Efficient induction of ginsenoside biosynthesis and alteration of ginsenoside heterogeneity in cell cultures of Panax notoginseng by using chemically synthesized 2-hydroxyethyl jasmonate

Chemically synthesized 2-hydroxyethyl jasmonate (HEJA) was for the first time employed to induce the ginsenoside biosynthesis and to manipulate the product heterogeneity in plant cell cultures. The dose response and timing of HEJA elicitation were investigated in cell suspension cultures of Panax notoginseng. The optimal concentration and timing of HEJA addition for both cell growth and ginsenoside accumulation was identified to be 200 μM added on day 4. It was interestingly found that HEJA could stimulate ginsenosides biosynthesis and change their heterogeneity more efficiently than methyl jasmonate (MJA), i.e., the total ginsenoside content and the Rb/Rg ratio increased about 60 and 30% with HEJA elicitation than that by MJA, respectively. The activity of Rb₁ biosynthetic enzyme, i.e., UDPG-ginsenoside Rd glucosyltransferase (UGRdGT), was also higher in the former case. A maximal production titer of ginsenoside Rg₁, Re, Rb₁, and Rd was 47.4±4.8, 52.3±4.4, 190±18, and 12.1±2.5 mg/l with HEJA elicitation, which was about 1.3-, 1.3-, 1.7-, and 2.1-fold than that using MJA, respectively. Early signal events in plant defense response, including oxidative burst and jasmonic acid (JA) biosynthesis, were also examined. Levels of H₂O₂ and NO in medium and l-phenylalanine ammonia lyase activity in cells were not affected by addition of MJA and HEJA. On the other hand, the JA content in cells was increased with external jasmonates elicitation, and it was inhibited with the addition of JA biosynthesis inhibitors. The results suggest that oxidative burst might not be involved in the jasmonates-elicited signal transduction pathway, and MJA and HEJA may induce the ginsenoside biosynthesis via induction of endogenous JA biosynthesis and key enzymes (such as UGRdGT) in the ginsenoside biosynthetic pathway of P. notoginseng cells. The information is useful for hyperproduction of plant-specific heterogeneous products.     

Efficient elicitation of ginsenoside biosynthesis in cell cultures ofPanax notoginseng by using self-chemically-synthesized jasmonates

A series of fluorine and hydroxyl containing jasmonate derivatives, which were chemically synthesized in our institute, were investigated for their effects on the biosynthesis and heterogeneity of ginsenosides in suspension cultures ofPanax notoginseng cells. Compared to the control (without addition of elicitors), 100 μM of each of the jasmonate was added on day 4 to the suspension cultures ofP. notoginseng cells. It was observed that, jasmonates greatly enhanced the ginsenoside content and the ratio of Rb group to Rg group (i.e. (Rb₁+Rd)/(Rg₁+Re)) in theP. notoginseng cells. Some of the synthetic jasmonates, such as pentafluoropropyl jasmonate (PFPJA), 2-hydroxyethyl jasmonate (HEJA) and 2-hydroxyethoxyethyl jasmonate (HEEJA), could promote the ginsenoside content to 2.55±0.11, 3.65±0.13 and 2.94±0.06 mg/100 mg DW, respectively, compared to that of 0.64±0.06 mg/100 mg DW for the control and 2.17±0.04 mg/100 mg DW by the commercially available methyl jasmonate (MJA); and they could change the respective Rb: Rg ratio to 1.60±0.04, 1.87±0.01 and 1.56±0.05, compared to that of 0.47±0.01 for the control and 1.42±0.06 by MJA. The results suggest that suitable esterification of MJA with fluorine or hydroxyl group could increase the elicitation activity to induce plant secondary metabolism. The information obtained from this study is useful for hyper-production of heterogeneous products by plant cell cultures.     

