Cloning and heterologous expression of UDP-glycosyltransferase genes from Bacillus subtilis and its application in the glycosylation of ginsenoside Rh1.

Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

Cloning and heterologous expression of UDP-glycosyltransferase genes from Bacillus subtilis and its application in the glycosylation of ginsenoside Rh1 S.L. Luo1, L.Z. Dang2, K.Q. Zhang1, L.M. Liang1 and G.H. Li1 1 Laboratory for Conservation and Utilization of Bio-resource, and Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University, Kunming, China 2 Technology Centre of Hongyun Honghe Tobacco (Group) Co., Ltd., Kunming, China

Significance and Impact of the Study: The Chinese traditional medicinal plant Panax is reported to have multiple health benefits. Its main active ingredient is saponin, and different saponins have different activity spectrum. In the study, three UDP-glycosyltransferase genes, ydhE1, yojK1 and yjiC1, were cloned from Bacillus subtilis CCTCC AB2012913 and the three genes were expressed in Escherichia coli BL21 (DE3). The enzyme YjiC1 was purified and converted ginsenoside Rh1 to 3-O-b-D-glucopyranosyl-6-O-b-D–glucopyranosyl-20(S)-protopanaxatriol in vitro. The compound is the first saponin possessing b-glucopyranosyl at both C-3 and C-6 sites. We showed that the in vitro biotransformation was effective, and the reaction condition was easy to control. Our research suggests that a diversity of saponins could be generated through efficient and directed enzymatic biotransformation.

Keywords 3-O-b-D-glucopyranosyl-6-O-b-D– glucopyranosyl-20(S)-protopanaxatriol, Bacillus subtilis, biotransformation, ginsenoside Rh1, UDP-glycosyltransferase. Correspondence Guohong Li, Laboratory for Conservation and Utilization of Bio-resource, and Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University, Kunming 650091, China. E-mail: [email protected] and Lizhi Dang, Technology Centre of Hongyun Honghe Tobacco (Group) Co., Ltd., Kunming 650202, China. E-mail:[email protected] 2014/1193: received 10 June 2014, revised 2 October 2014 and accepted 2 October 2014

Abstract Bacillus subtilis CCTCC AB 2012913 can transform ginsenoside Rh1 to 3-O-bD-glucopyranosyl-6-O-b-D–glucopyranosyl-20(S)-protopanaxatriol. Based on its genome sequence, strain B. subtilis 168 contains three UDP-glycosyltransferase genes. Here, we cloned the three UDP-glycosyltransferase genes (ydhE1, yojK1 and yjiC1) from B. subtilis CCTCC AB 2012913 and expressed in Escherichia coli BL21 (DE3) with His-tag. The crude enzyme extracts were assayed, respectively, for their activities to transform ginsenoside Rh1. Extracts containing enzymes YojK1 and YjiC1 could use ginsenoside Rh1 as a substrate to produce 3-O-b-D-glucopyranosyl-6-O-b-D–glucopyranosyl-20(S)protopanaxatriol, which had an additional glucopyranosyl linked with C-3 over the substrate. Enzyme YjiC1 was purified by affinity chromatography on NiNTA His Binding resin. The molecular mass of purified YjiC1 was c. 47 kDa as determined by SDS-PAGE. This is the first report of an in vitro biotransformation of ginsenoside Rh1 to 3-O-b-D-glucopyranosyl-6-O-b-D– glucopyranosyl-20(S)-protopanaxatriol using the recombinant UDPglycosyltransferase.

doi:10.1111/lam.12339

Introduction Glycosyltransferases (GT) are essential for the biosynthesis of glycosylated natural products. They catalyse the attachment of a sugar to an aglycon (Hu and Walker 2002; Luzhetskyy et al. 2008) and are important for the development of complex glycosylated natural products. Bacillus 72

sp. contains UDP-glycosyltransferases capable of using UDP-activated sugar moieties as the sugar donor and small molecules such as flavonoids and antibiotics as the sugar acceptors. Bacillus cereus 10987 contained four UDP-glycosyltransferases. One of them, BcGT-1, could carry out glycosylation reactions on flavonoids to give one or two reaction products (Ko et al. 2006). A

Letters in Applied Microbiology 60, 72--78 © 2014 The Society for Applied Microbiology

