Bioconversion of major ginsenosides Rg(1) to minor ginsenoside F(1) using novel recombinant ginsenoside hydrolyzing glycosidase cloned from Sanguibacter keddieii and enzyme characterization

This study focuses on the cloning, expression, and characterization of recombinant ginsenoside hydrolyzing glycosidase from Sanguibacter keddieii in order to biotransform ginsenosides efficiently. The gene, termed bglSk, consists of 1857bp and revealed significant homology to that of glycoside hydrolase family 3. The enzyme was over-expressed in Escherichia coli BL21 (DE3) using a GST-fused pGEX 4T-1 vector system. The over-expressed recombinant enzymes could convert six major ginsenosides Rb(1), Rb(2), Rc, Rd, Re and Rg(1) into more pharmacologically active rare ginsenosides such as C-Y, C-Mc, C-K, Rg(2)(S), and F(1). Especially, BglSk could completely convert the Rg(1) into F(1). The GST-fused BglSk was purified with GST·bind agarose resin and then characterized. The kinetic parameters for β-glucosidase had apparent K(m) values of 0.456±0.009 and 0.167±0.003mM and V(max) values of 30.2±0.7 and 4.1±0.1μmolmin(-1)mg of protein(-1) against p-nitrophenyl-β-d-glucopyranoside and Rb(1), respectively.     

Isolation and characterization of novel ginsenoside-hydrolyzing glycosidase from Microbacterium esteraromaticum that transforms ginsenoside Rb2 to rare ginsenoside 20(S)-Rg3

Ginsenoside Rb2 was transformed by recombinant glycosidase (Bgp2) into ginsenosides Rd and 20(S)-Rg3. The bgp2 gene consists of 2,430 bp that encode 809 amino acids, and this gene has homology to the glycosyl hydrolase family 2 protein domain. SDS-PAGE was used to determine that the molecular mass of purified Bgp2 was 87 kDa. Using 0.1 mg ml^?1 of enzyme in 20 mM sodium phosphate buffer at 40 °C and pH 7.0, 1.0 mg ml^?1 ginsenoside Rb2 was transformed into 0.47 mg ml^?1 ginsenoside 20(S)-Rg3 within 120 min, with a corresponding molar conversion yield of 65 %. Bgp2 hydrolyzed the ginsenoside Rb2 along the following pathway: Rb2 → Rd → 20(S)-Rg3. This is the first report of the biotransformation of ginsenoside Rb2 to ginsenoside 20(S)-Rg3 using the recombinant glycosidase.     

Purification of (S)-3-cyano-5-methylhexanoic acid from bioconversion broth using an acetone/ammonium sulfate aqueous two-phase system

(S)-3-Cyano-5-methylhexanoic acid ((S)-CMHA) is the key chiral intermediate of pregabalin. In this paper, an aqueous two-phase system (ATPS) was developed to extract (S)-CMHA from nitrilase-catalyzed bioconversion broth. Inorganic salts and hydrophilic solvents were screened to form ATPS, among which an acetone/ammonium sulfate ATPS was investigated in detail, including phase diagram, effect of phase composition and stability of (S)-CMHA. The maximum product recovery of 99.15% was obtained by an optimized ATPS system composed of 15% (w/w) ammonium sulfate and 35% (w/w) acetone with the removal of 99% cells and 86.27% proteins. The total (S)-CMHA yield reached 92.11% after back-extraction. The recycling use of ammonium sulfate was investigated, and 93.10% of salt in the salt-rich phase was recovered with the addition of methanol. The results demonstrated the efficiency of the two-step extraction process for separation of (S)-CMHA.     

