Search

A novel yellow-pigmented catalase- and oxidase-positive bacterial strain (designated NA20 T ) was isolated from wetland soil and characterized. Results of 16S rRNA and draft genome sequence analysis placed strain NA20 T within the genus Terrimonas of the family Chitinophagaceae . Strain NA20 T showed ≤97.1 % sequence similarity to members of the genus Terrimonas and the highest sequence similarity was found to Terrimonas lutea DY T (97.1%). The draft genome of strain NA20 T had a total length of 7 144 125 base pairs. A total of 5659 genes were identified, of which 5613 were CDS and 46 RNA genes were assigned a putative function. Mining the genomes revealed the presence of 225 carbohydrate genes out of 1334 genes. Strain NA20 T contained iso-C 15 : 0 , iso-C 15 : 0 G, iso-C 17 : 0 3-OH and summed feature 3 (C 16 : 1  ω 7 c and/or C 16 : 1  ω showed the conversion of protopanaxatriol-mix type major ginsenosides (Rb1, Rc and Rd) to minor ginsenosides F2 and weak conversion of Rh2 and C-K within 24 h. As a result, the genotypic, phenotypic and taxonomic analyses support the affiliation of NA20 c ) as major fatty acids. The predominant quinone was MK-7. The major polar lipids were phosphatidylethanolamine, one unknown polar lipid and one unknown aminophospholipid. Additionally, the functional analysis of NA20 T showed the conversion of protopanaxatriol-mix type major ginsenosides (Rb1, Rc and Rd) to minor ginsenosides F2 and weak conversion of Rh2 and C-K within 24 h. As a result, the genotypic, phenotypic and taxonomic analyses support the affiliation of NA20 T within the genus Terrimonas , for which the name Terrimonas ginsenosidimutans sp. nov. is proposed. The type strain is NA20 T (=KACC 22218 T =LMG 32198 T ).

Background and Objective: The ginsenoside compound K (C-K) (which is a de-glycosylated derivative of major ginsenosides) is effective in the treatment of cancer, diabetes, inflammation, allergy, angiogenesis, aging, and has neuroprotective, and hepatoprotective than other minor ginsenosides. Thus, a lot of studies have been focused on the conversion of major ginsenosides to minor ginsenosides using glycoside hydrolases but there is no study yet published for the bioconversion of minor ginsenosides into another high pharmacological active compound. Therefore, the objective of this study to identify a new gene (besides the glycoside hydrolases) for the conversion of minor ginsenosides C-K into another highly pharmacological active compound., Methods and Results: Lactobacillus brevis which was isolated from Kimchi has showed the ginsenoside C-K altering capabilities. From this strain, a novel potent decarboxylation gene, named HSDLb1, was isolated and expressed in Escherichia coli BL21 (DE3) using the pMAL-c5X vector system. Recombinant HSDLb1 was also characterized. The HSDLb1 consists of 774 bp (258 amino acids residues) with a predicted molecular mass of 28.64 kDa. The optimum enzyme activity was recorded at pH 6.0-8.0 and temperature 30 °C. Recombinant HSDLb1 effectively transformed the ginsenoside C-K to 12-β-hydroxydammar-3-one-20(S)-O-β-D-glucopyranoside (3-oxo-C-K). The experimental data proved that recombinant HSDLb1 strongly ketonized the hydroxyl (-O-H) group at C-3 of C-K via the following pathway: C-K → 3-oxo-C-K. In vitro study, 3-oxo-C-K showed higher solubility than C-K, and no cytotoxicity to fibroblast cells. In addition, 3-oxo-C-K induced the inhibitory activity of ultraviolet A (UVA) against matrix metalloproteinase-1 (MMP-1) and promoted procollagen type I synthesis. Based on these expectations, we hypothesized that 3-oxo-C-K can be used in cosmetic products to block UV radiations and anti-ageing agent. Furthermore, we expect that 3-oxo-C-K will show higher efficacy than C-K for the treatment of cancer, ageing and other related diseases, for which more studies are needed.

