Enhanced production of gypenoside LXXV using a novel ginsenoside-transforming β-glucosidase from ginseng-cultivating soil bacteria and its anti-cancer property

molecules

Article

Enhanced Production of Gypenoside LXXV Using a

Novel Ginsenoside-Transforming β-Glucosidase

from Ginseng-Cultivating Soil Bacteria and Its

Anti-Cancer Property

Chang-Hao Cui , Da Jung Kim , Suk-Chae Jung , Sun-Chang Kim ¹^,²^,³^,* and Wan-Taek Im ⁴^,*

1

2

1

Intelligent Synthetic Biology Center, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Korea;

seldoms@163.com (C.-H.C.); scjung@kaist.ac.kr (S.-C.J.)

Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 291 Daehak-ro,

2

Yuseong-gu, Daejeon 305-701, Korea; kimdj1404@kaist.ac.kr

KAIST Institute for Biocentury, Korea Advanced Institute of Science and Technology, 291 Daehak-ro,

3

Yuseong-gu, Daejeon 305-701, Korea

Department of Biological Sciences, Hankyong National University, 327 Chungang-no Anseong-si,

4

Kyonggi-do 456-749, Korea

*

Correspondence: sunkim@kaist.ac.kr (S.-C.K.); wandra@hknu.ac.kr (W.-T.I.); Tel.: +82-42-350-4450 (S.-C.K.);

82-31-670-5335 (W.-T.I.); Fax: +82-42-350-2619 (S.-C.K.); +82-31-670-5339 (W.-T.I.)

+

Academic Editor: Qing-Wen Zhang

Received: 14 March 2017; Accepted: 12 May 2017; Published: 19 May 2017

Abstract: Minor ginsenosides, such as compound K, Rg (S), which can be produced by deglycosylation

3

of ginsenosides Rb , showed strong anti-cancer effects. However, the anticancer effects of gypenoside

1

LXXV, which is one of the deglycosylated shapes of ginsenoside Rb , is still unknown due to the

1

rarity of its content in plants. Here, we cloned and characterized a novel ginsenoside-transforming

β-glucosidase (BglG167b) derived from Microbacterium sp. Gsoil 167 which can efﬁciently hydrolyze

gypenoside XVII into gypenoside LXXV, and applied it to the production of gypenoside LXXV at

the gram-scale with high speciﬁcity. In addition, the anti-cancer activity of gypenoside LXXV was

investigated against three cancer cell lines (HeLa, B16, and MDA-MB231) in vitro. Gypenoside

LXXV signiﬁcantly reduced cell viability, displaying an enhanced anti-cancer effect compared to

gypenoside XVII and Rb . Taken together, this enzymatic method would be useful in the preparation

1

of gypenoside LXXV for the functional food and pharmaceutical industries.

Keywords: gypenoside LXXV; gypenoside XVII; ginsenoside; deglycosylation; biotransformation;

Microbacterium sp. Gsoil 167

1

. Introduction

Ginseng (Panax ginseng C.A. Meyer) has been widely used to treat various diseases for thousands

of years in Asian countries and, nowadays, is used as a medicinal herb or functional food all

over the world [ ]. Ginsenosides are considered as the main active ingredients responsible for

pharmacological activities of the ginseng root, such as anti-cancer, anti-obesity, anti-inﬂammatory,

antioxidant, and hepatoprotective activities [ ]. The six major ginsenosides (Rb , Rb , Rc, Rd,

1,2

3–7

1

2

Re, and Rg ) constitute more than 80% of the total ginsenosides in ginseng root, and various minor

1

ginsenosides (Rg (S), Rh (S), F , compound K (C-K), Rg (S), Rh (S), F , protopanaxadiol (PPD) and

3

2

1

protopanaxatriol (PPT), and gypenoside XVII (GypXVII)) that are deglycosylated from the major

ginsenosides exist in smaller amounts in ginseng [ 11]. Major ginsenoside Rb is relatively abundant

in ginseng, and it can be deglycosylated into rare minor ginsenosides (F , Rg (S), Rh (S), CK, PPD)

8–

1

2

3

2

which have enhanced anti-cancer activities [12,13].

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GypXVII and gypenoside LXXV (GypLXXV), which have primarily been found in

Gynostemma pentaphyllum are also the deglycosylated products of major ginsenoside Rb₁[14]. However,

the pharmacological effects of GypXVII and GypLXXV is still unknown, because of their rare contents

in G. pentaphyllum or P. ginseng C.A. Meyer. Although various puriﬁcation methods were developed to

improve the yield of saponins from the raw plants, neither GypXVII nor GypLXXV have been puriﬁed

enough from raw plants for pharmacological study [15].

In this study, we isolated a novel ginsenoside-transforming β-glucosidase (BglG167b) from a

ginseng-cultivating soil bacteria. BglG167b was cloned and overexpressed in Escherichia coli and the

enzymatic properties and substrate speciﬁcities were investigated. This enzyme could hydrolyze

various ginsenosides (Rb , Rb , Rc, Rd, Rg (S), F , Re, Rg , GypXVII) through various cleavage

1

2

3

2

1

pathways, and has been applied for the production of GypLXXV from GypXVII at the gram-scale.

Eventually, the pure GypLXXV was used for the study of anti-cancer effects against three kinds of

cancer cells in vitro.