Simple and efficient preparation of ginsenoside (S)-Rg2 from ginsenoside Re by biotransformation with Cellulosimicrobium sp. 21

A novel ginsenoside-hydrolyzing strain was isolated from ginseng-cultivation soil in Changbai Mountain (China). The strain was identified as Cellulosimicrobium sp. 21 by 16S rDNA sequencing. Using the β-glucosidases secreted from Cellulosimicrobium sp. 21, protopanaxatriol-type ginsenoside Re was converted to the highly active neuroprotective molecule (S)-Rg2 by removal of the C-20-glucopyranosyl residue. The α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranose at the C-6 position of Rg2 was not further attacked by Cellulosimicrobium sp. 21, so the transformation shows high specificity. To simplify the transformation and product-preparation process, a simple and efficient transformation system was developed in a phosphate buffer system instead of organic media. The optimum conditions for transforming ginsenoside Re into Rg2 by Cellulosimicrobium sp. 21 were determined through single-factor experiments and response surface methodology. Under the optimized conditions: transformation buffer, 50 mM phosphate buffer, at pH: 7.00; temperature: 27.6°C; substrate concentration: 0.50 mg/ml; biotransformation period: 12 h; the biotransformation efficiency reached 89.8% (molar ratio) in 2-L reaction system. This simple biotransformation with high specificity and efficiency has potential for use in Rg2 preparation in the pharmaceutical industry.     

Determination of major ginsenosides in Panax quinquefolius (American ginseng) using high-performance liquid chromatography

In order to determine the active ingredients in root extracts of Panax quinquefolius (American ginseng), a gradient HPLC method involving UV photodiode array detection was applied to separate and quantify simultaneously the ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf and Rg1. All ginseng saponins were baseline-resolved under the selected conditions, and the detection limits were 1.0 microg/mL or less. The method has been applied to analyse ginsenosides extracted from American ginseng cultivated in both Wisconsin and Illinois. Ginsenosides Re and Rb1 were the two main ginseng saponins in the root. The amounts of Re in 5- and 7-year Illinois-cultivated samples were greater than those found in ginseng cultivated for 3 or 4 years in Wisconsin, whereas the levels of Rb1 were greater in the younger Wisconsin samples.     

20(S)-Ginsenoside Rh2 as aldose reductase inhibitor from Panax ginseng

The root of Panax ginseng C. A. Meyer (Araliaceae) is a well-known herbal medicine in East Asia. The major bioactive metabolites in this root are commonly identified as ginsenosides. A series of ginsenosides were determined for in vitro human recombinant aldose reductase. This Letter aims to clarify the structural requirement for aldose reductase inhibition. We discovered that only ginsenoside 20(S)-Rh2 showed potent against aldose reductase, with an IC₅₀ of 147.3 μM. These results implied that the stereochemistry of the hydroxyl group at C-20 may play an important role in aldose reductase inhibition. An understanding of these requirements is considered necessary in order to develop a new type of aldose reductase inhibitor. Furthermore, P. ginseng might be an important herbal medicine in preventing diabetic complications.     

Structural modification of ginsenoside Rh(2) by fatty acid esterification and its detoxification property in antitumor

Ginsenoside Rh(2), one of the most important ginsenosides with anticancer properties in red ginseng, has been developed as principal antitumor ingredient for clinical use. However, the cytotoxicity test in human hepatocyte cell line QSG-7701 (IC(50) 37.3μM) indicated that Rh(2) might show strong cytotoxic side-effect on the normal liver cells. For blunting the toxicity, Rh(2) was structurally modified by reacting with octanoyl chloride to give a dioctanoyl ester of Rh(2) (D-Rh(2)) in the present study. MTT assay in QSG-7701 cell line in vitro showed that the cytotoxicity of D-Rh(2) on human hepatocyte cells (IC(50) 80.5μM) was significantly lower than that of Rh(2). While antitumor xenograft assay in mice bearing H22 liver cancer cells in vivo showed that the antitumor activity of D-Rh(2) retained to be strong as that of Rh(2). According to previous pharmacokinetic studies, the fatty acid esterification of Rh(2) might be of detoxification reaction to cells. Additionally, D-Rh(2) showed significant enhancement on increasing thymus index at the dose of 10mg/kg compared with vehicle treated control group. Thus, D-Rh(2) might indirectly affect tumor growth by stimulating lymphocytes to become cytotoxic to tumor cells. Finally, our findings suggested that D-Rh(2), the fatty acid ester of Rh(2), might attenuate the side-effect by detoxification to human normal cell and could be a more potential candidate for developing as an antitumor drug.     

