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Purification and characterization of piceid-β-d -glucosidase from Aspergillus oryzae
Process Biochemistry 42 (2007) 83–88 www.elsevier.com/locate/procbio
Purification and characterization of piceid-b-D-glucosidase from Aspergillus oryzae Chunzhi Zhang *, Dai Li, Hongshan Yu, Bo Zhang, Fengxie Jin * College of Bio & Food Technology, Dalian Institute of Light Industry, Qinggong-yuan No. 1, Ganjingzi-qu, Dalian 116034, People’s Republic of China Received 21 April 2006; received in revised form 28 June 2006; accepted 10 July 2006
Abstract The piceid-b-D-glucosidase that hydrolyzes the b-D-glucopyranoside bond of piceid to release resveratrol was isolated from Aspergillus oryzae sp.100 strain, and the enzyme was purified and characterized. The enzyme was purified to one spot in SDS polyacrylamide gel electrophoresis, and its molecular weight was about 77 kDa. The optimum temperature of the piceid-b-D-glucosidase was 60 8C, and the optimum pH was 5.0. The piceid-b-D-glucosidase was stable at less than 60 8C, and pH 4.0–5.0. Ca2+, Mg2+ and Zn2+ ions have no significant effect on enzyme activity, but Cu2+ ion inhibits enzyme activity strongly. The Km value was 0.74 mM and the Vmax value was 323 nkat mg1 for piceid. # 2006 Elsevier Ltd. All rights reserved. Keywords: Purification; Characterization; Piceid-b-D-glucosidase; Aspergillus oryzae; Resveratrol
1. Introduction Resveratrol (3,5,40 -trihydroxystilbene, Fig. 1a) is a secondary metabolite of some plants and often as phytoalexin to prevent themselves from microbial pathogens and herbivorous animals [1]. It is originally found in the skins and seeds of the grapes, and contributes to the antioxidant potential of red wine. Recently, resveratrol has attracted more attention because of its widely physiological activity. Resveratrol has the activity of cardiovascular protection, owing to its oxidative modification of low-density lipoproteins [2,3], its ability to act as an antioxidant and as an inhibitor of platelet aggregation, and its action of phytoestrogens [4]. It has been shown that resveratrol inhibits the growth of several cancerous cell lines [5–7], or has the ability to cause apoptosis in these lines [8,9]. Resveratrol also has anti-inflammatory, anti-microbial, anti-HIV and some other effects [10–12]. Resveratrol is a polyphenol compound existing in a variety of plant species, including grapes, peanuts, mulberries and other plants, especially in medicinal plants of the Polygonum species (Polygonaceae). In general, resveratrol is obtained by extraction from natural sources. Because the amount of stilbenes that occurs
* Corresponding authors. Tel.: +86 411 86307737; fax: +86 411 86307737. E-mail addresses: [email protected] (C. Zhang), [email protected] (F. Jin). 1359-5113/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2006.07.019
in the roots of Polygonum cuspidatum (PC) is far more than the amount found in wine and other sources, the roots of PC is the best material offering resveratrol [13]. The main stilbenes in PC, however, are piceid (3,5,40 -trihydroxystilbene-3-O-b-D-glucopyranoside, Fig. 1b) and resveratroloside (3,5,40 -trihydroxystilbene-40 -O-b-D-glucopyranoside, Fig. 1c) rather than resveratrol [13]. For example, the amount of piceid in PC from Hanzhong region in China is about six times larger than that of resveratrol [14]. Therefore, resveratrol content in PC crude extraction will increase by adding the enzyme hydrolyzing resveratrol glucoside into the preparation of PC (Fig. 1). The enzyme, named as piceid-b-D-glucosidase from Aspergillus oryzae, which hydrolyzes the b-(1 ! 3)-D-glucopyranoside bond and transforms piceid to resveratrol, was isolated, purified and characterized in this paper. 2. Materials and methods 2.1. Materials The standard piceid and resveratrol were purchased from Tauto Biotech. Co. Ltd. (Shenzhen, China). The thin-layer chromatography (TLC) plate was the silica gel plate (Kieselgel 60 F-254, Merck). The microorganism, A. oryzae sp.100 (FFCDL-100) strain producing piceid-b-glucosidase, was isolated from traditional Chinese Koji (Daqu in Chinese), identified by Prof. FX Jin, College of Bio & Food Technology, Dalian Institute of Light Industry, and maintained in Food Fermentation Culture Collection of Dalian Institute of Light Industry (FFCDL).
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Fig. 1. Enzymatic transformation of stilbenes.
2.2. Enzyme analysis Piceid-b-glucosidase activity was measured using 0.3 mM piceid in 20 mM acetate buffer, pH 5.0 as the substrate. Enzyme solution 0.1 ml was added to the same volume of piceid solution and allowed to react at 60 8C for 20 min. Then butanol 0.2 ml was added to the reaction mixture to stop the reaction. The product resveratrol in the butanol layer was detected by TLC: developing solvent, CHCl3–MeOH–H2O (4.5:2:1, v/v), and the resveratrol on the silica plate was determined by scanning the TLC spots using a Shimadzu CS-930 [15]. One unit of enzyme activity was defined as the amount of enzyme producing 1 nmol of resveratrol per second (nkat).