A novel Gram-stain-positive, thin rod-shaped, non-motile, aerobic and creamy-white pigmented bacterium (designated strain WJ7-1 T ) was isolated from activated sludge sampled in Suwon, Republic of Korea. 16S rRNA gene sequence analysis indicated that the isolate belongs to the genus Humibacter , family Microbacteriaceae , with a sequence similarity of 97.9 % to its nearest neighbour Humibacter albus KACC 20986 T . Strain WJ7-1 T contained menaquinone (MK)-10 (16.0 %), 11 (48.5 %) and 12 (35.5 %) as major respiratory quinones. The predominant cellular fatty acids (>15 %) were anteiso-C 17 : 0 , iso-C 16 : 0 and anteiso-C 15 : 0 . The peptidoglycan of strain WJ7-1 T contained the diagnostic diamino acid ornithine and 2,4-diaminobutyric acid alanine. Alanine, glutamic acid and glycine were also present in the cell wall. The characteristic whole-cell sugars present were glucose, galactose, xylose and rhamnose. The polar lipids consisted of phosphatidylglycerol, diphosphatidylglycerol, an unidentified glycolipid and two unidentified phospholipids. Strain WJ7-1 T possessed ginsenoside-converting ( β -glucosidase) activity, which enabled it to transform ginsenoside protopanaxadiol-type Rb 1 (one of the dominant active components of ginseng) to compound K. The genome size of strain WJ7-1 T has 4.2 Mbp and the G+C content is 68.3 mol%. Average nucleotide identity, amino acid identity and digital DNA-DNA hybridization values between strain WJ7-1 T and the closely related strain were 79.8, 36.1 and 23.5 %, respectively, indicating that strain WJ7-1 T represents a novel species of the genus Humibacter . Strain WJ7-1 T could be distinguished from the other members of the genus Humibacter by a number of chemotaxonomic and phenotypic characteristics. Based on polyphasic taxonomic analysis, strain WJ7-1 T (=KACC 19729 T =LMG 30802 T ) represents a novel species within the genus Humibacter , for which the name Humibacter ginsenosidimutans sp. nov. is proposed.

A Gram-stain-negative, non-motile, non-spore-forming, aerobic, rod-shaped and yellow-pigmented bacterium, designated strain Gsoil 183 T , was isolated from ginseng-cultivation soil sampled in Pocheon Province, Republic of Korea. This bacterium was characterized to determine its taxonomic position by using a polyphasic approach. Strain Gsoil 183 T grew at 10-37 °C and at pH 5.0-9.0 on tryptic soy agar. Strain Gsoil 183 T had β -glucosidase activity, which was responsible for its ability to convert ginsenoside Rb1 (one of the dominant active components of ginseng) to F2. Based on 16S rRNA gene sequencing, strain Gsoil 183 T clustered with species of the genus Chryseobacterium and appeared to be closely related to Chryseobacterium sediminis LMG 28695 T (99.1 % sequence similarity), Chryseobacterium lactis NCTC 11390 T (98.6%), Chryseobacterium rhizoplanae LMG 28481 T (98.6%), Chryseobacterium oncorhynchi CCUG 60105 T (98.5%), Chryseobacterium viscerum CCUG 60103 T (98.4%) and Chryseobacterium joostei DSM 16927 T (98.3%). Menaquinone MK-6 was the predominant respiratory quinone and the major fatty acids were iso-C 15 : 0 , iso-C 17 : 0 -3OH and summed feature 3 (C 16 : 1  ω 6 c and/or C 16 : 1  ω 7 c ). The polar lipids were phosphatidylethanolamine, six unidentified glycolipids, five unidentified aminolipids and three unidentified lipids. The G+C content of the genomic DNA was 36.6 mol%. Digital DNA-DNA hybridization between strain Gsoil 183 T and the type strains of C. sediminis , C. lactis , C. rhizoplanae , C. oncorhynchi , C. viscerum and C. joostei resulted in values below 70 %. Strain Gsoil 183 T could be differentiated genotypically and phenotypically from the recognized species of the genus Chryseobacterium . The isolate therefore represents a novel species, for which the name Chryseobacterium panacisoli sp. nov. is proposed, with the type strain Gsoil 183 T (=KACC 15033 T =LMG 23397 T ).

The conversion of major ginsenosides into minor ginsenosides attracts a lot of interest because of their biological and pharmaceutical activities. Therefore, for the conversion of ginsenosides, finding a novel competent glycoside hydrolase-producing bacterial strain is useful for future research studies and the mass production of minor ginsenosides. Wastewater samples were collected and screened for novel glycoside hydrolase bacterial strains using Reasoner's 2A+aesculin agar medium. As a result, a novel glycoside hydrolase positive bacterial strain (SB-02 T ) was identified and subjected to a polyphasic taxonomic analysis. Based on genome analysis, strain SB-02 T was found to be affiliated with the family Chitinophagaceae and have less than 92.8 % sequence similarity to other members of the same family. Functional analysis indicated that SB-02 T was able to hydrolyse the ginsenosides Rb1, Rc and Rd to F2 and C-K. Due to the conversion of ginsenosides, the strain's genome was sequenced and the genes were annotated by the NCBI. The average amino acid identity and average nucleotide identity values between SB-02 T and the available reference genomes were 65.7 and 65.9 %, respectively. The novel isolate contained MK-7 as the predominant menaquinone, the major polyamine putrescine, and iso-C 15 : 0 , iso-C 15 : 1 G and iso-C 17 : 0 3-OH as major fatty acids. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Thus, based on the data presented here, strain SB-02 T represents a novel species within a new genus in the family Chitinophagaceae , for which the name Phnomibacter ginsenosidimutans gen. nov., sp. nov. is proposed. The type strain of Phnomibacter ginsenosidimutans is SB-02 T (=KACC 21266 T =LMG 31707 T ). The genome annotation of SB-02 T shows many glycoside hydrolase genes, which may be responsible for the efficient production of many kinds of minor ginsenosides and will be very helpful for future research (target gene cloning) and mass production of either F2 or C-K.

