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.     

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.     

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.     

Comparison of ginsenoside composition in native roots and cultured callus cells of Panax quinquefolium L.

In order to compare the ginsenoside composition in native Panax quinquefolium and in suspension cultured cells derived from root callus, HPLC–ESI-MSⁿ analysis was performed. Under the present HPLC–ESI-MSⁿ conditions, ten ginsenosides from native root were acquired in the positive and negative ion modes, namely Rg₁, Re, Ro, malonyl-Rb₁, Rf, Rb₁, Rc, Rb₂, Rb₃ and Rd. Only four ginsenosides (Rg1, Re, Rf and Rb1) were identified from callus cells. Radical scavenging activity of P. quinquefolium callus cells with 250 mg l^?1 methanolic extract on 1,1-diphenyl-2-picrylhydrazyl (DPPH) was 55.72 %, while only 6.31 % DPPH inhibition was obtained in native root.     

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.     

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.     

Effect of sugar concentration on ginsenoside biosynthesis in hairy root cultures of Panax quinquefolium cultivated in shake flasks and nutrient sprinkle bioreactor

The study assessed the influence of sugar concentration (10, 20, 30, 50, 70, 100, 120 g l^?1) on growth and ginsenoside biosynthesis in Panax quinquefolium hairy roots cultivated in shake flasks and a nutrient sprinkle bioreactor. The highest growth rate was achieved in medium containing 3–5 % sucrose. More than 70 g l^?1 or less than 20 g l^?1 sugar content in the medium induces significant inhibition of root growth when cultivated in shake flasks. The saponin content was determined using HPLC. The maximum yield (above 9 mg g^?1 d.w.) of the sum of six examined ginsenosides (Rb₁, Rb₂, Rc, Rd, Re and Rg₁) in hairy roots cultivated in shake flasks was obtained with 30 g l^?1 sucrose in the medium. The sucrose concentration in the medium was found to correlate with saponin content in bioreactor-cultured specimens. A higher level of protopanaxadiol derivatives was found for lower (20 and 30 g l^?1) sucrose concentrations; higher sucrose concentrations (50 and 70 g l^?1) in the medium stimulated a higher level of Rg group saponins.     

Production of saponins from <Emphasis Type="Italic">Panax ginseng</Emphasis> suspension and adventitious root cultures

Biomass growth and ginsenoside production in cell suspension and adventitious roots of Panax ginseng C.A. Meyer cultures cultivated both in Erlenmayer flasks and a 3 dm³ bioreactor were studied. The maximum content of ginsenosides was found in the suspension culture cultivated in the bioreactor (4.34 % dry mass), however the saponin content was limited to two major ginsenosides, Rb₁ and Rg₁. The production of ginsenosides in adventitious roots was lower (1.45 or 1.72 % dry mass), nevertheless, the full range of ginsenosides was detected.This work was supported by 521/02/P064, COST 843.10, ME671 and Z4 055 905 projects.     

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.     

Enhancement of ginsenoside biosynthesis in high-density cultivation of Panax notoginseng cells by various strategies of methyl jasmonate elicitation

A single addition of 200 M methyl jasmonate (MJA) to high-density cell cultures of Panax notoginseng enhanced ginsenoside production in both shake-flask (250 ml) and airlift bioreactor (ALR; 1 l working volume). Repeated elicitation with two additions of 200 M MJA during cultivation further induced the ginsenoside biosynthesis in both cultivation vessels. The content of ginsenosides Rg₁, Re, Rb₁ and Rd in the ALR was increased from, respectively, 0.18±0.01, 0.21±0.01, 0.21±0.02 and 0 mg per100 mg dry cell weight (DW) in untreated cell cultures (control) to 0.32±0.02, 0.36±0.02, 0.72±0.06 and 0.08±0.01 mg per100 mg DW with a single addition of MJA and further increased to 0.43±0.02, 0.46±0.03, 1.09±0.07 and 0.14±0.02 mg per100 mg DW with two additions of MJA. Interestingly, the activity of the Rb₁ biosynthetic enzyme (UDPG-ginsenoside Rd glucosyltransferase), was also increased with a single elicitation by MJA and increased again by a repeated elicitation, which coincided well with the trend in the increase in Rb₁ content. In order to further improve the cell density and ginsenoside production, a strategy of MJA repeated elicitation combined with sucrose feeding was adopted. The final cell density and total ginsenoside content in the ALR reached 27.3±1.5 g/l and 2.02±0.06 mg per100 mg DW; and the maximum production of ginsenoside Rg₁, Re, Rb₁ and Rd was 111.8±4.7, 117.2±4.6, 290.2±5.1 and 32.7±8.1 mg/l, respectively. The strategies demonstrated and the information obtained in this work are useful for the efficient large-scale production of bioactive ginsenosides by plant cell cultures.     

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.     

