by determining the amount of the appearance of naringenin
released from naringin; By comparison, the αꢀLꢀrhamnosidase can be measured by
determining either the degraded naringin or the released prunin, and the βꢀDꢀglucosidase can
be analyzed by measuring either the hydrolyzed prunin or the production of naringenin
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(Figure 1). However, naringenin had a very poor solubility in the aqueous solution,
which has caused a difficulty to directly determine the production of naringenin.
All the naringenin solutions were measured to have their detected concentrations
remarkably lower than their theoretical (or actually prepared) concentrations at any tested
temperatures, i.e., 50°C, 20°C and 4°C (Figure 3C). This result was consistent with previous
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results that naringenin had a very poor solubility in the aqueous solution,
and implied the
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naringenin generated in the enzymatic reaction was not be sufficiently dissolved in water. It
was reported that addition of methanol to a final concentration of 50% increased the solubility
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of naringenin in grapefruit juice.
A similar result was also found in our study that an
addition of 60ꢀ80% methanol achieved a simultaneous dissolution of 200 ꢁg/mL of naringin
and 100 ꢁg/mL of naringenin (Figure 4). However, the addition of methanol to dilute the
sample solutions by 4ꢀ5 times may cause difficulty in analyses of low concentrations of the
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analytes. These results indicate the released naringenin is hard to be directly determined due
to its poor solubility. Therefore, in order to ensure the accuracy of the determination of the
activities of naringinase and βꢀDꢀglucosidase, the production of naringenin was measured
through an indirect approach according to the consumption of naringin and prunin.
Given the case above, the concentration changes of naringin, prunin and naringenin
before and after the reactions are expressed in the following equations (1) and (2), and listed
in Table 2, which allow the calculation of the enzymatic activities. The concentration change
of substrate naringin could be used to determine the activity of αꢀLꢀrhamnosidase as shown in
equation (1), where the amount of consumed naringin (y+z) was obtained by taking its
remaining concentration (x) from its original concentration before the hydrolysis reaction
(x+y+z) to calculate the activity of αꢀLꢀrhamnosidase; Meanwhile, the amount of the
generated naringenin (z, equal to the concentration in micromole of the hydrolyzed prunin)
was indirectly obtained by subtracting the remaining naringin (x) and prunin (y) from the
original concentration of naringin (x+y+z) to measure the activity of naringinase. Substrate
prunin was used to determine the activity of βꢀDꢀglucosidase by measuring the consumed
prunin (equal to the production of naringenin) as shown in equation (2), where the amount of
consumed prunin (b, theoretically equal to the amount of the generated naringenin) was
obtained by taking the remaining concentration (a) from the original concentration (a+b).
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Rha
Glu
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(x + y + z) nar → x nar + (y + z) pru → x nar
+
y pru + z narg
(1)
(2)
Glu
2
(a + b) pru → a pru + b narg
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In the equation (1) and (2), nar, pru, narg, Rha and Glu were the abbreviations of
naringin, prunin, naringenin, αꢀLꢀrhamnosidase and βꢀDꢀglucosidase, respectively; x, y, z, a
and b were variables as defined in Table 2. (x+y+z) was the number of mM of the reaction
naringin before the enzymatic reaction for analyzing the activity of αꢀLꢀrhamnosidase, (y+z)
was the total amount (mM) of prunin that was released by the αꢀLꢀrhamnosidase after the
reaction, and z was the amount (mM) of naringenin that was released from prunin by the
βꢀDꢀglucosidase after the reaction. (a+b) was total amount (mM) of prunin before a reaction
for analyzing the activity of βꢀDꢀglucosidase; a was the amount (mM) of the remaining prunin
after the reaction, b was the amount (mM) of naringenin generated after the reaction.
Desirable Detective Range and RSD of the Measured Enzyme Activities. The
enzymatic activities were observed to have varying values depending on the dilution times
(Table 3). More times the enzyme solution was diluted, higher activity (product of the activity
of a diluted enzyme multiplied by the dilution time) of the solution was obtained. When the
enzyme was diluted more than 16 times, its activity had no significant changes along with the
increasing dilution times. This result indicated the dilutees had enzymatic activities linearly
related to the diluted times when diluted more than 16 times, which implied the desirable
applied ranges of the naringinase, αꢀLꢀrhamnosidase and βꢀDꢀglucosidase were 0.06ꢀ0.43
U/mL, 0.07ꢀ0.53 U/mL and 0.15ꢀ1.13 U/mL (Table 3), respectively. It suggests a proper
dilution is needed to get the accurate result when the enzyme activities go beyond the
upꢀlimits of these concentrations.