2.3. Protein concentration The concentration of protein was measured by the method of Bradford using bovine serum albumin as a standard protein [16].
buffer, pH 7.4. The purified enzyme was dried by freeze-drying and used for the study of enzymatic properties. The enzyme purity and molecular weight were estimated by the SDS polyacrylamide gel electrophoresis method using alactalbumin (14.4 kDa), trypsin inhibitor (20.1 kDa), ovalbumin (45 kDa), bovine serum albumin (66 kDa), and phosphorylase b (97 kDa) as standard proteins [17].
2.7. Effects of pH and temperature on piceid-b-D-glucosidase activity The optimal pH of the piceid-b-D-glucosidase was tested at 60 8C with different buffers at 20 mM. For the pH ranges of 3.0, 4.0–5.0, and 6.0–8.0, citrate, acetate, and phosphate buffers were used, respectively. The pH stability was determined by standard enzyme assay after pre-incubating the purified enzyme at different pH buffer at 4 8C for 1 h. Optimal temperature was tested between 30 and 90 8C by standard enzyme assay. Thermal stability was evaluated by incubation of the enzyme solution at different temperature for 1 h, before performing the standard enzymatic assay.
2.4. Microorganism incubation and crude enzyme extraction
3. Results The FFCDL-100 strain was incubated at 28 8C with shaking at 150 rpm in the medium containing 4% wheat bran extract and 2% plant extract of P. cuspidatum. After incubated for 72 h, the cells were removed by centrifugation and the supernatant was used in the enzyme purification procedure.
2.5. Ammonium sulfate fractionation Pellets of (NH4)2SO4 were slowly added to the supernatant with shaking to 50% saturation. After incubation at 4 8C for 1 h, the enzymatic extract was centrifuged. A second step of precipitation was performed adding ammonium sulfate to 80% saturation and incubating at 4 8C for 1 h. The final precipitate was collected by centrifugation and dissolved in 20 mM Tris–HCl (pH 7.4) for purification or dissolved in 20 mM acetate buffer (pH 5.0) for enzymatic properties.
2.6. Purification of piceid-b-D-glucosidase and estimation of molecular weight The crude protein in 20 mM Tris–HCl after ammonium sulfate fractionation was dialyzed against 20 mM Tris–HCl, pH 7.4. After removing the nondissolved fraction by centrifugation, the enzyme solution was fractionated on a column (Ø2.0 cm 10 cm) of DEAE-cellulose DE-52 (Whatman). The column was eluted stepwise with 60, 120, 180, 240, and 300 mM KCl in 20 mM Tris–HCl
3.1. Enzyme purification and molecular weight The piceid-b-D-glucosidase hydrolyzing piceid to resveratrol from A. oryzae was precipitated firstly by selective fractionation with ammonium sulfate between 50% and 80% saturation. And then the precipitates were centrifuged to collect the protein, and the crude protein was dissolved, dialyzed and fractionated on a DEAE-cellulose column and the result is shown in Fig. 2. The enzyme peak eluted by the step of 180 mM KCl solution had the enzyme activity of hydrolyzing piceid to resveratrol, and formed one spot in SDS polyacrylamide gel electrophoresis, confirming that this enzyme was a pure protein (lane 3 in Fig. 3). The pure enzyme (i.e. piceid-b-D-glucosidase) activity (yield) was 12.8% of total enzyme used in the purification, and the specific activity of pure enzyme was 55.0 nkat mg1, an increase of approximately 12.6-fold (Table 1). The enzyme peak eluted by the step of 120 mM KCl solution also had piceid hydrolysis activity, but was not a pure protein (lane 2 in Fig. 3).
C. Zhang et al. / Process Biochemistry 42 (2007) 83–88
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Fig. 2. Purification of piceid-b-glucosidase on DEAE-cellulose DE-52 column, Ø2.0 cm 10 cm; fraction, 3 ml/tube; elution buffer, 60, 120, 180, 240, and 300 mM KCl in 20 mM Tris–HCl buffer (pH 7.4).
The molecular weight of piceid-b-D-glucosidase’s subunit, estimated by SDS polyacrylamide gel electrophoresis, was approximately 77 kDa (Fig. 3). Gel filtration chromatography (Sephadex G-100) showed that the elution volume of piceid-bD-glucosidase was larger than that of Taq DNA polymerase (94 kDa), proved that the molecular weight of piceid-b-Dglucosidase was less than 94 kDa (data not shown). So we conclude that the piceid-b-D-glucosidase was a monomer and its molecular weight was 77 kDa. 3.2. Some properties of the piceid-b-D-glucosidase For determining the optimal pH, the activity was determined by carrying out standard assays at different pH values ranging from 3.0 to 8.0. It shows that the optimal pH is 5.0. To estimate the pH stability, the residual activity after an incubation of a liquor of the enzyme for 30 min at different pH was measured under standard condition, and the result shows the pure enzyme from A. oryzae sp.100 strain is stable in a narrow pH range from 4.0 to 5.0 (Fig. 4). For determining the optimal temperature, the activity was determined by carrying out standard assays at several temperatures. It shows that the optimal temperature of the pure enzyme is 60 8C. To estimate the thermal stability, the residual activity after an incubation of a liquor of the enzyme for 30 min at different temperature was measured under standard condition. It indicates that the pure enzyme is stable at less than 60 8C (Fig. 5).