2

. Results and Discussion

2

.1. Isolation and Characterization of Strain Microbacterium sp. Gsoil 167

Strain Gsoil 167 which has been isolated from ginseng cultivating soil was conﬁrmed to hydrolyze

Rg , Re and Rb into PPT and PPD, respectively (Data not shown), and was selected for the screening

1

of ginsenoside-transforming enzymes. Phylogenetic analysis based on 16S rRNA gene sequences

indicated that the isolate belongs to the genus Microbacterium in the phylum Actinobacteria, and is most

T

closely related to Microbacterium luteolum IFO 15074 (99.9% similarity).

2

.2. Fosmid Library Construction and Cloning of BglG167b

Genomic DNA from Microbacterium sp. Gsoil 167 was isolated by phenol-chloroform extraction

and used to generate a fosmid library.

β-D-glucosidase activity was detected from a fosmid vector

containing an inserted contig (9.4 kb). Twenty-three (23) putative ORFs of longer than 300 codons have

been found using an ORF FINDER [16] analysis. Among them, one was homologous to glycoside

hydrolase genes in glycoside hydrolase family 3 (GH3). This ORF, termed BglG167b, consisting of

2412 bp encoding 803 amino acids, was ampliﬁed via polymerase chain reaction (PCR) and then

inserted into the pGEX 4T-1 vector.

2

.3. Phylogenetic Analysis of BglG167b Sequences

The -glucosidase (BglG167b) has a predicted molecular mass of 90.3 kDa and a theoretical

β

pI value of 4.56 [17]. Analysis of the amino acid sequences of BglG167b indicated that it

was 79% identical to the glycoside hydrolase of Microbacterium paraoxydans (GenBank number

WP_018187396), which belongs to GH3. The enzyme from M. paraoxydans has not yet been

characterized. The nearest characterized glycoside hydrolase in GH3 in the CAZy database was

β-D-glucosidase from Agrobacterium tumefaciens, which was 40% identical to BglG167b based on amino

acid sequences [18]. The phylogenetic analysis resulting consensus tree is presented in Figure S1.

BglG167b clustered within subfamily 5 and formed a separate, well-supported clade (bootstrap of 100)

with cellobiase of Cellulomonas biazotea, uncultured bacterium

bacterium β-glucosidase [19–21].

β-glucosidase and uncultured rumen

Various ginsenoside-hydrolyzing

including two -glucosidases (Bgp1 and Bgp3) from Microbacterium esteraromaticum, a

rApy-H11) from Biﬁdobacterium longum H-1, a -glucosidase (BGL1) from Aspergillus niger,

-glucosidase (BglAm) from Actinosynnema mirum, a -glucosidase (BgpA) from Terrabacter

-glucosidase (BglSk) from Sanguibacter keddieii [22 29]. The relationship

between BglG167b and these ginsenoside-hydrolyzing β-glucosidases is presented in Figure S1.

β-glucosidases in GH3 have been previously characterized,

β

β-glucosidase

(

β

a

β

ginsenosidimutans, and a

β

–

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2

.4. Puriﬁcation of Recombinant BglG167b

The GST-fused BglG167b protein was puriﬁed using GST-Bind agarose resin. The recombinant

enzyme was puriﬁed by GST-Bind agarose resin, and then the puriﬁed protein was determined

using SDS-PAGE analysis (Figure 1). The molecular mass of the GST-BglG167b calculated via an

amino acid sequence was 116 kDa, which is similar to the mass detected by SDS-PAGE. In addition,

the recombinant GST-BglG167b contains 36.5 ± 1.4% of total soluble proteins in the cell lysate.

Figure 1. SDS-PAGE analysis of recombinant BglG167b. Lane M, molecular weight standard; lane 1,

crude extract of BL21 (DE3) carrying pGEX-BglG167b without induction; lane 2, soluble fraction in

crude extract of BL21 (DE3) carrying pGEX-BglG167b after induction; lane 3, GST-BglG167b enzyme

fraction after puriﬁcation with the GST-bind agarose resin; lane 4, puriﬁed recombinant BglG167b after

cleavage by thrombin2.5. Characterization of recombinant BglG167b.

The optimum temperature and pH were characterized using puriﬁed GST-BglG167b. The optimal

◦

temperature activity was 45 C; at 30 C and 37 C, the enzyme has 75.0% and 89.8% of relative activity,

◦

while thermo-stability was decreased signiﬁcantly from 45 C, and not detected after incubation at

5 C for 2 h (Figure 2A). The enzyme was stable at temperatures lower than 30 C, and about 86.4%

of the activity was lost after incubation at 37 C for 2 h (Figure 2A). The optimal temperature for

the BglG167b activity was similar to the

◦

4

◦

β

-glucosidase isolated from T. ginsenosidimutans (45 C) [22].

BglG167b had optimal pH activity at pH 7.0 in a sodium phosphate buffer; BglG167b is stable from

pH 6.0 to 9.0; at pH 5.0 to 10.0, the enzyme stability decreased swiftly, while at pH 5.0 and 10.0 the

enzyme activity decreased to 1.2% and 40.0% (Figure 2B). The enzyme is probably mesophilic and

stable at a neutral pH range. These optimal conditions are consistent with the soil environment from

which Microbacterium sp. Gsoil 167 was isolated. In addition, the near-neutral optimal pH and mild

optimal temperature of BglG167b are similar to those of other ginsenoside-hydrolyzing GH3 from

◦

bacteria [22

,

23,

27,

29,

30]. Although the optimum temperature of BglG167b for pNPGlc is 45 C, the

◦

ginsenoside-conversion reaction occurred at 37 C for the extension of stable transformation activity.