Regarding the repeatability of the analyzed enzymatic activities, RSDs of the fermented
broth and the purified enzymes were 8.25% and 7.33% for the naringinase; 4.29% and 4.54%
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79
for the αꢀLꢀrhamnosidase, and 3.33% and 4.64% for the βꢀDꢀglucosidase, respectively (Table
4). These results demonstrated good repeatability for determinations of activities of the
enzymes in both the fermented broth and their purified forms.
80
81
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03
Validation of the Determinations of the Enzyme Activities. Although some
naringinases, αꢀLꢀrhamnosidases and βꢀDꢀglucosidases have been characterized by the Davis
1
2ꢀ15, 24, 25, 30, 31
and pꢀnitro phenol methods,
their enzymatic kinetic parameters towards the
naringin hydrolysis were hardly studied based on the previous old methods. Our HPLC
method has facilitated to reveal those kinetics. Based on the Lineweaver–Burk plots, the
naringinase from A. niger had Vmax of 0.60 U/mL and K of 0.13 mM (Figure 5). The
m
αꢀLꢀrhamnosidase had Vmax of 1.51 U/mL and K of 0.28 mM, while the βꢀDꢀglucosidase had
m
Vmax of 9.63 U/mL and K
m
of 0.39 mM (Figure 5). The αꢀLꢀrhamnosidase was determined to
33
have a K similar to that of a previous study obtained by a HPLC method, indicating both
m
HPLC methods were accurate to analyze the kinetic parameters of αꢀLꢀrhamnosidase. The
βꢀDꢀglucosidase from A. niger was determined to have a much lower K (0.39 mM) than that
m
of a previous study (K 2.59 mM) analyzed based on the direct detection of naringenin,
m
verifying that direct analysis of the production of naringenin did cause inaccuracy to the
determination of the activity of βꢀDꢀglucosidase, whereas the indirect calculation of
naringenin production is necessary and more desirable to ensure the accuracy of the
determination of naringinase and βꢀDꢀglucosidase. It is evident that the present naringinase
had the K value (0.13 mM) much lower than that of a previous counterpart from A. niger
m
2
4
(1.9 mM) determined by the Davis method,
indicating the our improved method has
overcome the inaccuracy of the Davis method and provided an accurate and sensitive
determination of naringinase.
The K of this αꢀLꢀrhamnosidase (0.28 mM) was much smaller than those (about 2.8 mM)
m
of previous αꢀLꢀrhamnosidases from A. niger using the substrate pNPR, which indicates the
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317
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320
321
322
323
324
325
326
327
328
enzyme has a higher affinity to naringin than pNPR.
m
In addition, the K of our
βꢀDꢀglucosidases (0.39 mM) is much lower than that of a βꢀDꢀglucosidase extracted from A.
3
0
m
niger, which had K values of 1.03 mM for pNPG and 5.36 mM for cellobiose, indicating
the βꢀDꢀglucosidases had a higher affinity to naringin than to pNPG and cellobiose. In
addition, the αꢀLꢀrhamnosidase has a lower Vmax than the βꢀDꢀglucosidase, implying the
hydrolysis of naringin to prunin was the velocity limiting step of the naringin degradation,
3
3
which is similar to our previous study of the naringinase from A. aculeatus.
Recently, naringinase has been confirmed to be an enzyme complex consisting of two
33
subunits rather than a solo protein with two catalytic sites. Although a few naringinase
24, 25
fermentations were studied according to the Davis method,
the production of
αꢀLꢀrhamnosidase and βꢀDꢀglucosidase and their quantitative relationship during this process
are still not clear. The present HPLC method could help to monitor the production of
naringinase as well as its αꢀLꢀrhamnosidase and βꢀDꢀglucosidase (Figure 6). This study
revealed that A. niger excreted the αꢀLꢀrhamnosidase a little ahead of βꢀDꢀglucosidase, which
explained why the activity of naringinase was hardly observed in the initial 48 h of
fermentation (Figure 6). The αꢀLꢀrhamnosidase activity stopped its increase in the middle of
the fermentation (after 60 h). However, the βꢀDꢀglucosidase kept on rapid increase all over the
fermentation period, and its activity (3.45 U/mL) greatly outnumbered that of the
αꢀrhamnosidase (1.75 U/mL) at the end of fermentation (Figure 6). Since the naringinase was
affected by both the αꢀLꢀrhamnosidase and the βꢀDꢀglucosidase, it was reasonable to suggest
that increasing the production of the αꢀLꢀrhamnosidase and the βꢀDꢀglucosidase could
increase the production and/or activity of the naringinase. Nevertheless, the activity tendency
of αꢀLꢀrhamnosidase was not consistent to that of βꢀDꢀglucosidase, indicating these two
enzymes might be synthesized by two independent biochemical processes. Although the
3
4,
αꢀLꢀrhamnosidase and a βꢀDꢀglucosidase were purified from a fermented broth of A. niger,
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3
29
their synthetic pathways for the formation of the naringinase complex is not elucidated yet.
