Process Biochemistry 45 (2010) 897–903
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A highly selective ginsenoside Rb1 -hydrolyzing -d-glucosidase from Cladosporium fulvum Juan Gao a,1 , Xuesong Zhao a,b,1 , Haibo Liu a , Yuying Fan a , Hairong Cheng a , Fei Liang a , Xingxing Chen a , Nan Wang a , Yifa Zhou a,∗ , Guihua Tai a,∗ a b
Laboratory of Molecular Epigenetics of MOE, School of Life Sciences, Northeast Normal University, 5268 Renmin Street, Changchun 130024, Jilin Province, PR China College of Resource and Environment Engineering, Liaoning Technical University, Fuxin 123000, PR China
a r t i c l e
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Article history: Received 10 July 2009 Received in revised form 28 December 2009 Accepted 11 February 2010 Keywords: -Glucosidase Cladosporium fulvum (syn. Fulvia fulva) Ginsenoside Rb1 Ginsenoside Rd Biotransformation Selectivity
a b s t r a c t G-I, a highly selective -glucosidase, was purified from phytopathogenic fungus Cladosporium fulvum (syn. Fulvia fulva). G-I was a monomer with native molecular weight of 85 kDa and pI value of 4.2. The maximal activity to p-nitrophenyl--d-glucopyranoside (pNPG) occurred at pH 6.0 and 45 ◦ C at which the Km against pNPG was 0.18 mM and Vmax was 46.7 mol nitrophenol/min/mg. G-I was highly stable within pH 4.0–11.0 and below 40 ◦ C. It was inhibited by Co2+ , Cu2+ and Zn2+ (50 mM), but showed resistance to sodium dodecyl sulfonate (SDS, 250 mM). G-I was highly active against -linked disaccharide cellobiose, gentiobiose and sophorose, but exhibited very low activities against other aryl-glycosides, methyl-␣glycosides and disaccharides trehalose and sucrose. Moreover, G-I specifically hydrolyzed -(1 → 6)glucosidic linkage at the C-20 site of ginsenoside Rb1 to produce ginsenoside Rd, without attack on other -d-glucosidic linkages. The oligopeptide fragments of G-I were sequenced by nanoESI-MS/MS and showed similarity to the sequences from the glycoside hydrolase family 3. G-I is different to G-II (a glycosidase previously purified from the same fungus) in composition and molecular weight. It shows more stable and higher selectivity than G-II. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Panax ginseng C.A. Meyer (ginseng) is a well-known plant medicine in the world [1]. Ginsenosides are the major active components of ginseng [2]. Rd, a protopanaxadiol (PPD) type ginsenoside, has relative low content in ginseng (about 5%). However, the experimental results showed that it was more active than another protopanaxadiol ginsenoside Rb1 which has high content in ginseng (more than 20%) [3]. Rd can prevent kidney injury by chemical drugs [4], enhance the differentiation of neural stem cells [5] and prevent the contraction of blood vessels [6]. However, it is difficult to prepare Rd by isolation from ginseng because of their low content and by chemical synthesis from simple start materials because of their complex structure. A possible pathway for preparation of Rd is transformation of Rb1 . The major protopanaxadiol type ginsenoside Rb1 , has one more glucose residue than Rd (Fig. 1). Therefore, Rd can be prepared from Rb1 by removing one glucose residue using glycosidase. Several ginsenoside Rb1 -hydrolyzing -glucosidases have been iso-
∗ Corresponding authors. Tel.: +86 431 85098212; fax: +86 431 85098212. E-mail addresses:
[email protected] (Y. Zhou),
[email protected] (G. Tai). 1 These authors contributed equally to this paper. 1359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2010.02.016
