SGL 121 Attenuates Nonalcoholic Fatty Liver Disease through Adjusting Lipid Metabolism Through AMPK Signaling Pathway.

A ginsenoside F2-enhanced mixture (SGL 121) increases the content of ginsenoside F2 by biotransformation. In the present study, we investigated the effect of SGL 121 on nonalcoholic fatty liver disease (NAFLD) in vitro and in vivo. High-fat, high-carbohydrate-diet (HFHC)-fed mice were administered SGL 121 for 12 weeks to assess its effect on improving NAFLD. In HepG2 cells, SGL 121 acted as an antioxidant, a hepatoprotectant, and had an anti-lipogenic effect. In NAFLD mice, SGL 121 significantly improved body fat mass; levels of hepatic triglyceride (TG), hepatic malondialdehyde (MDA), serum total cholesterol (TC), high-density lipoprotein (HDL), and low-density lipoprotein (LDL); and activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). In HepG2 cells, induced by oxidative stress, SGL 121 increased cytoprotection, inhibited reactive oxygen species (ROS) production, and increased antioxidant enzyme activity. SGL 121 activated the Nrf2/HO-1 signaling pathway and improved lipid accumulation induced by free fatty acids (FFA). Sterol regulatory element-binding protein-1 (SREBP-1) and fatty acid synthase (FAS) expression was significantly reduced in NAFLD-induced liver and HepG2 cells treated with SGL 121. Moreover, SGL 121 activated adenosine monophosphate-activated protein kinase (AMPK), which plays an important role in the regulation of lipid metabolism. The effect of SGL 121 on the improvement of NAFLD seems to be related to its antioxidant effects and activation of AMPK. In conclusion, SGL 121 can be potentially used for the treatment of NAFLD.

High-Level Production of Bacteriotoxic Phospholipase A1 in Bacterial Host Pseudomonas fluorescens Via ABC Transporter-Mediated Secretion and Inducible Expression.

: Bacterial phospholipase A1 (PLA1) is used in various industrial fields because it can catalyze the hydrolysis, esterification, and transesterification of phospholipids to their functional derivatives. It also has a role in the degumming process of crude plant oils. However, bacterial expression of the foreign PLA1-encoding gene was generally hampered because intracellularly expressed PLA1 is inherently toxic and damages the phospholipid membrane. In this study, we report that secretion-based production of recombinant PlaA, a bacterial PLA1 gene, or co-expression of PlaS, an accessory gene, minimizes this harmful effect. We were able to achieve high-level PlaA production via secretion-based protein production. Here, TliD/TliE/TliF, an ABC transporter complex of Pseudomonas fluorescens SIK-W1, was used to secrete recombinant proteins to the extracellular medium. In order to control the protein expression with induction, a new strain of P. fluorescens, which had the lac operon repressor gene lacI, was constructed and named ZYAI strain. The bacteriotoxic PlaA protein was successfully produced in a bacterial host, with help from ABC transporter-mediated secretion, induction-controlled protein expression, and fermentation. The final protein product is capable of degumming oil efficiently, signifying its application potential.

Ginsenoside Rh2 Ameliorates Atopic Dermatitis in NC/Nga Mice by Suppressing NF-kappaB-Mediated Thymic Stromal Lymphopoietin Expression and T Helper Type 2 Differentiation.

Ginsenosides are known to have various highly pharmacological activities, such as anti-cancer and anti-inflammatory effects. However, the search for the most effective ginsenosides against the pathogenesis of atopic dermatitis (AD) and the study of the effects of ginsenosides on specific cytokines involved in AD remain unclear. In this study, ginsenoside Rh2 was shown to exert the most effective anti-inflammatory action on thymic stromal lymphopoietin (TSLP) and interleukin 8 in tumor necrosis factor-alpha and polyinosinic: polycytidylic acid induced normal human keratinocytes by inhibiting proinflammatory cytokines at both protein and transcriptional levels. Concomitantly, Rh2 also efficiently alleviated 2,4-dinitrochlorobenzene-induced AD-like skin symptoms when applied topically, including suppression of immune cell infiltration, cytokine expression, and serum immunoglobulin E levels in NC/Nga mice. In line with the in vitro results, Rh2 inhibited TSLP levels in AD mice via regulation of an underlying mechanism involving the nuclear factor κB pathways. In addition, in regard to immune cells, we showed that Rh2 suppressed not only the expression of TSLP but the differentiation of naïve CD4+ T-cells into T helper type 2 cells and their effector function in vitro. Collectively, our results indicated that Rh2 might be considered as a good therapeutic candidate for the alternative treatment of AD.