Manipulation of ginsenoside heterogeneity of <Emphasis Type="Italic">Panax notoginseng</Emphasis> cells in flask and bioreactor cultivations with addition of phenobarbital

Structure-similar ginsenosides have different or even totally opposite biological activities, and manipulation of ginsenoside heterogeneity is interesting and significant to biotechnological application. In this work, addition of 1 mM phenobarbital to cell cultures of Panax notoginseng at a relatively high inoculation size of 7.6 g dry cell weight (DW)/L enhanced the production of protopanaxatriol-type (Rg₁ + Re) ginsenosides in both shake flask and airlift bioreactor (ALR, 1 L working volume). The content of Rg₁ + Re in the ALR was increased from 42.5 ± 4.0 mg per gram DW in untreated cell cultures (control) to 56.4 ± 4.6 mg per gram DW with addition of 1.0 mM phenobarbital. The maximum productivity of Rg₁ + Re in the ALR reached 5.66 ± 0.38 mg L⁻¹ d⁻¹, which was almost 3.3-fold that of control. The maximum ratio of the detectable ginsenosides protopanaxatriol:protopanaxadiol (Rb₁) was 7.6, which was about twofold that of control. The response of protopanaxadiol 6-hydroxylase (P6H) activity to phenobarbital addition coincided with the above-mentioned change of ginsenoside heterogeneity (distribution). Phenobarbital addition is considered as a useful strategy for manipulating the ginsenoside heterogeneity in bioreactor with enhanced biosynthesis of protopanaxatriol by P. notoginseng cells.     

High Density Cultivation of Panax notoginseng Cell Cultures with Methyl Jasmonate Elicitation in a Centrifugal Impeller Bioreactor

True ginseng roots contain “active compounds” called ginsenosides. The enhanced production of useful bioactive ginsenosides by high‐density cell cultures of Panax notoginseng in a self‐developed centrifugal impeller bioreactor (CIB) was achieved by adding methyl jasmonic acid (MJA) during cultivation. The production of the major, individual ginsenosides Rg₁, Re and Rb₁ was significantly enhanced in both 3‐L and 30‐L CIBs. The production titer of Rg₁, Re and Rb₁ ginsenosides in the 30‐L CIB was improved from 42 ± 8, 42 ± 9 and 41 ± 6 mg/L without MJA elicitation, to 104 ± 6, 71 ± 5 and 95 ± 6 mg/L with MJA elicitation, respectively. The ratio of Rb/Rg was slightly improved by MJA treatment in a 3‐L CIB but no apparent difference was observed in a 30‐L CIB. This work is useful for the understanding of the effects of large‐scale production on the individual ginseng saponins produced by plant cell cultures     

Co-transformation of <Emphasis Type="Italic">Panax</Emphasis> major ginsenosides Rb<Subscript>1</Subscript> and Rg<Subscript>1</Subscript> to minor ginsenosides C–K and F<Subscript>1</Subscript> by <Emphasis Type="Italic">Cladosporium cladosporioides</Emphasis>

Rb₁ and Rg₁ are the major ginsenosides in protopanaxadiol and protopanaxatriol. Their content in ginsenosides was 23.8 and 17.6%, respectively. A total of 22 isolates of β-glucosidase producing microorganisms were isolated from the soil of a ginseng field using Esculin-R2A agar. Among these isolates, the strain GH21 showed the strongest activities to convert ginsenoside Rb₁ and Rg₁ to minor ginsenosides compound-K and F₁, respectively. Ginsenosides Rb₁ and Rg₁ bioconversion rates were 74.2 and 89.3%, respectively. Meanwhile, the results demonstrated that the ginsenoside Rg₁ could change the biotransformation pathway of ginsenoside Rb₁ by inhibiting the formation of the intermediate metabolite gypenoside-XVII. GH21 was identified as a Cladosporium cladosporioides species based on the internal transcribed spacers (ITS) ITS1-5.8S-ITS2 rRNA gene sequences constructed phylogenetic trees.     

Methyl jasmonate elicitation enhanced synthesis of ginsenoside by cell suspension cultures of Panax ginseng in 5-l balloon type bubble bioreactors

The effects of methyl jasmonate (MJ) elicitation on the cell growth and accumulation of ginsenoside in 5-l bioreactor suspension cultures of Panax ginseng were investigated. Ginsenoside accumulation was enhanced by elicitation by MJ (in the range 50–400 M); however, fresh weight, dry weight and growth ratio of the cells was strongly inhibited by increasing MJ concentration. The highest ginsenoside yield was obtained at 200 M MJ. In the second experiment, 200 M MJ was added on day 15 during the cultivation. The ginsenoside, Rb group, and Rg group ginsenoside content increased 2.9, 3.7, and 1.6 times, respectively, after 8 days of MJ treatment. Rb group gisnsenosides accumulated more than Rg group ginsenosides. Among Rb group ginsenosides, Rb1 content increased significantly by four times but the contents of Rb2, Rc and Rd increased only slightly. Among Rg group ginsenosides, Rg1 and Re showed 2.3-fold and 3.0-fold increments, respectively, whereas there was only a slight increment in Rf group ginsenosides. These results suggest that MJ elicitation is beneficial for ginsenoside production using 5-l bioreactor cell suspension cultures.     

