Gypenoside LXXV

Gypenoside XIII regulates lipid metabolism in HepG2 hepatocytes and ameliorates nonalcoholic steatohepatitis in mice.[Pubmed:38294255]

Kaohsiung J Med Sci. 2024 Mar;40(3):280-290.

Gypenoside XIII is isolated from Gynostemma pentaphyllum (Thunb.) Makino. In mice, G. pentaphyllum extract and Gypenoside LXXV have been shown to improve non-alcoholic steatohepatitis (NASH). This study investigated whether gypenoside XIII can regulate lipid accumulation in fatty liver cells or attenuate NASH in mice. We used HepG2 hepatocytes to establish a fatty liver cell model using 0.5 mM oleic acid. Fatty liver cells were treated with different concentrations of gypenoside XIII to evaluate the molecular mechanisms of lipid metabolism. In addition, a methionine/choline-deficient diet induced NASH in C57BL/6 mice, which were given 10 mg/kg gypenoside XIII by intraperitoneal injection. In fatty liver cells, gypenoside XIII effectively suppressed lipid accumulation and lipid peroxidation. Furthermore, gypenoside XIII significantly increased SIRT1 and AMPK phosphorylation to decrease acetyl-CoA carboxylase phosphorylation, reducing fatty acid synthesis activity. Gypenoside XIII also decreased lipogenesis by suppressing sterol regulatory element-binding protein 1c and fatty acid synthase production. Gypenoside XIII also increased lipolysis and fatty acid beta-oxidation by promoting adipose triglyceride lipase and carnitine palmitoyltransferase 1, respectively. In an animal model of NASH, gypenoside XIII effectively decreased the lipid vacuole size and number and reduced liver fibrosis and inflammation. These findings suggest that gypenoside XIII can regulate lipid metabolism in fatty liver cells and improve liver fibrosis in NASH mice. Therefore, gypenoside XIII has potential as a novel agent for the treatment of NASH.

Increasing brain glucose uptake by Gypenoside LXXV ameliorates cognitive deficits in a mouse model of diabetic Alzheimer's disease.[Pubmed:36325883]

Phytother Res. 2023 Feb;37(2):611-626.

We have previously reported that Gypenoside LXXV (GP-75), a novel natural PPARgamma agonist isolated from Gynostemma pentaphyllum, ameliorated cognitive deficits in db/db mice. In this study, we further investigated the beneficial effects on cognitive impairment in APP/PS1 mice and a mouse model of diabetic AD (APP/PS1xdb/db mice). Interestingly, intragastric administration of GP-75 (40 mg/kg/day) for 3 months significantly attenuated cognitive deficits in APP/PS1 and APP/PS1xdb/db mice. GP-75 treatment markedly reduced the levels of glucose, HbA1c and insulin in serum and improved glucose tolerance and insulin sensitivity in APP/PS1xdb/db mice. Notably, GP-75 treatment decreased the beta-amyloid (Abeta) burden, as measured by (11) C-PIB PET imaging. Importantly, GP-75 treatment increased brain glucose uptake as measured by (18) F-FDG PET imaging. Moreover, GP-75 treatment upregulated PPARgamma and increased phosphorylation of Akt (Ser473) and GLUT4 expression levels but decreased phosphorylation of IRS-1 (Ser616) in the hippocampi of both APP/PS1 and APP/PS1xdb/db mice. Furthermore, GP-75-induced increases in GLUT4 membrane translocation in primary hippocampal neurons from APP/PS1xdb/db mice was abolished by cotreatment with the selective PPARgamma antagonist GW9662 or the PI3K inhibitor LY294002. In summary, GP-75 ameliorated cognitive deficits in APP/PS1 and APP/PS1xdb/db mice by enhancing glucose uptake via activation of the PPARgamma/Akt/GLUT4 signaling pathways.