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Glycosylate of ginsenoside Rh1

UDP-glycosyltransferase from Bacillus subtilis Marburg strain 60015 successively transferred up to four sugar moieties to 1,2-diacylglycerol (Jorasch et al. 1998). However, there are few reports on the glycosylation of ginsenosides by UDP-glycosyltransferases. The bacterium B. subtilis 168, whose genome has been completely sequenced recently, contains three UDP-glycosyltransferases, yjiC, yojK and ydhE (Barbe et al. 2009). Sanchi (Panax notoginseng) is a famous traditional herb. Ginsenosides, such as ginsenoside Rb1, Re, Rg1, which are all glycosylated triterpenoids (saponins), are considered the main active compounds in P. notoginseng. These compounds have a wide range of bioactivities including promoting blood circulation, preventing the formation of blood clots (antithrombosis property), dissolving blood clots, enhancing the removal of cellular breakdown products and other debris from the blood circulation (Jiang and Qian 1995; Li and Chu 1999). Ginsenoside Rh1, with a triterpenoid of dammarane skeleton as on aglycon and one glucose molecule moiety at C-6, has a strong pharmacological activity including cytotoxic effect, anti-inflammatory, an active estrogenic ligand, neuroprotective effect and anti-allergic action (Park et al. 1998, 2004; Lee et al. 2003; Jung et al. 2010; Li et al. 2011), while it has a low water-solubility. From a chemical point of view, sugar conjugation results in both increased stability and water solubility (Lairson et al. 2008). The chemical synthesis of such sugar ligands is exceedingly difficult to carry out and therefore impractical to establish on a large scale (Luzhetskyy and Bechthold 2008). Recently, researchers used recombinant enzymes to transform ginsenosides at relatively high productivities, and the result had been efficiently applied in industrial use (Hong et al. 2012; Quan et al. 2012). In our previous work, a new triterpenoid saponin, 3-O-b-D-glucopyranosyl-6-O-b-D-glucopyranosyl-20(S)protopanaxatriol, was obtained through biotransformation by B. subtilis using ginsenoside Rh1 as the substrate (Fig. 1). The transformed product had an additional b-glucopyranosyl group linked with C-3 (Li et al. 2005), resulting in a new type of saponin possessing b-glucopyr-

HO OH

HO Oglc Ginsenoside Rh1

HO OH

anosyl at both C-3 and C-6. Here, we describe the cloning and heterologous expression of the UDP-glycosyltransferases genes from another strain B. subtilis CCTCC AB 2012913 and show its application to produce 3-O-b-Dglucopyranosyl-6-O-b-D-glucopyranosyl-20(S)-protopanaxatriol in vitro. Results and discussion Gene cloning and sequencing analysis of yjiC1, yojK1 and ydhE1 There are three UDP-glycosyltransferases YjiC (NP 3891041), YojK (NP 3898242) and YdhE (NP 3884532) contained in the genome of strain B. subtilis 168 (Barbe et al. 2009). Based on the published sequence to these three genes, we designed corresponding primers to amplify by PCR and to sequence the three genes encoding the UDP-glycosyltransferases from another strain B. subtilis CCTCC AB 2012913. According to the ExPASy program (http://web.expasy.org/compute_pi/), the genes in strain AB2012913 for YjiC1, YojK1 and YdhE1 are 1179, 1218 and 1188 bp long, and encode 439, 456 and 444 kDa proteins, respectively. The amino-acid sequence of YjiC1 showed low similarities to YojK1 (33%), and YdhE1 (32%), while YojK1 showed a 34% similarity to YdhE1 (Fig. 2). Heterologous expression of yjiC1, yojK1 and ydhE1 in Escherichia coli cells The three genes were successfully cloned from B. subtilis CCTCC AB 2012913 genome and inserted into the pET28a vector, and then overexpressed the corresponding putative UDP-glycosyltransferase in E. coli. After induced at a low temperature with isopropyl-b-D-thiogalactoside (IPTG), the E. coli cells were collected and sonicated on ice. The mixtures were then centrifuged to obtain supernatants, and the supernatants were stored until use. Recombinant E. coli cells carrying pET28a/yjiC1, pET28a/yojK1 or pET28a/ ydhE1 all produced soluble YjiC1, YojK1 and YdhE1, while E. coli BL21 (DE3) harbouring pET28a could not (Fig. 3). The molecular weights of recombinant proteins YjiC1, YojK1 and YdhE1 were c. 470, 490 and 470 kDa, respectively, consistent with the predicted molecular weight sum of YjiC1, YojK1, and YdhE1 plus the His-tag.