lated from Achatina fulica, Aspergillus niger, Paecilomyces Bainier and Fusobacterium K-60 [7–10]. However, these glycosidases lack high selectivity, which limits the industrial application of those -glucosidases in large-scale preparation of minor ginsenosides. More selective ginsenoside Rb1 -hydrolyzing -glucosidases are needed to be screened from suitable resources such as fungi. In our previous study, it was found that a phytopathogenic fungus Cladosporium fulvum could covert Rb1 to Rd [11]. Subsequently, two novel -glucosidases (G-I and G-II) which can selectively convert ginsenoside Rb1 to terminate at Rd were isolated from the culture filtrate of C. fulvum. G-II was purified and characterized to be a novel member of glycoside hydrolase family 3 [12]. In this paper, G-I was purified and characterized. Similar to G-II, G-I selectively converts Rb1 into Rd as a sole product. Both G-I and G-II would be useful in industrial application. 2. Materials and methods 2.1. Materials The substrates p-nitrophenyl--d-glucopyranoside (pNPG), p-nitrophenyl␣-d-glucopyranoside, p-nitrophenyl--d-mannopyranoside, p-nitrophenyl--dgalactopyranoside, p-nitrophenyl-␣-d-galactopyranoside, p-nitrophenyl-␣-dmannopyranoside, methyl-␣-d-glucopyranoside, methyl-␣-d-mannopyranoside, sophorose, d-(+)-trehalose and 4-methylumbelliferyl -d-glucopyranoside (MUG) were Sigma products (St. Louis, MO, USA). -Gentiobiose and d-(+)-cellobiose were
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Fig. 1. Structures of related protopanaxadiol type ginsenosides.
from Fluka AD (Switzerland). Ginsenoside Rb1 , Rb2 , Rc and Rd were purchased from Chengdu Mansite Biotechnology Co. Ltd., China. Molecular weight marker and pI Marker were from Bio-Rad Laboratory (Hercules, CA, USA) and Amersham Pharmacia (Uppsala, Sweden), respectively. Mono Q HR 5/5 column, Superose 6 10/300 GL column and phenyl sepharose CL-4B gels were from Amersham Pharmacia. DEAE–cellulose matrices and sepharose CL-6B gels were the products of Shanghai Hengxin Co. Ltd., China and Beijing Dingguo Biotechnology Co. Ltd., China, respectively. Shim-pack PREP-ODS (H) column and the HPLC system containing two LC-10ATvp pumps and a SPD-10Avp detector were from Shimadzu, Japan. All other reagents were of analytical or HPLC grade. 2.2. Microorganism and culture conditions The tomato pathogen C. fulvum was isolated from tomato locally, identified and granted by Jilin Academy of Agricultural Sciences. The strain was maintained on V8 juice agar medium. The spores were incubated in V8 juice liquid medium (per liter: 200 ml of V8 juice, 2 g of CaCO3 ) at 28 ◦ C with shaking (130 rpm) for 84 h as Zhao et al. described [12]. The culture broth was filtered through absorbent gauze and centrifuged at 10,000 × g for 20 min. The supernatant was used as crude enzyme preparation. 2.3. Purification of ˇ-glucosidase G-I from C. fulvum Unless otherwise specified, the purification steps were carried out at 20 ◦ C. The purification was monitored by measuring the absorbance at 280 nm and the -glucosidase activity of each fraction. 120 ml (bed volume) of DEAE–cellulose were added to approximate 6 l of crude enzyme preparation. The gel slurry was rotated overnight at 4 ◦ C and subsequently applied onto an empty column (2.8 cm × 25 cm). The unbound materials flowed through the column during packing column. The bound materials were eluted stepwise with 240 ml of 0, 0.25, 0.50, 0.75 and 1.0 M NaCl in buffer A (25 mM acetate buffer, pH 5.0) at a flow rate of 3 ml/min. The activity fractions eluted by 0.25 M NaCl were combined and referred to as G-I. G-I was further purified in this paper as described below. It was precipitated by ammonium sulfate (30–80%) at 0 ◦ C, then dissolved in buffer A and applied onto sepharose CL-6B column (3.0 cm × 90 cm). The column was equilibrated with 0.15 M NaCl in buffer A at 0.5 ml/min. The fractions with high enzyme activity (corresponding to 390–510 ml) were combined, adjusted to 1.0 M (NH4 )2 SO4 and loaded onto a phenyl sepharose CL-4B column (1.5 cm × 10.2 cm) pre-equilibrated with buffer B (buffer