Ultrahigh-performance liquid chromatography coupled to ion mobility quadrupole time-of-flight mass spectrometry profiling and unveiling the transformation of ginsenosides by the dual conditions of citric acid and high-pressure steaming.[Pubmed:35902380]

Rapid Commun Mass Spectrom. 2022 Oct 30;36(20):e9363.

RATIONALE: Many methods have been reported for the production of rare ginsenosides, including heat treatment, acid hydrolysis, alkaline hydrolysis, enzymatic hydrolysis, and microbial transformation. However, the conversion of original ginsenosides to rare ginsenosides under the dual conditions of citric acid and high-pressure steam sterilization has rarely been reported. METHODS: In this study, a method involving ultrahigh-performance liquid chromatography coupled to ion mobility quadrupole time-of-flight mass spectrometry was developed for analysis of chemical transformation of protopanaxatriol (PPT)-type ginsenosides Rg(1) and Re, protopanaxadiol (PPD)-type ginsenoside Rb(1) , and total ginsenosides in the dual conditions of citric acid and high-pressure steam sterilization. An internal ginsenoside database containing 126 known ginsenosides and 18 ginsenoside reference compounds was established to identify the transformation products and explore possible transformation pathways and mechanisms. RESULTS: A total of 54 ginsenosides have been preliminarily identified in the transformation products of PPD-type ginsenosides Rg(1) and Re, PPD-type ginsenoside Rb(1) , and total ginsenosides, and the possible transformation pathways were as follows: Rg(1) , Re --> 20(S)-Rh(12) , 20(R)-Rh(12) ; Rg(1) , Re --> 20(S)-Rh(1) , 20(R)-Rh(1) --> Rk(3) , Rh(4) , Rh(5) ; Rb(1) --> Gypenoside LXXV; Rb(1) --> 20(S)-Rg(3) , 20(R)-Rg(3) --> Rk(1) , Rg(5) ; Re --> 20(S)-Rg(2) , 20(R)-Rg(2) --> 20(S)-Rf(2) , 20(R)-Rf(2) , Rg(4) , F(4) . CONCLUSIONS: The results elucidated the possible transformation pathways and mechanisms of ginsenosides in the dual conditions of citric acid and high-pressure steam sterilization, which were helpful for revealing the mechanisms of ginsenosides and enhanced safety and quality control of pharmaceuticals and nutraceuticals. Meanwhile, a simple, efficient, and practical method was developed for the production of rare ginsenosides, which has the potential to produce diverse rare ginsenosides on an industrial scale.

Key Glycosyltransferase Genes of Panax notoginseng: Identification and Engineering Yeast Construction of Rare Ginsenosides.[Pubmed:35687875]

ACS Synth Biol. 2022 Jul 15;11(7):2394-2404.

Panax notoginseng is one of the most famous valuable medical plants in China, and its broad application in clinical treatment has an inseparable relationship with the active molecules, ginsenosides. Ginsenosides are glycoside compounds that have varied structures for the diverse sugar chain. Although extensive work has been done, there are still unknown steps in the biosynthetic pathway of ginsenosides. Here, we screened candidate glycosyltransferase genes based on the previous genome and transcriptome data of P. notoginseng and cloned the full length of 27 UGT genes successfully. Among them, we found that PnUGT33 could catalyze different ginsenoside substrates to produce higher polarity rare ginsenosides by extending the sugar chain. We further analyzed the enzymatic kinetics and predicted the catalytic mechanism of PnUGT33 by simulating molecular docking. After that, we reconstructed the biosynthetic pathway of rare ginsenoside Rg(3) and Gypenoside LXXV in yeast. By combining the Golden Gate method and overexpressing the UDPG biosynthetic genes, we further improved the yield of engineering yeast strain. Finally, the shake-flask culture yield of Rg(3) reached 51 mg/L and the fed-batch fermentation yield of Gypenoside LXXV reached 94.5 mg/L, which was the first and highest record.

