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Home , Daidzin, Genistin

Journal of Medicinal Plants Research Vol. 4(24), pp. 2684-2690, 18 December, 2010 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR09.570 ISSN 1996-0875 ©2010 Academic Journals

Full Length Research Paper

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

Yanmei Sun 1,2, Yongzhong Zhang 1,2 * Ning Liu 3, Dongmei Li 1 and Muzi Li 1

1Department of Applied Chemistry, Northeast Agricultural University, Harbin, 150030, China. 2Chinese Education Ministry’s and Provincial Key Laboratory of Biology, Harbin, 150030, China. 3Key Laboratory For dairy science of Ministry of Education, Harbin, 150030, China.

Accepted 23 November, 2010

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 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 into aglucones.

Key words: Lentinula edodes , glucosidase, hydrolysis, daidzin, genistin.

INTRODUCTION

Among the foods eaten by humans, contain the risk of cardiovascular diseases (Anthony et al., 1996; highest level of . Soy isoflavones exist in the Goodman-Gruen and Kritz-Silverstein, 2001), and form of aglucones (, and ) and improvement in strength (Cotter and Cashman, β-glucosides conjugates, which include the glucosides 2003; Weaver and Cheong, 2005). Numerous studies (daidzin, genistin and ), the malonylglucosides, have shown that the biological effects of isoflavone are and acetylglucosides (Kudou et al., 1991). The content of not due to the forms but mainly to their daidzin and genistin are high in soybean and their aglucones, such as daidzein and genistein. For example, aglucones (daidzein, genistein) are found in trace aglucone isomers are able to bind to receptor quantities. and hence mimic the functions in the human Soy isoflavones have been implicated in prevention of body (Setchell, 1998; Setchell and Cassidy, 1999), and certain (Cappelletti et al., 2000; Messina, 1999; thus prevent certain cancers (Fritsche and Steinhart, Miura et al., 2002; Ravindranath et al., 2004), lowering the 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 *Corresponding author. E-mail: [email protected]. Fax: hydrolyzed in the jejunum by means of β-glycosidase, and 86-0451-55103336. Tel: 13359993588, 86-0451-55191831. then probably was absorbed only as aglycones. The

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absorption of soybean isoflavones in human varies crystallization was allowed to take place at 5°C for 24 h. The crude individually for their different capabilities due crystals were collected by filtration yielding about 100 mg. The crude to ethnic backgrounds, dietary habit and activity of gut crystals were dissolved in 100 ml 10% aqueous methanol. The resulted solution was applied to the Sephadex LH-20 column (2.0 × β-glucosidase. One way to improve the bioavailability and 30 cm), eluted with 10% aqueous methanol solution, and the flow biological activity of soybean isoflavones is simply to out was crystallized using 80% ethanol to obtain about 10 mg pure increase the concentration of isoflavone aglucones in daidzin and 8 mg pure genistin. Purities were checked by the High soybean food. Performance Liquid Chromatographic (HPLC) method comparing β-glucosidase (EC 3.2.1.21, β-glucoside with the standard isoflavones. glucohydrolase) catalyzes compounds containing β-glucosidic linkages by splitting off the terminal Assay of enzyme activity nonreducing β-D- residues with the release of β-D-glucose. β-glucosidase can catalyze the hydrolysis of β-glucosidase activity was monitored for its hydrolysis capacity of a glycosidic linkages in aryl and alkyl β-glucosides as well synthetic substrate, p-NPG. The initial concentration of the substrate as β-oligoglucosides. β-glucosidase occurs widely used is 1 mM. In a standard assay, enzyme activity determined by measuring the p-NPG concentration after 10 min incubation with the through bacteria, fungus, plants, and animals. Some β-glucosidases from plants and bacteria have been used enzyme at 60 ℃ in 0.05 M phosphate-citrate buffer, pH 5.0. In detail, in biotransformation of isoflavone glucosides (Hsieh and 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 Graham, 2001; Pandjaitan et al., 2000), whereas their ml of β-glucosidase solution and further incubated for another 10 thermo stabilities are lower. In order to increase the utility min. The reaction was stopped by addition of 2.00 ml of 0.5 M of isoflavone glucosides in reaction system, we need an sodium carbonate. The light absorbance of the resulting yellow color enzyme with higher thermo stability and activity. Some was immediately measured at wavelength of 400 nm. The researchers (Xie et al., 2003; Tamio et al., 2004; Wu et al., concentration of hydrolyzed p-nitrophenol was determined by 2004; Ito et al., 2008) showed that β-glucosidases from referring to a calibration curve prepared concurrently in the same manner with 5 to 300 µM of p-nitrophenol. fungus have higher thermo stability and higher optimum temperature to hydrolyze isoflavone glucosides. However, a β-glucosidases from edible mushrooms should be a Purification of β-glucosidase better choice for safety. Zheng and Shetty (2000) showed that a crude β-glucosidase from a fungi Lentinula edodes , All steps were carried out under 20 ℃, unless otherwise stated. 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 Crude enzyme extraction