Ginsenoside Rd production from the major ginsenoside Rb<Subscript>1</Subscript> by <Emphasis Type="Italic">β</Emphasis>-glucosidase from <Emphasis Type="Italic">Thermus caldophilus</Emphasis>

Under optimum conditions (pH 5, 75°C, and 0.2 U purified enzyme ml⁻¹), 4 mg ginsenoside Rd was produced from 5 mg reagent-grade ginsenoside Rb₁ in 5 ml after 30 min by β-glucosidase from Thermus caldophilus GK24. Using a ginseng root extract containing 1 mg ginsenoside Rb₁ ml⁻¹ and 3.2 mg additional ginsenosides ml⁻¹, 1.23 mg ginsenoside Rd ml⁻¹ was produced after 18 h; the concentrations of ginsenosides Rb₁, Rb₂, and Rc used for ginsenoside Rd production were 0.77, 0.17, and 0.19 mg ml⁻¹, respectively.     

Growth and biosynthetic characteristics of ginseng (Panax japonicus var. repens) deep-tank cell culture in bioreactors

The aim of the work was to study the growth characteristics of cultured cells of Panax japonicus var. repens, an endemic plant of the Primorski Krai of Russia, grown in laboratory bioreactors and to determine the content of basic ginsenosides under these conditions. An increase of the inoculum size of the culture produced higher biomass accumulation and economic coefficient but slightly reduced the specific growth rate. An increase in the auxin concentration in a medium by adding 2,4-D practically did not affect growth characteristics of the culture but significantly reduced the size of cell aggregates. In all treatments tested, all major ginsenosides (Rb₁, Rc, Rb₂, Rd, Rf, Rg₁, and Re) were found in the culture. The total ginsenoside content was 2–3% per biomass dry weight. Meantime, ginsenosides of the Rg-series with protopanaxatriol as aglycone prevailed (70% of the total ginsenoside content). The culture conditions considerably affected the ratio of individual ginsenosides. In 2,4-D-containing medium, the preferential synthesis of Re ginsenoside was observed while both Rg₁ and Re were synthesized in other treatments.     

A new ginsenosidase from Aspergillus strain hydrolyzing 20-O-multi-glycoside of PPD ginsenoside

A ginsenosidase specifically hydrolyzing multi-20-O-glycosides of protopanaxadiol type ginsenosides such as ginsenoside Rb₁, Rb₃, Rb₂ and Rc, named ginsenosidase type II, was isolated and purified from Aspergillus sp.g48p strain. The molecular weight of the enzyme was 60 kDa. Ginsenosidase type II was demonstrated to hydrolyze multi-20-O-glycoside of protopanaxadiol type ginsenoside Rb₁, Rb₃, Rb₂ and Rc; i.e. the ginsenosidase type II hydrolyzes 20-O-β-glucoside of the ginsenoside Rb₁, 20-O-β-xyloside of ginsenoside Rb₃, 20-O-α-arabinoside(p) of ginsenoside Rb₂ and α-arabinoside(f) of ginsenoside Rc to produce mainly ginsenoside Rd, and small amount of Rg₃. However, it did not hydrolyze 3-O-β-glucosides of ginsenoside Rb₁, Rb₃, Rb₂ and Rc which was different with the ginsenosidase type I previously reported, either did not hydrolyze the glycosides of protopanaxatriol type ginsenoside such as ginsenoside Re, Rf and Rg₁, showing significant difference from all previously described glycosidases.     

Ginsenoside F1 production from ginsenoside Rg1 by a purified β-glucosidase from Fusarium moniliforme var. subglutinans

Fusarium moniliforme var. subglutinans was selected from among 100 strains of fungi for producing ginsenoside F₁ from ginsenoside Rg₁. The enzyme responsible was purified as a single 85 kDa band with a specific activity of 136 U mg⁻¹. It hydrolysed glucose-linked ginsenosides Rb₁, Rd and Rg₁ but not for other monosaccharide-linked ginsenosides, Rb₂, Rc, R₁, and Re. Under the optimum conditions of pH 6.0, 50°C, 30 U l⁻¹ of enzyme, and 5 mg Rg₁ ml⁻¹, 4 mg F₁ ml⁻¹ was produced after 4 h, with a molar yield of 100% and a productivity of 1 g l⁻¹ h⁻¹. This represents the highest productivity and conversion yield of F₁ yet reported.     

