Purification and characterization of a novel stable ginsenoside Rb1-hydrolyzing β-d -glucosidase from China white jade snail

Process Biochemistry 41 (2006) 1974–1980 www.elsevier.com/locate/procbio

Purification and characterization of a novel stable ginsenoside Rb1-hydrolyzing b-D-glucosidase from China white jade snail Hongwei Luan a,b, Xin Liu c, Xiaohui Qi a, Ying Hu a,b, Dacheng Hao a,b, Yu Cui c, Ling Yang a,* a

Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 116023 Dalian, China b Graduate School of the Chinese Academy of Sciences, Beijing, China c Dalian Medical University, 116027 Dalian, China Received 6 January 2006; received in revised form 6 April 2006; accepted 11 April 2006

Abstract A highly stable b-glucosidase from the viscera of China white jade snail was purified to apparent homogeneity. This purified glucosidase consisted of two identical subunits with a native molecular mass of approximately 230 kDa. The maximal activity to p-nitrophenyl-b-Dglucopyranoside ( pNPG) occurred at 70 8C and pH 5.6. Its most notable characteristic was stable over a wide pH range (3–11 at 30 8C for 24 h) and a relatively high temperature (60 8C for 24 h). The Km for pNPG was 0.338 mM and the Vmax was 0.25 mmol nitrophenol/min/mg glucosidase at pH 5.6 and 50 8C. Compared to its activity against pNPG (100%), the b-glucosidase exhibited low levels of activity against other aryl-glycosides (less than 1%). Additionally, the enzyme hydrolyzed the 20-C, b-(1 ! 6)-glucoside of ginsenoside Rb1 to produce ginsenoside Rd, but did not hydrolyze the other b-D-glucosidic bonds of Rb1. The properties of the enzyme could make it become a useful tool in biotransformation of glucosides. # 2006 Elsevier Ltd. All rights reserved. Keywords: b-D-Glucosidase; Thermo- and pH-stability; Ginsenoside Rb1; Ginsenoside Rd; Purification; China white jade snail

1. Introduction Ginseng, the root of Panax ginseng C.A. MEYER (Araliaceae), has been used as a folk medicine for thousands of years in East Asian countries and recently has also become popular in Western countries. The major active ingredients of ginseng have been demonstrated to be ginsenosides (ginseng saponins) [1–3]. In recent decades, many studies have focused on the minor ginsenosides, such as ginsenosides Rd, Rg3, Rh2 and compound K (C–K) because of their pharmaceutical activities. These minor ginsenosides, which are present in ginseng in small percentages, can be produced by hydrolysis of the sugar moieties of the major ginsenosides (Rb1, Rb2 and Rc). Therefore, many methods, such as heating [4], acid treatment [5], alkali treatment [6] and enzymatic conversion [7], have been developed for the production of these minor ginsenosides.

* Corresponding author. Tel.: +86 411 84379317; fax: +86 411 84676961. E-mail address: [email protected] (L. Yang). 1359-5113/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2006.04.011

However, due to non-specificity of the other three methods, enzymatic conversion is considered to be the most desirable. Of these minor ginsenosides, Rd has been suggested to have very useful physiological activity. For instance, it can prevent kidney injury by chemical drugs including cisplatin, cephaloridine and other anti-cancer drugs [8–10]. Rd is also shown to attenuate the oxidative damage, which may be responsible for the aging process [11]. In a recent study, Rd has been found to enhance the differentiation of neural stem cells, while other ginsenosides induce no differentiation of neurons [12]. However, the content of Rd is usually 4.3–11.9% of the total ginsenosides in the various ginseng samples [13]. Therefore, several biotransformation methods have been developed to obtain a large amount of Rd. In China, two enzymes have been found from the root of ginseng and Absidia sp. 90 (FFCDL-90) strain, which can transform ginsenoside Rc and gypenoside-5 to Rd, respectively [14,15]. Ginsenoside Rb1, another protopanaxadiol type ginsenoside, has a higher content (usually more than 20% of total ginsenosides) in the various ginseng samples [13]. Its structure can be easily converted to Rd by hydrolysis of one glucose moiety.

H. Luan et al. / Process Biochemistry 41 (2006) 1974–1980

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Fig. 1. Biotransformation pathways of ginsenoside Rb1.

