Purification and Characterization of a Ginsenoside Rb1-Hydrolyzing β-Glucosidase from Aspergillus niger KCCM 11239

Rb1-hydrolyzing β-glucosidase from Aspergillus niger KCCM 11239 was studied to develop a bioconversion process for minor ginsenosides. The specific activity of the purified enzyme was 46.5 times greater than that of the crude enzyme. The molecular weight of the native enzyme was estimated to be approximately 123 kDa. The optimal pH of the purified enzyme was pH 4.0, and the enzyme proved highly stable over a pH range of 5.0–10.0. The optimal temperature was 70 °C, and the enzyme became unstable at temperatures above 60 °C. The enzyme was inhibited by Cu2+, Mg2+, Co2+, and acetic acid (10 mM). In the specificity tests, the enzyme was found to be active against ginsenoside Rb1, but showed very low levels of activity against Rb2, Rc, Rd, Re, and Rg1. The enzyme hydrolyzed the 20-C,β-(1→6)-glucoside of ginsenoside Rb1 to generate ginsenoside Rd and Rg3, and hydrolyzed 3-C,β-(1→2)-glucoside to generate F2. The properties of the enzyme indicate that it could be a useful tool in biotransformation applications in the ginseng industry, as well as in the development of novel drug compounds.


Introduction
β-Glucosidases (β-D-glucoside glucohydrolase, EC 3.2.1.21)are a heterogeneous group of enzymes that are capable of cleaving the β-glucosidic linkages of aryl and alkyl β-glucosides, β-linked oligoglucosides, and several other oligosaccharides, with the release of glucose, generally in the β configuration [1,2].This enzyme is also commonly referred to as gentiobiase and cellobiase.The enzyme β-glucosidase may be considered to be ubiquitous, occurring widely throughout the bacteria, plant and animal kingdoms, although its functions differ broadly according to the type of organism in which it occurs [3].β-glucosidase performs important functions in a variety of enzymatic conversion processes, depending on the nature and diversity of the glycone or aglycone moiety of their substrates, since significant differences in the substrate specificity of β-glucosidases have been observed, even when they were derived from the same source [4][5][6][7].
The root of Panax ginseng C. A. Meyer (Araliaceae) is employed as a traditional medicine, and is used to treat a broad variety of ailments [8].The pharmacological effects of ginseng originate principally from ginsenosides (ginseng saponins).Thus far, more than 50 types of ginsenosides have been isolated and identified in the roots of Panax ginseng C. A. Meyer [9].The major ginsenosides, including ginsenoside Rb 1 , Rb 2 , and Rc, are in greater abundance in ginseng, whereas the minor ginsenosides, such as ginsenosides Rd, Rg 3 , Rh 2 , and compound K, are present at low levels in ginseng [10].Recently, several reports have shown that the minor ginsenosides have some remarkable pharmacological activities, and may constitute excellent candidates for use in future drug development.Therefore, many researchers have focused on the conversion of major ginsenosides to minor ginsenosides using acid or alkaline hydrolysis [11,12], heating [13], and enzymatic methods [14].Among these methods, biocatalytic approaches have become the predominant conversion modality, owing to their marked selectivity, mild reaction conditions, and environmental compatibility.
Aspergillus species are one of the most important microorganisms employed in biotechnological techniques for the production of extracellular enzymes.Among Aspergillus species, A. niger is considered rather attractive to the food industry, since A. niger has been recognized as GRAS (generally regarded as safe) by the FDA [15].In this study, an extracellular β-glucosidase, which specifically hydrolyzes ginsenoside Rb 1, was purified from A. niger KCCM 11239, and the properties of this purified β-glucosidase were characterized.Furthermore, other by-products after treatment with the enzyme were identified using high-performance liquid chromatography (HPLC).

Growth and β-Glucosidase Production
Changes in dry cell weight (DCW) and the β-glucosidase activity of A. niger KCCM 11239 on PDB medium at 30 °C were assessed under aerobic conditions.Figure 1 illustrates the relationship between β-glucosidase activity for the supernatant and cellular growth.As can be seen in Figure 1, the formation of extracellular β-glucosidase activity is growth-associated, ending when the stationary phase of growth is reached.A maximum β-glucosidase activity of cultivation medium was obtained after 16 days (specific activity, 9.9 U/mg).After that, the enzymatic activities were not changed during further culturing.Cultivation was, therefore, halted at 16 days.The culture supernatants were collected and employed as the source of enzyme purification.