Influences of polyunsaturated fatty acids (PUFAs) on growth and secondary metabolite accumulation in Panax ginseng C.A. Meyer adventitious roots cultured in air-lift bioreactors

The present study relates to different polyunsaturated fatty acids (PUFAs) which were used as elicitors to enhance biomass accumulation and ginsenoside production in Panax ginseng. Adventitious root cultures of ginseng were elicited with oleic and linolenic acid at 0, 1, 5, 10 or 50 µmol/l concentrations respectively. Elicitors were added to the medium of adventitious roots on the 40th day of culture and roots were harvested on day 47. Cultures supplemented with oleic acid decreased root biomass and ginsenoside accumulation. Cultures supplemented with 1 µmol/l linolenic acid enhanced ginsenoside accumulation, without the decrease of adventitious root biomass. Linolenic acid enhanced the biosynthesis of both protopanxatriols (2.95 ± 0.048 mg/g DW) and protopanxadiols (5.66 ± 0.043 mg/g DW) compared to that of control at (1.41 ± 0.002 mg/g DW) and (1.58 ± 0.006 mg/g DW) respectively. No changes in polysaccharides and phenolics content have been noticed upon elicitation with PUFAs. This is the first report on linolenic acid as an elicitor for ginsenoside accumulation in ginseng adventitious root cultures.     

Autotoxic Ginsenosides in the Rhizosphere Contribute to the Replant Failure of Panax notoginseng

MethodsThe autotoxicities were measured using seedling emergence bioassays and root cell vigor staining. The ginsenosides in the roots, soils, and root exudates were identified with HPLC-MS.ResultsThe seedling emergence and survival rate decreased significantly with the continuous number of planting years from one to three years. The root exudates, root extracts, and extracts from consecutively cultivated soils also showed significant autotoxicity against seedling emergence and growth. Ginsenosides, including R₁, Rg₁, Re, Rb₁, Rb₃, Rg₂, and Rd, were identified in the roots and consecutively cultivated soil. The ginsenosides, Rg₁, Re, Rg₂, and Rd, were identified in the root exudates. Furthermore, the ginsenosides, R₁, Rg₁, Re, Rg₂, and Rd, caused autotoxicity against seedling emergence and growth and root cell vigor at a concentration of 1.0 µg/mL.ConclusionOur results demonstrated that autotoxicity results in replant failure of Sanqi ginseng. While Sanqi ginseng consecutively cultivated, some ginsenosides can accumulate in rhizosphere soils through root exudates or root decomposition, which impedes seedling emergence and growth.     

Effects of ginsenosides on carbachol-stimulated formation of inositol phosphates in rat cortical cell cultures

We examined the effect of ginseng total saponins (GTS) on phosphoinositide metabolism stimulated by activation of muscarinic receptor using rat cortical cultures. Carbachol stimulated formation of [³H]inositol phosphates ([³H]InsPs) by 3.3-fold over basal level in [³H]inositol-prelabeled cells. Pretreatment of GTS inhibited formation of [³H]InsPs evoked by carbachol by 70%–90%. Addition of GTS alone had no effect on the basal formation of [³H]InsPs. The inhibitory effect of the GTS on carbachol-stimulated formation of [³H]InsPs was dose- and time-dependent. IC₅₀ was 6.0 ± 2.8 g/ml. We also examined the effect of GTS on [³H]InsP₁, [³H]InsP₂, or [³H]InsP₃ formation evoked by carbachol. Although GTS had no effect on the basal [³H]InsP₁, [³H]InsP₂, or [³H]InsP₃ formation, pretreatment of GTS inhibited [³H]InsP₁, [³H]InsP₂, or [³H]InsP₃ formation evoked by carbachol, respectively. Addition of individual ginsenosides such as ginsenoside Rb₁, Rc, Rd, Re, or Rg₂ had no effect on the basal formation of [³H]InsPs, whereas pretreatment of ginsenoside Rb₂, Rc, Rd, Re, Rf, Rg₁ or Rg₂ inhibited formation of [³H]InsPs evoked by carbachol by 79%–89%. The results suggest that the inhibitory effect of GTS and its individual ginsenosides on carbachol-stimulated formation of [³H]InsPs in cortical neurons could be one pharmacological action of Panax ginseng.     