Figure 2. Effects of pH (A) and temperature (B) on the stability and activity of recombinant BglG167b.

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The effects of metal ions, EDTA, DTT, and SDS on BglG167b activity were also studied (Table S1).

BglG167b activity was affected by 10 mM of -mercaptoethanol, which is a well-known thiol group

inhibitor. This suggested that sulfhydryl groups may be involved in the catalytic center of the enzyme.

β

+

2+

The enzyme was not affected by Na , K , Mn , and Mg . The chelating agent EDTA inhibits

BglG167b activity, which indicated that divalent cations may be required for enzymatic activity. The

2+

enzyme activity appeared to be strongly inhibited in the presence of Ca , SDS, Zn , Co , and Cu .

However, no dramatic positive effect on the activity of the BglG167b was found for the tested ions.

The substrate speciﬁcity of BglG167b was tested using 2.0 mM of p-nitrophenyl (pNP) and

o-nitrophenyl (oNP)-glycosides with

was active against glucose moiety of pNP-

It showed maximum activity towards pNP-

pNP-α-D-glucopyranoside and 22.9% relative activity towards oNP-β-D-glucopyranoside (Table S2).

α

and

β

conﬁgurations. The results showed that BglG167b

-D-glucopyranoside and oNP- -D-glucopyranoside.

-D-glucopyranoside, 8.1% relative activity towards

β

2

.5. Transformation Characteristics of BglG167b

For the veriﬁcation of the bioconversion pathways of the nine ginsenosides (Rb , Rb , Rc, Rd,

1

2

GypXVII, Rg (S), F , Re and Rg ) by BglG167b, the TLC analyses were performed at regular intervals.

3

2

1

GST-BglG167b could clearly transform nine ginsenosides, as shown by the R values of the TLC

f

analysis (Figure 3).

Figure 3. TLC analyses of time-course of ginsenoside bioconversion by BglG167b at an enzyme

concentration of 1.0 mg/mL. (

transformation of Rc; ( ) the transformation of Rd; (

transformation of Rg (S); ( ) the transformation of F ; (

A) the transformation of Rb ; (B) the transformation of Rb ; (C) the

1

2

D

E

) the transformation of gypenoside XVII; (

F

) the

G

H

) the transformation of Re; and (I

) the

3

2

transformation of ginsenoside Rg . Developing solvent: CHCl –CH OH–H O (70:30:10, v/v, lower

1

3

2

phase). Lane S, standards (Rb , Rd, F , C-K, PPT, PPD); Lane S1, standards (Rb , Rd, Rg (S), Rh (S),

1

2

1

3

2

PPD); Lane S2, standards (Rb , C-Y, PPD); Lane S3, standards (Rc, C-Mc, PPD); Lane S4, standards (Rb ,

2

1

GypXVII, GypLXXV, Rh (S), PPD); Lane S5, standards (Rg (S), Rh (S), PPD); Lane S6, standards (F ,

2

3

2

Rh (S), PPD); Lane S7, ginsenoside standards (Re, Rg (S), Rh (S), PPT); Lane S8, ginsenoside standards

2

1

(

Rg , Rh (S), PPT); Lane 1, control; Lane 2, 5 min; Lane 3, 30 min; Lane 4, 3 h; Lane 5, 10 h; Lane 6, 24 h;

1 1

Lane 7, 48 h.

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In PPD-type ginsenosides, Rb , Rd, GypXVII, Rb , and Rc have been converted by BglG167b.

1

2

For Rb biotransformation, three types of metabolites (Rd, Rg (S), PPD) were detected as time passed

1

3

(

Figure 4A). BglG167b successively hydrolyzed the outer and inner glucose moieties at position C20,

and the inner glucose at position C3. BglG167b transformed all GypXVII into GypLXXV in 5 min, and

continuously into C-K and PPD. The conversion speed of GypXVII was highest in all ginsenoside

transformations of Bgl167b and it is the ﬁrst enzyme in subfamily 5 which can hydrolyze GypXVII

into GypLXXV. For F transformation, BglG167b produced C-K from F , which means BglG167b

2

prefers to hydrolyze glucose moiety at the C3 position than C20 when each position has one glucose

moiety. According to the results of pNP and oNP substrates reactions, BglG167b could not hydrolyze

arabinopyranoside and arabinofuranoside attached at the outer position of C20, but could hydrolyze

two glucose moieties at C20. In PPT-type ginsenosides hydrolyzation, Re and Rg were transformed

1

into Rg (S) and Rh (S), respectively, which means BglG167b cannot hydrolyze the attached sugars at

2

1

C6, but glucose moieties at C3.

Figure 4. Transformation pathways of ginsenosides Rb , Rb , Rc, Rd, GypXVII, Rg (S), F , GypLXXV,

1

2

3

2

Re, and Rg by recombinant BglG167b, respectively.

1

Summarizing the transformation pathways, the proposed biotransformation pathways by

BglG167b for the ginsenosides are as follows: Rb₁Rd Rg (S) PPD; Rd Rg (S) PPD;

GypXVII GypLXXV C-K PPD; F₂C-K PPD; Rb₂C-Y; Rc C-Mc; Re Rg (S); Rg

Rh (S) via the stepwise hydrolysis of glucose moieties at the C20, and C3 positions of aglycon

→

3

→

2

1

→

1

(

Figure 4).

