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.     

Enzymatic biotransformation of ginsenoside Rb1 to 20(<Emphasis Type="Italic">S</Emphasis>)-Rg3 by recombinant β-glucosidase from <Emphasis Type="Italic">Microbacterium esteraromaticum</Emphasis>

Microbacterium esteraromaticum was isolated from ginseng field. The β-glucosidase gene (bgp1) from M. esteraromaticum was cloned and expressed in Escherichia coli BL21 (DE3). The bgp1 gene consists of 2,496 bp encoding 831 amino acids which have homology to the glycosyl hydrolase family 3 protein domain. The recombinant β-glucosidase enzyme (Bgp1) was purified and characterized. The molecular mass of purified Bgp1 was 87.5 kDa, as determined by SDS-PAGE. Using 0.1 mg ml⁻¹ enzyme in 20 mM sodium phosphate buffer at 37°C and pH 7.0, 1.0 mg ml⁻¹ ginsenoside Rb1 was transformed into 0.444 mg ml⁻¹ ginsenoside Rg3 within 6 h. The Bgp1 sequentially hydrolyzed the outer and inner glucose attached to the C-20 position of ginsenosides Rb1. Bgp1 hydrolyzed the ginsenoside Rb1 along the following pathway: Rb1 → Rd → 20(S)-Rg3. This is the first report of the biotransformation of ginsenoside Rb1 to ginsenoside 20(S)-Rg3 using the recombinant β-glucosidase.     

Enzymatic transformation of the major ginsenoside Rb2 to minor compound Y and compound K by a ginsenoside-hydrolyzing β-glycosidase from Microbacterium esteraromaticum

The ginsenoside-hydrolyzing β-glycosidase (Bgp3) derived from Microbacterium esteraromaticum transformed the major ginsenoside Rb2 to more pharmacologically active minor ginsenosides including compounds Y and K. The bgp3 gene consists of 2,271?bp encoding 756 amino acids which have homology to the glycosyl hydrolase family 3 protein domain. Bgp3 is capable of hydrolyzing beta-glucose links and arabinose links. HPLC analysis of the time course of ginsenoside Rb2 hydrolysis by Bgp3 (0.1?mg?enzyme?ml(-1) in 20?mM sodium phosphate buffer at 40?°C and pH 7.0) showed that the glycosidase first hydrolyzed the inner glucose moiety attached to the C-3 position and then the arabinopyranose moiety attached to the C-20 position. Thus, Bgp3 hydrolyzed the ginsenoside Rb2 via the following pathway: Rb2?→?compound Y?→?compound K.     

Production of aglycone protopanaxatriol from ginseng root extract using Dictyoglomus turgidum β-glycosidase that specifically hydrolyzes the xylose at the C-6 position and the glucose in protopanaxatriol-type ginsenosides

The hydrolytic activity of a recombinant β-glycosidase from Dictyoglomus turgidum that specifically hydrolyzed the xylose at the C-6 position and the glucose in protopanaxatriol (PPT)-type ginsenosides followed the order Rf > Rg₁ > Re > R₁ > Rh₁ > R₂. The production of aglycone protopanaxatriol (APPT) from ginsenoside Rf was optimal at pH 6.0, 80 °C, 1 mg ml^?1 Rf, and 10.6 U ml^?1 enzyme. Under these conditions, D. turgidum β-glycosidase converted ginsenoside R₁ to APPT with a molar conversion yield of 75.6 % and a productivity of 15 mg l^?1 h^?1 after 24 h by the transformation pathway of R₁ → R₂ → Rh₁ → APPT, whereas the complete conversion of ginsenosides Rf and Rg₁ to APPT was achieved with a productivity of 1,515 mg l^?1 h^?1 after 6.6 h by the pathways of Rf → Rh₁ → APPT and Rg₁ → Rh₁ → APPT, respectively. In addition, D. turgidum β-glycosidase produced 0.54 mg ml^?1 APPT from 2.29 mg ml^?1 PPT-type ginsenosides of Panax ginseng root extract after 24 h, with a molar conversion yield of 43.2 % and a productivity of 23 mg l^?1 h^?1, and 0.62 mg ml^?1 APPT from 1.35 mg ml^?1 PPT-type ginsenosides of Panax notoginseng root extract after 20 h, with a molar conversion yield of 81.2 % and a productivity of 31 mg l^?1 h^?1. This is the first report on the APPT production from ginseng root extract. Moreover, the concentrations, yields, and productivities of APPT achieved in the present study are the highest reported to date.     