A novel yellow-pigmented bacterial strain (designated BR5-29 T ), was isolated and its taxonomy was studied. Phylogenetic study based on the 16S rRNA and draft genome sequence placed the strain BR5-29 T in a distinct lineage within the family Chitinophagaceae, sharing ≤ 93.4% sequence similarity with members of the closely related genera Ferruginibacter, Flavisolibacter, Flavitalea and Niastella. The novel isolate showed the highest sequence similarity to the genus Ferruginibacter. The draft genome of strain BR5-29 T had a total length of 5,505,520 base pairs. A total of 4585 genes were identified, in which 4537 were CDS and 48 RNA genes were assigned a putative function. The genome annotation of BR5-29 T showed 225 carbohydrate genes which may be responsible for the conversion of major ginsenosides to minor ginsenosides. Strain BR5-29 T contained MK-7 as a predominant quinone, and iso-C 15:0 , iso-C 15:0 G, iso-C 17:0 3-OH, and C 16:1 ω7c and/or C 16:1 ω6c (summed feature 3) as major fatty acids. The polar lipids found in the strain BR5-29 T were phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), five unidentified polar lipids (L1-L5), two unidentified aminolipid and one unidentified aminophospholipid. Our pilot data demonstrate that the novel isolate shares the similar major polar lipid PE, major quinone MK-7 and major fatty acids with the described members of the family Chitinophagaceae. However, the low 16S rRNA gene sequence (< 93.4%), the little high amount of C 12:0 , iso-C 17:0 2-OH and iso-C 15:1 2-OH fatty acids, low DNA G + C content, and the presence of DPG, PG and two unidentified polar lipids (L1 and L3 differentiate the BR5-29 T from its closest phylogenetic neighbors. Thus, the isolate represents a novel genus and species in the family Chitinophagaceae for which the name Ginsengibacter hankyongi gen. nov., sp. nov. is proposed. The type strain is BR5-29 T (= KACC 19446 T  = LMG 30462 T ). Thus, we predict that this novel strain may prove useful for the future research analysis (target gene cloning) and mass production of Rg3.

In this study, a novel ginsenoside transforming bacterium, strain W1-2-3 T , was isolated from mineral water. The 16S rRNA gene sequence analysis showed that strain W1-2-3 T shares 93.7-92.2% sequence similarity with the members of the family Sphingomonadaceae and makes a group with Sphingoaurantiacus capsulatus YLT33 T (93.7%) and S. polygranulatus MC 3718 T (93.4%). The novel isolate efficiently hydrolyses the ginsenoside Rc to Rd. The genome comprises a single circular 2,880,809, bp chromosome with 3211 genes in total, and 1993 protein coding genes. The isolate was observed to grow at 10-37 °C and at pH 6-10 on R2A agar medium; maximum growth was found to occur at 25 °C and pH 7.0. Strain W1-2-3 T was found to contain ubiquinone-10 as the predominant quinone and the fatty acids C 16:1 , C 17:1 ω6c, C 14:0 2-OH, summed feature 3 (C 16:1 ω6c/C 16:1 ω7c) and summed feature 8 (C 18 : 1 ω6c/C 18:1 ω7c). The DNA G+C content was determined to be 65.9 mol%. Strain W1-2-3 T can be distinguished from the other members of the family Sphingomonadaceae by a number of chemotaxonomic and phenotypic characteristics. The major polar lipids of strain W1-2-3 T were identified as phosphatidylethanolamine, an unidentified glycolipid and an unidentified polar lipid. The major poly amine was found to be homospermidine. Based on polyphasic taxonomic analysis, strain W1-2-3 T is concluded to represent a novel species within a new genus, for which the name Hankyongella ginsenosidimutans gen. nov., sp. nov. is proposed. The type strain of Hankyongella ginsenosidimutans is W1-2-3 T (= KACC 18307 T  = LMG 28594 T ).