Enzymatic preparation of 20(S, R)-protopanaxadiol by transformation of 20(S, R)-Rg3 from black ginseng

20(S)-protopanaxadiol (PPD(S)) and 20(R)-protopanaxadiol (PPD(R)), the main metabolites of ginsenosides Rg3(S) and Rg3(R) in black ginseng, are potential candidates for anti-cancer therapy due to their pharmacological activities such as anti-tumor properties. In the present study, we report the preparation of PPD(S, R) by a combination of steaming and biotransformation treatments from ginseng. Aspergillus niger was isolated from soil and showed a strong ability to transform Rg3(S, R) into PPD(S, R) with 100% conversion. Furthermore, the enzymatic reactions were analyzed by reversed-phase HPLC, showing the biotransformation pathways: Rg3(S) → Rh2(S) → PPD(S) and Rg3(R) → Rh2(R) → PPD(R), respectively. In addition, 12 ginsenosides including 3 pairs of epimers, namely Rg3(S), Rg3(R), Rh2(S), Rh2(R), PPD(S) and PPD(R), were simultaneously determined by reversed-phase HPLC. Our study may be highly applicable for the preparation of PPD(S) and PPD(R) for medicinal purposes and also for commercial use.     

20(S)-protopanaxadiol and the ginsenoside Rh2 inhibit Na+ channel-activated depolarization and Na+ channel-dependent amino acid neurotransmitter release in synaptic fractions isolated from mammalian brain

The ginsenoside Rh(2) and its aglycone 20(S)-protopanaxadiol are known to inhibit the binding of [(3)H]batrachotoxinin 20alpha-benzoate to site 2 on voltage-gated sodium channels and electrophysiological investigations conducted by others have shown that ginsenosides cause voltage-dependent inhibition of reconstituted forms of the sodium channel. Here we describe the actions of Rh(2) and 20(S)-protopanaxadiol on sodium channel function and release of neurotransmitters resulting from activation of native sodium channels in synaptic preparations isolated from whole mouse brain. Rh(2) and 20(S)-protopanaxadiol inhibited veratridine-dependent (tetrodotoxin-suppressible) depolarization of synaptoneurosomes as determined using the rhodamine 6G method although 20(S)-protopanaxadiol was more potent as an inhibitor than Rh(2). Veratridine- (sodium channel-) dependent release of the neurotransmitters L-glutamate and GABA was almost fully inhibited by 20(S)-protopanaxadiol, however, less complete inhibition was observed with Rh(2). At its maximum inhibitory concentration, Rh(2) also produced release of l-glutamate and GABA from synaptosomes, in contrast to 20(S)-protopanaxadiol. We conclude that low to moderate micromolar concentrations of Rh(2) and 20(S)-protopanaxadiol inhibit sodium channel function and sodium channel-activated release of neurotransmitters. Apparently the ginsenoside Rh(2) cannot achieve complete inhibition of sodium channel-activated transmitter release because at high concentrations it also stimulates release.     

Enhanced poly(3-hydroxybutyrate) production in transgenic tobacco BY-2 cells using engineered acetoacetyl-CoA reductase

Highly active mutant of NADPH-dependent acetoacetyl-CoA reductase (PhaB) was expressed in Nicotiana tabacum cv. Bright Yellow-2 cultured cells to produce poly(3-hydroxybutyrate) [P(3HB)]. The mutated PhaB increased P(3HB) content by three-fold over the control, indicating that the mutant was a versatile tool for P(3HB) production. Additionally, the PhaB-catalyzed reaction was suggested to be a rate-limiting step of P(3HB) biosynthesis in tobacco BY-2 cells.     

Preparation of Single-Enantiomer Biofunctional Molecules with (S)-2-Methoxy-2-(1-naphthyl)propanoic Acid

(RS)-β-Ionol and (RS)-2-methyl-4-octanol were resolved by using (S)-2-methoxy-2-(1-naphthyl)propanoic acid [(S)-MαNP acid]. The specific stereochemistry of each MαNP ester was elucidated by 2D NMR analyses, and shielding by the 1-naphthyl group was observed in both the ¹H- and ¹³C-NMR spectra. Solvolysis of the individual (S)-MαNP esters gave four single-enantiomer alcohols. The normal-phase HPLC elution order of each MαNP ester is also discussed.     