In summary, an improved procedure was developed and evaluated to accurately analyze
30
31
32
33
34
35
36
37
38
39
40
41
42
43
the enzymatic activities of naringinase, αꢀLꢀrhamnosidase and βꢀDꢀglucosidase based on the
HPLC determination of naringin, prunin and naringenin. It is critical to contain about 200
ꢁg/mL of substrate (naringin and prunin) in the reaction solution, and measure the product
naringenin through the indirect calculation approach instead of the direct HPLC analysis. The
desirable ranges of the enzymes for the enzymatic reactions were 0.06ꢀ0.43 U/mL for
naringinase, 0.07ꢀ0.53 U/mL for αꢀL rhamnosidase, 0.15ꢀ1.13 U/mL for βꢀDꢀglucosidase. The
analyses of repeatability of RSDs of the enzymatic activities were below 9% for naringinase,
below 5% for both αꢀLꢀrhamnosidase and βꢀDꢀglucosidase. In addition, this new method
could help to study the fermented production and characteristics of naringinase. This HPLC
method provides accurate determinations of the activities of naringinase, αꢀLꢀrhamnosidase
and βꢀDꢀglucosidase, which overcomes the shortcomings of previous methods, and facilitates
further studies on the naringinase complex.
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44 ■ ASSOCIATED CONTENT
345
346
347
348
349
Supporting Information. This research was supported by grants from the National
Natural Science Foundation of China (No. 31371751), Science Foundation for Distinguished
Young Scholars of Xiamen City (No. 3502Z20126008), and Foundation for Innovative
Research Team of Jimei University (2010A006).
3
50 ■ AUTHOR INFORMATION
3
51
52
Corresponding Author. *Phone: 864ꢀ656ꢀ5702. Eꢀmail: fchen@clemson.edu.
Notes. The authors declare no competing financial interest.
3
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54 ■ REFERENCES
355
356
357
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359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
(1)
(2)
(3)
(4)
(5)
Puri, M.; Banerjee, U. C. Production, purification, and characterization of the
debittering enzyme naringinase. Biotechnol. Adv. 2000, 18, 207ꢀ217.
Yadav, V.; Yadav, P. K.; Yadav, S.; Yadav, K. D. S. αꢀLꢀRhamnosidase: a review.
Process Biochem. 2010, 45, 1226ꢀ1235.
Gray, G. M.; Olson, A. C. Hydrolysis of high levels of naringin in grape juice using a
hollow fiber naringinase reactor. J. Agric. Food Chem. 1981, 29, 1299ꢀ1301.
Tsen, H. Y.; Yu, G. K. Limonin and naringin removal from grapefruit juice with
naringinase entrapped in cellulose triacetate fibers. J. Food Sci. 1991, 56, 31ꢀ35.
Busto, M. D.; Meza, V.; Mateos, N. P. Immobilization of naringinase from Aspergillus
niger CECT 2088 in poly (vinyl alcohol) cryogels for the debittering of juices. Food
Chem. 2007, 104, 1177ꢀ1182.
(6)
(7)
(8)
(9)
Trummler, K.; Effenberger, F.; Syldatk, C. An integrated microbial/enzymatic process
for production of rhamnolipids and Lꢀ(+)ꢀrhamnose from rapeseed oil with
Pseudomonas sp. DSM 2874. Eur. J. of Lipid Sci. Technol. 2003, 105, 563ꢀ571.
Caldini, C.; Bonomi, F.; Pifferi, P. G.; Lanzarini, G.; Galante, Y. M. Kinetic and
immobilization studies on the fungal glycosidases for the aroma enhancement in wine.