The metabolic pathways of ginsenoside Rb , Rb , and Rc hydrolysis by BglG167b are similar

1

2

to

β-glucosidases from M. esteraromaticum and Flavobacterium johnsoniae, which is attributed to their

Molecules 2017, 22, 844

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high amino acid sequence similarity in subfamily 5, and they can provide Rb₁

→

Rg (S) conversions

3

via hydrolysis of the outer and inner glucose at the C20 position of Rb (Figure S1) [26

, ,31]. The

27

-glucosidases from T. ginsenosidimutans,

,25]. However, they simultaneously transform GypLXXV into C-K or

1

transformation of GypXVII into GypLXXV has been found by

A. mirum, and S. keddieii [22 23

β

,

PPD. On the other hand, the higher conversion ability of BglG167b makes it more efﬁcient for the

production of GypLXXV.

Previously, GypLXXV has been produced using microbial and enzymatic transformation, but the

low concentration (less than 2.0 mg/mL), long transformation time (60 h), and further deglycosylation

limited its application to the production of GypLXXV [22,32]. GypXVII has been produced at a

gram-scale from major ginsenoside Rb using BgpA [20], and used for the substrate for the production

1

of GypLXXV. BglG167b could efﬁciently transform GypXVII into GypLXXV at a higher concentration

(5.0 mg/mL) and in a shorter period (9 h).

2

.6. Scale-Up Production of GypLXXV and Puriﬁcation

The bacterial cells that harbor pGEX-BglG167b were incubated further for 18 h at 22 C after

◦

induction and were harvested via centrifugation when the culture reached an OD of 40 at 600 nm.

One-hundred twenty-ﬁve grams (125 g) of wet cells were harvested and the pellets were resuspended

in 10 volumes (w/v) of 100 mM sodium phosphate buffer (pH 7.0). The cells were disrupted

via ultrasonication and the supernatant was used as crude enzymes for the biotransformation of

the ginsenosides.

Gram-scale GypXVII has been produced using recombinant BgpA from ginsenoside Rb (data

1

not shown) [22]. The produced GypXVII (80.1% chromatography purity) was used as the substrate for

the gram-scale production of GypLXXV. The enzyme reaction occurred using the crude recombinant

BglG167b with GypXVII as the substrate with a concentration of 5.0 mg/mL. As shown in Figure 5,

all GypXVII was converted to GypLXXV within 9 h (Figure 5B). Most of the produced GypLXXV was

precipitated to form a solid, with a small quantity remaining dissolved in the supernatant.

Figure 5. HPLC results of the production of GypLXXV from GypXVII by BglG167b; (A) substrate

GypXVII; (B) the reaction mixture after nine-hour treatment with BglG167b; and (C) puriﬁed GypLXXV

using Prep-HPLC.

In order to remove the free sugar, enzyme, and salt, the mixture was centrifuged at 5000 rpm for

1

5 min. After a puriﬁcation step using column chromatography packed with HP20 resin, approximately

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3

BVs of the 95% ethanol eluent was evaporated in vacuo in order to create 5.7 g of GypLXXV.

Its chromatographic purity was 83.1% as determined via high-performance liquid chromatography

HPLC) (Figure 5). For the anti-cancer activity study, further puriﬁcation was performed using

(

Prep-HPLC. An ODS column is used as the stationary phase and 75% acetonitrile was used as

the mobile phase. GypLXXV was eluted from 150 mL to 280 mL, and after recycling two times

to remove impurity peaks, the compound with 97.8% purity was harvested. The recovery ratio

through the biotransformation process using GypXVII to GypLXXV reached 69.6% during the entire

bioprocess engineering.

Relatively abundant major ginsenosides were normally used as substrates for the production of

minor ginsenosides, and we have demonstrated several enhanced productions of rare minor ginsenoside

Rg (S), F , and Rg (S) using various ginsenoside-transforming recombinant enzymes [31,33,34]

3

2

from major ginsenosides. These various enzymatic methods were reported thus far for the

preparation of minor ginsenosides as a result of their higher conversion efﬁciency, fewer by-products,

superior environmental protection, and better stereo-speciﬁcity than physiochemical methods and

microbiological methods [35

of relatively abundant ginsenoside Rb [22

–

37]. Efforts have also been made to produce GypLXXV by deglycosylation

32]. Due to its rarity, GypXVII has not been used as a

,

1

substrate for the preparation of GypLXXV. In this study, recombinant BgpA which has been cloned

T

from Terrabacter ginsenosidimutans Gsoil 3082 with high substrate tolerance (Rb , 50 mg/mL) and

1

efﬁciency (5 h), was used for the gram-scale GypXVII preparation [22]. Previously, GypXVII has been

used for the production of F and CK [38,39]. However, no gram-scale production of GypLXXV, which

2

is important for industrial application, has been achieved before, and the gram-scale production of

GypLXXV showed a strong possibility for more large scale applications of BglG167b.