Conversion of major ginsenoside Rb1 to 20(S)-ginsenoside Rg3 by Microbacterium sp. GS514

Ginseng saponin, the most important secondary metabolite in ginseng, has various pharmacological activities. Many studies have been directed towards converting major ginsenosides to the more active minor ginsenoside, Rg3. Due to the difficulty in preparing ginsenoside Rg3 enzymatically, the compound has been mainly produced by either acid treatment or heating. A microbial strain GS514 was isolated from soil around ginseng roots in a field and used for enzymatic preparation of the ginsenoside Rg3. Blast results of the 16S rRNA gene sequence of the strain GS514 established that the strain GS514 belonged to the genus Microbacterium. Its 16S rRNA gene sequence showed 98.7%, 98.4% and 96.1% identity with those of M. esteraromaticum, M. arabinogalactanolyticum and M. lacticum. Strain GS514 showed a strong ability to convert ginsenoside Rb1 or Rd into Rg3. Enzymatic production of Rg3 occurred by consecutive hydrolyses of the terminal and inner glucopyranosyl moieties at the C-20 carbon of ginsenoside Rb1 showing the biotransformation pathway: Rb1-->Rd-->Rg3.     

Control of biogenic H(2)S production with nitrite and molybdate

The effects of the metabolic inhibitors, sodium nitrite and ammonium molybdate, on production of H₂S by a pure culture of the sulfate-reducing bacterium (SRB) Desulfovibrio sp. strain Lac6 and a consortium of SRB, enriched from produced water of a Canadian oil field, were investigated. Addition of 0.1 mM nitrite or 0.024 mM molybdate at the start of growth prevented the production of H₂S by strain Lac6. With exponentially growing cultures, higher levels of inhibitors, 0.25 mM nitrite or 0.095 mM molybdate, were required to suppress the production of H₂S. Simultaneous addition of nitrite and molybdate had a synergistic effect: at time 0, 0.05 mM nitrite and 0.01 mM molybdate, whereas during the exponential phase, 0.1 mM nitrite and 0.047 mM molybdate were sufficient to stop H₂S production. With an exponentially growing consortium of SRB, enriched from produced water of the Coleville oil field, much higher levels of inhibitors, 4 mM nitrite or 0.47 mM molybdate, were needed to stop the production of H₂S. The addition of these inhibitors had no effect on the composition of the microbial community, as shown by reverse sample genome probing. The results indicate that the efficiency of inhibitors in containment of SRB depends on the composition and metabolic state of the microbial community. Journal of Industrial Microbiology & Biotechnology (2001) 26, 350–355. Received 02 August 2000/ Accepted in revised form 17 April 2001     

A novel method of producing the key intermediate ASI-2 of ranirestat using a porcine liver esterase (PLE) substitute enzyme

(R)-2-amino-2-ethoxycarbonylsuccinimide (ASI-2) is a key intermediate used in the pharmaceutical industry and is valuable for the industrial synthesis of ranirestat, which is a potent aldose reductase inhibitor. ASI-2 was synthesized in a process combining chemical synthesis and bioconversion. Bioconversion in this study is a key reaction, since optically active carboxylic acid derivative ((R)-1-ethyl hydrogen 3-benzyloxycarbonylamino-3-ethoxycarbonylsuccinate, Z-MME-AE) is synthesized from a prochiral ester, diethyl 2-benzyloxycarbonylamino-2-ethoxycarbonylsuccinate, Z-MDE-AE, at a theoretical yield of 100%. Upon screening for microorganisms that asymmetrically hydrolyze Z-MDE-AE, Bacillus thuringiensis NBRC13866 was found. A novel esterase EstBT that produces Z-MME-AE was purified from Bacillus thuringiensis NBRC13866 and was stably produced in Escherichia coli JM109 cells. Using EstBT rather than porcine liver esterase (PLE), ASI-2 was synthesized with a 17% higher total yield by a novel method, suggesting that the esterase EstBT is a PLE substitute enzyme and therefore, may be of interest for future industrial applications.     