A containing 1.0 M (NH4 )2 SO4 and 0.15 M NaCl). The column was eluted first with 36 ml buffer B, then with a 180-ml linear gradient from 100% buffer B to 100% buffer A at a flow rate of 1.0 ml/min. The fractions corresponding to 126–174 ml were combined, dialyzed against buffer C (20 mM Tris–HCl buffer, pH 7.5) and loaded onto a Mono Q HR 5/5 column connected to Shimadzu HPLC system. The column was eluted at 1 ml/min with 0.1 M NaCl in buffer C for 15 min followed by a linear gradient of 0.1–0.3 M NaCl in buffer C for 46 min. The fractions collected between 24 and 30 min were combined and dialyzed against buffer A. After concentration, the sample was applied to a superose 6 10/300 GL column connected to Shimadzu HPLC system and eluted at 0.3 ml/min with 0.15 M NaCl in buffer A for 80 min. The fractions with enzyme activity (corresponding to 52–56 min) were recovered for determination of purity and properties. 2.4. Enzyme assays -Glucosidase activity was measured with pNPG as substrate [13]. The reaction mixture containing 10 mM pNPG, 25 mM acetate buffer (pH 6.0) and proper enzyme
solution was incubated at 37 ◦ C for 30 min. Then the reaction was terminated by adding 0.25 M NaOH and the absorbance was read at 405 nm. All other pnitrophenyl glucosides were measured under the same conditions. Activities against methyl-␣-glycosides, carboxymethyl cellulose (CMC) and non-reducing disaccharides trehalose and sucrose were assayed with dinitrosalicylic acid reagent at 520 nm for determining produced reducing sugars [14]. The activity for hydrolysis of reducing disaccharides including cellobiose, gentiobiose and sophorose was measured using HPLC [15]. Briefly, 10 mM disaccharides were incubated with proper enzyme solution in 25 mM acetate buffer (pH 6.0) at 37 ◦ C for 30 min. The reaction was stopped with 0.3 M NaOH, the products were derivatized with 1-phenyl-3-methyl5-pyrazolone (PMP) and then analyzed on Thermo ODS column (4.6 mm × 250 mm) connected to HPLC system, eluted with 81.6% PBS (0.1 M, pH 7.0) and 18.4% acetonitrile (v/v) at 1.0 ml/min, monitored by UV absorbance at 245 nm. The content of product was calculated according to peak area. The hydrolysis activities against ginsenosides were carried out by HPLC, too. The reaction mixture containing 10 mM ginsenoside, 25 mM acetate buffer (pH 6.0) and proper enzyme solution was incubated at 37 ◦ C for 30 min. The reaction was stopped by adding equal volume of n-butanol. The n-butanol phase was evaporated to dryness under vacuum and subsequently applied on HPLC. The Shim-pack PREP-ODS (H) column (4.6 mm × 250 mm, 5 m) was eluted at 1.0 ml/min with a linear gradient of acetonitrile–water from 35:65 (v/v) to 65:35 (v/v) within 30 min, monitored by the absorbance at 203 nm. The content of product was calculated according to peak area. One unit (U) of glucosidase activity was defined as the amount of enzyme liberating 1 nmol/min of p-nitrophenyl/reducing sugar/ginsenoside Rd under assay conditions.
2.5. Purity, molecular weight and isoelectric point determination The purity and molecular weight of G-I were estimated by both gel filtration chromatography and sodium dodecyl sulfonate polyacrylamide gel electrophoresis (SDS-PAGE). Gel filtration chromatography was carried out using a superose 6 10/300 GL column pre-calibrated with a molecular weight marker kit (MW-GF-1000 from Sigma) as described previously [12]. SDS-PAGE was performed on 8% resolving gel [16]. The gel was stained by 0.25% Coomassie Brilliant Blue G-250. The molecular weight was estimated using Protein Molecular Weight Marker ranging from 225 to 31 kDa. IEF-PAGE was performed on Bio-Rad Model 111 Mini IEF Cell apparatus. The gel was silver stained following the manufactory’s instruction (Bio-Rad). Isoelectric point was determined using standard pI markers.