Purification of β-Glucosidase
The β-glucosidase from the culture supernatant of A. niger KCCM 11239 was purified using ammonium sulfate precipitation, gel chromatography, and ion-exchange chromatography.The protein in the crude enzyme was precipitated with 30%-90% ammonium sulfate, and was subsequently separated via gel filtration chromatography on a Sephadex G-100 column.The results of β-glucosidase activity and protein from each fraction during the elution of the ammonium precipitate absorbed onto a Sephadex G-100 column are depicted in Figure 2. A fraction volume was 3 mL and an activity measurement revealed only a single peak on the column and major peaks at fraction numbers 8-10.The active fractions were pooled and purified further via anion-exchange chromatography on DEAE-Sephadex.The β-glucosidase activity was observed at fraction numbers 28-29 in 0.15-0.20 M NaCl.The results of β-glucosidase activity and the protein profile on DEAE-Sephadex column are shown in Figure 3.The β-glucosidase was purified by approximately 46.5-fold, with a total volume yield of 1.5% relative to the crude enzyme.The specific activity of the purified enzyme was measured at 460.5 U/mg of protein.The purification steps subsequent to the removal of cells and their results are summarized in Table 1.

Molecular Mass of the Purified Enzyme
The purified β-glucosidase appeared to be homogeneous according to the criteria of SDS-PAGE, as shown in Figure 4.The molecular weight of the purified β-glucosidase was estimated at approximately 123 kDa, based on calculations of its mobility using standard calibration proteins.The β-glucosidase from A. niger KCCM 11239 differs from those of other β-glucosidases generated by A. niger reported thus far, which were 46, 110, and 240 kDa [16][17][18].

Effects of pH and Temperature
The optimal pH of the purified β-glucosidase was studied at the range of pH 3.0-pH 10.0.The optimal pH of the purified β-glucosidase was 4.0.The activity of the enzyme was rapidly reduced at pH values above 7.0.The enzyme proved to be highly stable at pH values of 5.0-10.0 and retained more than 70% of original activity after the incubation of the enzyme for 8 h in buffer solution at pH 4.0 at room temperature (data not shown).
The effects of temperature on enzyme activity were evaluated at the optimal pH of 4.0.In our previous study, it appeared that the β-glucosidase activity was significantly affected at higher temperatures and for a longer time (data not shown).An incubation at above 60 °C for 2 h resulted in a 98% loss of activity.In enzymatic assay at various temperatures for 10 min, the purified enzyme showed maximum activity at a temperature of 70 °C, presuming that the enzyme treated with these conditions was denatured in the direction of increasing the activity.At above 70 °C, the enzymatic activity dramatically decreased.The other β-glucosidase derived from A. niger exhibited a similar optimum pH and temperature [17,18].

Effects of Metal Ions and Reagents
The effects of various metal ions and reagents on the purified enzyme activity are shown in Table 2.The enzyme activity was reduced to 79% ± 0.27% by 10 mM Cu 2+ , and slightly inhibited by Mg 2+ and Co 2+ .By way of contrast, Na + and Ca 2+ enhanced enzyme activity slightly at the same concentration.It has known that β-glucosidase from Cladosporium fulvum was inhibited strongly by Cu 2+ , and another enzyme from Melanocarpus sp.MTCC 3922 was also inhibited by Cu 2+ [10,19].In addition, it appeared that almost all reagents tested in this study did not inhibit enzyme activity except for acetic acid at 10 mM.On the other hand, an emulsifier, Triton X-100, activated the enzymatic activity although the mechanism has been clearly unknown yet.In particular, a chelating agent, EDTA, did not inhibit enzyme activity, thereby suggesting that metal ions are not essential for enzyme activation [20].a The concentration of all chemicals tested in this study was adjusted to10 mM; b The relative activity was determined via pre-incubation of the purified enzyme with each chemical at 25 °C for 30 min, followed by measurements of residual activity at 50 °C in the presence of the same metal ions and reagents with PNPG; c The activity against PNPG was considered 100%; d Results are presented as means ± standard deviations (n = 2, p < 0.05).
When ginsenoside Rb 1 was incubated, it was converted completely into Rd after 1 h.After 2 h, the ginsenoside Rd content decreased gradually, and the F 2 content increased gradually.After 12 h, both S-Rg 3 and R-Rg 3 began to be detected, and the content of total Rg 3 increased over time.This result indicated that the hydrolysis pathway of ginsenoside Rb 1 by β-glucosidase was Rb 1 →Rd→F 2 and Rb 1 →Rd→Rg 3 , purified enzyme hydrolyzed a β-(1→6)-glucosidic linkage at C-20, as well as a β-(1→2)-glucosidic linkage at C-3 (Figure 6).These results are similar to those of the pathway by which ginsenoside Rb 1 is converted to Rg 3 by Microbacterium sp.GS514 [9].Ginsenoside Rd has been shown to protect neurons against neurotoxic chemicals [21] and to prevent the contraction of blood vessels [22].F 2 is a potent inhibitor of acetylcholine-evoked secretion [23].Rg 3 induces a variety of pharmacological activities, such as tumor suppression [24], and hepatoprotective [25] and immune-enhancing effects [26].Therefore, the β-glucosidase obtained from A. niger KCCM 11239 is considered a good candidate for use in biotransformation applications for the ginseng industry, and for the development of new drugs.