L. edodes , and its activity in hydrolysis of genistin and 100 g of fresh fruit bodies of L. edodes mixed with 200 ml distilled daidzin. 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. MATERIALS AND METHODS The supernatants from the 1st and 2nd centrifugation were combined and acidified at pH 5.0 with 0.1 M HCl, and subjected to Materials another centrifugation at 4000 g for 10 min. The resulting supernatant served as the source of crude enzyme. 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 Ammonium sulfate fractionation chemical reagents were analytical grade of purity. Sephadex LH-20 was obtained from GE Healthcare. CM-Sephadex C-50 and Ammonium sulfate was added with stirring to the above supernatant Sephacryl S-300 HR were obtained from Amersham Biosciences. (crude enzyme extracts) to give 75% saturation at 4℃, and allowed 20% isoflavone powders was a gift from Heilongjiang Food research to stand for 24 h. The precipitated proteins were collected by Institute, China. 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. Isolation and identification of soybean isoflavone glucosides

A modified procedure of Matsuura and Obata (1993) was used for Column chromatography on CM-Sephadex C-50 isolation and fractionation of the isoflavone glucosides. Briefly, two grams of 20% isoflavones powders were added 180 ml acetone. About 6.25 ml of dialyzed enzyme solution was applied to a The filtrate of the acetone soluble fraction was concentrated to 5 ml, CM-Sephadex C-50 column (3.2 × 23 cm) equilibrated with 0.05 M followed by addition of 10 ml distilled water. The precipitate, after sodium acetate buffer, pH 4.0, at a flow rate of 110 ml/h. After elution filtration, was dissolved with 10 ml hot 80% ethanol, after which of the unbound proteins by washing with the same buffer, the

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Table 1. Purification of the L. edodes β-glucosidase.

Vol. Total protein Total activity Specific activity Yield Purification Purification step (ml) (mg) (units) (units/mg) (%) (-fold) Crude extract 35 15.8 11155 706 100 1.00

(NH4) 2SO 4 75%ppt 6.25 2.38 5900 2479 52.9 3.51 CM-Sephadex C-50 46 0.669 3875 5792 34.7 8.20 Sephacryl S-300 9 0.089 1667 18730 14.9 26.5