glcO Oglc 3-O-β-D-glucopyranosyl-6-O-β-Dglucopyranosyl-20(S)-protopanaxatriol

Figure 1 Transformation reaction from ginsenoside Rh1 to 3-O-b-Dglucopyranosyl-6-O-b-D–glucopyranosyl-20(S)-protopanaxatriol by Bacillus subtilis.

Assay using crude enzymes activities The solution fractions obtained above were used for assaying their ability to convert ginsenoside Rh1 to other products. In these assays, ginsenoside Rh1 and UDPG were used as substrates for crude enzyme extracts


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YojK1 YdhE1 YjiC1

1 --MANVLMIGFPGEGHINPSIGVMKELKSRGENITYYAVKEYKEKITALDIEFREYHDFR 1 --MKTVLILNFPAEGHVNPTLGITKAFSDKGYDVHYISTEKYKKRLEAAGATVHLHRDLL 1 MKKYHISMINIPAYGHVNPTLALVEKLCEKGHRVTYATTEEFAPAVQQAGGEALIYHTSL

YojK1 YdhE1 YjiC1

59 GDYFGKNATGDEERDFTEMLCAFLKACKDIATHIYEEVKHESYDYVIYDHHLLAGKVIAN 59 RTTPIHVGSPN---GILDFVKIHIKTSLDILQIVKDLSKSIQFDFVYYDKFG-AGELVRD 61 NIDPKQIREMME---KNDAPLSLLKESLSILPQLEELYKGDQPDLIIYDFVALAGKLFAE

YojK1 YdhE1 YjiC1

116 MLKLPRFSLCTTFAMNEEFAKEMMGAYMKGSLEDSPHYESYQQLAETLNADFQAEIKKPF 112 YLDIPGVSSSASFLFGEEHLKILP--LHPESGAPLELDQECEDLLAKMKETYGVAPKNLV 115 KLNVPVIKLCSSYAQNESFQLGNEDMLKKIKEAEAEFKAYLEQ---------EKLPAVSF

YojK1 YdhE1 YjiC1

172 DVFLADGDLTIVFTSRGFQPLAEQFGERYVFVGPSITERAGNNDFPFDQIDNENVLFISM 166 QFMNNKGELNVVYTSRYFQPESDRFGDECLFIGPSFPKRAEKTDFPIEQLKDEKVIYISM 162 EQLAVPEALNIVFMPKSFQIQHETFDDRFCFVGPSLGERKEQEGLLIDKD-DRPLMLISL

YojK1 YdhE1 YjiC1

228 GTIFNNQKQFFNQCLEVCKDFDGKVVLSIGKHIKTSELNDIPENFIVRPYVPQLEILKRA 222 GTVLDHTEDFFNLCIDAFSGFNGKVVIAAGEKADLTKLKQAPENFIIAPYVPQLEVLEQS 218 GTAFNAWPEFYKMCIKAFRDSSWQVIMSVGKTIDPESLEDIPANFTIRQSVPQLEVLEKA

YojK1 YdhE1 YjiC1

284 SLFVTHGGMNSTSEGLYFETPLVVIPMGGDQFVVADQVEKVGAGKVIKKEELSESLLKET 278 DVFITHGGMNSVNEGIHFSVPLVVMPHDKDQPMVAQRLSELHAGYVISKDEVNAQILKQA 273 DLFISHGGMNSTMEAMNAGVPLVVIPQMYEQELTANRVDELGLGVYLPKEEVTVSSLQEA

YojK1 YdhE1 YjiC1

340 IQEVMNNRSYAEKAKEIGQSLKAAGGSKKAADSILEAVKQKTQSANA 334 VDEVLRNDQYTAGIKKINQSFKECMDMEEVIERIDELIRQKNK---329 VQAVSSDQELLTRVKNMQKDVKEAGGAERAAAEIEAFMKKSAVPQ--

kDa

M

1

2

3

4

5

6

Figure 2 Alignment of three UDPglycosyltransferases (YojK1, YjiC1 and YdhE1) from Bacillus subtilis CCTCC AB 2012913.