Fig. 3. SDS-polyacrylamide gel electrophoresis of piceid-b-glucosidase. (1) Protein markers: a-lactalbumin (14.4 kDa); trypsin inhibitor (20.1 kDa); ovalbumin (45 kDa); bovine serum albumin (66 kDa); phophorylase b (97 kDa). (2) The enzyme eluted by 120 mM KCl. (3) The enzyme eluted by 180 mM KCl.
In addition, the effect of metallic ions on the piceid-b-Dglucosidase was studied. It shows that Ca2+, Mg2+ and Zn2+ ions have no significant effect on the activity in the concentration of 10 mM, while Cu2+ ion inhibits the enzyme activity strongly (Table 2). 3.3. Kinetic parameters of piceid-b-D-glucosidase Kinetic parameters of the piceid-b-D-glucosidase were estimated for piceid by using concentrations of 0.02– 0.64 mM. Activity was measured continuously as described above. Kinetic parameters were calculated from Lineweaver– Burk plots. The Km and Vmax values of the enzyme for piceid at
Table 1 Purification of piceid-b-D-glucosidase Step
Volume (ml)
Total activity (nkat)
Total protein (mg)
Specific activity (nkat mg1)
Yield (%)
Purification
Extraction (NH4)2SO4 selective fractionation DEAE-cellulose
150 12 3
855 657 110
196.3 65.9 2.0
4.36 9.97 55.0
100 76.8 12.9
1 2.3 12.6
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Fig. 6. Lineweaver–Burk plot of piceid-b-glucosidase reaction.
pH 5.0 and 60 8C were 0.74 mM and 323 nkat mg1, respectively (Fig. 6). Fig. 4. Optimal pH and pH stability of piceid-b-glucosidase.
3.4. Ingredients’ change in the enzymatic reaction The enzymatic reaction of piceid was carried out using piceid 5 mg/ml in 20 mM acetate buffer, pH 5.0 as the substrate. Piceid-b-D-glucosidase solution (pure enzyme eluted by 180 mM KCl) 1 ml was added to the same volume of piceid solution and allowed to react at 60 8C for 2 h with shaking. Then butanol 2 ml was added to the reaction mixture to stop the reaction. The product of enzymatic reaction in the butanol layer was detected by TLC (Fig. 7a). The enzymatic reaction of crude extraction of PC was carried out using the crude extraction of
Fig. 5. Optimal temperature and thermal stability of piceid-b-glucosidase.
Table 2 Effect of metallic ions on piceid-b-glucosidase (relative activity, %) Concentration (mM)
CaCl2
MgSO4
ZnSO4
CuSO4
0 1 10 20 100
100 100 82 67 58
100 100 100 100 95
100 100 90 83 83
100 55 10 0 0
Fig. 7. Transformation of piceid to resveratrol by the enzyme. (a) Piceid as the substrate and (b) crude extracts of PC as the substrate. (1) Resveratrol; (2) piceid; (3) emodin; (4) physcion; (5) rhein. (S) Substrate in the enzymatic reaction and (P) product in the enzymatic reaction.
C. Zhang et al. / Process Biochemistry 42 (2007) 83–88
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Table 3 Hydrolysis of different substrates by enzymes Substrates
Activity of piceid-b-D-glucosidase (nkat/ml)
Activity of b-glucosidase from almonds (nkat/ml)
p-Nitrophenyl-b-D-glucoside p-Nitrophenyl-b-D-galactoside p-Nitrophenyl-b-L-arabinoside p-Nitrophenyl-b-D-xyloside p-Nitrophenyl-a-L-arabinoside p-Nitrophenyl-a-L-rhamnoside Piceid
0.41 0.17 0.10 0.03 0 0 37.7
1.72 1.70 1.68 1.71 0.04 0 1.60
Table 4 Characteristic parameters of b-glucosidase from different Aspergillus species Enzyme origin
Km (mM) pNPG as substrate
Km (mM) piceid as substrate
pI
Optimum pH
pH stability
Optimum temperature (8C)
Temperature stability (8C)
Aspergillus phoenicis A. niger A. oryzae sp.100
0.58 0.48 0.92
– – 0.74
3.53 3.72 –
5.0 4.5–5.0 5.0
4.0–8.0 4.0–8.0 4.0–5.0
60 60 60
Up to 50 Up to 50 Up to 60
–: not tested.
