Journal of Agricultural and Food Chemistry
Article
sequence of P. furiosus β-glucosidase, celB gene (GenBank accession
no. AAC25555). Forward (5′-GGATCCATGAAGTTCTC-
CAAAAAAC-3′) and reverse primers (5′-GTCGACCTTTCTTG-
TAACAAATT-3′) were designed to introduce the BamHI and SalI
restriction sites (underline), respectively, and were synthesized by
Bioneer (Daejon, Korea). The amplified DNA fragment obtained by
PCR was purified and inserted into the pET24a(+) vector digested
with the same restriction enzymes. E. coli ER2566 strain was
transformed with the ligation mixture using an electroporator
(MicroPulser, Bio-Rad, Hercules, CA) and plated on LB agar
containing 25 μg/mL of kanamycin. A kanamycin-resistant colony
was selected, and plasmid DNA from the transformant was isolated
with a plasmid purification kit (Promega, Madison, WI). DNA
sequencing was conducted using a DNA analyzer (ABI Prism 3730xl,
Perkin-Elmer, Waltham, MA). Gene expression was evaluated by both
SDS-PAGE and enzyme activity.
Culture Conditions. P. furiosus was grown anaerobically at 90 °C
on a complex medium containing starch as the carbon and energy
source.24 E. coli cells containing the β-glucosidase/pET24a(+) gene
were cultivated in a 2000 mL flask containing 500 mL of Luria−
Bertani (LB) medium and 20 μg/mL kanamycin at 37 °C with shaking
at 200 rpm. When the optical density of bacteria reached 0.6 at
600 nm, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to
a final concentration of 0.1 mM to induce the expression of
β-glucosidase, and then the culture was incubated with shaking at
150 rpm at 16 °C for 4 h.
Enzyme Preparation. The grown cells were harvested and
disrupted by sonication on ice for 2 min in 50 mM phosphate buffer
(pH 7.0) containing 300 mM NaCl with the addition of 1 mg/mL
lysozyme. The unbroken cells and cell debris were removed by
centrifugation at 13000g for 20 min at 4 °C, and the supernatant
obtained was used as a crude extract. The enzyme solution of the
supernatant was applied to a His-trap affinity chromatography column
(Amersham Biosciences, Uppsala, Sweden) equilibrated with 50 mM
phosphate buffer (pH 7.0). The bound protein was then eluted at 4 °C
with the same buffer containing 250 mM imidazole at a flow rate of
1 mL/min. The active fractions were collected and dialyzed at 4 °C for
16 h against 50 mM citrate/phosphate buffer (pH 6.0). The resultant
solution was used as a purified enzyme. The purification step using the
column was conducted using a fast protein liquid chromatography
system (Bio-Rad) in a cold room at 4 °C.
Hydrolytic Activity. The hydrolytic reactions of P. furiosus β-
glucosidase were performed in 50 mM citrate/phosphate buffer
(pH 6.0) containing 0.017 U/mL enzyme and 1.0 mM p−nitrophenyl-
(pNP)-β-D-glucopyranoside or 0.2 mM isoflavone glycoside genistin,
daidzin, glycitin, malonyl genistin, malonyl daidzin, or malonyl glycitin for
5 min at 95 °C. The activity for pNP-β-D-glucopyranoside was deter-
mined by release of pNP. The absorbance at 415 nm was measured
after the reactions had been quenched by the addition of 200 mM
Na2CO3. One unit (U) of enzyme activity used for the hydrolysis of
pNP-β-D-glucopyranoside was defined as the amount of enzyme
required to liberate 1 μmol of pNP per minute at 95 °C and pH 6.0.
The activity for isoflavone glycoside was determined by release of
aglycon isoflavone. One unit of enzyme activity used for the hydrolysis
of isoflavone glycosides was defined as the amount of enzyme required
to liberate 1 μmol of genistein from genistin as a substrate per minute
at 95 °C and pH 6.0. The specific activity (U/mg) was defined as the
produced amount of NP or isoflavone aglycone as a product per
enzyme amount per unit of reaction time. The productivity (mM/h)
was defined as the increase in the concentration of isoflavone aglycone
as a product per unit of reaction time.
by the subunit molecular mass of 221952 Da. Protein concentrations
were determined according to the Bradford method using bovine
serum albumin as a standard protein.25
Effects of Metal Ions, pH, and Temperature. To investigate the
effect of metal ions on enzyme activity, the enzyme assay was carried
out after treatment with 1 mM ethylenediaminetetraacetic acid (EDTA)
at 4 °C for 1 h or after the addition of 1 mM of each metal ion such as
Co2+, Mn2+, Ba2+, Mg2+, Zn2+, Cu2+, Ni2+, Fe2+, or Ca2+. The reactions
were performed in 50 mM citrate/phosphate buffer (pH 6.0) containing
each metal ion at 95 °C.