7 Rh1

116·0 66·2 45·0

1

35·0

25·0 S 18·4

YjiC1

YojK1

YdhE1

Figure 4 TLC analysis of transformation of ginsenosides Rh1 by crude enzyme. S: standard. C: control. YjiC1: transformation of ginsenoside Rh1 by YjiC1. YojK1: transformation of ginsenoside Rh1 by YojK1. YdhE1: transformation of ginsenoside Rh1 by YdhE1. 1: compound 3-O-b-D-glucopyranosyl-6-O-b-D–glucopyranosyl-20(S)-protopanaxatriol.

14·4

Figure 3 Heterologous expression and solubility of YjiC1, YojK1 and YdhE1 in Escherichia coli. M: molecular mass markers. Lane 1: total protein samples from YjiC1 expression in BL21(DE3). Lane 2: the soluble fractions from expressed YjiC1 in BL21(DE3). Lane 3: total protein samples from YojK1 expression in BL21(DE3). Lane 4: the soluble fractions from expressed YojK1 in BL21(DE3). Lane 5: total protein samples from YdhE1 expression in BL21(DE3). Lane 6: the soluble fractions from expressed YdhE1 in BL21(DE3). Lane 7: induced cell samples carrying pET28a.

containing YjiC1, YojK1 or YdhE1 to test their transformation activity to produce compound 1 (3-O-b-D-glucopyranosyl-6-O-b-D-glucopyranosyl-20(S)-protopanaxatriol). The reaction products were analysed by TLC. The results indicated that ginsenoside Rh1 was converted to compound 1 by the crude extracts containing YjiC1 or 74

C

YojK1, while that containing YdhE1 showed no similar activity (Fig. 4). Moreover, YjiC1 converted ginsenoside Rh1 to compound 1 within 20 min, while YojK1 required more time and had weaker activity than YjiC1 (Fig. 4). Thus, YjiC1 was selected for further studies. Purification of YjiC1 by Ni-NTA His Bind resin The recombinant protein YjiC1 was purified from cellfree bacterial lysate by affinity chromatography on Ni-NTA His Bind resin. Most of the fusion protein was eluted after adding 150 mmol l1 imidazole as the elution buffer. The recombinant YjiC1 was purified to homogeneity and confirmed by SDS-PAGE, which


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(a) kDa M 116·0 66·2 45·0 35·0

1

2

3

4

(b)

25·0 18·4 Figure 5 Properties of the enzyme YjiC1 expression in BL21(DE3). (a) SDS-PAGE analysis of His-YjiC1 purification by Ni-NTA column. M: molecular mass markers; Lane 1: total protein samples; Lane 2: the insoluble fractions; Lane 3: the soluble fractions; Lane 4: purified YjiC1 protein. (b) Temperature optimization for the ginsenoside Rh1 biotransformation catalysed by YjiC1. (c) pH optimization for the ginsenoside Rh1 biotransformation catalysed by YjiC1. (d) Effect of metal ions on the activity of YjiC1.

(c)

(d)

3 4 5 6 7 8 9 10 1 Rh1

showed a molecular mass of c. 47 kDa, in agreement with those of the YjiC1 plus the His-tag (Fig. 5a). Enzyme characterization Based on the reaction temperature profile, YjiC1 showed activity between 15 and 50°C, with its maximal activity from 30 to 45°C (Fig. 5b). The relationship between enzymatic activity and pH was determined at 35°C in five buffer 50 mmol l1 glycine-HCl buffer (pH 30), citric acid-sodium citrate buffer (pH 40–50), sodium phosphate buffer (pH 60–70), Tris-HCl buffer (pH 80–90) and sodium carbonate buffer (pH 100). YjiC1 showed its maximal activity in the pH range of 60–90 (Fig. 5c). The effect of metal ions on YjiC1 activity was investigated at 35°C in 50 mmol l1 Tris-HCl (Fig. 5d). The enzyme activity appeared to be strongly inhibited in the presence of Zn2+, Cu2+, Fe2+, and the enzyme did not require Na+, K+ for activity and was significantly stimulated by Ca2+, Mg2+, Mn2+ and Co2+.