2.6. Native PAGE analysis with MUG-zymogram analysis For detection of in-gel -glucosidase activity, the purified G-I was analyzed by native PAGE using 8% separation gel under non-reducing conditions. After electrophoresis, the gel was washed for twice with 25 mM NaAC buffer (pH 6.0, buffer A) for 10 min and then overlaid with 2 mM MUG in buffer A at room temperature for 15 min. The fluorescent band was then visualized under UV light and proteins were stained with Coomassie Brilliant Blue G-250.
2.7. Protein sequencing The purified G-I was first analyzed by SDS-PAGE using 8% separation gel. The gel was stained with Coomassie Brilliant Blue G-250 then the band of G-I was in-gel digested with trypsin and the peptides were sequenced using nanoESI-MS/MS (QTOF2, Micromass, UK) at National Center of Biomedical Analysis, Chinese Academy of Military Medical Sciences. The obtained peptide sequences were compared with known sequences by BLAST2 (http://www.ebi.ac.uk/Tools/blast2) and submitted to get accession number.
2.8. Effects of temperature and pH on enzyme activity and stability The optimal pH of G-I was determined by testing the -glucosidase activity at 37 ◦ C at various pH values (25 mM Na2 HPO4 –citrate buffer, pH 2.5–8.0). The optimal temperature was determined between 20 and 60 ◦ C at optimum pH. Thermal stability of G-I was examined by measuring the residual activity after the enzyme was incubated for 2 h at different temperatures (20–60 ◦ C). The pH stability was examined by measuring the residue activity after incubating G-I at 4 ◦ C and 24 h in different solutions (pH 2.0–12.0). Following buffers were used: 25 mM Na2 HPO4 –citrate buffer (pH 2.0–8.0), 25 mM glycine–NaOH buffer (pH 8.0–11.0) and 25 mM Na2 HPO4 –NaOH buffer (pH 11.0–12.0), the remaining activity was determined under standard enzyme assay conditions [7].
2.9. Effects of metal ions and reagents The purified enzyme was pre-incubated with 0.05 M metal ions or 0.25 M reagents in 25 mM acetate buffer (pH 6.0) at 20 ◦ C for 30 min. Enzyme activities were then measured in the presence of the metal ions or reagents under standard assay conditions [7].
J. Gao et al. / Process Biochemistry 45 (2010) 897–903 2.10. Kinetic measurements The kinetic parameters were determined by measuring the initial reaction velocity at different pNPG concentrations ranging from 0.017 to 0.25 mM at optimal pH and temperature. Values for Km and Vmax were determined from Lineweaver–Burk plots [17]. 2.11. Time course of the hydrolysis of ginsenoside Rb1 by G-I The purified G-I (2 g) was incubated with ginsenoside Rb1 (final concentration: 2 mM) in 25 mM NaAC buffer (pH 6.0) at 37 ◦ C. Aliquots were withdrawn at suitable time intervals (0, 2, 4, 8, 24 h) and the reaction was stopped by adding equal volume n-butanol. The n-butanol phase was evaporated to dryness under vacuum and subsequently detected by HPLC as mentioned above. The control without enzyme was incubated under the same conditions.
3. Results 3.1. Purification of ˇ-glucosidase G-I After DEAE–cellulose chromatography (Fig. 2A), two glucosidase activity fractions (G-I and G-II) were obtained from the
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culture filtrates of C. fulvum. G-II eluted with 0.5 M NaCl has been discussed in previous paper [12]. In present paper, G-I eluted with 0.25 M NaCl was purified. G-I was precipitated by 30–80% ammonium sulfate and subsequently applied onto a preparative sepharose CL-6B column. As shown in Fig. 2B, there was an asymmetric glucosidase activity peak which was not strictly corresponding to the protein peak. The active fractions were combined and further purified by hydrophobic chromatography on phenyl sepharose CL-4B, eluted with 0.45 M ammonium sulfate to successfully separate G-I from the majority of the contaminating proteins (Fig. 2C). The purification was further carried out using two HPLC columns—mono Q HR 5/5 and superose 6 10/300 GL column (Fig. 2D and E). After these steps, the purity of the collected enzyme was examined by high-performance gel filtration chromatography and SDS-PAGE. The enzyme displayed a single peak on superose 6 10/300 GL column (Fig. 3A) and a single band on SDS-PAGE (Fig. 3B), which indicated that the enzyme had been purified to homogeneity. The purification factors and yields in each purification step are summarized in Table 1. The specific activity of the purified enzyme was 17563 U/mg protein.