Biotransformation pathways of ginsenoside Rb1 to compound K by β-glucosidases in fungus Paecilomyces Bainier sp. 229

Ginsenoside Rb1 is the most abundant ginsenoside in Panax (ginseng). The hydrolysis of this ginsenoside produces compound K, the biologically active ginsenoside of ginseng. We previously identified a fungus Paecilomyces Bainier sp. 229 (sp. 229), which can efficiently convert ginsenoside Rb1 to compound K. In this report, the ginsenoside hydrolyzing β-glucosidases were isolated from sp. 229 and the pathway of the biotransformation of ginsenoside Rb1 to compound K by sp. 229 was investigated. Based on reverse-phase HPLC and TLC analysis, we found the main metabolic pathway is as follows: ginsenoside Rb1 → ginsenoside Rd → ginsenoside F2 → compound K. Moreover, the results showed that there were other metabolic pathways: ginsenoside Rb1 → ginsenoside XVII → ginsenoside F2 → compound K and ginsenoside Rb1 → ginsenoside Rg3 → ginsenoside Rh2. These processes would allow the specific bioconversion of ginsenoside Rb1 to various ginsenosides using an appropriate combination of specific microbial enzymes.     

Fermentation of ginseng extracts by Penicillium simplicissimum GS33 and anti-ovarian cancer activity of fermented products

A total of 58 isolates of β-glucosidase-producing microorganisms were isolated from soil around the wild ginseng roots under forest using Esculin-R2A agar. Among these isolates, strain GS33 showed a strong ability to convert ginsenosides Rb1, Rb2, Rc, and Rd into F2, Rg3, C-K, and convert ginsenoside Rg1 into Rh1, and F1. Fermented ginseng products can inhibit ES-2 cells growth and the IC₅₀ value was 0.73 mg ml^?1. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain GS33 belongs to the genus Penicillium and is most closely related to Penicillium simplicissimum (99 %).     

Efficient biotransformation for preparation of pharmaceutically active ginsenoside Compound K by Penicillium oxalicum sp. 68

Pathogenic fungus Penicillium oxalicum sp. 68 was screened from soil and identified by ITS sequencing. The strain was found to be able to transform protopanaxadiol-type ginsenosides to produce a series of bioactive metabolites. Glycosidase from the culture of P. oxalicum sp. 68 was partially purified with a simple two-step procedure consisting of DEAE-cellulose chromatography and ammonium sulfate precipitation. Bioactive ginsenoside Compound K was prepared selectively and efficiently by biotransformation of ginsenosides Rb1, Rb2, Rc and Rd using the partially purified glycosidase. The optimal conditions for transforming Rb1 into Compound K were pH 4.0, 55 °C and 0.5 mg mL^?1 Rb1. The sole product is Compound K and the maximum yield reached 87.7 % (molar ratio). The transformation pathways of Rb1, Rb2, Rc and Rd are Rb1→Rd→F2→Compound K, Rb2→CO→CY→Compound K, Rc→Mb→Mc→Compound K and Rd→F2→Compound K, respectively. This biotransformation method showed great potential for preparing minor bioactive ginsenosides, especially Compound K, in the pharmaceutical industry because of its high specificity and favorable environmental compatibility.     