15 20 25 30 32 35 37 40 45 50 1 Rh1 Tm(°C)

K+ Na+ Mg2+ Ca2+ Co2+ Mn2+ Fe2+ Cu2+ Zn2+ 1 Rh1

pH

In the study, it was confirmed by biotransformation in vivo and enzymatic conversion in vitro that ginsenoside Rh1 transformed into 3-O-b-D-glucopyranosyl-6-O-b-Dglucopyranosyl-20 (S)-protopanaxatriol. Recombinant b-glycosidases (Quan et al. 2012) as well as GT (Jorasch et al. 1998; Ko et al. 2006) from micro-organisms have been used for biotransformation in vitro, but product of biotransformation is the first saponin possessing b-glucopyranosyl at both C-3 and C-6 sites. The Chinese traditional medicinal plant Panax is reported to have multiple health benefits. Its main active ingredient is saponin and different saponins have different activity spectrum. Our research suggests that a diversity of saponins could be generated through efficient and directed enzymatic biotransformation, so potential promising active saponins would be obtained by enzymatic biotransformation in vitro. Materials and methods Chemicals and reagents

Biotransformation of ginsenoside Rh1 to compound 1 by YjiC1 Compound 1 was prepared in vitro to confirm its structure. Seven milligram compound 1 was transformed from 1276 mg substrate ginsenoside Rh1 by purified His-YjiC1, and residual 1 mg ginsenoside Rh1 was isolated again when transformed at 05 h. The transformed yield of compound 1 within 05 h is 518%. Compound 1 was further identified by NMR (Data S1, see supporting information) and MS (FAB-MS m/z: 799 ([M-H]-), which showed the same data with compound 3-O-b-D-glucopyranosyl-6-O-b-Dglucopyranosyl-20 (S)-protopanaxatriol (Li et al. 2005).

UDP-glucose was obtained from Sigma. Restriction enzymes, T4 DNA lingase and Pfu DNA polymerase were purchased from Takara-Bio (Tokyo, Japan). The substrate ginsenoside Rh1 was prepared from P. notoginseng by our laboratory. The NMR spectra were carried out on Bruker DRX-500 NMR spectrometer. ESIMS was recorded on Finnigan LCQ-Advantage mass spectrometer. Column chromatography was performed with silica gel (200– 300 mesh; Qingdao Haiyang Chemical, Qingdao, China) and Sephadex LH-20 (Amersham Pharmacia, Uppsala, Sweden).


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Strains, plasmids, culture techniques and media Escherichia coli DH5a (Invitrogen, Carlsbad, CA, USA) and BL21(DE3) (Invitrogen) were used as hosts for plasmid construction and protein overexpression; pET28a (Novagen, Darmstadt, Germany) was used as vector for protein overexpression in E. coli. B. subtilis CCTCC AB 2012913 (China Center for Type Culture Collection, China) was used as the DNA donor. The bacterium was cultivated in Luria–Bertani (LB) medium (pH 70) containing 1% tryptone (Oxoid), 05% yeast extract (Oxoid), and 1% NaCl at 37°C. E. coli BL21 (DE3) was employed as host of overexpression vector. Cells of E. coli strains were also grown in LB medium at 37°C. Molecular cloning of the putative UDPglycosyltransferases genes (yjiC1, ydhE1 and yojK1) from Bacillus subtilis CCTCC AB 2012913 The genomic DNA was isolated from B. subtilis CCTCC AB 2012913 using a CTAB method (Simon et al. 1996). The genes encoding UDP-glycosyltransferases were amplified from the genomic DNA by polymerase chain reaction (PCR) using Pfu DNA polymerase. The genes yjiC1, yojK1 and ydhE1 were amplified using the following primers (with BamHI and SalI restriction sites in underlined): yjiC1F (50 -ATAGGATCCATGAAAAAGTACCATATTTCG A-30 ) and yjiC1R (50 -TTAGTCGACTTACTGCGGGACAG CGGATTTT-30 ); yojK1F (50 -ATAGGATCCATGGCTAAT GTATTAATG-30 ) and yojK1R (50 -TTAGTCGACTTAT GCATTTGCTGATTGAGTTT-30 ); ydhE1F (50 -ATAGGAT CCATGAAGACAGTATTGATTTTGAA-30 ) and ydhE1R (50 -TTAGTCGACTTATTTGTTTTTTTGGCGAAT-30 ). The primers for cloning of yjiC1, yojK1 and ydhE1 from B. subtilis CCTCC AB 2012913 were designed based on the yjiC, yojK and ydhE sequences of B. subtilis 168 (Barbe et al. 2009). The PCR mixtures contained 1 lg of total DNA, 10 nmol l1 of dNTPs, 10 lmol l1 of each primer and 25 U of Pfu DNA polymerase in 50 ll. Amplification was performed under the following conditions: 95°C for 5 min; 30 cycles of 95°C for 30 s, 55°C for 30 s and 72°C for 2 min; and a hold at 72°C for 10 min. DNA sequence analysis and construction of expression vector