Regardless of using either chemical or biological methods, the hydrolysis of Rb1 is difficult to terminate at Rd because other three glucosidic bonds may also be attacked. Rb1 can be hydrolyzed to Rg3, Rh2 and panaxadiol (PD) (via acid treatment), or gypenoside XVII, F2 and C–K (via biotransformation, Fig. 1). Therefore, it is important to find out a method to hydrolyze the b(1 ! 6)-glucosidic bond at the 20-C site of Rb1 to give Rd. Kim et al. [16] have found three strains owning this ability, but key enzymes for the conversion have not been purified and characterized. An Rb1-converting enzyme has been purified from human intestinal bacteria, but the major product is ginsenoside F2 [17]. In this study, for the first time we report a highly pH- and thermo-stable b-glucosidase from China white jade snail (from Achatina fulica), which can hydrolyze the b-(1 ! 6)-glucosidic bond at the 20-C site of Rb1 to give Rd. We describe the purification and biochemical properties of the novel b-Dglucosidase. 2. Materials and methods 2.1. Special chemicals The substrates, p-nitrophenyl-b-D-glucopyranoside ( pNPG), o-nitrophenylb-D-glucopyranoside (oNPG), p-nitrophenyl-a-L-arabinofuranoside ( pNP-a-Larabinofuranoside), pNP-b-L-arabinopyranoside, pNP-a-L-arabinopyranoside, pNP-b-D-cellobioside, pNP-a-D-galactopyranoside, pNP-b-D-galactopyranoside, pNP-b-D-fucopyranoside, pNP-b-L-fucopyranoside, pNP-a-L-fucopyranoside, pNP-b-D-maltoside, pNP-a-D-mannopyranoside, pNP-b-D-mannopyranoside, pNP-a-D-xylopyranoside, 4-methylumbelliferyl-b-D-glucopyranoside (MUG), as well as all alkyl-glucosides and disaccharides, were obtained from Sigma (St. Louis, MO, USA). The three artificial substrates, pNP-a-L-rhamnopyranoside, pNP-a-D-glucopyranoside and pNP-b-D-xylopyranoside, were the products of ICN Biomedical Inc. Ginsenosides Rb1 and Rd were obtained from Hongjiu Ginseng Co. Ltd., China, and gypenoside XVII, F2 and C–K were generously provided by Dr. Hideo Hasegawa (Fermenta Herb Institute Inc., Tokyo, Japan). DEAE Sepharose CL-6B, Q-Sepharose Fast Flow, Sephadex G-50, Sephacryl S-300 HR and Sephacryl S-400 HR were purchased from Pharmacia. Toyopearl Butyl 650C was obtained from TOSOH Corporation. The general chemicals used were of AR grade.

2.2. Enzyme source The acetone powder of viscera from China white jade snail was purchased from JingKeHongDa Biotechnology Co. Ltd., China. It was used as the source of b-glucosidase.

2.3. Enzyme assays For routine assays and monitoring of purification, b-glucosidase activity was measured using the artificial substrate pNPG. In brief, the reaction mixture

(total volume of 0.3 ml) contained 0.02 ml of 10 mM pNPG, 0.26 ml of 150 mM citrate–phosphate buffer (pH 5.6) and 0.02 ml of the enzyme solution. It was incubated at 50 8C for 20 min, then stopped by the addition of 0.25 M NaOH (1.5 ml) and the absorbance was read at 405 nm by UV–vis spectrophotometer (JASCO V-530, Japan). All other aryl-glucosides were assayed under the same conditions. One nkat of b-glucosidase activity is defined as the amount of enzyme liberating 1 nmol p-nitrophenol/s under these conditions. Enzyme activities against different alkyl-glucosides and disaccharides were determined by measuring the amount of glucose released from these substrates using the method of Leary et al. [18] with a glucose kit (Kehua Bio-engineering Co. Ltd., China). The amount of glucose released from cellobiose, gentiobiose, sophorose and trehalose was calculated by dividing by two because they are composed of two glucose molecules. Hydrolytic activities were measured as follows. Ginsenosides were dissolved in 150 mM citrate–phosphate buffer, pH 5.6, to a concentration of 8 mM as substrates. Enzyme and substrate solution were added to the above buffer and allowed a final volume of 0.1 ml to react at 50 8C for 20 min. Then 0.9 ml ethanol was added to the reaction mixture, and the supernatant containing product ginsenoside was obtained by centrifugation. The supernatant was analyzed by HPLC system (SHIMADZU, Japan), which consisted of an SCL-10A system controller, two LC-10AD pumps and a UV detector (203 nm). Separations were carried out through a 5m C18 column (Agilent, ZORBAX 80A, 4.6 mm
250 mm). The binary gradient employed (A) water and (B) acetonitrile according to the following profile: 0–20 min, 70–40% A, 30–60% B; 20–30 min, 40–10% A, 60–90% B. The flow rate was 1.0 ml/min. The amount of product ginsenoside Rd was calculated from the peak area. Protein was quantified according to the Bradford method [19] using bovine serum albumin as the standard. All chromatographic runs were monitored for protein at 280 nm.

