Hydrolysis of genistin and daidzin by a β-glucosidase purified from Lentinula edodes

We had studied the purification and characterization of β-glucosidase from Lentinula edodes, and its activity of hydrolyzing genistin and daidzin. The beta-glucosidase was extracted from an edible mushrooms L. edodes fruiting body and concentrated 26.5-fold by (NH4)2SO4 precipitation, followed by CM-Sephadex C-50 and Sephacryl S-300 HR chromatography. The purified enzyme showed a single 66 kd band on SDS-PAGE. The optimal enzyme activity occurred at 60°C and pH 4.0 in hydrolysis of genistin, daidzin and p-NPG. The enzyme activity was completely inhibited by 5 mM Ag + , Cu 2+ or Al 3+ , respectively. The enzyme had apparent the Km values of 0.347, 0.070 and 0.150 mM and Vmax values of 84878, 225 and 639 nkat•mg of protein-1 for the hydrolysis of p-NPG, genistin and daidzin, respectively, at 60°C and pH 5.0. All tested organic solvents inhibited the β-glucosidase activity on hydrolysis of genistin and daidzin. The hydrolysis efficiency of daidzin and genistin could come up to 97 and 92%, respectively with enough enzyme addition. These experiments demonstrate that β-glucosidase from L. edodes has high activities for hydrolysis of daidzin and genistin, and are likely to be used in the transformation of isoflavone glucosides into aglucones.


INTRODUCTION
Among the foods eaten by humans, soybeans contain the highest level of isoflavones.Soy isoflavones exist in the form of aglucones (daidzein, genistein and glycitein) and β-glucosides conjugates, which include the glucosides (daidzin, genistin and glycitin), the malonylglucosides, and acetylglucosides (Kudou et al., 1991).The content of daidzin and genistin are high in soybean and their aglucones (daidzein, genistein) are found in trace quantities.
Soy isoflavones have been implicated in prevention of certain cancers (Cappelletti et al., 2000;Messina, 1999;Miura et al., 2002;Ravindranath et al., 2004), lowering the risk of cardiovascular diseases (Anthony et al., 1996;Goodman-Gruen and Kritz-Silverstein, 2001), and improvement in bone strength (Cotter and Cashman, 2003;Weaver and Cheong, 2005).Numerous studies have shown that the biological effects of isoflavone are not due to the glucoside forms but mainly to their aglucones, such as daidzein and genistein.For example, aglucone isomers are able to bind to estrogen receptor and hence mimic the estradiol functions in the human body (Setchell, 1998;Setchell and Cassidy, 1999), and thus prevent certain cancers (Fritsche and Steinhart, 1999).In addition, some researchers have shown that isoflavone aglucones in soybean food are absorbed faster and in higher amounts than that of respective glucoside in humans (Izumi et al., 2000;Piskula et al., 1999;Setchell et al., 2002) suggesting that the glucosides must first be hydrolyzed in the jejunum by means of β-glycosidase, and then probably was absorbed only as aglycones.The absorption of soybean isoflavones in human varies individually for their different metabolism capabilities due to ethnic backgrounds, dietary habit and activity of gut β-glucosidase.One way to improve the bioavailability and biological activity of soybean isoflavones is simply to increase the concentration of isoflavone aglucones in soybean food.
β-glucosidase (EC 3.2.1.21,β-glucoside glucohydrolase) catalyzes compounds containing β-glucosidic linkages by splitting off the terminal nonreducing β-D-glucose residues with the release of β-D-glucose.β-glucosidase can catalyze the hydrolysis of glycosidic linkages in aryl and alkyl β-glucosides as well as β-oligoglucosides.β-glucosidase occurs widely through bacteria, fungus, plants, and animals.Some β-glucosidases from plants and bacteria have been used in biotransformation of isoflavone glucosides (Hsieh and Graham, 2001;Pandjaitan et al., 2000), whereas their thermo stabilities are lower.In order to increase the utility of isoflavone glucosides in reaction system, we need an enzyme with higher thermo stability and activity.Some researchers (Xie et al., 2003;Tamio et al., 2004;Wu et al., 2004;Ito et al., 2008) showed that β-glucosidases from fungus have higher thermo stability and higher optimum temperature to hydrolyze isoflavone glucosides.However, a β-glucosidases from edible mushrooms should be a better choice for safety.Zheng and Shetty (2000) showed that a crude β-glucosidase from a fungi Lentinula edodes, one species of edible Chinese mushrooms, has relatively higher thermal tolerance.In the present study, we report the purification and characterization of β-glucosidase from L. edodes, and its activity in hydrolysis of genistin and daidzin.

