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Purification and characterization of a ginsenoside Rb(1)-hydrolyzing β-glucosidase from Aspergillus niger KCCM 11239.

Chang KH, Jo MN, Kim KT, Paik HD - Int J Mol Sci (2012)

Bottom Line: In the specificity tests, the enzyme was found to be active against ginsenoside Rb(1), but showed very low levels of activity against Rb(2), Rc, Rd, Re, and Rg(1).The enzyme hydrolyzed the 20-C,β-(1→6)-glucoside of ginsenoside Rb(1) to generate ginsenoside Rd and Rg(3), and hydrolyzed 3-C,β-(1→2)-glucoside to generate F(2).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.

View Article: PubMed Central - PubMed

Affiliation: Division of Animal Life Science, Konkuk University, Seoul 143-701, Korea; E-Mails: khchang80@cj.net (K.H.C); ulul55@naver.com (M.N.J.).

ABSTRACT
Rb(1)-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 Cu(2+), Mg(2+), Co(2+), and acetic acid (10 mM). In the specificity tests, the enzyme was found to be active against ginsenoside Rb(1), but showed very low levels of activity against Rb(2), Rc, Rd, Re, and Rg(1). The enzyme hydrolyzed the 20-C,β-(1→6)-glucoside of ginsenoside Rb(1) to generate ginsenoside Rd and Rg(3), and hydrolyzed 3-C,β-(1→2)-glucoside to generate F(2). 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.

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Related in: MedlinePlus

Chromatogram of the crude enzyme on a Sephadex G-100 column. The enzyme was eluted with 0.02 M sodium acetate buffer (pH 4.0). The volume of each fraction was 3 mL. ▲, β-glucosidase activity (U/mL); ■, Absorbance at 280 nm.
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f2-ijms-13-12140: Chromatogram of the crude enzyme on a Sephadex G-100 column. The enzyme was eluted with 0.02 M sodium acetate buffer (pH 4.0). The volume of each fraction was 3 mL. ▲, β-glucosidase activity (U/mL); ■, Absorbance at 280 nm.

Mentions: 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.


Purification and characterization of a ginsenoside Rb(1)-hydrolyzing β-glucosidase from Aspergillus niger KCCM 11239.

Chang KH, Jo MN, Kim KT, Paik HD - Int J Mol Sci (2012)

Chromatogram of the crude enzyme on a Sephadex G-100 column. The enzyme was eluted with 0.02 M sodium acetate buffer (pH 4.0). The volume of each fraction was 3 mL. ▲, β-glucosidase activity (U/mL); ■, Absorbance at 280 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3472798&req=5

f2-ijms-13-12140: Chromatogram of the crude enzyme on a Sephadex G-100 column. The enzyme was eluted with 0.02 M sodium acetate buffer (pH 4.0). The volume of each fraction was 3 mL. ▲, β-glucosidase activity (U/mL); ■, Absorbance at 280 nm.
Mentions: 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.

Bottom Line: In the specificity tests, the enzyme was found to be active against ginsenoside Rb(1), but showed very low levels of activity against Rb(2), Rc, Rd, Re, and Rg(1).The enzyme hydrolyzed the 20-C,β-(1→6)-glucoside of ginsenoside Rb(1) to generate ginsenoside Rd and Rg(3), and hydrolyzed 3-C,β-(1→2)-glucoside to generate F(2).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.

View Article: PubMed Central - PubMed

Affiliation: Division of Animal Life Science, Konkuk University, Seoul 143-701, Korea; E-Mails: khchang80@cj.net (K.H.C); ulul55@naver.com (M.N.J.).

ABSTRACT
Rb(1)-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 Cu(2+), Mg(2+), Co(2+), and acetic acid (10 mM). In the specificity tests, the enzyme was found to be active against ginsenoside Rb(1), but showed very low levels of activity against Rb(2), Rc, Rd, Re, and Rg(1). The enzyme hydrolyzed the 20-C,β-(1→6)-glucoside of ginsenoside Rb(1) to generate ginsenoside Rd and Rg(3), and hydrolyzed 3-C,β-(1→2)-glucoside to generate F(2). 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.

Show MeSH
Related in: MedlinePlus