2

.7. Cytotoxic Effect of GypLXXV on Cancer Cells

In order to prove cytotoxic effects of GypLXXV on tumor cells, we compared the effect of Rb₁,

GypXVII, Rg (S) and GypLXXV on cell viability using three cancer cell lines (HeLa (cervical cancer

3

cell line), B16 (melanoma cell line), and MDA-MB231 (human breast cancer cell line)). Doxorubicin,

a well-known chemotherapy medication used to treat cancer, was used as a positive control. The cancer

cells were treated with 1.0–100

µM of pure doxorubicin, Rb , GypXVII, Rg (S), and GypLXXV for

1 3

4

8 h (Figure 6A–C). According to MTT assay results, treatment with Rg (S) and GypLXXV reduced

3

proliferation in a dose-dependent manner (Figure 6A–C). After 48 h of treatment, almost all cancer

cells were inhibited by GypLXXV and Rg (S) at 50 M. The LC₅₀of doxorubicin was lower than Rg (S)

µ

3

and GypLXXV on three cancer cells (Figure 6D). Though, as compared with LC50 doxorubicin and

Rg (S) (Figure 6D), GypLXXV showed considerable anti-proliferate activities higher than Rb and

3

1

GypXVII, and 63–78% of to ginsenoside Rg (S), which has shown strong anti-cancer effects in vitro

3

and in vivo in accordance with many studies [40].

Anticancer effects of minor ginsenosides (F , Rg (S), Rh (S), CK, and PPD) which were the

2

3

2

deglycosylated form of ginsenoside Rb , have been studied, and these minor ginsenosides showed

1

strong activity against various cancer cell lines [41,42]. Among them, Rg (S) appeared on the market

3

and has been applied to clinical therapy. GypLXXV has two glucose moieties attached at the C20

position of aglycon, whereas Rg (S) has two glucose moieties at C3 (Figure 4). The LC of GypLXXV

3

50

showed similar anti-cancer effects with Rg (S) (Figure 6D), and higher than Rb and GypXVII,

3

1

which have more attached glucose moieties (Figure 6A–C). It is the ﬁrst report of the anti-cancer

activity of GypLXXV, and the patent of the anti-cancer activity of GypLXXV against HeLa cells has

been registered by this team [43]. These results demonstrate that deglycosylation contributes to

improved anticancer activity, and the mechanism of increased anticancer effects requires further

structure-related approaches.

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Figure 6. The anti-cancer effects of GypLXXV, Rg (S), GypXVII and Rb on cell viability on (

A

) HeLa

3

1

cells; (B) B16 cells; (C) MDA-MB231; and (D) LC50 comparison of doxorubicin, Rg (S), and GypLXXV.

3

Cancer cells were incubated with various concentrations of GypLXXV for 48 h.

3

. Materials and Methods

3

.1. Materials

Ginsenoside standards that are over 98% purity, such as Rb , Rb , Rc, Rd, Rg (S), Rh (S), F ,

1

2

3

2

compound K (C-K), protopanaxadiol (PPD), Rg , Re, Rg (S), Rh (S), and protopanaxatriol (PPT), were

1

2

1

purchased from (Zelang Medical Technology Co., Ltd., Nanjing, China). HPLC grade acetonitrile was

obtained from Merck (Darmstadt, Germany). GypXVII, GypLXXV, and ginsenosides compound O

(

C-O), compound Y (C-Y), compound Mc (C-Mc ), and compound Mc (C-Mc), were prepared as

1 1

described by Cui et al. [23]. 5-bromo-4-chloro-3-indolyl β-D-glucopyranoside (X-Glc) was purchased

from Wako Co. Ltd. (Tokyo, Japan). The other chemicals used were a minimum of analytical reagent

grade. Microbacterium sp. Gsoil 167, was isolated from a soil sample of a ginseng ﬁeld, Pocheon

Province, South Korea, and cultivated on R2A agar (Becton, Dickinson and Company, Franklin Lakes,

◦

NJ, USA) under aerobic conditions at 30 C and used for the gene cloning experiment. Escherichia coli

BL21 (DE3), and pGEX 4T-1 plasmid (GE Healthcare, Chicago, IL, USA) were used as host and

expression vector sources, respectively. The E. coli cells harboring the BglG167b-containing recombinant

plasmid for protein expression was cultivated in a Luria-Bertani (LB) medium supplemented with

ampicillin (100 mg/L).

3

.2. Fosmid Library Construction and Fosmid Sequencing

A CopyControl^™Fosmid Production kit (Epicentre Technologies, Madison, WI, USA) was used

to isolate the ginsenoside-hydrolyzing glycosidase genes from Microbacterium sp. Gsoil 167. A fosmid

library was constructed following the manufacturer’s protocol. Infected E. coli was transferred onto

X-Glc (5-bromo-4-chloro-3-indolyl

chloramphenicol and then incubated at 37 C for 16 h. The blue color colonies were selected as putative

β

-D-glucopyranoside) LB plates supplemented with 12.5

µ

g/mL

◦

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ginsenoside-hydrolyzing candidates. After conﬁrmation of the ginsenoside-hydrolyzing activity by a

TLC assay, one clone was selected for fosmid sequencing. Fosmid DNA was puriﬁed and sequenced

by Macrogen Co. Ltd. (Seoul, Korea). The ﬁnal sequences assembly procedure was conducted by

the SeqMan program in the DNASTAR package (DNASTAR, Madison, WI, USA), which yielded a

9

.4-kb contig.