An endophytic bacterium isolated from Panax ginseng C.A. Meyer enhances growth,reduces morbidity,and stimulates ginsenoside biosynthesis

Ginseng (Panax ginseng C.A. Meyer) is known for its therapeutically useful ginsenosides that have anticancer and other pharmacological effects. However, its low levels in plants and the high costs of chemical synthesis make ginsenosides commercially non-viable; as such, strategies for increasing ginsenoside yield are of great interest. The present study reports the isolation of eight novel endophytic bacteria from ginseng leaves, the highest ginsenoside concentration of microbial transformed strain was identified as Paenibacillus polymyxa. Inoculation of ginseng plants with P. polymyxa by foliar application combined with irrigation enhanced plant growth parameters, reduced morbidity, and increased plant concentration of the ginsenosides (Rg₁, Re, Rf, Rb₁, Rg₂, Rb₂, Rb₃, and Rd) in field experiments. These results indicate that P. polymyxa isolated from ginseng is a beneficial endophytic bacterium with biocontrol properties that can enhance the yield and quality of this medicinal plant.     

Bioconversion of ginsenosides Rb(1), Rb(2), Rc and Rd by novel β-glucosidase hydrolyzing outer 3-O glycoside from Sphingomonas sp. 2F2: cloning, expression, and enzyme characterization

A new β-glucosidase gene (bglSp) was cloned from the ginsenoside converting Sphingomonas sp. strain 2F2 isolated from the ginseng cultivating filed. The bglSp consisted of 1344 bp (447 amino acid residues) with a predicted molecular mass of 49,399 Da. A BLAST search using the bglSp sequence revealed significant homology to that of glycoside hydrolase superfamily 1. This enzyme was overexpressed in Escherichia coli BL21 (DE3) using a pET21-MBP (TEV) vector system. Overexpressed recombinant enzymes which could convert the ginsenosides Rb₁, Rb₂, Rc and Rd to the more pharmacological active rare ginsenosides gypenoside XVII, ginsenoside C-O, ginsenoside C-Mc₁ and ginsenoside F₂, respectively, were purified by two steps with Amylose-affinity and DEAE-Cellulose chromatography and characterized. The kinetic parameters for β-glucosidase showed the apparent K_m and V_max values of 2.9 ± 0.3 mM and 515.4 ± 38.3 μmol min⁻¹ mg of protein⁻¹ against p-nitrophenyl-β-d-glucopyranoside. The enzyme could hydrolyze the outer C3 glucose moieties of ginsenosides Rb₁, Rb₂, Rc and Rd into the rare ginsenosides Gyp XVII, C-O, C-Mc₁ and F₂ quickly at optimal conditions of pH 5.0 and 37 °C. A little ginsenoside F₂ production from ginsenosides Gyp XVII, C-O, and C-Mc₁ was observed for the lengthy enzyme reaction caused by the side ability of the enzyme.     

Conversion of Major Ginsenoside Rb1 to Ginsenoside F2 by Caulobacter leidyia

Ginsenoside Rb₁ is the most predominant ginsenoside in Panax species (ginseng) and the hydrolysis of this ginsenoside produces pharmaceutically active compounds. Caulobacter leidyia GP45, one of the isolates having strong β-glucosidase-producing activity, converted ginsenoside Rb₁ to the active metabolites by 91%. The structures of the resultant metabolites were identified by NMR. Ginsenoside Rb₁ had been consecutively converted to ginsenoside Rd (1), F₂ (2) and compound K (3) via the hydrolyses of 20-C β-(1→6)-glucoside, 3-C β-(1→2)-glucoside, and 3-C β-glucose of ginsenoside Rb₁.     