Fig. 2. Purification of -glucosidase G-I by chromatographies: (A) DEAE–cellulose; (B) sepharose CL-6B; (C) phenyl sepharose CL-4B; (D) mono Q HR 5/5; (E) superose 6 10/300 GL. () Represents -glucosidase activity; ( or —) represents absorption at 280 nm; (—) represents salt gradient. Solid bars represent the fractions pooled for further purification.
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Fig. 3. Determination of purity and molecular weight of G-I by high-performance gel filtration chromatography and SDS-PAGE. (A) Gel filtration chromatography on Superose 6 10/300 GL column: () represents -glucosidase activity; (—) represents absorption at 280 nm. (B) SDS-PAGE on 8% resolving gel: lane 1, crude extract; lane 2, DEAE–cellulose; lane 3, ammonium sulfate precipitation; lane 4, sepharose CL-6B; lane 5, phenyl sepharose CL-4B; lane 6, mono Q HR 5/5; lane 7, purified G-I on superose 6 10/300; M, molecular weight markers.
3.2. Molecular weight, zymogram analysis and isoelectric point of G-I The molecular weight of G-I was estimated to be 85 kDa by both gel filtration chromatography (Fig. 3A) and SDS-PAGE (Fig. 3B), indicating that the enzyme was a monomer. With native PAGE and MUG-zymogram analysis (Fig. 4A), the single band stained by Coomassie Brilliant Blue G-250 was at the same location as the -glucosidase activity band. The IEF-PAGE analysis demonstrated that the -glucosidase G-I from C. fulvum was an acidic protein with pI value of 4.2 (Fig. 4B). 3.3. Amino acid sequence analysis Three peptide sequences of G-I (1: LVAHEENVR; 2: VGKDEGFAKAGGLSR; and 3: LPLEAGESGTATFNVR) were obtained after trypsin digestion and analysis with nanoESI-MS/MS. The peptide sequences were subjected to the UniProt Knowledgebase (European Bioinformatics Institute) to find homologues against all the protein sequences using the WU-BLAST2 network service. One peptide sequence of G-I (LPLEAGESGTATFNVR) showed high homology to some fungal -glucosidases or hypothetical proteins in glycoside hydrolase family 3 (Fig. 5). Two peptides from G-I (LVAHEENVR and VGKDEGFAKAGGLSR) showed no significant homology to known sequences. The protein sequence data reported in this paper will appear in the UniProt Knowledgebase under the accession number P85516. 3.4. Effects of pH and temperature The effects of pH and temperature on enzyme activity were studied with pNPG as substrate. G-I displayed maximum activity
Fig. 4. MUG-zymogram and IEF-PAGE analysis of G-I: (A) MUG-zymogram analysis of G-I by MUG staining: lane 1, BSA as negative control; 2: purified G-I; protein bands were stained with Coomassie Brilliant Blue G-250 (left) and -glucosidase activity band was stained with MUG (right). (B) IEF-PAGE analysis of G-I: 1: markers; 2: purified G-I.