Triterpenoid-biosynthetic UDP-glycosyltransferases from plants.

Triterpenoid saponins are naturally occurring structurally diverse glycosides of triterpenes that are widely distributed among plant species. Great interest has been expressed by pharmaceutical and agriculture industries for the glycosylation of triterpenes. Such modifications alter their taste and bio-absorbability, affect their intra-/extracellular transport and storage in plants, and induce novel biological activities in the human body. Uridine diphosphate (UDP)-glycosyltransferases (UGTs) catalyze glycosylation using UDP sugar donors. These enzymes belong to a multigene family and recognize diverse natural products, including triterpenes, as the acceptor molecules. For this review, we collected and analyzed all of the UGT sequences found in Arabidopsis thaliana as well as 31 other species of triterpene-producing plants. To identify potential UGTs with novel functions in triterpene glycosylation, we screened and classified those candidates based on similarity with UGTs from Panax ginseng, Glycine max, Medicago truncatula, Saponaria vaccaria, and Barbarea vulgaris that are known to function in glycosylate triterpenes. We highlight recent findings on UGT inducibility by methyl jasmonate, tissue-specific expression, and subcellular localization, while also describing their catalytic activity in terms of regioselectivity for potential key UGTs dedicated to triterpene glycosylation in plants. Discovering these new UGTs expands our capacity to manipulate the biological and physicochemical properties of such valuable molecules.

Gypenoside LXXV Promotes Cutaneous Wound Healing In Vivo by Enhancing Connective Tissue Growth Factor Levels Via the Glucocorticoid Receptor Pathway.

Cutaneous wound healing is a well-orchestrated event in which many types of cells and growth factors are involved in restoring the barrier function of skin. In order to identify whether ginsenosides, the main active components of Panax ginseng, promote wound healing, the proliferation and migration activities of 15 different ginsenosides were tested by MTT assay and scratched wound closure assay. Among ginsenosides, gypenoside LXXV (G75) showed the most potent wound healing effects. Thus, this study aimed to investigate the effects of G75 on wound healing in vivo and characterize associated molecular changes. G75 significantly increased proliferation and migration of keratinocytes and fibroblasts, and promoted wound closure in an excision wound mouse model compared with madecassoside (MA), which has been used to treat wounds. Additionally, RNA sequencing data revealed G75-mediated significant upregulation of connective tissue growth factor (CTGF), which is known to be produced via the glucocorticoid receptor (GR) pathway. Consistently, the increase in production of CTGF was confirmed by western blot and ELISA. In addition, GR-competitive binding assay and GR translocation assay results demonstrated that G75 can be bound to GR and translocated into the nucleus. These results demonstrated that G75 is a newly identified effective component in wound healing.

Pro-angiogenic Ginsenosides F1 and Rh1 Inhibit Vascular Leakage by Modulating NR4A1.

Vascular endothelial growth factor (VEGF) plays a key role in angiogenesis, but VEGF-induced angiogenesis is often accompanied by a vascular permeability response. Ginsenosides are triterpenoid saponins from the well-known medicinal plant, ginseng, and have been considered a candidate for modulating angiogenesis. Here, we systemically investigated the effects of 10 different ginsenosides on human umbilical vein endothelial cells and newly identified that two PPT-type ginsenosides, F1 and Rh1 induce the migration and proliferation of endothelial cells. Interestingly, RNA transcriptome analysis showed that gene regulation induced by VEGF in endothelial cells is distinct from that of ginsenoside F1 and Rh1. In addition, F1 and Rh1 significantly inhibited vascular leakage both in vitro and in vivo, which are induced by vascular endothelial growth factor. Furthermore, comparative transcriptome analysis revealed that these effects of F1 and Rh1 on vascular leakage restoration are mainly caused by changes in VEGF-mediated TNFα signaling via NFκB, particularly by the suppression of expression and transcriptional activity of NR4A1 by F1 and Rh1, even in the presence of VEGF. These findings demonstrate that ginsenosides F1 and Rh1 can be a promising herbal remedy for vessel normalization in ischemic disease and cancer and that NR4A1 is the key target.