2.4. Purification of glucosidase All procedures of the b-glucosidase purification were carried out at room temperature. The following buffers were used: buffer A, 50 mM Tris–HCl (pH 8.2); buffer B, 50 mM Tris–HCl (pH 7.5); buffer C, buffer B containing 1.5 M ammonium sulfate. The crude enzyme (acetone powder) was dissolved with buffer A and then loaded onto a DEAE Sepharose CL-6B column (F 3.5 cm
10 cm) equilibrated with the same buffer. After washing with two column volumes of buffer A, a 1000-ml linear gradient elution of 0–0.6 M NaCl was undertaken. The fractions (574–728 ml) were pooled and diluted to 300 ml with buffer B. This solution was directly loaded onto a Q-Sepharose Fast Flow column (F 1.0 cm
30 cm), which was pretreated with buffer B. Proteins bound to the matrix were washed with a 400-ml linear gradient of 0.25–0.6 M NaCl. The activity fractions eluted between 0.39 and 0.42 M NaCl were combined (84 ml) for further purification. Solid ammonium sulfate was added to the sample solution to a final concentration of 1.5 M. Then the sample was applied to a Toyopearl Butyl 650C column (F 1.0 cm
15 cm), which was pretreated with buffer C. Proteins were eluted with a 200-ml linear gradient of 1.5–0 M (NH4)2SO4. The b-glucosidase active fractions between 0.9 and 0.6 M (NH4)2SO4 were pooled (32 ml) and desalted through a Sephadex G-50 column following vacuum freeze-drying (lyophilization). The enzyme was further purified by gel filtration on a Sephacryl S-400 HR column (F 1.6 cm
70 cm), cm), running at 0.67 ml/min with distilled water. Fractions with b-glucosidase

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activity were collected and the purity was estimated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE).

2.5. Electrophoresis and molecular mass estimation SDS-PAGE was carried out according to Laemmli [20] in a Mini-Protean III dual-slab cell electrophoresis unit (Bio-Rad) on 8% polyacrylamide gel. The gel was stained by Coomassie Brilliant Blue R250. Denatured molecular weight was estimated using an HMW-SDS Marker Kit (Pharmacia). Activity staining of b-glucosidase in the slab gel was done with 0.1% MUG on a 7.5% native PAGE. Molecular mass of b-glucosidase was estimated by both SDS-PAGE and gel filtration on a column of Sephacryl S-300 HR (F 1.0 cm
40 cm). The gel filtration column was equilibrated with 20 mM Na2HPO4–citrate buffer (pH 7.0) and calibrated by elution of standard protein markers which included thyroglobulin (669 kDa), catalase (250 kDa), bovine serum albumin (66 kDa), ovalbumin (44 kDa) and cytochrome C (12 kDa).

2.6. Effects of temperature and pH on enzyme activity and stability The enzyme activity was studied within pH 3.0–12.0 using the following buffers: 50 mM Na2HPO4–citrate (pH 2.4–8.0), 50 mM Tris–HCl (pH 7.0–9.0),

50 mM glycine–NaOH (pH 8.0–11.0) and 50 mM Na2HPO4–NaOH buffer (pH 11.0–12.0). The temperature effect was studied between 30 and 90 8C at optimum pH. The pH stability of the b-glucosidase was studied within pH 2.4–12.0, using the above buffers. The temperature stability was studied between 30 and 80 8C.

2.7. Time course of hydrolysis of ginsenoside Rb1 The enzyme in 150 mM citrate–phosphate buffer, pH 5.6, was incubated with Rb1 (final concentration: 2 mM) at 50 8C. Aliquots were withdrawn at suitable time intervals and the reaction was terminated by the addition of ninefold volumes of ethanol. The reaction product was detected by HPLC as mentioned above. The control tube of no enzyme was incubated under the same conditions.

2.8. Kinetic studies The Km and Vmax values were determined by Lineweaver–Burk plot [21] towards substrates pNPG and ginsenoside Rb1. To estimate kinetic constants as accurately as possible, the enzyme reactions were carried out under initial velocity conditions and in a substrate concentration range that is widely across the Km (0.2–3 Km).