column was developed with a linear gradient of 0-1.2M NaCl in the HPLC analysis of isoflavones same 0.05M sodium acetate buffer, pH4.0. Eluted fractions were collected as 9.2 ml individuals. Five active fractions (No.52-56, 46 ml) The analysis of isoflavones was conducted by an HPLC, P680, were mixed, dialyzed to desalt and concentrated to a final volume 5 equipped with a UVD170S detector (Dionex), using a reverse-phase ml by dialyzing against polyethyleneglycol. 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 Sephacryl S-300 HR gel filtration with a 20 l injection volume. Samples were detected at 260 nm and quantified by external standards. The above 5 ml dialyzed and concentrated enzyme was applied to a preparative Sephacryl S-300 HR gel filtration column (1.5 × 80 cm) Other analytical methods that had been equilibrated with 300 ml of 50 mM phosphate-citrate buffer at (pH 5.0). Elution was performed with 200 ml of the same The amount of protein was estimated by the method of Lowry et al. buffer at a flow rate of 18 ml/h and 3 ml individual fractions were (1951) with bovine serum albumin as a standard. Protein in the collected for the enzyme activity assay. Active fractions (No.25-27, 9 column effluents was monitored by measuring A-280 nm. The K ml) were combined and stored as purified enzyme. m and V max values were determined by the double-reciprocal plot method of Lineweaver and Burk.

Enzyme activity on isoflavones RESULTS AND DISCUSSION The daidzin- and genistin- hydrolyzing activity was measured as follows: the reaction mixture was prepared using the 400 l of 1 mM Purification of β-glucosidase from L. edodes substrate solution and 100 l 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. Table 1 summarizes the results of the purification of a The daidzin and genistin hydrolyzing activity effected by metal β-glucosidase from L. edodes . The enzyme was purified ions or organic solvents was measured as follows: the reaction 26.5-fold to homogeneity with an overall recovery of mixture was prepared using the 200 l of 2 mM substrate solution 14.9% and an enzymatic activity 18730 U•mg of protein-1. (pH 4.0), the 200 l of different concentration metal ions or organic β-glucosidase active fractions were separated into two solvents which diluted by 0.05 M phosphate-citrate buffer (pH 4.0) peaks by chromatography on a CM-Sephadex C-50 and 100 l enzyme solution. The mixture was incubated for 10 min at 60ºC, after which 2 ml methanol was added to stop the reaction. column (Figure 1). The mixture was filtered through a 0.22 m membrane for the Some activities eluted as a single peak was found in the analysis of aglucones by High Performance Liquid Chromatography non-binding fractions (peak A in Figure 1). Most of the (HPLC). A unit of enzyme activity was defined as the amount of β-glucosidase activity was found in the bound fractions enzyme that would liberate 1 M of isoflavone aglucone/min or eluted at 0.6 M NaCl. (peak B in Figure 1). Fractions of p-nitrophenol/min. The enzymatic activity was expressed as units/mg protein of the enzyme. 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 Hydrolysis of the mixture of genistin and daidzin purification steps. SDS-PAGE analysis of the purified enzyme indicated the presence of a single band 0.2 g of 20% isoflavones powders were mixed with 30 ml 0.05 M corresponding to a molecular weight of 66 kd, when phosphate-citrate buffer, pH 4.0. After the mixture was heated up to stained with Coomassie brilliant blue (Figure 2). 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 Characterization of β-glucosidase completely dissolved, and then filtered through a 0.22 m membrane for HPLC analysis. The purified enzyme showed maximum activity at pH 4.0

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Fraction No. 0 10 20 30 40 50 60 35 120 1.2 30 activity 100 1.0 25 80 0.8 20 B 60 0.6 15 40 0.4 NaCl(M) 10 Activity(U/ml) Protein content(mV) Protein 5 A 20 0.2

0 0 0.0 0 60 120 180 240 300 360 Time(min)

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

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 Figure 2. SDS-PAGE of the purified β-glucosidase from Trichoderma harzianum type C-4 by enzyme from L. edodes. The enzyme was eletrophoresed at pH 8.3 on a 12% Yun et al. (2001). In order to get an activator of metal ions, acrylamide gel and stained with we studied the effects of metal ions on the activity of the Coomassie brilliant blue R-250. Lanes: purified enzyme. In contrast with the previous report (Yun 1, molecular weight standard; 2, et al., 2001), surprisingly, neither of Mg 2+ and Zn 2+ ions purified enzyme. increases the enzyme activity.