Enhanced Production of Gypenoside LXXV Using a Novel Ginsenoside-Transforming ß-Glucosidase from Ginseng-Cultivating Soil Bacteria and Its Anti-Cancer Property.

Minor ginsenosides, such as compound K, Rg3(S), which can be produced by deglycosylation of ginsenosides Rb1, showed strong anti-cancer effects. However, the anticancer effects of gypenoside LXXV, which is one of the deglycosylated shapes of ginsenoside Rb1, is still unknown due to the rarity of its content in plants. Here, we cloned and characterized a novel ginsenoside-transforming ß-glucosidase (BglG167b) derived from Microbacterium sp. Gsoil 167 which can efficiently hydrolyze gypenoside XVII into gypenoside LXXV, and applied it to the production of gypenoside LXXV at the gram-scale with high specificity. In addition, the anti-cancer activity of gypenoside LXXV was investigated against three cancer cell lines (HeLa, B16, and MDA-MB231) in vitro. Gypenoside LXXV significantly reduced cell viability, displaying an enhanced anti-cancer effect compared to gypenoside XVII and Rb1. Taken together, this enzymatic method would be useful in the preparation of gypenoside LXXV for the functional food and pharmaceutical industries.

Comparative analysis of the expression level of recombinant ginsenoside-transforming ß-glucosidase in GRAS hosts and mass production of the ginsenoside Rh2-Mix.

The ginsenoside Rh2, a pharmaceutically active component of ginseng, is known to have anticancer and antitumor effects. However, white ginseng and red ginseng have extremely low concentrations of Rh2 or Rh2-Mix [20(S)-Rh2, 20(R)-Rh2, Rk2, and Rh3]. To enhance the production of food-grade ginsenoside Rh2, an edible enzymatic bioconversion technique was developed adopting GRAS host strains. A ß-glucosidase (BglPm), which has ginsenoside conversion ability, was expressed in three GRAS host strains (Corynebacterium glutamicum, Saccharomyces cerevisiae and Lactococus lactis) by using a different vector system. Enzyme activity in these three GRAS hosts were 75.4%, 11.5%, and 9.3%, respectively, compared to that in the E. coli pGEX 4T-1 expression system. The highly expressed BglPm_C in C. glutamicum can effectively transform the ginsenoside Rg3-Mix [20(S)-Rg3, 20(R)-Rg3, Rk1, Rg5] to Rh2-Mix [20(S)-Rh2, 20(R)-Rh2, Rk2, Rh3] using a scaled-up biotransformation reaction, which was performed in a 10-L jar fermenter at pH 6.5/7.0 and 37°C for 24 h. To our knowledge, this is the first report in which 50 g of PPD-Mix (Rb1, Rb2, Rb3, Rc, and Rd) as a starting substrate was converted to ginsenoside Rg3-Mix by acid heat treatment and then 24.5-g Rh2-Mix was obtained by enzymatic transformation of Rg3-Mix through by BglPm_C. Utilization of this enzymatic method adopting a GRAS host could be usefully exploited in the preparation of ginsenoside Rh2-Mix in cosmetics, functional food, and pharmaceutical industries, thereby replacing the E. coli expression system.

Two ginseng UDP-glycosyltransferases synthesize ginsenoside Rg3 and Rd.

Ginseng is a medicinal herb that requires cultivation under shade conditions, typically for 4-6 years, before harvesting. The principal components of ginseng are ginsenosides, glycosylated tetracyclic terpenes. Dammarene-type ginsenosides are classified into two groups, protopanaxadiol (PPD) and protopanaxatriol (PPT), based on their hydroxylation patterns, and further diverge to diverse ginsenosides through differential glycosylation. Three early enzymes, dammarenediol-II synthase (DS) and two P450 enzymes, protopanaxadiol synthase (PPDS) and protopanaxatriol synthase (PPTS), have been reported, but glycosyltransferases that are necessary to synthesize specific ginsenosides have not yet been fully identified. To discover glycosyltransferases responsible for ginsenoside biosynthesis, we sequenced and assembled the ginseng transcriptome de novo and characterized two UDP-glycosyltransferases (PgUGTs): PgUGT74AE2 and PgUGT94Q2. PgUGT74AE2 transfers a glucose moiety from UDP-glucose (UDP-Glc) to the C3 hydroxyl groups of PPD and compound K to form Rh2 and F2, respectively, whereas PgUGT94Q2 transfers a glucose moiety from UDP-Glc to Rh2 and F2 to form Rg3 and Rd, respectively. Introduction of the two UGT genes into yeast together with PgDS and PgPPDS resulted in the de novo production of Rg3. Our results indicate that these two UGTs are key enzymes for the synthesis of ginsenosides and provide a method for producing specific ginsenosides through yeast fermentation.

