Characterization of the ginsenoside-transforming recombinant β-glucosidase from Actinosynnema mirum and bioconversion of major ginsenosides into minor ginsenosides.

Cui, Chang-Hao,Kim, Sun-Chang,Im, Wan-Taek Springer International 2013 Applied microbiology and biotechnology Vol.97 No.2

<P>This study focused on the cloning, expression, and characterization of ginsenoside-transforming recombinant β-glucosidase from Actinosynnema mirum KACC 20028(T) in order to biotransform ginsenosides efficiently. The gene, termed as bglAm, encoding a β-glucosidase (BglAm) belonging to the glycoside hydrolase family 3 was cloned. bglAm consisted of 1,830 bp (609 amino acid residues) with a predicted molecular mass of 65,277 Da. This enzyme was overexpressed in Escherichia coli BL21(DE3) using a GST-fused pGEX 4T-1 vector system. The recombinant BglAm was purified with a GSTbind agarose resin and characterized. The optimum conditions of the recombinant BglAm were pH 7.0 and 37 C. BglAm could hydrolyze the outer and inner glucose moieties at the C3 and C20 of the protopanaxadiol-type ginsenosides (i.e., Rb(1) and Rd, gypenoside XVII) to produce protopanaxadiol via gypenoside LXXV, F(2), and Rh(2)(S) with various pathways. BglAm can effectively transform the ginsenoside Rb(1) to gypenoside XVII and Rd to F(2); the K (m) values of Rb(1) and Rd were 0.69??0.06 and 0.45??0.02 mM, respectively, and the V (max) values were 16.13??0.29 and 51.56??1.35 μmol min(-1) mg(-1) of protein, respectively. Furthermore, BglAm could convert the protopanaxatriol-type ginsenoside Re and Rg(1) into Rg(2)(S) and Rh(1)(S) hydrolyzing the attached glucose moiety at the C6 and C20 positions, respectively. These various ginsenoside-hydrolyzing pathways of BglAm may assist in producing the minor ginsenosides from abundant major ginsenosides.</P>

Enzymatic Biotransformation of Ginsenoside Rb1 and Gypenoside XVII into Ginsenosides Rd and F2 by Recombinant β-glucosidase from F lavobacterium johnsoniae

Hao Hong,Chang-Hao Cui,Jin-Kwang Kim,Feng-Xie Jin,Sun-Chang Kim,Wan-Taek Im 고려인삼학회 2012 Journal of Ginseng Research Vol.36 No.4

This study focused on the enzymatic biotransformation of the major ginsenoside Rb1 into Rd for the mass production of minor ginsenosides using a novel recombinant β-glucosidase from Flavobacterium johnsoniae. The gene (bglF3) consisting of 2,235 bp (744 amino acid residues) was cloned and the recombinant enzyme overexpressed in Escherichia coli BL21(DE3) was characterized. This enzyme could transform ginsenoside Rb1 and gypenoside XVII to the ginsenosides Rd and F2, respectively. The glutathione S-transferase (GST) fused BglF3 was purifi ed with GST-bind agarose resin and characterized. The kinetic parameters for β-glucosidase had apparent Km values of 0.91±0.02 and 2.84±0.05 mM and Vmax values of 5.75±0.12 and 0.71±0.01 μmol·min-1·mg of protein-1 against p-nitrophenyl-β-D-glucopyranoside and Rb1, respectively. At optimal conditions of pH 6.0 and 37°C, BglF3 could only hydrolyze the outer glucose moiety of ginsenoside Rb1 and gypenoside XVII at the C-20 position of aglycon into ginsenosides Rd and F2, respectively. These results indicate that the recombinant BglF3 could be useful for the mass production of ginsenosides Rd and F2 in the pharmaceutical or cosmetic industry.