Materials
The fresh fruiting bodies of L. edodes were purchased from local food market.p-nitrophenyl-β-glucoside (p-NPG), the standard isoflavones (daidzin, genistin, daidzein, genistein) and the standard molecular weight marker were purchased from Sigma.Other chemical reagents were analytical grade of purity.Sephadex LH-20 was obtained from GE Healthcare.CM-Sephadex C-50 and Sephacryl S-300 HR were obtained from Amersham Biosciences.20% isoflavone powders was a gift from Heilongjiang Food research Institute, China.

Isolation and identification of soybean isoflavone glucosides
A modified procedure of Matsuura and Obata (1993) was used for isolation and fractionation of the isoflavone glucosides.Briefly, two grams of 20% isoflavones powders were added 180 ml acetone.The filtrate of the acetone soluble fraction was concentrated to 5 ml, followed by addition of 10 ml distilled water.The precipitate, after filtration, was dissolved with 10 ml hot 80% ethanol, after which Sun et al. 2685 crystallization was allowed to take place at 5°C for 24 h.The crude crystals were collected by filtration yielding about 100 mg.The crude crystals were dissolved in 100 ml 10% aqueous methanol.The resulted solution was applied to the Sephadex LH-20 column (2.0 × 30 cm), eluted with 10% aqueous methanol solution, and the flow out was crystallized using 80% ethanol to obtain about 10 mg pure daidzin and 8 mg pure genistin.Purities were checked by the High Performance Liquid Chromatographic (HPLC) method comparing with the standard isoflavones.

Assay of enzyme activity
β-glucosidase activity was monitored for its hydrolysis capacity of a synthetic substrate, p-NPG.The initial concentration of the substrate used is 1 mM.In a standard assay, enzyme activity determined by measuring the p-NPG concentration after 10 min incubation with the enzyme at 60℃ in 0.05 M phosphate-citrate buffer, pH 5.0.In detail, a 1.60 ml sample of 1 mM p-NPG in 0.05 M phosphate citrate buffer (pH 5.0) was preincubated at 60ºC for 10 min, then added with 0.40 ml of β-glucosidase solution and further incubated for another 10 min.The reaction was stopped by addition of 2.00 ml of 0.5 M sodium carbonate.The light absorbance of the resulting yellow color was immediately measured at wavelength of 400 nm.The concentration of hydrolyzed p-nitrophenol was determined by referring to a calibration curve prepared concurrently in the same manner with 5 to 300 µM of p-nitrophenol.

Purification of β-glucosidase
All steps were carried out under 20℃, unless otherwise stated.

Crude enzyme extraction
100 g of fresh fruit bodies of L. edodes mixed with 200 ml distilled water.The mixture was broken into fine pieces in a juicer for 5 min, and then stirred slowly for 30 min.The slurry was centrifuged at 4000 g for 3 min.The resulting supernatant was saved.The pellet was mixed with 50 ml distilled water, and centrifuged again as above.
The supernatants from the 1st and 2nd centrifugation were combined and acidified at pH 5.0 with 0.1 M HCl, and subjected to another centrifugation at 4000 g for 10 min.The resulting supernatant served as the source of crude enzyme.

Ammonium sulfate fractionation
Ammonium sulfate was added with stirring to the above supernatant (crude enzyme extracts) to give 75% saturation at 4℃, and allowed to stand for 24 h.The precipitated proteins were collected by centrifugation at 4000 g for 30 min, dissolved in 30 ml, 0.05 M sodium acetate buffer (pH 4.0), and then dialyzed against the same buffer for 36 h.

Column chromatography on CM-Sephadex C-50
About 6.25 ml of dialyzed enzyme solution was applied to a CM-Sephadex C-50 column (3.2 × 23 cm) equilibrated with 0.05 M sodium acetate buffer, pH 4.0, at a flow rate of 110 ml/h.After elution of the unbound proteins by washing with the same buffer, the

Sephacryl S-300 HR gel filtration
The above 5 ml dialyzed and concentrated enzyme was applied to a preparative Sephacryl S-300 HR gel filtration column (1.5 × 80 cm) that had been equilibrated with 300 ml of 50 mM phosphate-citrate buffer at (pH 5.0).Elution was performed with 200 of the same buffer at a flow rate of 18 ml/h and 3 ml individual fractions were collected for the enzyme activity assay.Active fractions (No.25-27, 9 ml) were combined and stored as purified enzyme.