Mucilaginibacter ginsenosidivorax sp. nov., with ginsenoside converting activity isolated from sediment.

A Gram-reaction-negative, strictly aerobic, non-motile, non-spore-forming, and rod-shaped bacterial strain designated KHI28(T) was isolated from sediment in Gapcheon (river) and its taxonomic position was investigated using a polyphasic approach. Strain KHI28(T) grew at 10-42°C and at pH 5.5-8.5 on R2A and nutrient agar without additional NaCl as a supplement. Strain KHI28(T) possessed ß-glucosidase activity, which was responsible for its ability to transform ginsenosides Rb1 and Re (ones of the dominant active components of ginseng) to C-K and Rg2, respectively. On the basis of 16S rRNA gene sequence similarity, strain KHI28(T) was shown to belong to the family Sphingobacteriaceae and to be related to Mucilaginibacter dorajii DR-f4(T) (97.9% sequence similarity), M. polysacchareus DRP28(T) (97.3%), and M. lappiensis ANJLI2(T) (97.2%). The G+C content of the genomic DNA was 45.8%. The predominant respiratory quinone was MK-7 and the major fatty acids were summed feature 3 (comprising C16:1 ω6c and/or C16:1 ω7c), iso-C15:0 and C16:0. DNA and chemotaxonomic data supported the affiliation of strain KHI28(T) to the genus Mucilaginibacter. Strain KHI28(T) could be differentiated genotypically and phenotypically from the recognized species of the genus Mucilaginibacter. The isolate therefore represents a novel species, for which the name Mucilaginibacter ginsenosidivorax sp. nov. is proposed, with the type strain KHI28(T) (=KACC 14955(T) =LMG 25804(T)).

Mass production of the ginsenoside Rg3(S) through the combinative use of two glycoside hydrolases.

The ginsenoside Rg3(S), which is one of the exceptional components of Korean red ginseng extract, has been known to have anti-cancer, anti-metastatic, and anti-obesity effects. An enzymatic bioconversion method was developed to obtain the ginsenoside Rg3(S) with a high specificity, yield, and purity. Two glycoside hydrolases (BglBX10 and Abf22-3) were employed to produce Rg3(S) as a 100g unit. The conversion reaction transformed ginsenoside Rc to Rd using Abf22-3, followed by Rb1 and Rd to Rg3(S), using BglBX10. It was performed in a 10L jar fermenter at pH 6.0 and 37°C for 24h, with a high concentration of 50mg/ml of purified ginsenoside mixture obtained from ginseng roots. Finally, 144g of Rg3(S) was produced from 250g of root extract with 78±1.2% chromatographic purity. These results suggest that this enzymatic method would be useful in the preparation of ginsenoside Rg3(S) for the functional food and pharmaceutical industries.

Nocardioides panaciterrulae sp. nov., isolated from soil of a ginseng field, with ginsenoside converting activity.

A Gram-positive, coccoid to rod-shaped, non-spore-forming bacterium, designated Gsoil 958(T), was isolated from soil of a ginseng field located in Pocheon province in South Korea. This bacterium was characterized in order to determine its taxonomic position by using a polyphasic approach. Strain Gsoil 958(T) was observed to grow well at 25-30 °C and at pH 7.0 on R2A and nutrient agar without NaCl supplementation. Strain Gsoil 958(T) was determined to have ß-glucosidase activity and the ability to transform ginsenoside Rb1 (one of the dominant active components of ginseng) to F2 via gypenoside XVII and Rd. On the basis of 16S rRNA gene sequence similarity, strain Gsoil 958(T) was shown to belong to the family Nocardioidaceae and related most closely to Nocardioides koreensis MSL-09(T) (97.6 % 16S rRNA gene sequence similarity), Nocardioides aquiterrae GW-9(T) (97.0 %), and Nocardioides sediminis MSL-01(T) (97.0 %). The sequence similarities with other validly named species within the genus Nocardioides were less than 96.8 %. Strain Gsoil 958(T) was characterized chemotaxonomically as having LL-2,6-diaminopimelic acid in the cell-wall peptidoglycan, MK-8(H4) as the predominant menaquinone, and iso-C16:0, iso-C16:1 H, iso-C14:0, iso-C15:0 were identified as the major fatty acids. The G + C content of genomic DNA was determined to be 70.8 mol %. The chemotaxonomic properties and phenotypic characteristics supported the affiliation of strain Gsoil 958(T) to the genus Nocardioides. The results of both physiological and biochemical tests allowed for differentiation of strain Gsoil 958(T) from the recognized Nocardioides species. Therefore, strain Gsoil 958(T) is considered to represent a novel species of the genus Nocardioides, for which the name Nocardioides panaciterrulae sp. nov. is proposed, with the type strain Gsoil 958(T) (KACC 14271(T) = KCTC 19471(T) = DSM 21350(T)).