Fig. 2. Purification of b-glucosidase from China white jade snail. (A) Ion-exchange chromatography on DEAE Sepharose CL-6B. (B) Ion-exchange chromatography on Q-Sepharose Fast Flow. (C) Hydrophobic interaction chromatography on Toyopearl Butyl 650C. (D) Gel filtration chromatography on Sephacryl S-400 HR. (&), (&) and (- - -) represent b-glucosidase, protein (A280) and the salt gradient, respectively. Solid bars represent the fractions pooled for further purification or characterization.

H. Luan et al. / Process Biochemistry 41 (2006) 1974–1980

Fig. 3. HPLC analysis of products of ginsenoside Rb1 hydrolyzed by different activity fractions from DEAE Sepharose column. A reaction mixture (800 ml) containing 1 mmol Rb1 and 0.0755 nkat b-glucosidase (using pNPG as the substrate) in 50 mM sodium phosphate–citrate buffer, pH 5.5, was incubated at 50 8C. The samples 1–6 correspond to hydrolysis for 0 h by the crude enzyme, 2 h by the crude enzyme, 2 h by fraction I, 2 h by fraction II, 2 h by fraction III and 2 h by fraction IV, respectively. The peaks a–d correspond to Rb1, Rd, gypenoside XVII and F2, respectively.

3. Results and discussion 3.1. Purification and some properties of b-glucosidase A b-glucosidase from China white jade snail was purified as described in Section 2. Using DEAE Sepharose column, glucosidase activities were eluted in several peaks, which were combined respectively (Fig. 2A). Among these fractions, only fraction IV can convert Rb1 into a single product identified as Rd, while the other three converted it into multiple products— Rd, gypenoside XVII and F2 (Fig. 3). The results suggested that there must be other glucosidases in the crude enzyme which can hydrolyze the b-(1 ! 2)-glucosidic bond at the 3-C site of Rb1 and Rd. Therefore, fraction IV was selected to be further purified and other fractions were stored at 20 8C for later study. Fraction IV was purified by the combination of QSepharose, Toyopearl Butyl 650C and Sephacryl S-400 column. In these steps, the b-glucosidase was eluted as a major activity peak (Fig. 2B–D). Finally, the b-glucosidase was purified approximately 580-fold with a yield of 2.4% relative to the crude enzyme. The purified enzyme had a specific activity of 2484 nkat/mg protein. A summary of the purification result is shown in Table 1.

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Fig. 4. Electrophoresis of b-glucosidase. (a) SDS-PAGE. Lane 1, molecular weight marker; lane 2, purified enzyme. (b) Non-denatured PAGE. Lane 3, activity staining; lane 4, Coomassie Blue staining.

The purified enzyme running on both the non-denatured and denatured PAGE produced a single band when stained with Coomassie Blue. Moreover, activity staining to the nondenatured gel also produced one single band which was coincident with the one stained with Coomassie Blue (Fig. 4). These results indicted that the b-glucosidase from white jade snail was purified to homogeneity by a four-step columnchromatographic procedure. Molecular mass of the bglucosidase, estimated by SDS-PAGE and gel filtration, was 115 and 239 kDa, respectively, which suggested that this enzyme is comprised of two identical subunits of about 115 kDa, which is similar to several other b-glucosidases isolated from microorganisms [22–24]. However, it was different from two b-D-glucosidases obtained from other snails (Achatina achatina and Helix pomatia), whose molecular masses were 41 and 300 kDa, respectively [25,26]. 3.2. Effects of temperature and pH on the activity and stability of b-glucosidase This b-glucosidase exhibited a sharp optimum at 70 8C while only 7% of its maximum activity was retained at 90 8C. Thermal stability tests showed that the enzyme retained its full activity between 30 and 60 8C after 24 h at pH 5.6 (Fig. 5). When preincubated at 70 8C, it was stable for 1 h (data not

Table 1 Purification of b-D-glucosidase from white jade snail Purification steps

Total protein (mg)

Total activity (nKat)

Specific activity (nkat/mg)

Yield (%)

Purification fold

Crude enzyme DEAE Sepharose CL-6B Q-Sepharose Fast Flow Toyopearl Butyl 650C Sephacryl S-400