Enhanced production of recombinant rabies virus glycoprotein (rRVGP) by Drosophila melanogaster S2 cells through control of culture conditions

Culture conditions that affect product quality are important to the successful operation and optimization of recombinant protein production. The objective of this study was to optimize culture conditions for growth of recombinant Drosophila melanogaster S2 cells (S2AcRVGP) in order to enhance the production of rRVGP. The addition of DMSO and glycerol to the medium and growth at a reduced temperature (22 °C) were the culture condition variations selected to be tested. Experimental cultures were first performed in serum-free Sf900 II medium in 250 ml Schott flasks. The most promising conditions identified in these experiments were also tested on a higher scale in a 3l bioreactor. In the Schott flasks experiments, all the changes in culture conditions resulted in an increase of rRVGP production. The protein concentration was 3.6-fold higher with addition of 1% DMSO and 1% glycerol and 9.3-fold higher when the cells were cultured at 22 °C instead of the standard 28 °C. The maximum concentration of rRVGP reached was 591 μg l⁻¹. In bioreactor experiments, with control of pH at 6.20 and DO at 50%, the reduced culture temperature (22 °C) was the strategy that promoted the highest glycoprotein production, 928 μg l⁻¹.     

Inoculum size and auxin concentration influence the growth of adventitious roots and accumulation of ginsenosides in suspension cultures of ginseng ( Panax ginseng C.A. Meyer)

The effect of the root-inoculum size and axuin concentration on growth of adventitious roots and accumulation of ginsenosides were studied during suspension cultures of ginseng (Panax ginseng C.A. Meyer). Of the various concentrations of indole-3-butyric acid (IBA) and γ-naphthaleneacetic acid (NAA) used as supplementary growth regulators along with Murashige and Skoog medium, 25 μM IBA was found suitable for lateral root induction and growth, as well as accumulation of ginsenosides. Inoculum size of 5 g L⁻¹ was found suitable for optimal biomass (10.5 g L⁻¹ dry biomass) and ginsenosides (5.4 mg g⁻¹ DW) accumulation. Of the various length of root inocula tested (chopped to 1–3, 4–6, 7–10 mm and un-chopped), root inocula of 7–10 mm was found suitable for biomass and ginsenoside accumulation.     

A novel process for the production of high-purity galactooligosaccharides (GOS) using consortium of microbes

Galactooligosaccharides (GOS) are nondigestible dietary fibers which have a beneficial effect on human health by promoting the growth of probiotic bacteria in the gut. In addition, other health benefits have been reported from oligosaccharides consumption such as stimulation of intestinal mobility, colon cancer prevention, mineral absorption as well as protection against certain pathogenic bacterial infections. The goal of this research was to develop an efficient biotransformation system using a consortium of microbes for the production of ≥85% pure GOS and reusing the cell biomass in repeated cycles of biotransformation. Production of GOS by lactose transgalactosylation using whole cells of Sporobolomyces singularis MTCC 5491 as a source of β-galactosidase and monosaccharides utilization by yeast isolate (NUTIDY007) were studied. For increasing the purity of GOS, growth and bioconversion parameters on the transgalactosylation by the whole cells were investigated. Further, continuous production of GOS was studied in a reactor with microfiltration membrane system. A maximum GOS purity of 42% was achieved using single culture of S. singularis. Under optimized conditions, single culture of S. singularis produced a maximum of 56% pure GOS. Addition of second culture to the reaction mixture for utilization of glucose significantly increased the GOS purity from 56% to ≥85%. The product consisted of tri- to penta-galactooligosaccharides. Trisaccharides were the main component of the reaction mixture. A maximum productivity of 10.9?g/L/hr was obtained under the optimum conditions.     