Production of ginsenoside aglycons and Rb1 deglycosylation pathway profiling by HPLC and ESI-MS/MS using Sphingobacterium multivorum GIN723

Using enrichment culture, Sphingobacterium multivorum GIN723 (KCCM80060) was isolated as having activity for deglycosylation of compound K and ginsenoside F1 to produce ginsenoside aglycons such as S-protopanaxadiol (PPD(S)) and S-protopanaxatriol (PPT(S)). Through BLAST search, purified enzyme from S. multivorum GIN723 was revealed to be the outer membrane protein. The purified enzyme from S. multivorum GIN723 has unique specificity for the glucose moiety. However, it has activity with PPD and PPT group ginsenosides such as ginsenosides Rb1, Rb2, Rb3, Rc, F2, CK, Rh2, Re, and F1. From these results, it was predicted that the enzyme has activity on several ginsenosides. Therefore, the biotransformation pathway from Rb1, which is a major, highly glycosylated compound of ginseng, was analyzed using high-performance liquid chromatography and electrospray ionization mass spectrometry/mass spectrometry. The dominant biotransformation pathway from Rb1 to PPD(S) was determined to be Rb1 → Gp-XVII → Gp-LXXV → CK → PPD(S). S. multivorum GIN723 can be used as a whole cell biocatalyst because its activity as whole cells is nine times higher than its activity as cell extracts. The specific activity of whole cells is 2.89 nmol/mg/min in the production of PPD(S). On the other hand, the specific activity of cell extracts is 0.32 nmol/mg/min. The productivity of this enzyme in whole cell form is 500 mg/1 l of cultured cell. Its optimum reaction condition is 10 mM of calcium ions added to a phosphate buffer with a pH of 8.5.     

Nitrogen and phosphorus as the factors affecting ginsenoside production in hairy root cultures of <Emphasis Type="Italic">Panax quinquefolium</Emphasis> cultivated in shake flasks and nutrient sprinkle bioreactor

The study assesses the influence of different concentrations of nitrogen and phosphorus sources on ginsenoside biosynthesis in Panax quinquefolium hairy roots cultivated in shake flasks and a nutrient sprinkle bioreactor. The saponin content was determined using HPLC. The maximum yield (12.45 mg g^?1 dw) of the sum of six examined ginsenosides (Rb1, Rb2, Rc, Rd, Re and Rg1) in hairy roots cultivated in shake flasks was achieved in modified Gamborg B-5 medium containing 0.83 mM l^?1 phosphate, 12.4 mM l^?1 nitrate and 0.5 mM l^?1 ammonium. The yield itself was 1.93 times higher than that achieved in standard Gamborg medium. The modified medium also favourably influenced the biosynthesis of studied saponins in bioreactor cultures. The saponin content (35.11 mg g^?1 d.w.) was 2.75-times higher than that achieved in control medium.     

Production of ginsenosides Rg1 and Rh1 by hydrolyzing the outer glycoside at the C-6 position in protopanaxatriol-type ginsenosides using β-glucosidase from Pyrococcus furiosus

The specific activity of a recombinant β-glucosidase from Pyrococcus furiosus for protopanaxatriol (PPT)-type ginsenosides followed the order Rf > R₁ > Re > R₂ > Rg₂, which were converted to Rh₁, Rg₁, Rg₁, Rh₁, and Rh₁, respectively. No activity was observed with Rg₁ and Rh₁. Thus, P. furiosus β-glucosidase hydrolyzed the outer glycoside at the C-6 position in PPT-type ginsenosides whereas the enzyme did not hydrolyze the inner glucoside at the C-6 position and the glucoside at the C-20 position. The activity for Rf was optimal at 95 °C, pH 5.5, 5 mM ginsenoside, and 32 U enzyme l^?1. Under these conditions, P. furiosus β-glucosidase completely converted from R₁ to Rg₁ after 10 h, with a productivity of 0.4 g l^?1 h^?1 and completely converted Rf to Rh₁ after 1.2 h, with a productivity of 2.74 g l^?1 h^?1.     