PC 50 mg/ml in 20 mM acetate buffer, pH 5.0 as the substrate. Crude enzyme (fractionated precipitation by ammonium sulfate) from A. oryzae sp.100 strain 10 ml was added to the 100 ml of extraction solution and allowed to react at 60 8C for 2 h with shaking. Then butanol 110 ml was added to the reaction mixture to stop the reaction. The product of enzymatic reaction in the butanol layer was detected by TLC (Fig. 7b). It was shown that all the piceid was transformed to resveratrol in the experimental condition. 4. Discussion Resveratrol, a polyphenol compound found in grapes and red wines, is a prominent anti-cancer agent. It provides a protective effect against breast cancer, gastric cancer and colonic cancer through its phytoestrogenic properties, inhibition of protein kinase C and anti-proliferative effect, respectively [18–20]. It protects against the development of cardiovascular diseases by inhibiting squalene monooxygenase, a rate-limiting enzyme in cholesterol biosynthesis [21]. It also has some other activities according to the recent reports [22,23]. All this has attracted more and more attentions. Many attempts have been made to increase the content of resveratrol in wine using the technology of enzymatic hydrolysis or fermentation with transgenic yeasts [24,25]. In this study, the stilbene glycosidase, i.e. piceid-b-Dglucosidase, was purified and characterized. The stilbene glycoside hydrolase, hydrolyzed the b-(1 ! 3)-D-glucopyranoside bond and transformed piceid to resveratrol. The molecular weight was about 77 kDa. Nearly 100% of piceid was conversed into resveratrol in 2 h by the enzyme under the condition of substrate concentration, 5 mg/ml; temperature, 60 8C; pH, 5.0. The enzyme has the advantages of higher activity releasing resveratrol, lower cost and easier to obtain. To realize the transformation from piceid to resveratrol, bglucosidase (EC 3.2.1.21) from almonds and piceid-b-D-
glucosidase from A. oryzae sp.100 strain were used. bGlucosidase from almonds had a higher activity hydrolyzing pnitrophenyl-b-D-glucoside ( pNPG) and a lower activity hydrolyzing piceid. On the contrary, piceid-b-D-glucosidase from A. oryzae sp.100 strain had a higher activity hydrolyzing piceid and a lower activity hydrolyzing pNPG (Table 3). Piceidb-D-glucosidase from A. oryzae sp.100 had no significant differences from the b-glucosidase from A. phoenicis and A. niger in optimum pH, pH stability, optimum temperature and temperature stability [26], but the kinetic parameters showed that piceid-b-D-glucosidase had a stronger affinity for piceid than for pNPG.(Table 4). In our opinion, the fungus and its cultural conditions resulted in the efficient hydrolyzation of piceid. To obtain the enzyme hydrolyzing piceid, the extracts of P. cuspidatum (contains piceid) were added in enzyme fermentation, which induced the production of piceid-b-Dglucosidase that fit to piceid hydrolyzation. And the more properties of piceid-b-D-glucosidase and the hydrolysis of resveratroloside to resveratrol by the enzyme will be further studied in the future. Acknowledgements The authors wish to thank Mr. Kou and Ms. Gao for their friendly assistance. This work was supported by the Ministry of Education of Liaoning Province of China (2004D212) and National Science of Foundation of China (NSFC). References [1] Bennet RC, Wallsgrove RM. Secondary metabolites in plant defence mechanisms. Tansley Review No. 72. New Phytol 1994;127:617–33. [2] Steinberg D. Antioxidants and atherosclerosis. A current assessment. Circulation 1991;84:1420–5. [3] Cao ZX, Li YB. Potent induction of cellular antioxidants and phase 2 enzymes by resveratrol in cardiomyocytes: protection against oxidative and electrophilic injury. Eur J Pharmacol 2004;489:39–48.
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[4] Kopp P, Resveratrol. a phytoestrogen found in red wine. A possible explanation for the conundrum of the ‘French paradox’? Eur J Endocrinol 1998;138:619–20. [5] Damianaki A, Bakogeorgou E, Kampa M, Notas G, Hatzoglou A, Panagiotou S, et al. Potent inhibitory action of red wine polyphenols on human breast cancer cells. J Cell Biochem 2000;78:429–41. [6] Schneider Y, Vincent F, Duranton B, Badolo L, Gosse F, Bergmann C, et al. Anti-proliferative effect of resveratrol, a natural component of grapes and wine, on human colonic cancer cells. Cancer Lett 2000;158: 85–91. [7] Bruno R, Ghisolfi L, Priulla M, Nicolin A, Bertelli A. Wine and tumors: study of resveratrol. Drugs Exp Clin Res 2003;29:257–61. [8] Surh YJ, Hurh YJ, Kang JY, Lee E, Kong G, Lee SJ. Resveratrol, an antioxidant present in red wine, induces apoptosis in human promyelocytic leukaemia (HL-60) cells. Cancer Lett 1999;140:1–10. [9] Ahmad N, Adhami VM, Afaq F, Feyes DK, Mukhtar H. Resveratrol causes WAF-1/p21-mediated G1-phase arrest of cell cycle and induction of apoptosis in human epidermoid carcinoma A431 cells. Clin Cancer Res 2001;7:1466–73. [10] Subbaramaiah K, Chung WJ, Michaluart P, Telang N, Tanabe T, Inoue H, et al. Resveratrol inhibits cyclooxygenase-2 transcription and activity in phorbol ester-treated human mammary epithelial cells. J Bio Chem 1998;273:21875–82. [11] Heredia A, Davis C, Redfield R. Synergistic inhibition of HIV-1 in activated and resting peripheral blood mononuclear cells, monocytederived macrophages, and selected drug-resistant isolates with nucleoside analogues combined with a natural product, resveratrol. J Acquir Immune Defic Syndr 2000;25:246–55. [12] Gao X, Deeb D, Media J, Divine G, Jiang H, Chapman RA, et al. Immunomodulatory activity of resveratrol: discrepant in vitro and in vivo immunological effects. Biochem Pharmacol 2003;66:2427–35. [13] Vastano BC, Chen Y, Zhu N, Ho CT, Zhou Z, Rosen RT. Isolation and Identification of Stilbenes in Two Varieties of Polygonum cuspidatum. J Agric Food Chem 2000;48:253–6. [14] Zhou JJ, Zhang HJ, Yang PJ, Li HN. Determination of resveratrol glucoside and resveratrol in radix and rhizome of Polygonum cuspidatum yielded in Hanzhong region. Zhongcaoyao 2002;33:414–6 (Chinese).