Anaphylactic Rare Saponins Separated from Panax notoginseng Saponin and a Proteomic Approach to Their Anaphylactic Mechanism.[Pubmed:35310026]

Evid Based Complement Alternat Med. 2022 Mar 11;2022:7565177.

In recent years, many traditional Chinese medicine injections based on Panax notoginseng saponin (PNS) have been reported to cause anaphylaxis. Previous studies on the anaphylactic saponins of PNS and their mechanism are inadequate. In this study, potential anaphylactic saponins were obtained by the separation of PNS and preparation of each individual component through comprehensive techniques, such as liquid chromatography, preparative chromatography, HPLC, NMR, and MS. The anaphylactic abilities of these saponins were tested using RBL-2H3 cells via a beta-hexosaminidase release rate test. The results for the mechanism of anaphylaxis were obtained by a proteomic analysis using RBL-2H3 cells. The results indicate that, among all the saponins prepared, Gypenoside LXXV and notoginsenoside T5 showed strong anaphylactic abilities and notoginsenoside ST-4 and ginsenoside Rk3 showed weak anaphylactic abilities. These 4 saponins can induce anaphylaxis via direct stimulation of effector cells. The gene oncology enrichment analysis results showed that, among these saponins, only Gypenoside LXXV was related to organelles of the endoplasmic reticulum and Golgi apparatus and biological processes in response to organic cyclic compounds. Four proteins in RBL-2H3 cells with the accession numbers A0A0G2JWQ0, D3ZL85, D4A5G8, and Q8K3F0 were identified as crucial proteins in the anaphylactic process. This research will help traditional Chinese medicine injection manufacturers strengthen their quality control and ensure the safety of anaphylactic saponins.

A novel natural PPARgamma agonist, Gypenoside LXXV, ameliorates cognitive deficits by enhancing brain glucose uptake via the activation of Akt/GLUT4 signaling in db/db mice.[Pubmed:35192202]

Phytother Res. 2022 Apr;36(4):1770-1784.

Targeting the PPARgamma might be a potential therapeutic strategy for diabetes-associated cognitive decline (DACD). In this study, Gypenoside LXXV (GP-75), a dammarane-type triterpene compound isolated from Gynostemma pentaphyllum, was found to be a novel PPARgamma agonist using a dual-luciferase reporter assay system. However, whether GP-75 has protective effects against DACD remains unknown. Interestingly, intragastric administration of GP-75 (40 mg/kg/day) for 12 weeks significantly attenuated the cognitive deficit in db/db mice. GP-75 treatment significantly improved the glucose tolerance and lipid metabolism, and suppressed neuroinflammation. Notably, GP-75 treatment dramatically increased the uptake of glucose by the brain, as detected by (18) F-FDG PET. Incubation of primary cortical neurons with GP-75 significantly increased 2-deoxyglucose uptake. In addition, GP-75 treatment markedly increased the p-Akt (Ser 473)/total Akt levels and the expression levels of PPARgamma and GLUT4, while decreasing the levels of p-IRS-1 (Ser 616)/total IRS-1. Importantly, all of these protective effects mediated by GP-75 were abolished by cotreatment with the PPARgamma antagonist, GW9662. However, GP-75-mediated PPARgamma upregulation was not affected by coincubation with the phosphatidylinositol 3-kinase inhibitor, LY294002. Collectively, GP-75 might be a novel PPARgamma agonist that ameliorates cognitive deficit by enhancing brain glucose uptake via the activation of Akt/GLUT4 signaling in db/db mice.

Characterization of a Group of UDP-Glycosyltransferases Involved in the Biosynthesis of Triterpenoid Saponins of Panax notoginseng.[Pubmed:35107265]

ACS Synth Biol. 2022 Feb 18;11(2):770-779.