Enzyme activity on isoflavones
The daidzin-and genistin-hydrolyzing activity was measured as follows: the reaction mixture was prepared using the 400 µl of 1 mM substrate solution and 100 µl enzyme solution.The mixture was incubated for 10 min at a series of temperatures and pHs, after which 2 ml methanol was added to stop the reaction.
The daidzin and genistin hydrolyzing activity effected by metal ions or organic solvents was measured as follows: the reaction mixture was prepared using the 200 µl of 2 mM substrate solution (pH 4.0), the 200 µl of different concentration metal ions or organic solvents which diluted by 0.05 M phosphate-citrate buffer (pH 4.0) and 100 µl enzyme solution.The mixture was incubated for 10 min at 60ºC, after which 2 ml methanol was added to stop the reaction.
The mixture was filtered through a 0.22 µm membrane for the analysis of aglucones by High Performance Liquid Chromatography (HPLC).A unit of enzyme activity was defined as the amount of enzyme that would liberate 1 µM of isoflavone aglucone/min or p-nitrophenol/min.The enzymatic activity was expressed as units/mg protein of the enzyme.

Hydrolysis of the mixture of genistin and daidzin
0.2 g of 20% isoflavones powders were mixed with 30 ml 0.05 M phosphate-citrate buffer, pH 4.0.After the mixture was heated up to 60ºC, 10 ml purified β-glucosidase of 200, 800, or 2000 units was added.The mixture was immediately put on a gyratory shaker (100 rpm) at 60ºC for incubation.The reaction was stopped by addition of the 40 ml methanol.The mixture was diluted with methanol until it completely dissolved, and then filtered through a 0.22 µm membrane for HPLC analysis.

HPLC analysis of isoflavones
The analysis of isoflavones was conducted by an HPLC, P680, equipped with a UVD170S detector (Dionex), using a reverse-phase C18 column (250 × 46 mm, DIKMA) with a mobile phase of water and methanol (50:50, v/v) and at a flow rate of 1 ml/min and at 4°C.Injection was performed by an ASI-100 automated sample injector with a 20 injection volume.Samples were detected at 260 nm and quantified by external standards.

Other analytical methods
The amount of protein was estimated by the method of Lowry et al. (1951) with bovine serum albumin as a standard.Protein in the column effluents was monitored by measuring A-280 nm.The Km and Vmax values were determined by the double-reciprocal plot method of Lineweaver and Burk.

Purification of β-glucosidase from L. edodes
Table 1 summarizes the results of the purification of a β-glucosidase from L. edodes.The enzyme was purified 26.5-fold to homogeneity with an overall recovery of 14.9% and an enzymatic activity 18730 U•mg of protein -1 .β-glucosidase active fractions were separated into two peaks by chromatography on a CM-Sephadex C-50 column (Figure 1).Some activities eluted as a single peak was found in the non-binding fractions (peak A in Figure 1).Most of the β-glucosidase activity was found in the bound fractions eluted at 0.6 M NaCl.(peak B in Figure 1).Fractions of peak B on the CM-Sephadex C-50 were subjected to gel filtration on Sephacryl S-300 HR gel filtration column.Only one form of β-glucosidase was detected during the purification steps.SDS-PAGE analysis of the purified enzyme indicated the presence of a single band corresponding to a molecular weight of 66 kd, when stained with Coomassie brilliant blue (Figure 2).

Characterization of β-glucosidase
The purified enzyme showed maximum activity at pH 4.0  and at 60°C by using p-NPG.The enzyme was stable from pH 4.0 to 5.0 for 20 h at 4°C and was stable below 55°C for 20 min at pH 5.0.The thermal stability and the temperature optimum of the purified enzyme were similar to the crude enzyme tested by Zheng and Shetty (2000).The purified β-glucosidase was thermal stable, its heat tolerance was higher than most reported β-glucosidases, and was also fairly stable over a pH range of 3.0 to 6.5 at 2°C (~20 h).In contrast, the purified enzyme exhibited a maximum activity at pH 4.0, which was different from reported pH 3.5 by Zheng and Shetty (2000).The difference might be arisen from the different enzymatic purity or variety of L. edodes.
The effects of metal ions on the activity of the enzyme are shown in Table 2.The activity of the purified enzyme was completely abolished by adding Ag + , Cu 2+ and Al 3+, especially when metal ions final concentration was increased up to 5.0 mM.In contrast, addition of Ba 2+ , Ca 2+ , Mg 2+ , Zn 2+ and Mn 2+ ions have no significant effect on the activity of the purified enzyme.It had been demonstrated that Mn 2+ and Zn 2+ could increase the activity of the β-glucosidase from Trichoderma harzianum type C-4 by Yun et al. (2001).In order to get an activator of metal ions, we studied the effects of metal ions on the activity of the purified enzyme.In contrast with the previous report (Yun et al., 2001), surprisingly, neither of Mg 2+ and Zn 2+ ions increases the enzyme activity.
The kinetic parameters of the purified β-glucosidase were achieved by analysis of the hydrolysis reaction with different concentration of p-NPG substrate (0.025 to 100 mM).The K m and V max values were calculated from