[15] Bae EA, Park SY, Kim DH. Constitutive b-glucosidases hydrolyzing ginsenoside Rb1 and Rb2 from human intestinal bacteria. Biol Pharm Bull 2000;23:1481–5. [16] Bradford MM. A rapid and sensitive method for quanti.cation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:203–13. [17] Weber K, Pringle JR, Osborn M. Measurement of molecular weights by electrophoresis on SDS-acrylamide gel method. Enzymol 1971;26:3–27. [18] Le Corre L, Fustier P, Chalabi N, Bignon YJ, Bernard-Gallon D. Effects of resveratrol on the expression of a panel of genes interacting with the BRCA1 oncosuppressor in human breast cell lines. Clin Chim Acta 2004;344:115–21. [19] Atten MJ, Attar BM, Milson T, Holian O. Resveratrol-induced inactivation of human gastric adenocarcinoma cells through a protein kinase Cmediated mechanism. Biochem Pharmacol 2001;62:1423–32. [20] SchneiderY, Vincent F, Duranton B, Badolo L, Gosse F, Bergmann C, et al. Anti-proliferative effect of resveratrol, a natural component of grapes and wine, on human colonic cancer cells. Cancer Lett 2000;158:85–91. [21] Laden BP, Porter TD. Resveratrol inhibits human squalene monooxygenase. Nutr Res 2001;21:747–53. [22] Morin C, Zini R, Albengres E, Bertelli AA, Bertelli A, Tillement JP. Evidence for resveratrol-induced preservation of brain mitochondria functions after hypoxia-reoxygenation. Drugs Exp Clin Res 2003;29: 227–33. [23] Feng YH, Zhu YN, Liu J, Ren YX, Xu JY, Yang YF, et al. Differential regulation of resveratrol on lipopolysacchride-stimulated human macrophages with or without IFN-gamma pre-priming. Int Immunopharmacol 2004;4:713–20. [24] Loredana La Torre G, Lagana G, Bellocco E, Vilasi F, Salvo F, Dugo G. Improvement on enzymatic hydrolysis of resveratrol glucosides in wine. Food Chem 2004;85:259–66. [25] Gonzalez-Candelas L, Gil JV, Lamuela-Raventos RM, Ramon D. The use of transgenic yeasts expressing a gene encoding a glycosyl-hydrolase as a tool to increase resveratrol content in wine. Int J Food Microb 2000;59: 179–83. [26] Ja¨ger S, Brumbauer A, Fehe´r E, Re´czey K, Kiss L. Production and characterization of b-glucosidase from different Aspergillus strains. World J Microbiol Biochem 2001;17:455–61.
Purification and characterization of piceid-b-D-glucosidase from Aspergillus oryzae Chunzhi Zhang *, Dai Li, Hongshan Yu, Bo Zhang, Fengxie Jin * College of Bio & Food Technology, Dalian Institute of Light Industry, Qinggong-yuan No. 1, Ganjingzi-qu, Dalian 116034, People’s Republic of China Received 21 April 2006; received in revised form 28 June 2006; accepted 10 July 2006
Abstract The piceid-b-D-glucosidase that hydrolyzes the b-D-glucopyranoside bond of piceid to release resveratrol was isolated from Aspergillus oryzae sp.100 strain, and the enzyme was purified and characterized. The enzyme was purified to one spot in SDS polyacrylamide gel electrophoresis, and its molecular weight was about 77 kDa. The optimum temperature of the piceid-b-D-glucosidase was 60 8C, and the optimum pH was 5.0. The piceid-b-D-glucosidase was stable at less than 60 8C, and pH 4.0–5.0. Ca2+, Mg2+ and Zn2+ ions have no significant effect on enzyme activity, but Cu2+ ion inhibits enzyme activity strongly. The Km value was 0.74 mM and the Vmax value was 323 nkat mg1 for piceid. # 2006 Elsevier Ltd. All rights reserved. Keywords: Purification; Characterization; Piceid-b-D-glucosidase; Aspergillus oryzae; Resveratrol
1. Introduction Resveratrol (3,5,40 -trihydroxystilbene, Fig. 1a) is a secondary metabolite of some plants and often as phytoalexin to prevent themselves from microbial pathogens and herbivorous animals [1]. It is originally found in the skins and seeds of the grapes, and contributes to the antioxidant potential of red wine. Recently, resveratrol has attracted more attention because of its widely physiological activity. Resveratrol has the activity of cardiovascular protection, owing to its oxidative modification of low-density lipoproteins [2,3], its ability to act as an antioxidant and as an inhibitor of platelet aggregation, and its action of phytoestrogens [4]. It has been shown that resveratrol inhibits the growth of several cancerous cell lines [5–7], or has the ability to cause apoptosis in these lines [8,9]. Resveratrol also has anti-inflammatory, anti-microbial, anti-HIV and some other effects [10–12]. Resveratrol is a polyphenol compound existing in a variety of plant species, including grapes, peanuts, mulberries and other plants, especially in medicinal plants of the Polygonum species (Polygonaceae). In general, resveratrol is obtained by extraction from natural sources. Because the amount of stilbenes that occurs