3

.3. Phylogenetic Analysis of BglG167b

A homology search was performed with BLAST program provided by NCBI database

(blast.ncbi.nlm.nih.gov). Sequences of the characterized glycosyl hydrolases were obtained from the

CAZY database [44], and multiple alignments were performed using the CLUSTAL_X program [45].

Gaps were modiﬁed in the BioEdit program [46], and evolutionary distances were calculated

according to the Kimura two-parameter model [47]. A phylogenetic tree was constructed using the

neighbor-joining method in the MEGA5 program, with bootstrap values based on 1000 replicates [48].

3

.4. Molecular Cloning, Expression, and Puriﬁcation of Recombinant BglG167b

The fosmid DNA sequence was analyzed using the ORF Finder program on the NCBI website

(

www.ncbi.nlm.nih.gov/gorf ). Predicted ORFs were subjected to similarity searches using the BLAST

program, which identiﬁed two putative open reading frames (ORFs) for -glucosidases. One of

β

them, BglG167b, which belonged to GH3 and consisted of 803 predicted amino acids, showed

ginsenoside-transforming activities. To clone the gene encoding BglG167b, PCR ampliﬁcation was

conducted using primers containing BamHI and XhoI (BglG167b) restriction sites, respectively (bolded).

0

The primer sequences were as follows: bglC167bF 5 -GGT TCC GCG TGG ATC CAA CGC CCG

0

CCC GTC CGA TTC CAC C-3 and bglC167bR 5 -GAT GCG GCC GCT CGA GTC ATG CCT GCT

CCC AGT CCA CCG A-3 . The ampliﬁed DNA fragment obtained from the PCR was puriﬁed

0

and inserted into the pGEX 4T-1 using an EzCloning Kit (Enzynomics Co. Ltd., Daejeon, Korea).

The recombinant pGEX-BglG167b was transformed into E. coli BL21 (DE3). The recombinant plasmid

◦

harboring E. coli BL21 (DE3) was grown in an LB-ampicillin medium at 37 C until the culture

reached an OD₆₀₀of 0.6, and then the recombinant protein expression was induced through 0.1 mM

isopropyl-

β

-D-thiogalactopyranoside (IPTG). The bacterial cells were incubated for a further 18 h

◦

at 18 C and harvested via centrifugation at 13,000 rpm for 15 min at 4 C. The cells were washed

twice with a solution consisting of 50 mM sodium phosphate, 5 mM EDTA, and 1% Triton X-100

(pH 7.0); then, they were resuspended in 50 mM sodium phosphate (pH 7.0). The cells were lysed

via ultrasonication (Vibra-cell, Sonics and Materials Inc., Newtown, CA, USA). The insoluble debris

◦

was removed via centrifugation at 13,000 rpm for 15 min at 4 C in order to obtain the crude cell

extract. The GST-tagged BglG167b was puriﬁed using the GST bind agarose resin according to the

manufacturer’s protocol (Elpisbiotech Co. Ltd., Daejeon, Korea). The purity of the protein was assessed

using 10% SDS-PAGE and an EZ-Gel staining solution (Daeillab Co. Ltd., Seoul, Korea).

3

.5. Effect of pH, Temperature, Metal Ions, and Chemical Reagents on Enzyme Activity

The speciﬁc activity of puriﬁed BglG167b was determined using pNP-

pNPGlc) as a substrate in pH 7.0, 50 mM sodium phosphate buffer at 37 C. Reactions were performed

β

-D-glucopyranoside

◦

(

for 10 min by the addition of Na CO (ﬁnal concentration, 500 mM), and the release of p-nitrophenol

2

3

was measured directly using a microplate reader at 405 nm (Bio-Rad Model 680; Bio-Rad., Hercules,

CA, USA). One unit of activity was deﬁned as the amount of protein required to generate 1 mol of

p-nitrophenol per min. Protein concentrations were determined using the BCA protein assay protocol

µ

(Pierce, Rockford., IL, USA). All assays were performed in triplicate.

The effect of pH on enzymatic activity was determined as described [22]. The pH stability of

recombinant BglG167b was determined by quantifying enzymatic activity after incubation in each

◦

buffer for 24 h at 4 C. The effect of temperature was tested by incubating the enzyme at various

◦

temperatures ranging from 4 C to 65 C at the optimum pH for 10 min containing 2.0 mM pNPGlc.

Molecules 2017, 22, 844

10 of 15

The thermostability of the enzyme was determined by incubating the enzyme in 50 mM sodium

phosphate buffer (pH 7.0) for 2 h at different temperatures. After chilling the sample on ice for 10 min,

activity was determined using pNPGlc.

The effects of metals and other chemicals on BglG167b activity were also determined. BglG167b

activity was tested in the presence of 1 or 10 mM (ﬁnal concentration) of CaCl , CoCl , CuCl , KCl,

2

◦

MgCl , NaCl, ZnCl , dithiothreitol (DTT), EDTA, or SDS for 30 min at 25 C. The remaining activity

2

was determined and expressed as a percentage of the activity obtained in the absence of the compound.

Substrate preference was determined using 2.0 mM chromogenic o-nitrophenyl (oNP) and

◦

p-nitrophenyl (pNP) as surrogate substrates (all from Sigma) at 37 C for 10 min, with one unit

being deﬁned as the release of 1 µmol p-nitrophenol or o-nitrophenol per min.