Bioconversion of ginsenoside Rc into Rd by a novel α-l-arabinofuranosidase, Abf22-3 from Leuconostoc sp. 22-3: cloning, expression, and enzyme characterization

A novel α-l-arabinofuranosidase (Abf22-3) that could biotransform ginsenoside Rc into Rd was obtained from the ginsenoside converting Leuconostoc sp. strain 22-3, isolated from the Korean fermented food kimchi. The gene, termed abf22-3, consisting of 1,527 bp and encoding a protein with a predicted molecular mass of 58,486 Da was cloned into the pMAL-c2x (TEV) vector. A BLAST search using the Abf22-3’s amino acid sequence revealed significant homology to that of family 51 glycoside hydrolases. The over-expressed recombinant Abf22-3 in Escherichia coli BL21 (DE3) catalyzed the hydrolysis of the arabinofuranoside moiety attached to the C-20 position of ginsenoside Rc under optimal conditions of pH 6.0 and 30 °C. This result indicated that Abf22-3 selectively converts ginsenoside Rc into Rd, but did not catalyze the hydrolysis of glucopyranosyl groups from Rc or other ginsenosides such as Rb₁ and Rb₂. Over-expressed recombinant enzymes were purified by two steps with amylose-affinity and DEAE-cellulose chromatography and then characterized. The kinetic parameters for α-l-arabinofuranosidase showed apparent K_m and V_max values of 0.95 ± 0.02 μM and 1.2 ± 0.1 μmol min^?1 mg of protein^?1 against p-nitrophenyl-α-l-arabinofuranoside, respectively. Using a purified MBP–Abf22-3 (10 μg/ml), 0.1 % of ginsenoside Rc was completely converted to ginsenoside Rd within 20 min.     

A novel synthetic fluoro-containing jasmonate derivative acts as a chemical inducing signal for plant secondary metabolism

A novel fluoro-containing jasmonate derivative was chemically synthesized and evaluated as a potential elicitor with respect to the induction of plant defense responses and the biosynthesis of plant secondary metabolites. A bioactive taxuyunnanine C (Tc)-producing cell line of Taxus chinensis was taken as a model plant cell system. The presence of novel synthesized pentafluoropropyl jasmonate (PFPJA) induced two early and important events in plant defense responses, including an oxidative burst and activation of l-phenylalanine ammonia lyase. In addition, PFPJA was found to significantly increase Tc accumulation, without any inhibition of cell growth. Moreover, Tc accumulation was increased more in the presence of PFPJA compared with methyl jasmonate (MJA) and previously reported trifluoroethyl jasmonate (TFEJA). For example, addition of 100 M PFPJA on day 7 led to a high Tc content (38.2±0.3 mg/g) at day 21, while the Tc content was 29.3±0.3 mg/g and 34.9±0.9 mg/g with the addition of 100 M MJA and TFEJA, respectively. Quantitative structure–activity analysis of fluoro-containing jasmonates suggests that the increase in the fluoro-groups introduced into the carboxyl side-chain of MJA resulted in a higher stimulatory activity for Tc biosynthesis, which corresponds well with the markedly increased lipophilicity after fluorine introduction. These results indicate that newly synthesized fluoro-containing PFPJA can act as a powerful chemical inducing signal for secondary metabolism in plant cell cultures.     

Highly selective biotransformation of ginsenoside Rb<Subscript>1</Subscript> to Rd by the phytopathogenic fungus<Emphasis Type="Italic"> Cladosporium fulvum</Emphasis> (<Emphasis Type="Italic">syn</Emphasis>.<Emphasis Type="Italic"> Fulvia fulva</Emphasis>)

Fourteen phytopathogenic fungi were tested for their ability to transform the major ginsenosides to the active minor ginsenoside Rd. The transformation products were identified by TLC and HPLC, and their structures were assigned by NMR analysis. Cladosporium fulvum, a tomato pathogen, was found to transform major ginsenoside Rb₁ to Rd as the sole product. The following optimum conditions for transforming Rd by C. fulvum were determined: the time of substrate addition, 24 h; substrate concentration, 0.25 mg ml⁻¹; temperature, 37°C; pH 5.0; and biotransformation period, 8 days. At these optimum conditions, the maximum yield was 86% (molar ratio). Further, a preparative scale transformation with C. fulvum was performed at a dose of 100 mg of Rb₁ by a yield of 80%. This fungus has potential to be applied on the preparation for Rd in pharmaceutical industry.     

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

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 GST·bind 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., Rb₁ and Rd, gypenoside XVII) to produce protopanaxadiol via gypenoside LXXV, F₂, and Rh₂(S) with various pathways. BglAm can effectively transform the ginsenoside Rb₁ to gypenoside XVII and Rd to F₂; the K _m values of Rb₁ 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 Rg₁ into Rg₂(S) and Rh₁(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.