UDP-glycosyltransferase (UGT)-mediated glycosylation is a common modification in triterpene saponins, which exhibit a wide range of bioactivities and important pharmacological effects. However, few UGTs involved in saponin biosynthesis have been identified, limiting the biosynthesis of saponins. In this study, an efficient heterologous expression system was established for evaluating the UGT-mediated glycosylation process of triterpene saponins. Six UGTs (UGTPn17, UGTPn42, UGTPn35, UGTPn87, UGTPn19, and UGTPn12) from Panax notoginseng were predicted and found to be responsible for efficient and direct enzymatic biotransformation of 21 triterpenoid saponins via 26 various glycosylation reactions. Among them, UGTPn87 exhibited promiscuous sugar-donor specificity of UDP-glucose (UDP-Glc) and UDP-xylose (UDP-Xyl) by catalyzing the elongation of the second sugar chain at the C3 or/and C20 sites of protopanaxadiol-type saponins with a UDP-Glc or UDP-Xyl donor, as well as at the C20 site of protopanaxadiol-type saponins with a UDP-Glc donor. Two new saponins, Fd-Xyl and Fe-Xyl, were generated by catalyzing the C3-O-Glc xylosylations of notoginsenoside Fd and notoginsenoside Fe when incubated with UGTPn87. Moreover, the complete biosynthetic pathways of 17 saponins were elucidated, among which notoginsenoside L, vinaginsenoside R16, Gypenoside LXXV, and gypenoside XVII were revealed in Panax for the first time. A yeast cell factory was constructed with a yield of Rh2 at 354.69 mg/L and a glycosylation ratio of 60.40% in flasks. Our results reveal the biosynthetic pathway of a group of saponins in P. notoginseng and provide a theoretical basis for producing rare and valuable saponins, promoting their industrial application in medicine and functional foods.

Pharmaceutical Efficacy of Gypenoside LXXV on Non-Alcoholic Steatohepatitis (NASH).[Pubmed:33050067]

Biomolecules. 2020 Oct 8;10(10):1426.

Ginsenosides have offered a wide array of beneficial roles in the pharmacological regulation of hepatic metabolic syndromes, including non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), and obesity. Of the numerous ginsenosides, Rg3 has been widely investigated, but there have been few studies of gypenosides (Gyp). Particularly, no study on Gyp LXXV has been reported to date. Here, to firstly explore the pharmacological effects of Gyp LXXV against NASH and the related mechanism, methionine- and choline-deficient (MCD) diet-induced NASH mice and hepatic cells (stellate cells, hepatic macrophages, and hepatocytes) were selected. Gyp LXXV exhibited markedly alleviated MCD diet-induced hepatic injury, inflammation, and fibrosis by down-regulating hepatic fibrosis markers such as alpha-smooth muscle actin(alpha-SMA), collagen1, transforming growth factors-beta (TGF-beta1), tumor necrosis factor-alpha (TNF-alpha), MCP-1, interleukin (IL)-1beta, nuclear factor kappaB (NFkappaB), and GRP78. Remarkably, histopathological studies confirmed that 15 mg/kg of Gyp LXXV administration to MCD diet-induced mice led to effective prevention of liver injury, lipid accumulation, and activation of hepatic macrophages, indicating that Gyp LXXV might be a potential anti-NASH drug.

Gypenoside LXXV Promotes Cutaneous Wound Healing In Vivo by Enhancing Connective Tissue Growth Factor Levels Via the Glucocorticoid Receptor Pathway.[Pubmed:31018484]

Molecules. 2019 Apr 23;24(8):1595.

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.

Enhanced Production of Gypenoside LXXV Using a Novel Ginsenoside-Transforming beta-Glucosidase from Ginseng-Cultivating Soil Bacteria and Its Anti-Cancer Property.[Pubmed:28534845]

Molecules. 2017 May 19;22(5):844.