Ramlibacter ginsenosidimutans sp. nov., with ginsenoside-converting activity.

A novel beta-proteobacterium, designated BXN5-27(T), was isolated from soil of a ginseng field of Baekdu Mountain in China, and was characterized using a polyphasic approach. The strain was Gram-staining-negative, aerobic, motile, non-spore-forming, and rod shaped. Strain BXN5-27(T) exhibited beta-glucosidase activity that was responsible for its ability to transform ginsenoside Rb1 (one of the dominant active components of ginseng) to compound Rd. Phylogenetic analysis based on 16S rRNA gene sequences showed that this strain belonged to the family Comamonadaceae; it was most closely related to Ramlibacter henchirensis TMB834(T) and Ramlibacter tataouinensis TTB310(T) (96.4% and 96.3% similarity, respectively). The G+C content of the genomic DNA was 68.1%. The major menaquinone was Q-8. The major fatty acids were C16:0, summed feature 4 (comprising C16:1 omega7c and/or iso-C15:0 2OH), and C17:0 cyclo. Genomic and chemotaxonomic data supported the affiliation of strain BXN5-27(T) to the genus Ramlibacter. However, physiological and biochemical tests differentiated it phenotypically from the other established species of Ramlibacter. Therefore, the isolate represents a novel species, for which the name Ramlibacter ginsenosidimutans sp. nov. is proposed, with the type strain being BXN5-27(T) (= DSM 23480(T) = LMG 24525(T) = KCTC 22276(T)).

Nocardioides ginsengagri sp. nov., isolated from the soil of a ginseng field.

A Gram-reaction-positive, rod-shaped, non-motile, non-spore-forming bacterium (strain BX5-10(T)) was isolated from the soil of a ginseng field on Baekdu Mountain in Jilin district, China. The taxonomic position of this bacterium was determined in an investigation based on a polyphasic approach. On the basis of 16S rRNA gene sequence analysis, strain BX5-10(T) was shown to belong to the family Nocardioidaceae and to be most closely related to Nocardioides plantarum NCIMB 12834(T) (96.5% sequence similarity), Nocardioides dokdonensis KCTC 19309(T) (96.2%) and Nocardioides fonticola NAA-13(T) (95.1%). Strain BX5-10(T) was characterized chemotaxonomically as having ll-2,6-diaminopimelic acid in its cell-wall peptidoglycan, MK-8(H(4)) as the predominant menaquinone and C(18:1)ω9c, C(16:0) and C(17:1)ω8c as its major fatty acids. The G+C content of the genomic DNA was 70.3 mol%. The novel strain could be differentiated genotypically and phenotypically from all recognized species of the genus Nocardioides. Based on the results of the phylogenetic analyses and the genotypic and phenotypic data, a novel species, Nocardioides ginsengagri sp. nov., is proposed. The type strain is BX5-10(T) (=KCTC 19467(T)=DSM 21362(T)).

Double anti-angiogenic and anti-inflammatory protein Valpha targeting VEGF-A and TNF-alpha in retinopathy and psoriasis.