Ginsenosides 20(S)-protopanaxadiol and Rh2 reduce cell proliferation and increase sub-G1 cells in two cultured intestinal cell lines, Int-407 and Caco-2

Ginsenosides derived from 20(S)-protopanaxatriol (PT) and 20(S)-protopanaxadiol (PD) groups had similar characteristic cytotoxic effects on the growth of two intestinal cells lines, Int-407 and Caco-2. Pure Rh2, a ginsenoside structurally related to PD, inhibited intestinal cell growth at greater than twice the concentration of PD, while Rh1, a ginsenoside structurally related to aglycone PT, had no cytotoxic effect. Concentrations causing growth inhibition of 50% of cells (LC50) for the compounds PD, PT, and Rh2 were 23, 26, and 53 microg/mL, respectively, for Int-407 cells. In comparison, the LC50 for PD and PT was determined to be 24 microg/mL, and that for Rh2 was 55 microg/mL in Caco-2 cells. A standardized North American ginseng extract with a known ginsenosides composition did not induce cytotoxicity in either of the intestinal cell lines. Cell cycle analysis showed characteristically different (P = 0.05) effects of ginsenosides PD, Rh2, and PT in both cell lines. Rh2 treatment of Int-407 caused a significantly (P = 0.05) higher production of sub-G1 (apoptotic) cells (35% +/- 1%) compared with untreated cells (14% +/- 0.3%) after 24 h. PD and Rh2 treatments were both significantly (P < 0.05) higher in apoptotic cells than in untreated cells after 48 and 72 h. Similar results were obtained for treatment of Caco-2 cells. Lactate dehydrogenase (LDH) activity in both cell lines was similar for PD and Rh2 and higher (P = 0.05) than for PT treatment at most time periods. These results show a specific structure-function relationship for bioactive ginsenosides in two contrasting intestinal cell types.     

Identifying two moth species (Lepidoptera: Ditrysia) from Saudi Arabia using mitochondrial 16S rRNA sequences

The mitochondrial genetic markers are considered useful tools for discrimination between more closely related lepidopteran taxa. Therefore, the present study aimed to investigate the role of mitochondrial (mt) 16 s rRNA gene in the determination of the taxonomic position for two moth species within Ditrysia clade. Maximum likelihood analysis has indicated a well-supported dendrogram based on the Tamura-Nei model for the recovered lepidopterans. The mt 16 s rRNA query sequences from 24 species within seven families were analyzed. This analysis and bootstrap confidence revealed two major clades representing Glossata suborder within Lepidoptera, with a close relationship of Noctuoidea + (Pyraloidea (Hesperioidea + Papilionoidea)). The subfamily Heliothinae forming a sister group with Risobinae (Noctinae + Hadeninae). In addition, there is a clear observation about the close relation between Phycitinae + Galleriinae within Pyraloidea and Cyrestinae + Limenitidinae within Papilionoidea. The present study supported that the Helicoverpa and Meroptera species are the first accounts of these genera inhabiting Saudi Arabia.     

Interaction of internal Ba2+ with a cloned Ca(2+)-dependent K+ (hslo) channel from smooth muscle

We have studied potassium currents through a cloned Ca(2+)-dependent K+ channel (hslo) from human myometrium. Currents were recorded in inside- out macropatches from membranes of Xenopus laevis oocytes. In particular, the inactivation-like process that these channels show at high positive potentials was assessed in order to explore its molecular nature. This current inhibition conferred a bell shape to the current- voltage curves. The kinetic and voltage dependence of this process suggested the possibility of a Ba2+ block. There were the following similarities between the inactivation process observed at zero-added Ba2+ and the internal Ba2+ block of hslo channels: (a) in the steady state, the voltage dependence of the current inhibition observed at zero-added Ba2+ was the same as the voltage dependence of the Ba2+ block; (b) the time constant for recovery from current decay at zero- added Ba2+ was the same as the time constant for current recovery from Ba2+ blockade; and (c) current decay was largely suppressed in both cases by adding a Ba2+ chelator [(+)-18-crown-6-tetracarboxylic acid] to the internal solution. In our experimental conditions, we determined that the Kd for the complex chelator-Ba2+ is 1.6 x 10(-10) M. We conclude that the current decay observed at zero-added Ba2+ to the internal solution is due to contaminant Ba2+ present in our solutions (approximately 70 nM) and not to an intrinsic gating process. The Ba2+ blocking reaction in hslo channels is bimolecular. Ba2+ binds to a site (Kd = 0.36 +/- 0.05 mM at zero applied voltage) that senses 92 +/- 25% of the potential drop from the internal membrane surface.