Growth and Enzyme Production
A. niger KCCM 11239 was purchased from the Korean Culture Center of Microorganisms (KCCM, Seoul, Korea).The fungus was grown on potato dextrose agar at 30 °C for 4 days, and the stock culture was maintained at 4 °C.Erlenmeyer flasks were filled to 20% of their volume with potato broth, and were then inoculated with 5 day-old cultures.The cultures were grown under shaking conditions at 200 rpm for 16 days at 30 °C.During the shake flask culturing, a few glass beads were added to prevent mycelial clumping, and to achieve homogeneous growth.At the specified intervals, the dry weight and enzyme activity of the cells were evaluated.

Assay of Enzyme Activity
β-Glucosidase activity was evaluated via a colorimetric method using PNPG as a substrate.The reaction mixture, which contained 1 mL of 5 mM PNPG and 100 µL of enzyme solution, was incubated for 10 min at 50 °C.The reaction was subsequently terminated via the addition of 1 mL of 0.5 M sodium hydroxide, and the absorption of the released ρ-nitrophenyl (PNP) was measured at 400 nm using a standard calibration curve.One unit (U) of β-glucosidase activity was defined as the quantity of enzyme required to liberate 1 μmole of PNP min −1 under standard conditions [27].

Protein Measurement
Protein measurement was conducted with BCA reagent according to the method of Smith et al. [28], using bovine serum albumin as a standard protein.

Purification of β-Glucosidase
All steps of β-glucosidase purification were conducted at 4 °C.The crude enzyme from a 16 day-old culture was harvested via 30 min of centrifugation at 8000× g, after which the supernatant was precipitated by ammonium sulfate (70%) at 0 °C, and then dissolved in buffer A (20 mM sodium acetate buffer, pH 4.0).The solution was dialyzed exhaustively against the same buffer for 24 h.The dialyzed protein was concentrated and loaded on a Sephadex G-100 column (1.5 cm × 30 cm) equilibrated with buffer A and eluted with 120 mL of the same buffer at a flow rate of 1.0 mL/min.The active fractions were pooled to make a total volume of 44 mL, which was then dialyzed and concentrated prior to ion-exchange chromatography.The active fractions from gel filtration chromatography were applied to a DEAE Sephadex column (3 cm × 15 cm) equilibrated with buffer A. The enzyme was eluted with a linear gradient of 150 mL NaCl from 0 to 0.30 M in the same buffer.The fractions eluted by 0.15 M to 0.20 M NaCl exhibited enzyme activity.The active fractions were pooled, exhaustively dialyzed against buffer A, and were then subjected to molecular weight determinations.

Assay of Optimal pH and pH Stability
The optimal pH was assessed in a pH range of 2.0-10.0 using the following buffer systems: 100 mM sodium citrate buffer (pH 3.0), 100 mM sodium acetate buffer (pH 4.0-6.0), 100 mM sodium phosphate buffer (pH 7.0-8.0), 100 mM glycine-NaOH buffer (pH 9.0) and 100 mM sodium hydroxide buffer (pH 10.0).The pH stability of the purified enzyme solution (0.5 mg/mL) was measured according to the residual activity following 8 h of pre-incubation of the enzyme in the above buffers at 50 °C.