* Corresponding authors. Tel.: +86 411 86307737; fax: +86 411 86307737. E-mail addresses: [email protected] (C. Zhang), [email protected] (F. Jin). 1359-5113/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2006.07.019
in the roots of Polygonum cuspidatum (PC) is far more than the amount found in wine and other sources, the roots of PC is the best material offering resveratrol [13]. The main stilbenes in PC, however, are piceid (3,5,40 -trihydroxystilbene-3-O-b-D-glucopyranoside, Fig. 1b) and resveratroloside (3,5,40 -trihydroxystilbene-40 -O-b-D-glucopyranoside, Fig. 1c) rather than resveratrol [13]. For example, the amount of piceid in PC from Hanzhong region in China is about six times larger than that of resveratrol [14]. Therefore, resveratrol content in PC crude extraction will increase by adding the enzyme hydrolyzing resveratrol glucoside into the preparation of PC (Fig. 1). The enzyme, named as piceid-b-D-glucosidase from Aspergillus oryzae, which hydrolyzes the b-(1 ! 3)-D-glucopyranoside bond and transforms piceid to resveratrol, was isolated, purified and characterized in this paper. 2. Materials and methods 2.1. Materials The standard piceid and resveratrol were purchased from Tauto Biotech. Co. Ltd. (Shenzhen, China). The thin-layer chromatography (TLC) plate was the silica gel plate (Kieselgel 60 F-254, Merck). The microorganism, A. oryzae sp.100 (FFCDL-100) strain producing piceid-b-glucosidase, was isolated from traditional Chinese Koji (Daqu in Chinese), identified by Prof. FX Jin, College of Bio & Food Technology, Dalian Institute of Light Industry, and maintained in Food Fermentation Culture Collection of Dalian Institute of Light Industry (FFCDL).
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Fig. 1. Enzymatic transformation of stilbenes.
2.2. Enzyme analysis Piceid-b-glucosidase activity was measured using 0.3 mM piceid in 20 mM acetate buffer, pH 5.0 as the substrate. Enzyme solution 0.1 ml was added to the same volume of piceid solution and allowed to react at 60 8C for 20 min. Then butanol 0.2 ml was added to the reaction mixture to stop the reaction. The product resveratrol in the butanol layer was detected by TLC: developing solvent, CHCl3–MeOH–H2O (4.5:2:1, v/v), and the resveratrol on the silica plate was determined by scanning the TLC spots using a Shimadzu CS-930 [15]. One unit of enzyme activity was defined as the amount of enzyme producing 1 nmol of resveratrol per second (nkat).
2.3. Protein concentration The concentration of protein was measured by the method of Bradford using bovine serum albumin as a standard protein [16].
buffer, pH 7.4. The purified enzyme was dried by freeze-drying and used for the study of enzymatic properties. The enzyme purity and molecular weight were estimated by the SDS polyacrylamide gel electrophoresis method using alactalbumin (14.4 kDa), trypsin inhibitor (20.1 kDa), ovalbumin (45 kDa), bovine serum albumin (66 kDa), and phosphorylase b (97 kDa) as standard proteins [17].
2.7. Effects of pH and temperature on piceid-b-D-glucosidase activity The optimal pH of the piceid-b-D-glucosidase was tested at 60 8C with different buffers at 20 mM. For the pH ranges of 3.0, 4.0–5.0, and 6.0–8.0, citrate, acetate, and phosphate buffers were used, respectively. The pH stability was determined by standard enzyme assay after pre-incubating the purified enzyme at different pH buffer at 4 8C for 1 h. Optimal temperature was tested between 30 and 90 8C by standard enzyme assay. Thermal stability was evaluated by incubation of the enzyme solution at different temperature for 1 h, before performing the standard enzymatic assay.
2.4. Microorganism incubation and crude enzyme extraction
3. Results The FFCDL-100 strain was incubated at 28 8C with shaking at 150 rpm in the medium containing 4% wheat bran extract and 2% plant extract of P. cuspidatum. After incubated for 72 h, the cells were removed by centrifugation and the supernatant was used in the enzyme purification procedure.
2.5. Ammonium sulfate fractionation Pellets of (NH4)2SO4 were slowly added to the supernatant with shaking to 50% saturation. After incubation at 4 8C for 1 h, the enzymatic extract was centrifuged. A second step of precipitation was performed adding ammonium sulfate to 80% saturation and incubating at 4 8C for 1 h. The final precipitate was collected by centrifugation and dissolved in 20 mM Tris–HCl (pH 7.4) for purification or dissolved in 20 mM acetate buffer (pH 5.0) for enzymatic properties.