at pH 6.0 and 45 ◦ C (Fig. 6). Thermal stability and pH stability of the enzyme at different pHs and temperatures were monitored and the enzyme were found to be fairly stable over a wide pH range of 4.0–11.0 after pre-incubation at 4 ◦ C for 24 h and at temperatures
Table 1 Purification of G-I from C. fulvum. Purification steps
Proteina (mg)
Crude extract DEAE–cellulose 30–80% (NH4 )2 SO4 Sepharose CL-6B Phenyl sepharose CL-4B Mono Q Superose 6 HR 10/300
2050.40 311.20 29.00 8.29 3.29 0.91 0.47
a b
Total activity (Ub ) 90,170 32,038 23,781 10,618 10,375 9,758 8,255
Specific activity (U/mg)
Purification fold
Yield (%)
44 103 820 1281 3154 10723 17563
1 2 19 29 72 244 399
100 36 26 12 12 11 9
Protein contents were determined by Coomassie Brilliant Blue G-250 method using bovine serum albumin as standard. One unit (U) of -d-glucosidase was defined as the amount of enzyme liberating 1 nmol of p-nitrophenyl/min under standard assay conditions.
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Fig. 5. Multiple sequence alignment of peptide sequences of G-I: the peptide sequences of G-I are aligned with those of Chaetomium globosum, Magnaporthe grisea, Trichoderma reesei and Trichoderma viride. The running total number of amino acids is shown on the right. “*” Means that the residues are identical in all sequences in the alignment. “:” Means that conserved substitutions are observed. “·” Means that semi-conserved substitutions are observed. Table 2 The effects of metal ions and reagents on the activity of G-I. Metal ions or reagents
Relative activity (%)a
None NaCl KCl CaCl2 BaCl2 MnCl2 MgSO4 CuCl2 ZnCl2 HgCl2 CoCl2 SDS EDTA
100 113 108 111 98 121 112 41 49 93 71 101 83
a b c
± ± ± ± ± ± ± ± ± ± ± ± ±
0.00b , c 7.50 0.57 0.14 3.89 5.02 1.41 0.00 1.06 0.34 0.06 3.89 6.86
Reaction conditions: pH 6.0, 30 min, 37 ◦ C, pNPG as substrate. The activity assayed in the absence of cations or reagents was taken as 100%. Results are presented as means ± standard deviations (n = 3).
below 40 ◦ C after incubation for 2 h. It was completely inactivated on incubation at pH 12 for 24 h and 50 ◦ C for 2 h (Fig. 6). 3.5. Effects of metal ions and reagents As shown in Table 2, the activity of G-I was inhibited by Co2+ , Cu2+ and Zn2+ , but slightly activated by Na+ , K+ , Ca2+ , Mn2+ and Mg2+ . Ba2+ and Hg2+ did not influence the activity significantly. SDS, a strong denaturant which inhibits the activity of most enzymes, did not inhibit the activity of G-I at 0.25 M concentration. EDTA, a chelating agent, only slightly inhibited the activity of G-I at 0.25 M concentration. 3.6. Determination of kinetic parameters Kinetic parameters of the purified G-I were determined from Lineweaver–Burk plots with pNPG as substrate. The Km and Vmax
Fig. 7. HPLC analysis of the hydrolysis products of ginsenosides by G-I: The reaction mixture (800 l) containing 2.0 mM of ginsenoside, 25 mM acetate buffer (pH 6.0) and 2 g G-I was incubated at 37 ◦ C for up to 24 h. Aliquots (100 l) were withdrawn at suitable intervals and subjected to HPLC. The control was incubated under the same conditions (results are presented as means ± standard deviations (n = 3)).
values were calculated to be 0.18 mM and 46.7 mol/min/mg, respectively. 3.7. Substrate specificity The enzyme showed considerable activity against ginsenoside Rb1 but no activity against ginsenoside Rb2 , Rc and Rd. When the activity of G-I against pNPG was defined as 100%, the activity against Rb1 was 22.10%. As seen in Fig. 7, after 2 h incubation with G-I, HPLC analysis indicated that over 40% of Rb1 was transformed to Rd; after 24 h, all Rb1 was converted. In contrast, ginsenoside Rb2 , Rc and
Fig. 6. Effect of pH (A) and temperature (B) on activity () and stability () of G-I: (A) the optimal pH of G-I was determined by ranging pH from 2.0 to 8.0 at 37 ◦ C. The maximum activity obtained was defined as 100%. The pH stability of G-I was determined by pre-incubating G-I in different pH at 4 ◦ C for 24 h and then determining the percentage of residual activity under standard assay conditions. The activity of G-I without pre-incubating was defined as 100%. (B) Thermal stability of G-I was assayed by pre-incubating G-I for 2 h at optimal pH (6.0) from 20 to 60 ◦ C and then the residual activities were determined under the standard assay conditions. The maximum activity observed and the original activity without pre-incubation was defined as 100%, respectively (results are presented as means ± standard deviations (n = 3)).