Enzyme Microb. Technol. 1994, 16, 286ꢀ291.
Yoshinobu, T.; Takashi, K.; Takahisa, N.; Hiroshi, T.; Shigetaka, O. Prevention of
hesperidin crystal formation in canned mandarin orange syrup and clarified orange
juice by hesperidin glycosides. Food Sci. Technol. Int. 1995, 1, 29ꢀ33.
Elujoba, A. A.; Hardman, R. Diosgenin production by acid and enzymatic hydrolysis
of fenugreek. Fitoterapia 1987, 58, 299ꢀ303.
(10) Monti, D.; Pisvejcova, A.; Kren, V.; Lama, M.; Riva, S. Generation of an
17
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 18 of 32
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
αꢀlꢀrhamnosidase library and its application for the selective derhamnosylation of
natural products. Biotechnol. Bioeng. 2004, 87, 763ꢀ771.
(11) Feng, B.; Ma, B.; Kang, L.; Xiong, C.; Wang, S. The microbiological transformation
of steroidal saponins by Curvularia lunata. Tetrahedron 2005, 61, 11758ꢀ1163.
(12) Yu, H.; Gong, J.; Zhang, C.; Jin, F. Purification and characterization of
ginsenosideꢀlꢀrhamnosidase. Chem. Pharm. Bull. 2002, 50, 175ꢀ178.
(13) Romero, C.; Manjon, A.; Bastida, J.; Iborra, J. L. A method for assaying rhamnosidase
activity of naringinase. Anal. Biochem. 1985, 149, 566–571.
(14) Manzanares, P.; Graff, L. H. D.; Visser, J. Purification and characterization of an
αꢀLꢀrhamnosidase from Aspergillus niger. FEMS Microbiol. Lett. 1997, 157, 279ꢀ283.
(15) Himmel, M. E.; Adney, W. S.; Fox, J. W.; Mitchell, D. J.; Baker, J. O. Isolation and
characterization of two forms of βꢀDꢀglucosidase from Aspergillus niger. Appl.
Biochem. Biotechnol. 1993, 39-40, 213ꢀ225.
(16) Davis, D. W. Determination of flavonones in citrus juice. Anal. Chem. 1947, 19,
476–478.
(17) Habelt, K.; Pittner, F. A rapid method for the determination of naringin, prunin, and
naringin applied to the assay of naringinase. Anal. Biochem. 1983,.134, 393–397.
(18) Chien, P. J.; Sheu, F.; Shyu, Y. T. Monitoring enzymatic debittering in grapefruit juice
by high performance liquid chromatography. J. Food Drug Anal. 2001, 9, 115ꢀ120.
(19) Ribeiro, I. A. C.; Ribeiro, M. H. L. Naringin and naringenin determination and control
in grapefruit juice by a validated HPLC method. Food Control 2008, 19, 432–438.
(20) Chang, H.; Lee, Y.; Bae, H.; Huh, J.; Nam, S.; Sohn, H.; Lee, H. J.; Lee, S.
Purification and characterisation of Aspergillus sojae naringinase: The production of
prunin exhibiting markedly enhanced solubility with in vitro inhibition of HMGꢀCoA
reductase. Food Chem. 2011, 124, 234ꢀ241.
18
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4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
03
(21) Ribeiro, I. A. C.; Ribeiro, M. H. L. Kinetic modelling of naringin hydrolysis using a
bitter sweet alfaꢀrhamnopyranosidase immobilized in kꢀcarrageenan. J. Mol. Cat. B
Enz. 2008, 51, 10ꢀ18.
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
(22) 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,
C1ꢀC7.
(23) Patil, S. R.; Dayanand, A. Optimization of process for the production of fungal
pectinases from deseeded sunflower head in submerged and solidꢀstate conditions.
Bioresource Technol. 2006, 97, 2340ꢀ2344.
(24) Puri, M.; Kalra, S. Purification and characterization of naringinase from a newly
isolated strain of Aspergillus niger 1344 for transformation of flavonoids. World J.
Microbiol. Biotechnol. 2005, 21, 753ꢀ758.
(25) 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, 471ꢀ473.
(26) Pulley, G. N. Solubility of naringin in water. Ind. .Eng. Chem. Anal. Ed. 1936, 8,
360ꢀ360.
(27) Hendrickson, R.; Kesterson, J. W. Purification of naringin. P. Fl. St. Hortic. Soc. 1956,
69, 149ꢀ152.