3

.6. Biotransformation Activity of Ginsenosides Using BglG167b

The initial biotransformation experiments using the major ginsenosides Rb and Re as substrates

1

revealed that the GST-fused enzyme does not affect the activities of BglG167b. Therefore, the fusion

protein (GST-BglG167b) was used to determine the speciﬁcity and selectivity of the enzymes for the

hydrolysis of the glucose moieties attached at the C3 and C20 positions in the seven PPD ginsenosides

and two PPT ginsenosides. The puriﬁed enzyme at a concentration of 1.0 mg/mL in 50 mM of sodium

phosphate buffer (pH 7.0) were reacted with an equal volume of Rb , Rb , Rc, Rd, GypXVII, Rg (S), F ,

1

2

3

2

Re, and Rg solution at a concentration of 2 mg/mL in 50 mM of sodium phosphate buffer (pH 7.0)

1

◦

at 37 C. The samples were taken at regular intervals and analyzed via TLC after pretreatment (see

analytic methods). For TLC analysis, an equal volume of water-saturated n-butanol was added to stop

the reaction, and the reactant present in the n-butanol fraction was determined.

3

.7. Gram-Scale GypLXXV Production Using Recombinant BglG167b

Preparation of Recombinant BglG167b Using High Cell Density Culture

For the production of the recombinant BglG167b, the LB medium supplemented with ampicillin

(100 mg/mL) was used to cultivate the E. coli BL21 (DE3) harboring pGEX-BglG167b in a 10 L tank

reactor (Hanil science Co., Seoul, Korea) with a 5 L working volume at a 500 rpm stirring speed.

The pH value of the medium was adjusted to 7.0 by the addition of (29%, v/v) of NH OH and/or

4

◦

(

37%, v/v) HCl. The culture was incubated at 37 C until the culture reached an OD600 of 3.0.

Cells harboring pGST-BglG167b were induced at 18 C for 18 h and were then harvested via

centrifugation at 5000 rpm for 20 min at 4 C. Cell pellets were resuspended in 10 volumes (w/v) of

◦

5

0 mM sodium phosphate buffer (pH 7.0) and disrupted by sonication, and the supernatant was used

as the crude enzyme for ginsenoside biotransformation.

GypXVII had been produced from Rb₁using recombinant BgpA (17). The scaled-up

biotransformation of GypXVII into GypLXXV was performed in a 5.0 L glass bottle (2.0 L working

◦

volume) under optimal conditions (shaking 200 rpm for 8 h at pH 7.0 and 37 C). The reaction consisted

of crude recombinant 1.0 L BglG167b, 10 g of GypXVII in 1.0 L of 50 mM sodium phosphate buffer.

Samples were collected at regular intervals and HPLC was used to monitor the biotransformation of

GypXVII to GypLXXV, respectively.

3

.8. Puriﬁcation of GypLXXV

Following the 2.0 L reaction of GypXVII with BglG167b, the mixture was cooled at 4 C and

◦

centrifuged at 5000 rpm for 20 min. The supernatants and precipitates were processed separately in

order to purify the samples. The precipitate was also dissolved in 5.0 L of 70% ethanol solution twice

and ﬁltered using a ﬁlter paper (Advantec., Tokyo, Japan). The ethanol extracts were combined with

the supernatant and adjusted to be a 45% ethanol solution. The column chromatography (3170 (L)

28 (D) mm; Doointech, Korea) packed with HP20 resin (Mitsubishi Chemical Corp., Tokyo, Japan)

was adopted in order to remove the impurities, except the ginsenosides. The free sugar molecules and

×

1

Molecules 2017, 22, 844

11 of 15

unwanted hydrophilic compounds from the HP-20 that were adsorbed in beads were washed with

three bed volumes (BV) of water, and the adsorbed ginsenosides were ﬁnally eluted using 3 BVs of

95% ethanol. The ethanol eluent was evaporated in vacuo. The resulting powder was dissolved in

1

00% methanol and analyzed via HPLC.

GypLXXV was further puriﬁed using preparative HPLC (JAL Recycling Preparative HPLC

LC-9210II NEXT, Japan Analytical Industry Co., Tokyo, Japan). The preparative HPLC run was

performed using a pre-packed column (JAIGEL-ODS-AP-L, 20 mm i.d. 500 mm) purchased from

×

Japan Analytical Industry Co. (Tokyo, Japan). The mobile phase was 85% acetonitrile, and the ﬂow

rate was 7.0 mL/min. The sample solution was made by dissolving a sufﬁcient quantity of crude

GypLXXV in 85% acetonitrile to give a ﬁnal concentration of 30 mg/mL and 10 mL was loaded for

the puriﬁcation. GypLXXV was returned to the column after passing through the detector two times

to achieve a higher puriﬁcation. The elutionfrom 70 min to 77 min was collected, evaporated and

analyzed by HPLC.

3

.9. Cell Culture

Cancer cells were maintained in Dulbecco’s Modiﬁed Eagle’s Medium (DMEM) with 10% FBS,

◦

mmol/L glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37 C in a humidiﬁed 5%

2

CO atmosphere. Appropriate amounts of the resulting GypLXXV in DMSO were added to the culture

2

medium at the indicated ﬁnal concentrations.