Highly selective hydrolysis for the outer glucose at the C-20 position in ginsenosides by β-glucosidase from Thermus thermophilus and its application to the production of ginsenoside F2 from gypenoside XVII

β-Glucosidase from Thermus thermophilus has specific hydrolytic activity for the outer glucose at the C-20 position in protopanaxadiol-type ginsenosides without hydrolysis of the inner glucose. The hydrolytic activity of the enzyme for gypenoside XVII was optimal at pH 6.5 and 90 °C, with a half-life of 1 h with 3 g enzyme l^?1 and 4 g gypenoside XVII l^?1. Under the optimized conditions, the enzyme converted the substrate gypenoside XVII to ginsenoside F₂ with a molar yield of 100 % and a productivity of 4 g l^?1 h^?1. The conversion yield and productivity of ginsenoside F₂ are the highest reported thus far among enzymatic transformations.     

Biochemical characterization of an extracellular polyextremophilic α-amylase from the halophilic archaeon Halorubrum xinjiangense

An extracellular haloalkaliphilic thermostable α-amylase producing archaeon was isolated from the saltwater Lake Urmia and identified as Halorubrum xinjiangense on the basis of morphological, biochemical, and molecular properties. The enzyme was purified to an electrophoretically homogenous state by 80 % cold ethanol precipitation, followed by affinity chromatography. The concentrated pure amylase was eluted as a single peak on fast protein liquid chromatography. The molecular mass of the purified enzyme was about 60 kDa, with a pI value of 4.5. Maximum amylase activity was at 4 M NaCl or 4.5 M KCl, 70 °C, and pH 8.5. The K _m and V _max of the enzyme were determined as 3.8 mg ml^?1 and 12.4 U mg^?1, respectively. The pure amylase was stable in the presence of SDS, detergents, and organic solvents. In addition, the enzyme (20 U) hydrolyzed 69 % of the wheat starch after a 2-h incubation at 70 °C in an aqueous/hexadecane two-phase system.     

Lactobacillus ginsenosidimutans sp. nov., isolated from kimchi with the ability to transform ginsenosides

Biotransformation of ginsenosides was examined using lactic acid bacteria isolated from several kinds of kimchi. A Gram-positive, facultatively anaerobic, non-motile, non-spore-forming, and rod-shaped lactic acid bacterial strain, designated EMML 3041^T, was determined to have ginsenoside-converting activity and its taxonomic position was investigated using a polyphasic approach. Strain EMML 3041^T displayed β-glucosidase activity that was responsible for its ability to transform ginsenoside Rb₁ (one of the dominant active components of ginseng) to F₂ via gypenoside XVII, ginsenoside Rb₂ to compound Y via compound O, ginsenoside Rc to compound Mc via compound Mc₁, and ginsenoside Rd to ginsenoside F₂. On the basis of the 16S rRNA gene sequence similarity, strain EMML 3041^T was shown to belong to the genus Lactobacillus and is closely related to Lactobacillus versmoldensis KU-3^T (98.3 % sequence similarity). Polyphasic taxonomy study confirmed that the strain EMML 3041^T represents a novel species, for which the name Lactobacillus ginsenosidimutans sp. nov. is proposed, with EMML 3041^T (=KACC 14527^T = JCM 16719^T) as the type strain.     

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.     

UFEIII, a fibrinolytic protease from the marine invertebrate, Urechis unicinctus

A new serine protease with fibrinolytic activity from a marine invertebrate, Urechis unicinctus, was purified to electrophoretic homogeneity using column chromatography. SDS-PAGE of the purified enzyme showed a single polypeptide chain with MW ~20.8 kDa. Its N-terminal sequence was IIGGSQAAITSY. The purified enzyme, UFEIII, was stable at pH 6–10 below 60 °C with an optimum pH of 8.5 at approx. 55 °C. The enzyme activity was significantly inhibited by PMSF and SBTI suggesting that it was a serine protease. In fibrin plate assays, UFEIII was contained 1.46 × 10³ U (urokinase units) mg^?1 total fibrinolytic activity, which consisted of 692 U mg^?1 direct fibrinolytic activity and 769 U mg^?1 plasminogen-activator activity. K_m and V_max values for azocasein were 1 mg ml^?1 and 43 μg min^?1 ml^?1, respectively.     