2446 32.7 6.2 0.72 0.10

10485 833.3 628.5 321.7 248.4

4.29 25.5 101 447 2484

100 7.9 6.0 3.1 2.4

1.0 5.9 23.5 104 579

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Fig. 5. Effects of temperature on the enzyme activity (*) and stability ((*), incubation time: 24 h). The maximum activity observed at 70 8C and the original activity without preincubation were taken as 100%, respectively.

shown), and 40% of its initial activity was still retained after 24 h. The enzyme activity decreased significantly when preincubation temperature reached 80 8C, and only 14% activity was retained after 5 min. These results strongly proved that this b-glucosidase was very stable in a wide range of temperature from 30 8C up to at least 70 8C. Its thermo-stability was even better than those of three glucosidases from thermophilic organisms [24,27,28]. The b-glucosidase exhibited optimal activity within pH 4.8– 6.4. Outside this range, activity was rapidly lost. Optimum pH of the enzyme was 5.6 (Fig. 6). The effects of pH on stability of the enzyme are shown in Fig. 7. Preincubated at 4 and 30 8C for 24 h, the enzyme was stable within a wide pH range (2.4–11 and 3–11, respectively), but at pH 12 only 12 and 4% of its initial activities were retained (Fig. 7A and B). When preincubation temperature was increased to 50 8C, more than 90% of the original activities were retained within pH 4–9 for 2 h. Out of this range, activity decreased observably (Fig. 7C). Interestingly, in all three temperature conditions, there was always a pH value at which the activity measured was around 120% of the control (pH 3 at 4 8C; pH 4 at 30 and 50 8C). The reason is still unknown, but the enzyme is indeed of good pH stability. bGlucosidases with such properties are uncommon. A b-

Fig. 6. Effect of pH on the activity of b-glucosidase. Enzyme assay was conducted in different buffer systems of pH 3–12 at 50 8C, and the maximum activity observed at pH 5.6 was taken as 100%.

glucosidase from mushroom Termitomyces clypeatus [29] showed a similar pH stability range (2–10), but it was only preincubated for 1 h at 40 8C. Another enzyme from Mucor miehei YH-10 also exhibited this kind of property: it was stable within pH 2–10 at 4 8C for 24 h [30]. Other b-glucosidases such as the one from Thermomyces lanuginosus-SSBP (pH 5–12) [24] and the one from Apis mellifera (pH 3.5–9.5) [31] also displayed a relatively wide pH stability, but the preincubation time of the former was not given and the latter was preincubated for only 20 min. Furthermore, the b-glucosidase from H. pomatia [26] showed a narrow pH stability range (pH 4–7). The mechanisms of temperature stability and/or pH stability have been reported in several studies [32–34]. These mechanisms, although not identical, are all involved in structure and amino acid composition. Because the novel enzyme from white jade snail possesses both thermo- and pHstability, it may be one of the most suitable model proteins used to gain deeper insights into the protein stability at extreme conditions. Another interesting issue of this enzyme is its source—snail, a species which grows in the mild environment and is easier to grow. The reason that the snail produces the extreme enzyme worths further studies.

Fig. 7. Effects of pH on the stability of b-glucosidase at different temperatures. It was determined by detecting the residual activity after preincubating the bglucosidase for 24 h at 4 8C (A), 30 8C (B) or 2 h at 50 8C (C) in pH 2.4–8 sodium phosphate–citrate buffer (D), pH 7–9 Tris–HCl buffer (~), pH 8–11 glycine–NaOH buffer (*) and pH 11–12 sodium phosphate–NaOH buffer (*), respectively. The original b-glucosidase activity without preincubation was taken as 100%.

H. Luan et al. / Process Biochemistry 41 (2006) 1974–1980 Table 2 Relative activity of snail b-glucosidase on various substrates Substrate

Activity (%) a

Substrate

Activity (%)a

pNPG oNPG Cellobioseb Sophoroseb Gentiobioseb Lactoseb Sucroseb Trehaloseb

100 67.2 78.7 72.0 93.6 3.5 4.7 3.1

Methyl b-glucopyranosideb Hexyl b-glucopyranoside b Heptyl b-glucopyranoside b Decyl b-glucopyranoside b Dodecyl b-glucopyranoside b MUGb Ginsenoside Rb1 c Ginsenoside Rdc

4.9 46.7 53.2 60.4 40.7 38.3 6.9 0

a

Activity expressed relative to activity measured on pNPG (100%). Released glucose measured by the glucose oxidase-phenol 4-aminophenazone peroxidase system. c Released aglycon measured by HPLC. b