The kinetic parameters of the purified β-glucosidase were achieved by analysis of the hydrolysis reaction with and at 60°C by using p-NPG. The enzyme was stable different concentration of p-NPG substrate (0.025 to 100 from pH 4.0 to 5.0 for 20 h at 4°C and was stable below mM). The Km and Vmax values were calculated from

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

Relative activity (%) Relative activity (%) Metal ions (Metal ions1.0 mM final concentration) (Metal ions final concentration 5.0 mM) Control 100 100 Ba 2+ 94 85 Al 3+ 70 15 Ca 2+ 92 67 Mg 2+ 91 86 Cu 2+ 78 24 Ag + 0 ― Zn 2+ 97 ― Mn 2+ 98 86

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

Relative activity (%) at solvent conc. of Substrate Solvents 0% v/v 10% v/v 20% v/v 30% v/v Methanol 100 66.8 8.2 3.3 Ethanol 100 51.8 5.2 3.9 Genistin Acetone 100 56.8 5.8 3.2 Acetyl ester 100 4.6 — —

Methanol 100 76.9 5.8 4.6 Ethanol 100 38.1 1.4 0.7 Daidzin Acetone 100 51.3 17.8 0.1 Acetyl ester 100 4.2 — —

double- reciprocal plots. The enzyme had apparent the K m The kinetic parameters of the purified β-glucosidase values of 0.346 mM and V max values of 84878 nkat•mg of were achieved by adding either genistin (0.017 to 0.32 protein -1 for the hydrolysis of p-NPG. 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 Kinetic study on isoflavones the Km 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 The purified enzyme displayed maximum activity pH 4.0 genistin and daidzin, respectively. The best substrate and at 60°C at by using daidzin or genistin. The result was among the three tested substrates (p-NPG, genistin and similar to that of using the substrate of p-NPG. Reports daidzin) was p-NPG. Although, the purified β-glucosidase from other researches also suggested that some organic has a higher affinity to genistin than to daidzin and p-NPG, solvents could increase activity of β-glucosidases (Yan the maximal reaction rate of p-NPG hydrolysis was higher and Lin, 1997). Considering the solubility of genistin and than that of genistin and daidzin under the same catalytic daidzin were low, it would be also very desirous of adding conditions. Meanwhile, the turnover rate ( kcat ) of daidzin some organic solvents to improve their concentration in was about 3-fold higher that of genistin. system of enzymatic reaction. The effects of organic We found that the Kms of β-glucosidase from the L. solvents on the activity of enzyme are shown in Table 3. edodes on hydrolysis of genistin and daidzin were lower Organic solvents were added to reaction mixture at a final than that purified from soybean, in which the Km values of concentration of 10, 20 and 30%. All tested organic genistin and daidzin were 0.13 and 0.27 mM (Matsuura et solvents inhibited the enzymatic activity on hydrolysis of al., 1995). The V max values for the hydrolysis of genistin genistin and daidzin (Table 3). was roughly close to that of wheat seedlings (420 nkat•mg

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

Isoflavone content (mg/100 ml reaction solution) Hydrolysis yield (%) Amount of enzyme (unit) Time Daidzin Genistin Daidzein Genistein Daidzin Genistin 200 6 h 13.1 32.1 14.2 12.2 64.7 39.5

0 min 37.1 52.6 5.58 5.84 20.2 15.4 15 min 17.7 43.2 11.5 5.55 53.2 18.3 800 30 min 11.0 34.5 15.5 10.7 70.5 34.0 45 min 8.92 32.1 16.7 12.2 75.8 38.3 1 h 7.44 27.9 17.6 14.6 80.0 46.9 6 h 4.34 9.66 19.4 25.5 88.2 81.6

2000 6 h 1.11 4.16 21.4 28.7 97.0 92.0

The activity measured without any organic solvent addition was considered 100%. Average of three replicates.

of protein -1, Sue et al., 2000). REFERENCES

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