2.6. Purification of piceid-b-D-glucosidase and estimation of molecular weight The crude protein in 20 mM Tris–HCl after ammonium sulfate fractionation was dialyzed against 20 mM Tris–HCl, pH 7.4. After removing the nondissolved fraction by centrifugation, the enzyme solution was fractionated on a column (Ø2.0 cm 10 cm) of DEAE-cellulose DE-52 (Whatman). The column was eluted stepwise with 60, 120, 180, 240, and 300 mM KCl in 20 mM Tris–HCl
3.1. Enzyme purification and molecular weight The piceid-b-D-glucosidase hydrolyzing piceid to resveratrol from A. oryzae was precipitated firstly by selective fractionation with ammonium sulfate between 50% and 80% saturation. And then the precipitates were centrifuged to collect the protein, and the crude protein was dissolved, dialyzed and fractionated on a DEAE-cellulose column and the result is shown in Fig. 2. The enzyme peak eluted by the step of 180 mM KCl solution had the enzyme activity of hydrolyzing piceid to resveratrol, and formed one spot in SDS polyacrylamide gel electrophoresis, confirming that this enzyme was a pure protein (lane 3 in Fig. 3). The pure enzyme (i.e. piceid-b-D-glucosidase) activity (yield) was 12.8% of total enzyme used in the purification, and the specific activity of pure enzyme was 55.0 nkat mg1, an increase of approximately 12.6-fold (Table 1). The enzyme peak eluted by the step of 120 mM KCl solution also had piceid hydrolysis activity, but was not a pure protein (lane 2 in Fig. 3).
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Fig. 2. Purification of piceid-b-glucosidase on DEAE-cellulose DE-52 column, Ø2.0 cm 10 cm; fraction, 3 ml/tube; elution buffer, 60, 120, 180, 240, and 300 mM KCl in 20 mM Tris–HCl buffer (pH 7.4).
The molecular weight of piceid-b-D-glucosidase’s subunit, estimated by SDS polyacrylamide gel electrophoresis, was approximately 77 kDa (Fig. 3). Gel filtration chromatography (Sephadex G-100) showed that the elution volume of piceid-bD-glucosidase was larger than that of Taq DNA polymerase (94 kDa), proved that the molecular weight of piceid-b-Dglucosidase was less than 94 kDa (data not shown). So we conclude that the piceid-b-D-glucosidase was a monomer and its molecular weight was 77 kDa. 3.2. Some properties of the piceid-b-D-glucosidase For determining the optimal pH, the activity was determined by carrying out standard assays at different pH values ranging from 3.0 to 8.0. It shows that the optimal pH is 5.0. To estimate the pH stability, the residual activity after an incubation of a liquor of the enzyme for 30 min at different pH was measured under standard condition, and the result shows the pure enzyme from A. oryzae sp.100 strain is stable in a narrow pH range from 4.0 to 5.0 (Fig. 4). For determining the optimal temperature, the activity was determined by carrying out standard assays at several temperatures. It shows that the optimal temperature of the pure enzyme is 60 8C. To estimate the thermal stability, the residual activity after an incubation of a liquor of the enzyme for 30 min at different temperature was measured under standard condition. It indicates that the pure enzyme is stable at less than 60 8C (Fig. 5).
Fig. 3. SDS-polyacrylamide gel electrophoresis of piceid-b-glucosidase. (1) Protein markers: a-lactalbumin (14.4 kDa); trypsin inhibitor (20.1 kDa); ovalbumin (45 kDa); bovine serum albumin (66 kDa); phophorylase b (97 kDa). (2) The enzyme eluted by 120 mM KCl. (3) The enzyme eluted by 180 mM KCl.
In addition, the effect of metallic ions on the piceid-b-Dglucosidase was studied. It shows that Ca2+, Mg2+ and Zn2+ ions have no significant effect on the activity in the concentration of 10 mM, while Cu2+ ion inhibits the enzyme activity strongly (Table 2). 3.3. Kinetic parameters of piceid-b-D-glucosidase Kinetic parameters of the piceid-b-D-glucosidase were estimated for piceid by using concentrations of 0.02– 0.64 mM. Activity was measured continuously as described above. Kinetic parameters were calculated from Lineweaver– Burk plots. The Km and Vmax values of the enzyme for piceid at
Table 1 Purification of piceid-b-D-glucosidase Step
Volume (ml)
Total activity (nkat)
Total protein (mg)
Specific activity (nkat mg1)
Yield (%)
Purification
Extraction (NH4)2SO4 selective fractionation DEAE-cellulose
150 12 3
855 657 110
196.3 65.9 2.0
4.36 9.97 55.0
100 76.8 12.9
1 2.3 12.6
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Fig. 6. Lineweaver–Burk plot of piceid-b-glucosidase reaction.
pH 5.0 and 60 8C were 0.74 mM and 323 nkat mg1, respectively (Fig. 6). Fig. 4. Optimal pH and pH stability of piceid-b-glucosidase.
3.4. Ingredients’ change in the enzymatic reaction The enzymatic reaction of piceid was carried out using piceid 5 mg/ml in 20 mM acetate buffer, pH 5.0 as the substrate. Piceid-b-D-glucosidase solution (pure enzyme eluted by 180 mM KCl) 1 ml was added to the same volume of piceid solution and allowed to react at 60 8C for 2 h with shaking. Then butanol 2 ml was added to the reaction mixture to stop the reaction. The product of enzymatic reaction in the butanol layer was detected by TLC (Fig. 7a). The enzymatic reaction of crude extraction of PC was carried out using the crude extraction of
Fig. 5. Optimal temperature and thermal stability of piceid-b-glucosidase.