Pathological angiogenesis usually involves disrupted vascular integrity, vascular leakage, and infiltration of inflammatory cells, which are governed mainly by VEGF-A and TNF-α. Although many inhibitors targeting either VEGF-A or TNF-α have been developed, there is no single inhibitor molecule that simultaneously targets both molecules. Here, we designed and generated a novel chimeric decoy receptor (Valpha) that can simultaneously bind to VEGF-A and TNF-α and block their actions. In this experimental design, we have shown that Valpha, which is an effective synchronous blocker of VEGF-A and TNF-α, can drastically increase treatment effectiveness through its dual-blocking characteristics. Valpha contains the VEGF-A-binding domain of VEGFR1, the TNF-α-binding domain of TNFR2, and the Fc domain of IgG1. Valpha exhibited strong binding characteristics for its original counterparts, VEGF-A and TNF-α, but not for the extracellular matrix, resulting in a highly favorable pharmacokinetic profile in vivo. Compared with VEGF-Trap or Enbrel, both of which block either VEGF-A or TNF-α, singularly, Valpha is a highly effective molecule for reducing abnormal vascular tufts and the number of F4/80(+) macrophages in a retinopathy model. In addition, Valpha showed superior relief effects in a psoriasis model with regard to epidermal thickness and the area of blood and lymphatic vessels. Thus, the simultaneous blocking of VEGF-A and TNF-α using Valpha is an effective therapeutic strategy and cost-efficient for treatment of retinopathy and psoriasis.

Tumebacillus ginsengisoli sp. nov., isolated from soil of a ginseng field.

A gram-reaction-positive, rod-shaped, spore-forming bacterium, designated Gsoil 1105(T), was isolated from soil of a ginseng field in Pocheon Province in South Korea and characterized in order to determine its taxonomic position. Comparative analysis of the 16S rRNA gene sequence showed that the isolate belongs to the order Bacillales, showing the highest level of sequence similarity with respect to Tumebacillus permanentifrigoris Eur1 9.5(T) (94.6 %). The phylogenetic distances from other described species with validly published names within the order Bacillales were greater than 9.0 %. Strain Gsoil 1105(T) had a genomic DNA G+C content of 55.6 mol% and menaquinone 7 (MK-7) as the major respiratory quinone. The major fatty acids were iso-C(15 : 0) and anteiso-C(15 : 0). On the basis of its phenotypic properties and phylogenetic distinctiveness, strain Gsoil 1105(T) represents a novel species of the genus Tumebacillus, for which the name Tumebacillus ginsengisoli sp. nov. is proposed. The type strain is Gsoil 1105(T) ( = KCTC 13942(T)  = DSM 18389(T)).

Identification and characterization of a novel Terrabacter ginsenosidimutans sp. nov. beta-glucosidase that transforms ginsenoside Rb1 into the rare gypenosides XVII and LXXV.

A new beta-glucosidase from a novel strain of Terrabacter ginsenosidimutans (Gsoil 3082(T)) obtained from the soil of a ginseng farm was characterized, and the gene, bgpA (1,947 bp), was cloned in Escherichia coli. The enzyme catalyzed the conversion of ginsenoside Rb1 {3-O-[beta-D-glucopyranosyl-(1-2)-beta-D-glucopyranosyl]-20-O-[beta-D-glucopyranosyl-(1-6)-beta-D-glucopyranosyl]-20(S)-protopanaxadiol} to the more pharmacologically active rare ginsenosides gypenoside XVII {3-O-beta-D-glucopyranosyl-20-O-[beta-D-glucopyranosyl-(1-6)-beta-D-glucopyranosyl]-20(S)-protopanaxadiol}, gypenoside LXXV {20-O-[beta-v-glucopyranosyl-(1-6)-beta-D-glucopyranosyl]-20(S)-protopanaxadiol}, and C-K [20-O-(beta-D-glucopyranosyl)-20(S)-protopanaxadiol]. A BLAST search of the bgpA sequence revealed significant homology to family 3 glycoside hydrolases. Expressed in E. coli, beta-glucosidase had apparent K(m) values of 4.2 +/- 0.8 and 0.14 +/- 0.05 mM and V(max) values of 100.6 +/- 17.1 and 329 +/- 31 micromol x min(-1) x mg of protein(-1) against p-nitrophenyl-beta-D-glucopyranoside and Rb1, respectively. The enzyme catalyzed the hydrolysis of the two glucose moieties attached to the C-3 position of ginsenoside Rb1, and the outer glucose attached to the C-20 position at pH 7.0 and 37 degrees C. These cleavages occurred in a defined order, with the outer glucose of C-3 cleaved first, followed by the inner glucose of C-3, and finally the outer glucose of C-20. These results indicated that BgpA selectively and sequentially converts ginsenoside Rb1 to the rare ginsenosides gypenoside XVII, gypenoside LXXV, and then C-K. Herein is the first report of the cloning and characterization of a novel ginsenoside-transforming beta-glucosidase of the glycoside hydrolase family 3.