To evaluate the effects of pH and temperature on β-glucosidase
activity, the temperatures were varied from 75 to 98 °C at pH 6.0, and
the pH values were varied from 4.5 to 7.0 using 50 mM citrate/
phosphate buffer (pH 4.5−7.0) at 95 °C. The effect of temperature on
enzyme stability was monitored as a function of incubation time by
applying the enzyme solution at six different temperatures (70, 75, 80,
85, 90, and 95 °C) in 50 mM citrate/phosphate buffer (pH 6.0).
Samples were withdrawn at time intervals and then assayed in 50 mM
citrate/phosphate buffer (pH 6.0) containing 0.2 mM genistin at
95 °C for 5 min. The half-life of the enzyme was calculated using
Sigma Plot 9.0 software (Systat Software, San Jose, CA).
Preparation of Soybean Flour Extract. The extraction method
for soybean flour was used on the basis of the extraction method for
soy flour.20 The extraction conditions were optimized by varying
soybean flour concentration (10−25%, w/v), methanol concentra-
tion (50−100%, v/v), temperature (60−90 °C), and extraction time
(1−5 h). The highest amount of isoflavones was obtained after
extraction with 6 volumes of 80% methanol by stirring for 3 h at 80 °C
for 3 g of soybean flour. Thus, these conditions were used for extracting
soybean flour.
Time Courses for the Hydrolysis of Genistin, Daidzin, and
Glycitin. The hydrolysis of the isoflavone glycosides genistin, daidzin,
and glycitin from a reagent and a soybean flour extract was investigated
using P. furiosus β-glucosidase. The hydrolytic reactions were
performed at 95 °C in 50 mM citrate/phosphate buffer (pH 6.0)
containing 1.65 U/mL enzyme and 1 mM reagent grade genistin,
daidzin, or glycitin for 120, 160, or 200 min, respectively, and 33 U/mL
enzyme and 7.5% (w/v) soybean flour extract for 160 min.
Analytical Methods. Isoflavones were assayed using the isoflavone
standards and an HPLC system (Agilent 1100, Santa Clara, CA) equipped
with a UV detector at 254 nm and a C18 column (50 × 4.6 mm,
YMC, Kyoto, Japan). The column eluted at 35 °C with a gradient of
solvent A (3% acetic acid in water) and solvent B (3% acetic acid in
mixture of water and acetonitrile of 50:50 (v/v)) from 75:25 to 40:60
for 12 min and then from 40:60 to 75:25 for 3 min. The flow rate was
1.5 mL/min. The substrates daidzin, glycitin, genistin, malonyl daidzin,
malonyl glycitin, and malonyl genistin were detected with retention
times of 3.4, 3.8, 4.3, 4.6, 4.8, and 5.1 min, respectively. The products
daidzein, glycitein, genistein, malonyl daidzein, malonyl glycitein, and
malonyl genistein were detected with retention times of 5.4, 5.8, 7.2,
7.6, 8.0, and 8.6 min, respectively. The isoflavones in the reaction
samples formed from different substrates and soybean flour extract
were identified as the same retention times with the isoflavone
standards. The amounts of isoflavones in soybean flour extract were
determined using linear calibration curves relating the peak areas to
the concentrations of isoflavone standards.
RESULTS
■
Gene Cloning and Purification, Molecular Mass
Determination of P. furiosus β-Glucosidase. A gene
encoding P. furiosus β-glucosidase, with the same sequence as
reported in GenBank (accession no. AAC25555), was cloned
and expressed in E. coli. The enzyme was purified from crude
extract obtained from harvested cells as a soluble protein by
His-trap affinity chromatography. The β-glucosidase was purified
with a purification of 7.2-fold, a yield of 62%, and a specific activity
of 330 U/mg for genistin. After IPTG induction, the specific
activity of P. furiosus β-glucosidase was constant at approximately
Kinetic Parameters. Various concentrations of pNP-β-D-gluco-
pyranoside (from 0.1 to 3 mM), genistin (from 0.1 to 1.5 mM),
daidzin (from 0.1 to 2.5 mM), and glycitin (from 0.05 to 2.5 mM)
were used to determine the kinetic parameters of the enzyme. The
reactions were performed in citrate/phosphate buffer (pH 6.0) at
95 °C for 5 min. The amounts of isoflavone aglycones were detected
by HPLC assay. Km (mM) and kcat (1/s) were determined by the
Lineweaver−Burk plot from the Michaelis−Menten equation. To
calculate the catalytic constant, kcat, the amount of protein was divided
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dx.doi.org/10.1021/jf204432g | J. Agric.Food Chem. 2012, 60, 1535−1541