Table 2 Effect of metallic ions on piceid-b-glucosidase (relative activity, %) Concentration (mM)
CaCl2
MgSO4
ZnSO4
CuSO4
0 1 10 20 100
100 100 82 67 58
100 100 100 100 95
100 100 90 83 83
100 55 10 0 0
Fig. 7. Transformation of piceid to resveratrol by the enzyme. (a) Piceid as the substrate and (b) crude extracts of PC as the substrate. (1) Resveratrol; (2) piceid; (3) emodin; (4) physcion; (5) rhein. (S) Substrate in the enzymatic reaction and (P) product in the enzymatic reaction.
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Table 3 Hydrolysis of different substrates by enzymes Substrates
Activity of piceid-b-D-glucosidase (nkat/ml)
Activity of b-glucosidase from almonds (nkat/ml)
p-Nitrophenyl-b-D-glucoside p-Nitrophenyl-b-D-galactoside p-Nitrophenyl-b-L-arabinoside p-Nitrophenyl-b-D-xyloside p-Nitrophenyl-a-L-arabinoside p-Nitrophenyl-a-L-rhamnoside Piceid
0.41 0.17 0.10 0.03 0 0 37.7
1.72 1.70 1.68 1.71 0.04 0 1.60
Table 4 Characteristic parameters of b-glucosidase from different Aspergillus species Enzyme origin
Km (mM) pNPG as substrate
Km (mM) piceid as substrate
pI
Optimum pH
pH stability
Optimum temperature (8C)
Temperature stability (8C)
Aspergillus phoenicis A. niger A. oryzae sp.100
0.58 0.48 0.92
– – 0.74
3.53 3.72 –
5.0 4.5–5.0 5.0
4.0–8.0 4.0–8.0 4.0–5.0
60 60 60
Up to 50 Up to 50 Up to 60
–: not tested.
PC 50 mg/ml in 20 mM acetate buffer, pH 5.0 as the substrate. Crude enzyme (fractionated precipitation by ammonium sulfate) from A. oryzae sp.100 strain 10 ml was added to the 100 ml of extraction solution and allowed to react at 60 8C for 2 h with shaking. Then butanol 110 ml was added to the reaction mixture to stop the reaction. The product of enzymatic reaction in the butanol layer was detected by TLC (Fig. 7b). It was shown that all the piceid was transformed to resveratrol in the experimental condition. 4. Discussion Resveratrol, a polyphenol compound found in grapes and red wines, is a prominent anti-cancer agent. It provides a protective effect against breast cancer, gastric cancer and colonic cancer through its phytoestrogenic properties, inhibition of protein kinase C and anti-proliferative effect, respectively [18–20]. It protects against the development of cardiovascular diseases by inhibiting squalene monooxygenase, a rate-limiting enzyme in cholesterol biosynthesis [21]. It also has some other activities according to the recent reports [22,23]. All this has attracted more and more attentions. Many attempts have been made to increase the content of resveratrol in wine using the technology of enzymatic hydrolysis or fermentation with transgenic yeasts [24,25]. In this study, the stilbene glycosidase, i.e. piceid-b-Dglucosidase, was purified and characterized. The stilbene glycoside hydrolase, hydrolyzed the b-(1 ! 3)-D-glucopyranoside bond and transformed piceid to resveratrol. The molecular weight was about 77 kDa. Nearly 100% of piceid was conversed into resveratrol in 2 h by the enzyme under the condition of substrate concentration, 5 mg/ml; temperature, 60 8C; pH, 5.0. The enzyme has the advantages of higher activity releasing resveratrol, lower cost and easier to obtain. To realize the transformation from piceid to resveratrol, bglucosidase (EC 3.2.1.21) from almonds and piceid-b-D-
glucosidase from A. oryzae sp.100 strain were used. bGlucosidase from almonds had a higher activity hydrolyzing pnitrophenyl-b-D-glucoside ( pNPG) and a lower activity hydrolyzing piceid. On the contrary, piceid-b-D-glucosidase from A. oryzae sp.100 strain had a higher activity hydrolyzing piceid and a lower activity hydrolyzing pNPG (Table 3). Piceidb-D-glucosidase from A. oryzae sp.100 had no significant differences from the b-glucosidase from A. phoenicis and A. niger in optimum pH, pH stability, optimum temperature and temperature stability [26], but the kinetic parameters showed that piceid-b-D-glucosidase had a stronger affinity for piceid than for pNPG.(Table 4). In our opinion, the fungus and its cultural conditions resulted in the efficient hydrolyzation of piceid. To obtain the enzyme hydrolyzing piceid, the extracts of P. cuspidatum (contains piceid) were added in enzyme fermentation, which induced the production of piceid-b-Dglucosidase that fit to piceid hydrolyzation. And the more properties of piceid-b-D-glucosidase and the hydrolysis of resveratroloside to resveratrol by the enzyme will be further studied in the future. Acknowledgements The authors wish to thank Mr. Kou and Ms. Gao for their friendly assistance. This work was supported by the Ministry of Education of Liaoning Province of China (2004D212) and National Science of Foundation of China (NSFC). References [1] Bennet RC, Wallsgrove RM. Secondary metabolites in plant defence mechanisms. Tansley Review No. 72. New Phytol 1994;127:617–33. [2] Steinberg D. Antioxidants and atherosclerosis. A current assessment. Circulation 1991;84:1420–5. [3] Cao ZX, Li YB. Potent induction of cellular antioxidants and phase 2 enzymes by resveratrol in cardiomyocytes: protection against oxidative and electrophilic injury. Eur J Pharmacol 2004;489:39–48.
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