Assay of Optimal Temperature and Thermal Stability
The optimal temperature was determined by measuring the enzymatic activities with PNPG as a substrate at a range of temperatures from 20 °C to 80 °C in 20 mM sodium acetate buffer (pH 4.0).The thermal stability of the purified enzyme solution (0.5 mg/mL) was evaluated according to the residual activity after 2 h of pre-incubation of the enzyme in a temperature range from 20 °C to 80 °C.

Effects of Metal Ions and Reagents
The effects of metal ions and reagents on β-glucosidase activity were determined via the addition of 10 mM of various divalent (Fe 2+ , Cu 2+ , CO 2+ , Mg 2+ , Zn 2+ , Ca 2+ , and Mn 2+ ) and monovalent cations (K + , Na + ), as well as reagents (SDS, EDTA, Triton X-100, glycerol, acetic acid, ethanol, and methanol) to the reaction mixtures prior to 30 min incubation at 25 °C.The enzyme activities were subsequently measured in the presence of the metal ions or reagents under standard assay conditions [30].

Substrate (Ginsenosides) Specificity
Ginsenosides (Rb 1 , Rb 2 , Rc, Re, Rd, and Rg 1 ) were dissolved in distilled water to a concentration of 0.5 mg/mL as substrates.The mixture of ginsenosides (200 μL) and purified enzyme (200 μL, 460 U) was incubated for 30 min at 50 °C.The reaction was then halted via the addition of an equal volume of n-butanol.After centrifugation, the n-butanol phase was evaporated to dryness under vacuum and dissolved in methanol.The amount and pattern of hydrolyzed ginsenosides was detected via HPLC.The injection volume was 20 μL and a reverse phase column was used (C 18 , 4.6 × 150 mm, particle size 5 μm).All peaks were determined according to their absorbance at 203 nm.The mobile phase utilized gradient conditions with solvents A (acetonitrile:water = 100:0) and B (acetonitrile:water = 14:86).The solvent A and B ratios were as follows: [20% A (0 min)]; 20% A (5 min); 30% A (10 min); 30% A (15 min); 60% A (20 min); 60% A (23 min); and 0% A (25 min)], with a 1.2 mL/min flow rate.The amount of product was calculated according to its peak area [31].

Conclusions
A yield of extracellular β-glucosidase from A. niger KCCM 11239 was maximized when the stationary phase of growth was reached.Through several purification steps, the β-glucosidase was purified by approximately 46.5-fold (a specific activity of the enzyme: 460.5 U/mg of protein) and the molecular weight of β-glucosidase was estimated to be approximately 123 kDa.The enzyme activity was reduced to 79% ± 0.27% and 86.00% ± 4.25% by 10 mM of both Cu 2+ and acetic acid, respectively, but 10 mM of Triton X-100 increased the enzymatic activity as much as 10%.The enzyme had a maximum activity at 70 °C and a range of pH 9.0-10.0.The hydrolysis rate of ginsenoside Rb 1 to both S-Rg 3 and R-Rg 3 was very low.The enzymatic hydrolysis pathway of ginsenoside Rb 1 was Rb 1 →Rd→F 2 and Rb 1 →Rd→Rg 3, thus suggesting that the purified enzyme hydrolyzed both the β-(1→6)-glucosidic linkage at C-20 and the β-(1→2)-glucosidic linkage at C-3. Rg 3 has been known to have a potent bio-functional effect on human health.Therefore, β-glucosidase obtained from A. niger KCCM 11239 is considered to be worthy of application to the biotransformation technique for the development of new pharmaceutical medicine in the ginseng industry.

Figure 5 .
Figure 5. High-performance liquid chromatography (HPLC) analysis of the time-course product of ginsenoside Rb 1 hydrolyzed by the purified β-glucosidase.S means ginsenoside standards.

Table 1 .
Summary of purification of the β-glucosidase produced by Aspergillus niger KCCM 11239.

Table 2 .
The effects of metal ions and reagents on the activity of β-glucosidase from Aspergillus niger KCCM 11239.