Minor ginsenosides, such as compound K, Rg(3)(S), which can be produced by deglycosylation of ginsenosides Rb(1), showed strong anti-cancer effects. However, the anticancer effects of Gypenoside LXXV, which is one of the deglycosylated shapes of ginsenoside Rb(1), is still unknown due to the rarity of its content in plants. Here, we cloned and characterized a novel ginsenoside-transforming beta-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 Rb(1). Taken together, this enzymatic method would be useful in the preparation of Gypenoside LXXV for the functional food and pharmaceutical industries.

An amino acid at position 512 in beta-glucosidase from Clavibacter michiganensis determines the regioselectivity for hydrolyzing gypenoside XVII.[Pubmed:25820645]

Appl Microbiol Biotechnol. 2015 Oct;99(19):7987-96.

A recombinant beta-glucosidase from Clavibacter michiganensis specifically hydrolyzed the outer and inner glucose linked to the C-3 position in protopanaxadiol (PPD)-type ginsenosides and the C-6 position in protopanaxatriol (PPT)-type ginsenosides except for the hydrolysis of Gypenoside LXXV (GypLXXV). The enzyme converted gypenoside XVII (GypXVII) to GypLXXV by hydrolyzing the inner glucose linked to the C-3 position. The substrate-binding residues obtained from the GypXVII-docked homology models of beta-glucosidase from C. michiganensis were replaced with alanine, and the amino acid residue at position 512 was selected because of the changed regioselectivity of W512A. Site-directed mutagenesis for the amino acid residue at position 512 was performed. W512A and W512K hydrolyzed the inner glucose linked to the C-3 position and the outer glucose linked to the C-20 position of GypXVII to produce GypLXXV and F2. W512R hydrolyzed only the outer glucose linked to the C-20 position of GypXVII to produce F2. However, W512E and W512D exhibited no activity for GypXVII. Thus, the amino acid at position 512 is a critical residue to determine the regioselectivity for the hydrolysis of GypXVII. These wild-type and variant enzymes produced diverse ginsenosides, including GypXVII, GypLXXV, F2, and compound K, from ginsenoside Rb1. To the best of our knowledge, this is the first report of the alteration of regioselectivity on ginsenoside hydrolysis by protein engineering.

Characterization of the ginsenoside-transforming recombinant beta-glucosidase from Actinosynnema mirum and bioconversion of major ginsenosides into minor ginsenosides.[Pubmed:22911093]

Appl Microbiol Biotechnol. 2013 Jan;97(2):649-59.

This study focused on the cloning, expression, and characterization of ginsenoside-transforming recombinant beta-glucosidase from Actinosynnema mirum KACC 20028(T) in order to biotransform ginsenosides efficiently. The gene, termed as bglAm, encoding a beta-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 degrees C. BglAm could hydrolyze the outer and inner glucose moieties at the C3 and C20 of the protopanaxadiol-type ginsenosides (i.e., Rb(1) and Rd, gypenoside XVII) to produce protopanaxadiol via Gypenoside LXXV, F(2), and Rh(2)(S) with various pathways. BglAm can effectively transform the ginsenoside Rb(1) to gypenoside XVII and Rd to F(2); the K (m) values of Rb(1) 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 mumol min(-1) mg(-1) of protein, respectively. Furthermore, BglAm could convert the protopanaxatriol-type ginsenoside Re and Rg(1) into Rg(2)(S) and Rh(1)(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.

Highly selective microbial transformation of major ginsenoside Rb1 to gypenoside LXXV by Esteya vermicola CNU120806.[Pubmed:22805203]

J Appl Microbiol. 2012 Oct;113(4):807-14.