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Table 3 Relative activity of G-I.a . Substrate
Relative activity (%)
p-Nitrophenyl--d-glucopyranoside p-Nitrophenyl--d-galactopyranoside p-Nitrophenyl--d-mannopyranoside p-Nitrophenyl-␣-d-glucopyranoside p-Nitrophenyl-␣-d-galactopyranoside p-Nitrophenyl-␣-d-mannopyranoside Methyl-␣-d-glucopyranoside Methyl-␣-d-mannopyranoside Ginsenoside Rb1 Ginsenoside Rb2 Ginsenoside Rc Ginsenoside Rd Cellobiose Gentiobiose Sophorose Sucrose Trehalose CMC
100 2.10 0.34 0.44 0.13 0.63 2.0 2.9 22.10 0 0 0 100.0 132.1 98.4 4.3 3.9 0.24
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.00b 0.52 0.48 0.32 0.09 0.20 0.46 1.08 0.51 0.00 0.00 0.00 0.00 3.86 0.89 0.53 0.73 0.00
a Absorption caused by released p-nitrophenyl or reducing sugars (DNS method) was measured at 405 or 520 nm. The hydrolysis of cellobiose, gentiobiose and sophorose were determined by HPLC. The relative activities against pNPG or cellobiose were taken as controls (100%), respectively. The hydrolysis product of ginsenosides was measured by HPLC. b Results are presented as means ± standard deviations (n = 3).
Rd were not converted after 24 h incubation. The transformation product of Rb1 was purified by HPLC and then characterized by 13 C NMR spectroscopy (data not shown). All signals in the 13 C NMR spectrum were identical with those of Rd reported in literatures [12,18–20]. When using aryl-glycosides as substrates, G-I exhibited high specificity for -d-glucoside compared to other p-nitrophenyl glycosides and methyl glycosides (Table 3). If the activity against pNPG was defined as 100%, the activities were less than 3% against other p-nitrophenyl--glycosides, p-nitrophenyl-␣-glycosides and methyl-␣-glycosides. G-I showed high activities against gentiobiose (glucose--(1 → 6)-glucose), sophorose (glucose--(1 → 2)glucose) and cellobiose (glucose--(1 → 4)-glucose) as well. The relative activities against gentiobiose and sophorose were 132.1 and 98.4%, respectively, when the activity against cellobiose was defined as 100%. To ␣-linked disaccharides sucrose (glucose-␣, (1 → 2)-fructose) and trehalose (glucose-␣, ␣-(1 → 1)-glucose), G-I showed less than 5% of relative activities. In addition, G-I had little activity to carboxymethyl cellulose. 4. Discussion It is well known that fungi are good resources of -glucosidase. Many -glucosidases from fungi have been isolated and purified. In our previous study, a novel ginsenoside Rb1 -hydrolyzing -d-glucosidase G-II from C. fulvum was purified and characterized [12]. In this study, another ginsenoside Rb1 -hydrolyzing -d-glucosidase G-I secreted by C. fulvum was successfully purified to homogeneity. G-I is a monomer with a molecular weight of about 85 kDa, which is similar with -glucosidase from Trichoderma reesei, Sclerotinia sclerotiorum and Aspergillus oryzae [21–23], but different from G-II. G-II consists of two identical subunits and its molecular weight is about 180 kDa. Most -glucosidases from fungi possessed acidic isoelectric point. The pI value of G-I was 4.2, which was consistent with most fungi -glucosidases, such as G-II (pI 4.4) [12], -glucosidase from Clavibacter michiganense (pI 4.6) [24] and -glucosidase from T. reesei (pI 4.8) [21]. H+ and metal ions usually cause the changes of enzyme activities. G-I was stable within pH range 4.0–11.0, which is similar to G-II. G-II was stable within pH range 5.0–11.0. Like G-II, G-I was