Novel enzymatic elimination method for the chromatographic purification of ginsenoside Rb₃ in an isomeric mixture

Cui, Chang-Hao,Fu, Yaoyao,Jeon, Byeong-Min,Kim, Sun-Chang,Im, Wan-Taek The Korean Society of Ginseng 2020 Journal of Ginseng Research Vol.44 No.6

Background: The separation of isomeric compounds from a mixture is a recurring problem in chemistry and phytochemistry research. The purification of pharmacologically active ginsenoside Rb₃ from ginseng extracts is limited by the co-existence of its isomer Rb₂. The aim of the present study was to develop an enzymatic elimination-combined purification method to obtain pure Rb₃ from a mixture of isomers. Methods: To isolate Rb₃ from the isomeric mixture, a simple enzymatic selective elimination method was used. A ginsenoside-transforming glycoside hydrolase (Bgp2) was employed to selectively hydrolyze Rb₂ into ginsenoside Rd. Ginsenoside Rb₃ was then efficiently separated from the mixture using a traditional chromatographic method. Results: Chromatographic purification of Rb₃ was achieved using this novel enzymatic elimination-combined method, with 58.6-times higher yield and 13.1% less time than those of the traditional chromatographic method, with a lower minimum column length for purification. The novelty of this study was the use of a recombinant glycosidase for the selective elimination of the isomer. The isolated ginsenoside Rb₃ can be used in further pharmaceutical studies. Conclusions: Herein, we demonstrated a novel enzymatic elimination-combined purification method for the chromatographic purification of ginsenoside Rb₃. This method can also be applied to purify other isomeric glycoconjugates in mixtures.

Chemical constituents from the aerial parts of Melandrium firmum

Chang Hao Zhang,Gao Li,Jie Luo,Tian Li,Yong Cui,Mei Jin,Da Lei Yao,Ming Shan Zheng,Zhen-Hua Lin,Jiong Mo Cui 대한약학회 2015 Archives of Pharmacal Research Vol.38 No.10

Two new anthraquinones, melrubiellin C (1) andmelrubiellin D (2), were isolated from the aerial parts of Melandriumfirmum Rohrbach, together with eight known compounds(3–10). The structures of these compounds wereelucidated using 1Dand 2DNMR(COSY,HMQC,HMBCandNOESY) experiments. All isolated compounds were tested fortheir cytotoxicity against NCI-H460, Hep G2, MKN-28 andA-549 cells.Of these 10 compounds,1 and 2 exhibitedmoderatecytotoxicity with IC50 values ranging from 9.54 to 32.41 lM.

Identification and Characterization of a <i>Mucilaginibacter</i> sp. Strain QM49 β-Glucosidase and Its Use in the Production of the Pharmaceutically Active Minor Ginsenosides (<i>S</i>)-Rh₁ and (<i>S</i>)-Rg₂

Cui, Chang-Hao,Liu, Qing-Mei,Kim, Jin-Kwang,Sung, Bong-Hyun,Kim, Song-Gun,Kim, Sun-Chang,Im, Wan-Taek American Society for Microbiology 2013 Applied and environmental microbiology Vol.79 No.19

<P>Here, we isolated and characterized a new ginsenoside-transforming β-glucosidase (BglQM) from <I>Mucilaginibacter</I> sp. strain QM49 that shows biotransformation activity for various major ginsenosides. The gene responsible for this activity, <I>bglQM</I>, consists of 2,346 bp and is predicted to encode 781 amino acid residues. This enzyme has a molecular mass of 85.6 kDa. Sequence analysis of BglQM revealed that it could be classified into glycoside hydrolase family 3. The enzyme was overexpressed in <I>Escherichia coli</I> BL21(DE3) using a maltose binding protein (MBP)-fused pMAL-c2x vector system containing the tobacco etch virus (TEV) proteolytic cleavage site. Overexpressed recombinant BglQM could efficiently transform the protopanaxatriol-type ginsenosides Re and Rg<SUB>1</SUB> into (<I>S</I>)-Rg<SUB>2</SUB> and (<I>S</I>)-Rh<SUB>1</SUB>, respectively, by hydrolyzing one glucose moiety attached to the C-20 position at pH 8.0 and 30°C. The <I>K<SUB>m</SUB></I> values for <I>p</I>-nitrophenyl-β-<SMALL>d</SMALL>-glucopyranoside, Re, and Rg<SUB>1</SUB> were 37.0 ± 0.4 μM and 3.22 ± 0.15 and 1.48 ± 0.09 mM, respectively, and the <I>V</I><SUB>max</SUB> values were 33.4 ± 0.6 μmol min<SUP>−1</SUP> mg<SUP>−1</SUP> of protein and 19.2 ± 0.2 and 28.8 ± 0.27 nmol min<SUP>−1</SUP> mg<SUP>−1</SUP> of protein, respectively. A crude protopanaxatriol-type ginsenoside mixture (PPTGM) was treated with BglQM, followed by silica column purification, to produce (<I>S</I>)-Rh<SUB>1</SUB> and (<I>S</I>)-Rg<SUB>2</SUB> at chromatographic purities of 98% ± 0.5% and 97% ± 1.2%, respectively. This is the first report of gram-scale production of (<I>S</I>)-Rh<SUB>1</SUB> and (<I>S</I>)-Rg<SUB>2</SUB> from PPTGM using a novel ginsenoside-transforming β-glucosidase of glycoside hydrolase family 3.</P>