AIMS: This study examined the biotransformation pathway of ginsenoside Rb(1) by the fungus Esteya vermicola CNU 120806. METHODS AND RESULTS: Ginsenosides Rb(1) and Rd were extracted from the root of Panax ginseng. Liquid fermentation and purified enzyme hydrolysis were employed to investigate the biotransformation of ginsenoside Rb(1) . The metabolites were identified and confirmed using NMR analysis as gypenoside XVII and Gypenoside LXXV. A mole yield of 95.4% Gypenoside LXXV was obtained by enzymatic conversion (pH 5.0, temperature 50 degrees C). Ginsenoside Rd was used to verify the transformation pathway under the same reaction condition. The product Compound K (mole yield 49.6%) proved a consecutive hydrolyses occurred at the C-3 position of ginsenoside Rb(1) . CONCLUSIONS: Strain CNU 120806 showed a high degree of specific beta-glucosidase activity to convert ginsenosides Rb(1) and Rd to Gypenoside LXXV and Compound K, respectively. The maximal activity of the purified glucosidase for ginsenosides transformation occurred at 50 degrees C and pH 5.0. Compared with its activity against pNPG (100%), the beta-glucosidase exhibited quite lower level of activity against other aryl-glycosides. Enzymatic hydrolysate, Gypenoside LXXV and Compound K were produced by consecutive hydrolyses of the terminal and inner glucopyranosyl moieties at the C-3 carbon of ginsenoside Rb(1) and Rd, giving the pathway: ginsenoside Rb(1) --> gypenoside XVII --> Gypenoside LXXV; ginsenoside Rd-->F(2) -->Compound K, but did not hydrolyse the 20-C, beta-(1-6)-glucoside of ginsenoside Rb(1) and Rd. SIGNIFICANCE AND IMPACT OF THE STUDY: The results showed an important practical application on the preparation of Gypenoside LXXV. Additionally, this study for the first time provided a high efficient preparation method for Gypenoside LXXV without further conversion, which also gives rise to a potential commercial enzyme application.

Kinetics of a cloned special ginsenosidase hydrolyzing 3-O-glucoside of multi-protopanaxadiol-type ginsenosides, named ginsenosidase type III.[Pubmed:22450790]

J Microbiol Biotechnol. 2012 Mar;22(3):343-51.

In this paper, the kinetics of a cloned special glucosidase, named ginsenosidase type III hydrolyzing 3-O-glucoside of multi-protopanaxadiol (PPD)-type ginsenosides, were investigated. The gene (bgpA) encoding this enzyme was cloned from a Terrabacter ginsenosidimutans strain and then expressed in E. coli cells. Ginsenosidase type III was able to hydrolyze 3-O-glucoside of multi-PPD-type ginsenosides. For instance, it was able to hydrolyze the 3-O-beta-D-(1-->2)-glucopyranosyl of Rb1 to gypenoside XVII, and then to further hydrolyze the 3-O-beta-D-glucopyranosyl of gypenoside XVII to Gypenoside LXXV. Similarly, the enzyme could hydrolyze the glucopyranosyls linked to the 3-O- position of Rb2, Rc, Rd, Rb3, and Rg3. With a larger enzyme reaction Km value, there was a slower enzyme reaction speed; and the larger the enzyme reaction Vmax value, the faster the enzyme reaction speed was. The Km values from small to large were 3.85 mM for Rc, 4.08 mM for Rb1, 8.85 mM for Rb3, 9.09 mM for Rb2, 9.70 mM for Rg3(S), 11.4 mM for Rd and 12.9 mM for F2; and Vmax value from large to small was 23.2 mM/h for Rc, 16.6 mM/h for Rb1, 14.6 mM/h for Rb3, 14.3 mM/h for Rb2, 1.81mM/h for Rg3(S), 1.40 mM/h for Rd, and 0.41 mM/h for F2. According to the Vmax and Km values of the ginsenosidase type III, the hydrolysis speed of these substrates by the enzyme was Rc>Rb1>Rb3>Rb2>Rg3(S)>Rd>F2 in order.

Identification and characterization of a novel Terrabacter ginsenosidimutans sp. nov. beta-glucosidase that transforms ginsenoside Rb1 into the rare gypenosides XVII and LXXV.[Pubmed:20622122]

Appl Environ Microbiol. 2010 Sep;76(17):5827-36.

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.