inhibited by Cu2+ and Zn2+ ions, but G-I was more stable with ions compared with G-II. Substrate specificity of glycosidases is a very important factor for the industrial application of biotransformation. Similar to G-II, G-I had high specificity to p-nitrophenyl -d-glucosides compared to other p-nitrophenyl glycosides. However, when using disaccharides as substrate, the relative activity against gentiobiose (-(1 → 6)-glucosidic linkage) of G-I was 132.1%, which is higher than that of G-II (97.3%) [12], which indicated that G-I was more specific to -(1 → 6)-glucosidic linkage than G-II. Therefore, G-I might have more potential in industrial preparation of minor ginsenoside Rd than G-II. Although G-I showed no activity on -(1 → 2)-glucosidic linkage in C-3 site of ginsenoside Rb1 and Rd, it exhibited high activtiy against sophorose (-(1 → 2)-glucosidic linkage). These results were observed in other two ginsenoside Rb1 -hydrolyzing -d-glucosidases [7,12]. The reason for this might be that the spatial conformation of ginsenoside molecule blocked the attack of enzymes to -(1 → 2)-glucosidic linkage. G-I and G-II were purified from the same fungus, they both belonged to glycoside hydrolase family 3, and showed similar catalytical properties against ginsenosides. There are also some differences between them especially in their stability and selectivity. G-I was more stable compared to G-II. G-I has a broader pH stable range from pH 4.0–11.0. Stabilities of pH and temperature are especially important in industrial application. The good pH stability of G-I makes this enzyme more potential in ginsenoside Rd production. Moreover, G-I showed better stability in the presence of metal ions. The activity of G-I was not affected by most ions, some ions even slightly improve the activity of G-I. It is noticed that SDS, a strong denaturing reagent, did not inhibit the activity of G-I, which is quite special in known -glucosidases. Besides, G-I is more specific against -1, 6-glucosidic linkage in disaccharides compared to G-II. Good specificity is also an important factor in enzymatic engineering. Its substrate specificity would be useful in the biotransformation process in other glucosides. Based on above advantages, we think G-I more potential in ginsenoside Rd production. G-I would be further studied to lay a foundation for the application in Rd preparation. In conclusion, we successfully purified another novel ginsenoside Rb1 -hydrolyzing -d-glucosidase G-I from phytopathogenic fungus C. fulvum by a six-step procedure. G-I can transform ginsenoside Rb1 into Rd with high selectivity and yield. The amino acid sequence homology analysis showed that G-I may be a novel member of glycoside hydrolase family 3. G-I would be potential in industrial preparation of Rd. Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 30770489 and 30973857), the Natural Science Foundation of Jilin Province (Nos. 20070710 and 200905106). References [1] Attele AS, Wu JA, Yuan CS. Ginseng pharmacology: multiple constituents and multiple actions. Biochem Pharmacol 1999;58:1685–93. [2] Park JD, Rhee DK, Lee YH. Biological activities and chemistry of saponins from Panax ginseng C.A. Meyer. Phytochem Rev 2005;4:159–75. [3] Ji QC, Harkey MR, Henderson GL, Gershwin ME, Stern JS, Hackman RM. Quantitative determination of ginsenosides by high-performance liquid chromatography–tandem mass spectrometry. Phytochem Anal 2001;12:320–6. [4] Lee JK, Choi SS, Lee HK, Han KJ, Han EJ, Suh HW. Effects of ginsenoside Rd and decursinol on the neurotoxic responses induced by kainic acid in mice. Planta Med 2003;69:230–4. [5] Shi Q, Hao Q, Bouissac J, Lu Y, Tian S, Luu B. Ginsenoside-Rd from panax notoginseng enhances astrocyte differentiation from neural stem cells. Life Sci 2005;76:983–95.
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