Kinetic study on isoflavones
The purified enzyme displayed maximum activity pH 4.0 and at 60°C at by using daidzin or genistin.The result was similar to that of using the substrate of p-NPG.Reports from other researches also suggested that some organic solvents could increase activity of β-glucosidases (Yan and Lin, 1997).Considering the solubility of genistin and daidzin were low, it would be also very desirous of adding some organic solvents to improve their concentration in system of enzymatic reaction.The effects of organic solvents on the activity of enzyme are shown in Table 3. Organic solvents were added to reaction mixture at a final concentration of 10, 20 and 30%.All tested organic solvents inhibited the enzymatic activity on hydrolysis of genistin and daidzin (Table 3).
The kinetic parameters of the purified β-glucosidase were achieved by adding either genistin (0.017 to 0.32 mM) or daidzin (0.050 to 0.45 mM) as substrate into the reaction mixture.The K m and V max values were calculated from double-reciprocal plots.The enzyme had apparent the K m values of 0.070 and 0.150 mM and V max values of 225 and 639 nkat•mg of protein -1 for the hydrolysis of genistin and daidzin, respectively.The best substrate among the three tested substrates (p-NPG, genistin and daidzin) was p-NPG.Although, the purified β-glucosidase has a higher affinity to genistin than to daidzin and p-NPG, the maximal reaction rate of p-NPG hydrolysis was higher than that of genistin and daidzin under the same catalytic conditions.Meanwhile, the turnover rate (k cat ) of daidzin was about 3-fold higher that of genistin.
We found that the K m s of β-glucosidase from the L. edodes on hydrolysis of genistin and daidzin were lower than that purified from soybean, in which the K m values of genistin and daidzin were 0.13 and 0.27 mM (Matsuura et al., 1995).The V max values for the hydrolysis of genistin was roughly close to that of wheat seedlings (420 nkat•mg The activity measured without any organic solvent addition was considered 100%.Average of three replicates.

Hydrolysis of mixture of genistin and daidzin
(Table 4) showed the results of hydrolysis of the mixture of isoflavones contained genistin and daidzin by the purified β-glucosidase from L. edodes.The hydrolysis rate of daidzin by the β-glucosidase was significantly faster than that of genistin.When 2000 units of purified β-glucosidase were added to the reaction mixture, and incubated on a gyratory shaker at 60°C for 6 h, the hydrolysis efficiency of daidzin and genistin could come up to 97 and 92%.This indicated that the enzyme could hydrolyze them completely in a relatively short incubation by given enough amount of enzyme.

Conclusion
Taken together, we purified a β-glucosidase from L. edode, and studied its enzymatic kinetics, thermal stability and ions sensitivities.Compared with other β-glucosidase from other species, the β-glucosidase from L. edode has advantages to be applied to food industry for production of isoflavone aglucones, especially combining the consideration of source cost and safety.

Figure 1 .
Figure 1.Ion-exchange chromatography on CM-sephadex C-50 of crude material after ammonium sulfate precipitation at pH 4.0.

Figure 2 .
Figure 2. SDS-PAGE of the purified enzyme from L. edodes.The enzyme was eletrophoresed at pH 8.3 on a 12% acrylamide gel and stained with Coomassie brilliant blue R-250.Lanes: 1, molecular weight standard; 2, purified enzyme.

Table 2 .
Effects of metal ions on the β-glucosidase.

Table 3 .
Effects of organic solvents on the activity of the L. edodes β-glucosidase.

Table 4 .
The results of hydrolysis of the mixture contained genistin and daidzin by the purified β-glucosidase.