3

.10. MTT Assay

The in vitro chemosensitivity was measured by dimethyl thiazolyl-2,5-diphenyltetrazolium

®

bromide (MTT) assay (CellTiter 96 Non-Radioactive Cell Proliferation Assay kit, Promega, Madison,

4

WI, USA). Brieﬂy, Cancer cells were inoculated into 96-well plates in 100

µ

L medium (10 cells) per

well and allowed to attach and grow for 24 h. To determine the anti-proliferative effect of GypLXXV,

Rg (S), Rb and GypXVII, various concentrations of ginsenosides diluted with the FBS-free medium

3

1

◦

were added into the wells. Then the cells were exposed to drugs for 48 h at 37 C, after which, MTT

was added to each well and cultured for an additional 4 h. The formed formazan was dissolved in

100 µL of solubilization/stop solution after aspirating the culture medium. The plates were shaken

mechanically for 5 min and incubated for 1 h. After shearing for each well the optical density was

immediately read on a microplate reader at a wavelength of 595 nm. Results are expressed as LC₅₀

which was analyzed by GraphPad Prism 5 program.

3

.11. Analytic Methods

3

.11.1. Thin Layer Chromatography (TLC) Analysis

The thin layer chromatography (TLC) was performed using 60F₂₅₄silica gel plates (Merck & Co.,

Munchen, Germany) with CHCl –CH OH–H O (70:30:10, lower phase) as the solvent. The spots on

3

2

the TLC plates were identiﬁed through comparisons with standard ginsenoside after visualization was

◦

made by spraying 10% (v/v) H SO , followed by heating at 110 C for 5 min.

2

4

3

.11.2. High-Performance Liquid Chromatography (HPLC) Analysis

The HPLC analysis of the ginsenosides was performed using an HPLC system (Younglin Co.

Ltd., Anyang-si, Korea). The separation was performed on a Prodigy ODS(2) C₁₈column (5

µ

m,

4.6 mm i.d.). The mobile phases were A (acetonitrile) and B (water). The gradient elution started

1

50

2.5

×

4.6 mm i.d.; Phenomenex Inc., Torrance, CA, USA) with a guard column (Eclipse XDB C , 5 µ

18

×

with 32% solvent A, and was changed to the following: from 0–8 min, A was increased from 32 to

5%; from 8–12 min, A was increased from 65 to 100%; from 12–15 min, A was constant at 100%; from

5–15.1 min, A was decreased from 100 to 32%; from 15.1–25 min, A was constant at 32% The ﬂow rate

6

1

Molecules 2017, 22, 844

12 of 15

was 1.0 mL/min, and the detection was measured by monitoring the absorbance at 203 nm and with

an injected volume of 25 µL.

3

.12. Nucleotide Sequence Accession Numbers

The sequence for the BglG167b gene from Microbacterium sp. Gsoil 167 was deposited into

GenBank/EMBL/DDBJ under accession numbers JX960414.

4

. Conclusions

In summary, we have cloned and characterized ginsenoside-transforming

β-glucosidase

belonging to the glycoside hydrolases superfamily 3 from Microbacterium sp. Gsoil 167 from the

◦

ginseng-cultivating soil. This had optimum reaction conditions at 37 C and pH 7.0. BglG167b could

convert various ginsenosides (Rb , Rb , Rc, Rd, GypXVII, Rg (S), F , Re, and Rg ) through the selective

1

2

3

2

1

hydrolysis of only the glucose moieties. Among them, this enzyme efﬁciently hydrolyzes one inner

glucose moiety at the C3 position of PPD type ginsenosides (F and GypXVII). The recombinant

2

BglG167b was applied to produce GypLXXV on the gram-scale and scale-up of production using

10 g of the GypXVII resulting in 5.7 g of GypLXXV with 97.8% chromatography purity and 69.6%

recovery. The anti-cancer activity of this compound was primarily determined in vitro. GypLXXV

showed strong cancer cell growth inhibition of cancer cells, and the activity is similar with the

well-known anti-cancer ginsenoside-Rg (S) which showed its possibility for the application. Therefore,

3

the properties of BglG167b could be a useful tool in the research and production of GypLXXV in the

cosmetics, functional food, and pharmaceutical industries.

Supplementary Materials: Supplementary materials are available online.

Acknowledgments: This work was supported by the Intelligent Synthetic Biology Center of Global Frontier

Project funded by the Ministry of Science, ICT and Future Planning (2014M3A6A8066437), Republic of Korea.

Author Contributions: The list authors contributed to this work as follows: Chang-Hao Cui, Sun-Chang Kim and

Wan-Taek Im conceived and designed the experiments; Chang-Hao Cui, performed the experiments and analyzed

the data; Da Jung Kim and Suk-Chae Jung contributed reagents/materials/analysis tools; Chang-Hao Cui and

Wan-Taek Im wrote the paper. Sun-Chang Kim and Wan-Taek Im acquired funding for the research. All authors

read and approved the ﬁnal manuscript.

Conﬂicts of Interest: The authors declare no competing ﬁnancial interest.

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Sample Availability: Samples of the compounds Gypenoside XVII and Gypenoside LXXV are available from

the authors.

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2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access

article distributed under the terms and conditions of the Creative Commons Attribution

CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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Article Doi

Enhanced production of gypenoside LXXV using a novel ginsenoside-transforming β-glucosidase from ginseng-cultivating soil bacteria and its anti-cancer property

DOI: 10.3390/molecules22050844

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Article abstract of DOI:10.3390/molecules22050844

Full text of DOI:10.3390/molecules22050844

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