These transformants were grown in 5 ml LB medium supplemented with kanamycin (50 lg ml1) at 37°C, 180 rev min1 for 12 h individually. For large-scale cultivation, the preculture (10 ml) was transferred to a 250-ml shaking flask containing 100 ml LB medium supplemented with 50 lg ml1 kanamycin, and then the cultures were incubated at 37°C, 180 rev min1. After 4 h cultivation, the diluted cultures were grown to OD600=04-06, then 02 mmol l1 IPTG was added and the bacteria were incubated for additional 16 h at 20°C, 180 rev min1 and harvested by centrifuging at 5000 g for 10 min at 4°C. The cells were washed twice with 20 mmol l1 Tris-HCl buffer (pH 80) and then resuspended in the same buffer. The cells were sonicated on ice 10 times for 10 s each with 10 s intervals (where one cycle corresponded to 10 kHz for 5 s, followed by an interval of 10 s) after adding lysozyme (10 lg ml1) to lyse E. coli cells. The debris was removed by centrifugation (12 000 g, at 4°C for 10 min), and the supernatant was collected and storage at 4°C to use for subsequent crude enzymes assays. Analysis of crude enzymes activities Transformation of ginsenoside Rh1 was performed in 200 ll of a reaction mixture comprising 50 mmol l1 Tris-HCl (pH 80), 05 mmol l1 ginsenoside Rh1, 25 mmol l1 UDP-glucose, and the 50 ll crude enzymes obtained above with IPTG inducement. At the same time, a negative control was performed with the same reaction mixture but added induced E. coli lysate harbouring pET28a. The reaction was started by the addition of enzyme. After incubation at 35°C for 2 h for YojK1 and YdhE1, 20 min for YjiC1, the reaction was stopped by the addition of 200 ll of n-butanol with vigorous mixing and centrifugation. The supernatant contained ginsenoside Rh1 and transformed products were analysed by TLC. Each experiment was repeated three times with three replicates. TLC analysis of ginsenosides

The amplified fragment (about 1200 bp) was digested with BamHI and SalI and then cloned into pET28a (Novagen) to obtain recombinant plasmid pET28a/yjiC1, pET28a/yojK1, pET28a/ydhE1, which were then sequenced. Three plasmids were transformed into E. coli BL21(DE3) separately.

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The product extracted in the organic phase was collected and evaporated to dryness at room temperature. The samples were redissolved in methanol and spotted on a TLC plate, with the developing solvent CHCl3 : CH3OH : H2O (45 : 15 : 02, v/v/v). Spots on the TLC plates were detected by spraying plates with 5% H2SO4 followed by heating at 110°C for 10 min.