Microbial transformation of ginsenoside Rg3 to ginsenoside Rh2 by <Emphasis Type="Italic">Esteya vermicola</Emphasis> CNU 120806

In the present investigation, we successfully employed a cell-free extract of Esteya vermicola CNU 120806 to convert ginsenoside Rg3 to Rh2. Three important factors including pH, temperature and substrate concentration were optimized for the preparation of Rh₂. The optimal condition was obtained as follows: 50°C, pH 5.0 and substrate concentration of 3 mg ml⁻¹. The yield of conversion was up to 90.7%. In order to identify the specificity of the β-glucosidase activity of Esteya vermicola CNU 120806, ginsenoside Re (protopanaxatriol saponins) was treated under the same reaction system. Interestingly, no new metabolite was generated, which elucidated that the enzymatic process only occurred by hydrolysis of the terminal glucopyranosyl moieties at the C-3 carbon of ginsenoside Rg₃. The crude enzyme extract can be used for commercial ginsenoside Rh₂ preparation.     

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.     

A novel neutral xylanase with high SDS resistance from Volvariella volvacea: characterization and its synergistic hydrolysis of wheat bran with acetyl xylan esterase

A neutral xylanase (XynII) from Volvariella volvacea was identified and characterized. Unlike other modular xylanases, it consists of only a single GH10 catalytic domain with a unique C-terminal sequence (W-R-W-F) and a phenylalanine and proline-rich motif (T-P-F-P-P-F) at N-terminus, indicating that it is a novel GH10 xylanase. XynII exhibited optimal activity at pH 7 and 60 °C and stability over a broad range of pH 4.0–10.0. XynII displayed extreme highly SDS resistance retaining 101.98, 92.99, and 69.84 % activity at the presence of 300 mM SDS on birchwood, soluble oat spelt, and beechwood xylan, respectively. It remained largely intact after 24 h of incubation with proteinase K at a protease to protein ratio of 1:50 at 37 °C. The kinetic constants K _m value towards beechwood xylan was 0.548 mg ml^?1, and the k _cat/K _m ratio, reflecting the catalytic efficiency of the enzyme, was 126.42 ml mg^?1 s^?1 at 60 °C. XynII was a true endo-acting xylanase lacking cellulase activity. It has weak activity towards xylotriose but efficiently hydrolyzed xylans and xylooligosaccharides larger than xylotriose mainly to xylobiose. Synergistic action with acetyl xylan esterase (AXEI) from V. volvacea was observed for de-starched wheat bran. The highest degree of synergy (DS 1.42) was obtained in sequential reactions with AXEI digestion preceding XynII. The high SDS resistance and intrinsic stability suggested XynII may have potential applications in various industrial processes especially for the detergent and textile industries and animal feed industries.     

Effect of red and blue light emitting diodes “CRB-LED” on in vitro organogenesis of date palm (<Emphasis Type="Italic">Phoenix dactylifera</Emphasis> L.) cv. Alshakr

The objective of the present study is to determine the effect of light source on enhancement of shoot multiplication, phytochemicals, as well as, antioxidant enzyme activities of in vitro cultures of date palm cv. Alshakr. In vitro-grown buds were cultured on Murashige and Skoog (MS) medium and incubated under a conventional white fluorescent light (control), and combinations of red + blue light emitting diode (18:2) (CRB-LED). Results revealed that the treatment of CRB-LED showed a significant increase in the number of shoots compared with the white florescent light. Total soluble carbohydrate “TSCH” (7.10 mg g^?1 DW.), starch (1.63 mg g^?1 DW.) and free amino acids (2.90 mg g^?1 DW.) were significantly higher in CRB-LED (p < 0.05). Additionally, CRB-LED induced a higher peroxidase activity (25.50 U ml^?1) compared with the white fluorescent light treatment (19.74 U ml^?1) as control treatment. Potassium, magnesium and sodium contents in (3.62, 13.99 and 2.76 mg g^?1 DW.) were increased in in vitro shoots under CRB-LED treatment in comparison with fluorescent light (p < 0.05). Protein profile showed the appearance of newly bands with the molecular weight of 38 and 60 kDa at the treatment CRB-LED compared with control treatment. Our results demonstrate the positive effects of CRB-LED light during the course of date palm tissue cultures.     

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.