(28) Tommasini, S.; Raneri, D.; Ficarra, R.; Calabro, M. L.; Stancanelli, R.; Ficarra, P.
Improvement in solubility and dissolution rate of flavonoids by complexation with
betaꢀcyclodextrin. J. Pharmaceut. Biomed. Anal. 2004, 35, 379–387.
(29) Xu, G.; Liu, D.; Chen, J.; Ye, X.; Ma, Y.; Shi, J. Juice components and antioxidant
capacity of citrus varieties cultivated in China. Food Chem. 2008, 106, 545ꢀ551.
(30) Dekker, R. F. H. Kinetic, inhibition, and stability properties of a commercial
19
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Page 20 of 32
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
βꢀDꢀglucosidase (cellobiase) preparation from Aspergillus niger and its suitability in
the hydrolysis of lignocellulose. Biotechnol. Bioeng. 1986, 28, 1438ꢀ1442.
(31) Mccleary, B. V.; Harrington, J. Purification of βꢀdꢀglucosidase from Aspergillus niger.
Methods Enzymol. 1988, 160, 575ꢀ583.
(32) Spagna, G.; Barbagallo, R. N.; Martino, A.; Pifferi, P. G. A Simple method of purifying
glycosidases: αꢀLꢀrhamnopyranosidase from Aspergillus niger to increase the aroma
of Moscato wine. Enzyme Microb. Technol. 2000, 27(7), 522ꢀ530.
(33) Chen, Y. L.; Ni, H.; Chen, F.; Cai, H. N.; Li, L. J.; Su W. J. Purification and
characterization of a naringinase from Aspergillus aculeatus JMUdb058. J. Agric.
Food Chem. 2013, 61, 931–938.
(34) Ni, H.; Cai, H.; Xiao, A.; Chen, F.; You, Q.; Wang, Y. Improved purification of
αꢀLꢀrhamnosidase from Aspergillus niger naringinase. World J. Microbiol. Biotechnol.
2011, 27, 2539ꢀ2544.
(35) Ni, H.; Cai, H. N.; Chen. F.; You, Q.; Xiao, A. F.; Wang, Y. Q. Purification and
characterization of βꢀDꢀglucosidase from Aspergillus niger naringinase. J. Food
Biochem. 2012, 36, 395ꢀ404.
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Figure captions
Figure number Caption
Figure 1.
Chromatograms of the reaction mixture: (A) before and (B) after
the naringinase hydrolysis ,and (C) standards of naringin, prunin
and naringenin. Enzymatic hydrolysis of naringin is composed of
two reactions: (A) At first αꢀLꢀrhamnosidase breaks naringin down
into prunin and rhamnose, and (B) subsequently βꢀDꢀglucosidase
splits prunin to release naringenin and glucose.
Figure 2.
Figure 3.
Calibration curves of naringin, prunin and naringenin
Solublities of (A) naringin, B (prunin) and (C) naringenin in the
reaction solution (10 mM citric acid buffer) at 4, 20 and 50 °C.
Results are means of three independent determinations.
Figure 4.
Figure 5
Effect of methanol concentration on the determinations of naringin
and naringenin. The detected values were linked by a solid line,
compared with the theoretical values in a dotted line.
Lineweaver–Burk plots of the naringinase, αꢀLꢀrhamnosidase and
βꢀDꢀglucosidase from A. niger. The naringinase has Vmax of 0.60
U/mL (the reciprocal of 1.6742) and K
of 0.2235 divided by 1.6742), the αꢀLꢀrhamnosidase has Vmax of
.51 U/mL (the reciprocal of 0.664) and K of 0.28 mM (the
m
of 0.13 mM (the quotient
1
m
quotient of 0.1882 divided by 0.664), and the βꢀDꢀglucosidase has
Vmax of 9.63 U/mL (the reciprocal of 0.1038) and K of 0.39 mM
m
(the quotient of 0.04 divided by 0.1038).
Figure 6
The activities of naringinase,
αꢀLꢀrhamnosidase
and
βꢀDꢀglucosidase produced in a submerged fermentation of A. niger
during a fermentation period of 192 h. The final activity of
βꢀDꢀglucosidase (3.45 U/mL) was about two times of those of the
αꢀrhamnosidase (1.75 U/mL) and the naringinase (1.58 U/mL).
4
4
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48
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Table 1.