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Heterologous expression and purification of YjiC1 After induction with the same procedure described above, the recombinant E. coli cells that contained YjiC1 were harvested by centrifugation (5000 g, at 4°C for 10 min). All subsequent operations were conducted at 0 to 4°C. The cells were washed with 20 mmol l1 Tris-HCl buffer (pH 80), and were resuspended in buffer D (20 mmol l1 Tris-HCl (pH 80) containing 20 mmol l1 imidazole and 05 mol l1 NaCl), and then were sonicated on ice 15 min for 5 s each at 10 s intervals after adding lysozyme (10 lg ml1) to lyse E. coli cells. The cell debris was removed by centrifugation at 12 000 g for 30 min. The supernatant was applied to a Ni-NTA His Bind resin column (2 ml) equilibrated with buffer D. The column was washed with buffer D, and the enzyme was eluted with buffer E (20 mmol l1 Tris-HCl (pH 80) containing 150 mmol l1 imidazole and 05 mol l1 NaCl). The active fractions were collected. The enzyme solution was dialysed at 4°C against 20 mmol l1 TrisHCl buffer (pH 80) to remove imidazole. The protein homogeneity was assessed by 12% SDS-PAGE followed by Coomassie blue staining. The protein concentration was determined by the Bradford method (Bradford 1976) using bovine serum albumin as a standard. Characterization of purified YjiC1 The specific activity of purified YjiC1 was determined in the reaction mixture (02 ml) containing 50 mmol l1 buffer, 05 mmol l1 ginsenoside Rh1, 25 mmol l1 UDPG and 6 lg purified YjiC1. Reactions were stopped by adding n-butanol (2 9 200 ll). The n-butanol phases were collected by centrifugation and used directly for TLC analysis. The effect of temperature on enzymatic activity was tested after incubation of the YjiC1 enzyme in 50 mmol l1 Tris-HCl (pH 80) buffer at various temperatures ranging from 15 to 50°C for 20 min. To determine optimal pH for the YjiC1, ginsenoside Rh1 glycosylation activity was studied at optimal temperature 35°C in each buffer at various pH (30 to 100) for 20 min. The effect of metals on YjiC1 activity was tested in the presence of 5 mmol l1 (final concentration) NaCl, KCl, MgCl2.6H2O, CaCl2, MnSO4.H2O, CoCl2.6H2O, FeSO4.7H2O, CuSO4.5H2O, or ZnSO4.H2O for 20 min at optimal temperature 35°C and pH 70. Preparation and purification of the reaction product The reaction mixture (20 ml) for YjiC1 contained 1 mg of the purified enzyme, 25 mmol l1 UDP-glucose, 05 mmol l1 ginsenoside Rh1 and 5 mmol l1 Mn2+. The reaction mixture was incubated at 35°C for 05 h


and extracted three times with n-butanol, and then centrifuged to remove the proteins. The supernatant was concentrated to dryness. A yellow crude extract (30 mg) was obtained and dissolved in methanol. The crude extract was isolated on a silica gel G column (1 g) using chloroform : methanol : H2O (15 : 3 : 1, v/v/v) as the solvent, obtaining three main fractions A1-A3. Fraction A2 (12 mg) was chromatographed over a Sephadex LH20 column (20 g) with methanol to yield reaction product (1, 7 mg). Nucleotide sequence accession numbers The nucleotide sequence data reported in this paper were deposited in GenBank with accession numbers were yjiC1 (JX982974), yojK1 (JX982975) and ydhE1 (JX982973). Acknowledgements The work was supported by the ‘973’ Program of China (2013CB127505, 2012CB722208) and the NSFC (31360028), the Young Academic and Technical Leader of Yunnan Province (2010CI023), the Natural Science Foundation of Yunnan Province (2013FB003) and Program of Tobacco Corporation (HYHH2012CL04). Conflict of Interest No conflict of interest is declared. References Barbe, V., Cruveiller, S., Kunst, F., Lenoble, P., Meurice, G., Sekowska, A., Vallenet, D., Wang, T. et al. (2009) From a consortium sequence to a unified sequence: the Bacillus subtilis 168 reference genome a decade later. Microbiology 155, 1758–1775. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248–254. Hong, H., Cui, C.H., Kim, J.K., Jin, F.X., Kim, S.C. and Im, W.T. (2012) Enzymatic biotransformation of ginsenoside Rb1 and gypenoside XVII into ginsenosides Rd and F2 by recombinant b-glucosidase from Flavobacteriumjohnsoniae. J Ginseng Res 36, 418–424. Hu, Y. and Walker, S. (2002) Remarkable structural similarities between diverse glycosyltransferases. Chem Biol 9, 1287–1296. Jiang, K.Y. and Qian, Z.N. (1995) Effects of Panax notoginseng saponins on posthypoxic cell damage of neurons in vitro. Acta Pharmacol Sin 16, 399–402. Jorasch, P., Wolter, F.P., Z€ahringer, U. and Heinz, E.A. (1998) A UDP glucosyltransferase from Bacillus subtilis


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Supporting Information Additional Supporting Information may be found in the online version of this article: Data S1 Supporting information.


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