3.3. Substrate specificity and hydrolysis of ginsenosides The survey with various p-nitrophenyl glycosides indicated that this enzyme was quite specific to b-D-glucoside. Compared to its activity against pNPG (100%), the activities against other aryl-glycosides (as shown in Section 2) were less than 1%. Additionally, we also used various disaccharides, ginsenosides Rb1 and Rd, as well as different alkyl-glucosides as substrates to study the substrate specificity (Table 2). Among disaccharides tested, the enzyme showed high activity to b-glucosidic linkage and no activity to others, such as lactose (galactose-b-(1 ! 4)-glucose), sucrose (glucose-a, b-(1 ! 2)fructose) and trehalose (glucose-a, a-(1 ! 1)-glucose). To the substrates with b-glucosidic linkage, the order of hydrolysis abilities was gentiobiose (b-(1 ! 6)) > cellobiose (b(1 ! 4)) > sophorose (b-(1 ! 2)), but the differences among them were not more than 25%. Because ginsenoside Rb1 contains four b-glucosidic linkages including a 20-C, b-(1 ! 6) and a 3-C, b-(1 ! 2) linkage, the enzyme should theoretically hydrolyze Rb1 into other ginsenosides besides Rd. Therefore, a time course

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experiment was done. The result is shown in Fig. 8. When Rb1 was used as the substrate, only one product was detected and identified as Rd using standards and LC–MS (data not shown). The amount of Rd increased with the extension of the reaction time, while other hydrolysis products such as gypenoside XVII, ginsenoside F2, C–K as well as PPD were not detected after incubation at 50 8C for 24 h. Because glucose was a competitive inhibitor of this enzyme (data not shown), we at first supposed that the glucoses produced from the reaction could inhibit the enzyme activity to hydrolyze Rd further. Subsequently, Rd was used as a substrate to verify the supposition. Unexpectedly, the enzyme did not hydrolyze Rd after incubation at 50 8C for 48 h (Fig. 8), which suggested that the b-glucosidase from white jade snail can only hydrolyze b(1 ! 6)-glucosidic linkage of ginsenoside Rb1 and cannot hydrolyze b-(1 ! 2)-glucosidic linkage and other glucosidic bonds, although it can hydrolyze sophorose (b-(1 ! 2)glucosidic linkage). The reason of the conflicting results might be either that spatial conformation of Rd may block the attack of the enzyme to 3-C, b-(1!2)-glucosidic linkage, or that the affinity was decreased between the enzyme and Rd. The related studies are in progress. These results hinted that it was not credible to estimate hydrolysis ability of an enzyme to some natural substrates by using the results from some substitutes due to the effect of the aglycone, and it was necessary to use genuine substrates to validate. Additionally, when using different alkyl-glucosides as substrates, the hydrolysis ability was increased with the extension of C-chain except dodecyl b-glucopyranoside. This result suggested that the active center of the enzyme should be relatively hydrophobic and tryptophan and/or phenylalanine may participate in the formation of the binding site. 3.4. Kinetic parameters The Michaelis–Menten kinetic parameters, Km and Vmax, were 0.338 mM and 0.25 mmol/min/mg to pNPG and 0.276 mM and 0.0148 mmol/min/mg to ginsenoside Rb1 at pH 5.6 and 50 8C, respectively. The results indicated that the enzyme had a higher affinity with Rb1 than that with pNPG, while the reaction rate of catalyzing Rb1 was approximately 14fold lower in comparing Vmax/Km. In summary, we have successfully purified a b-glucosidase from China white jade snail. The enzyme is of good thermoand pH-stability and exhibits a high degree of substrate specificity. It can only hydrolyze b-(1 ! 6)-glucosidic linkage of ginsenoside Rb1 and convert Rb1 to Rd, and cannot hydrolyze b-(1 ! 2)-glucosidic linkage and other glucosidic bonds. With these characteristics, it can be potentially used in the biotransformation process of other glucosides. Acknowledgements

Fig. 8. HPLC analysis of product of ginsenosides Rb1 and Rd hydrolyzed by the b-glucosidase. The HPLC and reaction conditions were performed as described in Section 2. The peaks were identified using both standards and LC–MS. The arrows ‘a’ and ‘b’ correspond to ginsenosides Rb1 and Rd, respectively.

We thank Prof. Hongbin Xiao (Dalian Institute of Chemical Physics, China) for helping in LC–MS analysis of the hydrolysis products. We also thank Dr. Hideo Hasegawa for the gifts of ginsenosides standards. The work was funded by the

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