The mechanism of acid-catalyzed conversion of ginsenoside Rf and two new 25-hydroxylated ginsenosides

Ginsenoside Rf is known to have higher chemical stability than other ginsenosides and until lately, the constituents in which it would convert were not known. Only in recent times, it was found that ginsenoside Rf converted to (20E)-Rg₉, (20Z)-Rg₉, Rg₁₀, and 20(R)-Rf. During my continued studies to update the chemical profile of red ginseng, two new ginsenosides converted from ginsenoside Rf, 25-hydroxylated ginsenosides, were discovered. These two new converted ginsenosides, namely (20E),25(OH)-ginsenoside Rg₉ (1), and (20Z),25(OH)-ginsenoside Rg₉ (2), together with ginsenosides (20E)-Rg₉ (3), (20Z)-Rg₉ (4), Rg₁₀ (5), and 20(R)-Rf (6) were isolated from a reaction mixture of ginsenoside Rf in an acid-catalyzed reaction. Their chemical structures (1 and 2) were elucidated by NMR and Mass spectral methods. Compounds 1 and 2 were presumably generated by hydration of (20E)-, and (20Z)-ginsenoside Rg₉. The presence of these six converted ginsenosides was confirmed by UPLC/TOF-MS method in red ginseng. On the basis of these results, I deduced the overall conversion mechanism of ginsenoside Rf and evaluated the significance of ginsenoside Rf as a characteristic mark substance of Panax ginseng.     

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     

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.     

Improvement of ginsenoside production by <Emphasis Type="Italic">Panax ginseng</Emphasis> adventitious roots induced by γ-irradiation

In order to evaluate effects of γ-rays on adventitious root formation and ginsenoside production, embryogenic calli induced from cotyledon explants of Panax ginseng C.A. Meyer were treated with γ-rays of 0, 10, 30, 50, 70, and 100 Gy. The highest frequency of adventitious root formation of 75 % occurred at γ-irradiation of 30 Gy, which is considered adequate dosage for selecting mutant cell lines. Five mutated adventitious roots (MAR)3-lines out of the propagation of 142 adventitious root lines treated with 30 Gy were selected based a 100-fold increase in proliferation rate compared to control adventitious roots (CAR) and content of the seven major ginsenosides (Rb₁, Rb₂, R_c, R_d, R_e, R_f, and Rg₁) was determined. In the CAR and four of the MAR3-lines (except for MAR3-109), the Rb/Rg ratio was greater than 1.0, thereby indicating altered ginsenoside composition in these root lines. The HPLC analysis of the MAR3-13 and MAR3-26 lines confirmed different ginsenoside profiles, including the three unidentified ginsenoside candidates, Gm₁, Gm₂, and Gm₃. The ginsenosides of the MAR3-13 and MAR3-26 lines showed high hydroxyl and superoxide radical scavenging activities.     

Quantitative Comparison of Ginsenosides and Polyacetylenes in Wild and Cultivated American Ginseng

Quantitative comparison of seven ginsenosides in wild and cultivated American ginseng revealed that the Rg₁/Rd ratio presented a significantly large difference between cultivated and type‐I (one of the defined chemotypes) wild American ginseng, facilitating this ratio as a characteristic marker for differentiating these two groups. Similarly, the ratio (Rg₁+Re)/Rd, and the ratio of protopanaxatriol (PPT)‐type ginsenosides to protopanaxadiol (PPD)‐type ginsenosides showed a large difference between these two groups. On the other hand, type‐II wild samples were found to have high Rg₁/Rb₁ and Rg₁/Re ratios and low panaxydol/panaxynol ratio, which is entirely different from Type‐I American ginseng, but is very similar to that of Asian ginseng. This not only suggests that the chemotype should be taken into consideration properly when using these parameters for differentiating American and Asian ginseng, but also indicates that type‐II wild American ginseng may have distinct pharmacological activities and therapeutic effects.     

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.     

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.     

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.     

β-Glucosidase from Penicillium aculeatum hydrolyzes exo-, 3-O-, and 6-O-β-glucosides but not 20-O-β-glucoside and other glycosides of ginsenosides

A novel β-glucosidase from Penicillium aculeatum was purified as a single 110.5-kDa band on SDS–PAGE with a specific activity of 75.4 U?mg^?1 by salt precipitation and Hi-Trap Q HP and Resource Q ion exchange chromatographies. The purified enzyme was identified as a member of the glycoside hydrolase 3 family based on its amino acid sequence. The hydrolysis activity for p-nitrophenyl-β-d-glucopyranoside was optimal at pH 4.5 and 70 °C with a half-life of 55 h. The enzyme hydrolyzed exo-, 3-O-, and 6-O-β-glucosides but not 20-O-β-glucoside and other glycosides of ginsenosides. Because of the novel specificity, this enzyme had the transformation pathways for ginsenosides: Rb₁?→?Rd?→?F₂?→?compound K, Rb₂?→?compound O?→?compound Y, Rc?→?compound Mc₁?→?compound Mc, Rg₃?→?Rh₂?→?aglycone protopanaxadiol (APPD), Rg₁?→?F₁, and Rf?→?Rh₁?→?aglycone protopanaxatriol (APPT). Under the optimum conditions, the enzyme converted 0.5 mM Rb_2, Rc, Rd, Rg₃, Rg₁, and Rf to 0.49 mM compound Y, 0.49 mM compound Mc, 0.47 mM compound K, 0.23 mM APPD, 0.49 mM?F₁, and 0.44 mM APPT after 6 h, respectively.