Enzymatic Biotransformation of Ginsenoside Rb1 and Gypenoside XVII into Ginsenosides Rd and F2 by Recombinant β-glucosidase from Flavobacterium johnsoniae

Hong, Hao,Cui, Chang-Hao,Kim, Jin-Kwang,Jin, Feng-Xie,Kim, Sun-Chang,Im, Wan-Taek The Korean Society of Ginseng 2012 Journal of Ginseng Research Vol.36 No.4

This study focused on the enzymatic biotransformation of the major ginsenoside Rb1 into Rd for the mass production of minor ginsenosides using a novel recombinant ${\beta}$-glucosidase from Flavobacterium johnsoniae. The gene (bglF3) consisting of 2,235 bp (744 amino acid residues) was cloned and the recombinant enzyme overexpressed in Escherichia coli BL21(DE3) was characterized. This enzyme could transform ginsenoside Rb1 and gypenoside XVII to the ginsenosides Rd and F2, respectively. The glutathione S-transferase (GST) fused BglF3 was purified with GST-bind agarose resin and characterized. The kinetic parameters for ${\beta}$-glucosidase had apparent $K_m$ values of $0.91{\pm}0.02$ and $2.84{\pm}0.05$ mM and $V_{max}$ values of $5.75{\pm}0.12$ and $0.71{\pm}0.01{\mu}mol{\cdot}min^{-1}{\cdot}mg$ of $protein^{-1}$ against p-nitrophenyl-${\beta}$-D-glucopyranoside and Rb1, respectively. At optimal conditions of pH 6.0 and $37^{\circ}C$, BglF3 could only hydrolyze the outer glucose moiety of ginsenoside Rb1 and gypenoside XVII at the C-20 position of aglycon into ginsenosides Rd and F2, respectively. These results indicate that the recombinant BglF3 could be useful for the mass production of ginsenosides Rd and F2 in the pharmaceutical or cosmetic industry.

The Effects of Monolayer Subculture on the Phenotype and Growth of Degenerated Human Intervertebral Disc Cells

( Ji Hao Cui ),( Ho Geun Chang ),( Yong Chan Kim ) 한국조직공학·재생의학회 2012 조직공학과 재생의학 Vol.9 No.2

This study investigated the changes in the growth and phenotype of human degenerated intervertebral disc cells depending on the frequency of subculture in vitro monolayer culture system. The annulus fibrosus (AF) and nucleus pulposus (NP) cells were cultured in a monolayer manner. At each subculture , we performed an analysis of the morphological changes, the adhesion rate, the proliferation rate and the viability. The expressions of type I, II collagen and proteoglycan were analyzed at the mRNA gene level. All three times of subculture were performed. Both the AF and NP cells gradually showed a fibroblast-like spindle shape from their irregular, polygonal shape while undergoing the third time of subculture. The cell proliferation was the highest at the second subculture time. The viability was markedly lower before the first subculture, but there was no significant difference of viability between the three times of subculture. On RT-PCR, the type II collagen expression was gradually decreased and almost unexpressed at the third subculture, but the type I collagen expression was gradually increased in the NP cells. In the AF cells, the type I collagen expression did not show a significant difference depending on the frequency of subculture. Type II collagen was not expressed from the second time of subculture. The expression of proteoglycan was gradually decreased in both the AF and NP cells and it was almost unexpressed at the third time of subculture. In conclusions, after three times of subculture, the disc cells had completely changed their original growth and phenotypic characteristics.