4
4
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52
Recoveries and RSDs of the concentrations of naringin, prunin and naringenin after the
treatment under the enzymatic reaction condition
Naringin
Prunin
Rec. RSD
Naringenin
Rec. RSD
Con.
ꢁg/mL)
Rec. RSD
(%) (%)
Con.
(ꢁg/mL)
450.5
350.0
250.3
150.8
50.1
Con.
(
(%)
(%) (ꢁg/mL)
(%)
98.8
(%)
1.9
2.6
2.9
1.7
2.2
4
3
2
1
50.2
50.1
50.2
50.3
99.5
1.4
101.3 2.6
450.1
350.2
250.0
150.3
49.8
100.2 0.6
100.0 1.7
98.9
99.1
1.8
2.2
102.8
103.6
99.4
99.7
98.0
2.8
2.4
102.8 1.5
101.1 3.3
5
0.6
100.6
4
4
53
54
Results were means of three independent determinations.
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Table 2.
4
4
Calculation of the theoretical concentrations of naringin, prunin and naringenin before and
after the enzymatic reactions
Reaction with naringin for Reaction with prunin
determination of naringinase and for determination of
αꢀLꢀrhamnosidase (mM)
naringin prunin
βꢀDꢀglucosidase (mM)
naringenin
naringenin prunin
Before reaction
After reaction
x+y+z
0
0
a+b
0
x
y
z*
a
b
459
460
461
462
463
464
465
(x+y+z) was the number of mM of naringin before reaction, x mM of naringin and y mM of
prunin could be measured after the reaction, and z mM of naringenin could be obtained by
subtracting y and z from (x+y+z). (a+b) was the number of mM of prunin, a mM of prunin
could be determined after the reaction, thus the b mM of naringenin could be calculated by
taking a from (a+b).
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Table 3.
4
4
68
69
Effect of diluted times of the enzyme solutions on the activity changes of naringinase,
αꢀLꢀrhamnosidase and βꢀDꢀglucosidase
Naringinase
Diluted
solution
U/mL)
Control 2.15±0.17 2.15±0.17
αꢀLꢀrhamnosidase
Diluted
βꢀDꢀglucosidase
Diluted
Diluted
times
Sample
U/mL)
Sample
Sample
solution
(U/mL)
solution
(U/mL)
(
(U/mL)
(U/mL)
(
e
d
c
b
a
a
a
a
e
e
d
c
2.26±0.07 2.26±0.07
3.24±0.13 3.24±0.13
3.10±0.13 6.19±0.26
2.20±0.17 8.80±0.43
d
2
4
8
1.98±0.12 3.95±0.23
1.39±0.11 5.55±0.43
0.80±0.02 6.44±0.15
0.43±0.01 6.91±0.18
0.21±0.01 6.81±0.24
0.11±0.01 7.20±0.40
0.06±0.00 7.35±0.61
2.06±0.11 4.12±0.22
c
1.43±0.03 5.71±0.10
b
b
a
a
a
a
0.84±0.04 6.74±0.29
0.53±0.02 8.43±0.37
0.26±0.01 8.29±0.37
0.14±0.00 8.71±0.29
0.07±0.00 8.33±0.31
1.85±0.07 14.76±0.54
1.13±0.05 18.01±0.77
0.59±0.02 18.79±0.59
0.29±0.01 18.72±0.87
0.15±0.01 18.81±0.85
a
a
a
a
1
6
2
4
3
6
1
28
470
471
472
473
474
Results are means of three independent determinations. The listed activity of the sample
was the product of the measured activity of a diluted enzyme solution multiplied by its
a, b, c
corresponding dilution time.
in superscript within the same column mean significant
difference (P<0.05).
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Table 4.
Test of repeatability of the enzyme acitivities
Enzyme
RSD
Sample
Enzyme
activity (U/mL) (%)
Naringinase
αꢀLꢀrhamnosidase
βꢀDꢀglucosidase
Naringinase
0.20
0.22
0.43
0.21
0.24
0.59
8.25
4.29
3.33
7.33
4.54
4.64
Fermented
broth
Purified
enzyme
αꢀLꢀrhamnosidase
βꢀDꢀglucosidase
4
77
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naringin
naringinase
αꢀLꢀrhamnosidase
βꢀDꢀglucosidase
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78
B
+
+
prunin
naringenin
glucose
rhamnose
4
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C
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80
81
Figure 1.
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Figure 2.
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91
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Figure 3.
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Figure 4.
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