Characterization of Recombinant β-Glucosidase from Arthrobacter chlorophenolicus and Biotransformation of Ginsenosides Rb1, Rb2, Rc, and Rd

박명근,Chang-Hao Cui,Sung Chul Park,박슬기,김진광,Mi-Sun Jung,정석채,김선창,임완택 한국미생물학회 2014 The journal of microbiology Vol.52 No.5

The focus of this study was the cloning, expression, andcharacterization of recombinant ginsenoside hydrolyzingβ-glucosidase from Arthrobacter chlorophenolicus with anultimate objective to more efficiently bio-transform ginsenosides. The gene bglAch, consisting of 1,260 bp (419 aminoacid residues) was cloned and the recombinant enzyme, overexpressedin Escherichia coli BL21 (DE3), was characterized. The GST-fused BglAch was purified using GST·Bind agaroseresin and characterized. Under optimal conditions (pH 6.0and 37°C) BglAch hydrolyzed the outer glucose and arabinopyranosemoieties of ginsenosides Rb1 and Rb2 at the C20position of the aglycone into ginsenoside Rd. This was followedby hydrolysis into F2 of the outer glucose moiety ofginsenoside Rd at the C3 position of the aglycone. Additionally,BglAch more slowly transformed Rc to F2 via C-Mc1(compared to hydrolysis of Rb1 or Rb2). These results indicatethat the recombinant BglAch could be useful for theproduction of ginsenoside F2 for use in the pharmaceuticaland cosmetic industries.

Tumebacillus ginsengisoli sp. nov., isolated from soil of a ginseng field.

Baek, Sang-Hoon,Cui, Yingshun,Kim, Sun-Chang,Cui, Chang-Hao,Yin, Chengri,Lee, Sung-Taik,Im, Wan-Taek Society for General Microbiology 2011 International journal of systematic and evolutiona Vol.61 No.7

<P>A gram-reaction-positive, rod-shaped, spore-forming bacterium, designated Gsoil 1105(T), was isolated from soil of a ginseng field in Pocheon Province in South Korea and characterized in order to determine its taxonomic position. Comparative analysis of the 16S rRNA gene sequence showed that the isolate belongs to the order Bacillales, showing the highest level of sequence similarity with respect to Tumebacillus permanentifrigoris Eur1 9.5(T) (94.6???%). The phylogenetic distances from other described species with validly published names within the order Bacillales were greater than 9.0???%. Strain Gsoil 1105(T) had a genomic DNA G+C content of 55.6 mol% and menaquinone 7 (MK-7) as the major respiratory quinone. The major fatty acids were iso-C(15???:???0) and anteiso-C(15???:???0). On the basis of its phenotypic properties and phylogenetic distinctiveness, strain Gsoil 1105(T) represents a novel species of the genus Tumebacillus, for which the name Tumebacillus ginsengisoli sp. nov. is proposed. The type strain is Gsoil 1105(T) (???=???KCTC 13942(T) ???=???DSM 18389(T)).</P>

Gram-Scale Production of Ginsenoside F1 Using a Recombinant Bacterial β-Glucosidase

( Dong-shan An ),( Chang-hao Cui ),( Muhammad Zubair Siddiqi ),( Hong Shan Yu ),( Feng-xie Jin ),( Song-gun Kim ),( Wan-taek Im ) 한국미생물생명공학회(구 한국산업미생물학회) 2017 Journal of microbiology and biotechnology Vol.27 No.9

Naturally occurring ginsenoside F1 (20-O-β-D-glucopyranosyl-20(S)-protopanaxatriol) is rare. Here, we produced gram-scale quantities of ginsenoside F1 from a crude protopanaxatriol saponin mixture comprised mainly of Re and Rg1 through enzyme-mediated biotransformation using recombinant β-glucosidase (BgpA) cloned from a soil bacterium, Terrabacter ginsenosidimutans Gsoil 3082^T. In a systematic step-by-step process, the concentrations of substrate, enzyme, and NaCl were determined for maximal production of F1. At an optimized NaCl concentration of 200 mM, the protopanaxatriol saponin mixture (25 mg/ml) was incubated with recombinant BgpA (20 mg/ml) for 3 days in a 2.4 L reaction. Following octadecylsilyl silica gel column chromatography, 9.6 g of F1 was obtained from 60 g of substrate mixture at 95% purity, as assessed by chromatography. These results represent the first report of gram-scale F1 production via recombinant enzyme-mediated biotransformation.