In this study, we used a novel α-L-arabinopyranosidase (AbpBs) obtained from ginsenoside-converting Blastococcus saxobsidens that was cloned and expressed in Escherichia coli BL21 (DE3), and then applied it in the biotransformation of ginsenoside Rb 2 into Rd. The gene, termed AbpBs , consisting of 2,406 nucleotides (801 amino acid residues), and with a predicted translated protein molecular mass of 86.4 kDa, was cloned into a pGEX4T-1 vector. A BLAST search using the AbpBs amino acid sequence revealed significant homology with a family 2 glycoside hydrolase (GH2). The over-expressed recombinant AbpBs in Escherichia coli BL21 (DE3) catalyzed the hydrolysis of the arabinopyranose moiety attached to the C-20 position of ginsenoside Rb2 under optimal conditions (pH 7.0 and 40°;C). Kinetic parameters for α-Larabinopyranosidase showed apparent K m and V max values of 0.078 ± 0.0002 micrometer and 1.4 ± 0.1 μmol/min/mg of protein against p -nitrophenyl-α-L-arabinopyranoside. Using a purified AbpBs (1 μg/ml), 0.1% of ginsenoside Rb 2 was completely converted to ginsenoside Rd within 1 h. The recombinant AbpBs could be useful for high-yield, rapid, and low-cost preparation of ginsenoside Rd from Rb 2 .

Background: Several studies have reported that ginsenoside Rg3( S ) is effective in treating metastatic diseases, obesity, and various cancers, however, its presence in white ginseng cannot be estimated, and only a limited amount is present in red ginseng. Therefore, the use of recombinant glycosidases from a Generally Recognized As Safe (GRAS) host strain is a promising approach to enhance production of Rg3( S ), which may improve nutritional activity, human health, and quality of life., Method: Lactobacillus ginsenosidimutans EMML 3041 T , which was isolated from Korean fermented pickle (kimchi), presents ginsenoside-converting abilities. The strain was used to enrich the production of Rg3( S ) by fermenting protopanaxadiol (PPD)-mix-type major ginsenosides (Rb1, Rb2, Rc, and Rd) in four different types of food-grade media (1, MRS; 2, Basel Food-Grade medium; 3, Basel Food-Grade medium-I, and 4, Basel Food-Grade medium-II). Due to its tendency to produce Rg3( S ), the presence of glycoside hydrolase in Lactobacillus ginsenosidimutans was proposed, the whole genome was sequenced, and the probable glycoside hydrolase gene for ginsenoside conversion was cloned., Results: The L. ginsenosidimutans EMML 3041 T strain was whole genome sequenced to identify the target genes. After genome sequencing, 12 sets of glycoside hydrolases were identified, of which seven sets (α,β-glucosidase and α,β-galactosidase) were cloned in Escherichia coli BL21 (DE3) using the pGEX4T-1 vector system. Among the sets of clones, only one clone (BglL.gin-952) showed ginsenoside-transforming abilities. The recombinant BglL.gin-952 comprised 952 amino acid residues and belonged to glycoside hydrolase family 3. The enzyme exhibited optimal activity at 55 °C and a pH of 7.5 and showed a promising conversion ability of major ginsenoside Rb1→Rd→Rg3( S ). The recombinant enzyme (GST-BglL.gin-952) was used to mass produce Rg3( S ) from major ginsenoside Rb1. Scale-up of production using 50 g of Rb1 resulted in 30 g of Rg3( S ) with 74.3% chromatography purity., Conclusion: Our preliminary data demonstrated that this enzyme would be beneficial in the preparation of pharmacologically active minor ginsenoside Rg3( S ) in the functional food and pharmaceutical industries.

Use of recombinant glycosidases is a promising approach for the production of minor ginsenosides, e.g., Compound K (CK) and F 1 , which have potential applications in the food industry. However, application of these recombinant enzymes for food-grade preparation of minor ginsenosides are limited by the lack of suitable expression hosts and low productivity. In this study, Corynebacterium glutamicum ATCC13032, a GRAS strain that has been used extensively for the industrial-grade production of additives for foodstuffs, was employed to express a novel β-glucosidase (MT619) from Microbacterium testaceum ATCC 15829 with high ginsenoside-transforming activity. A cellulose-binding module was additionally fused to the N-terminus of MT619 for immobilization on cellulose, which is an abundant and safe material. Via one-step immobilization, the fusion protein in cell lysates was efficiently immobilized on regenerated amorphous cellulose at a high density (maximum 984 mg/g cellulose), increasing the enzyme concentration by 286-fold. The concentrated and immobilized enzyme showed strong conversion activities against protopanaxadiol- and protopanaxatriol-type ginsenosides for the production of CK and F 1 . Using gram-scale ginseng extracts as substrates, the immobilized enzyme produced 7.59 g/L CK and 9.42 g/L F 1 in 24 h. To the best of our knowledge, these are the highest reported product concentrations of CK and F 1 , and this is the first time that a recombinant enzyme has been immobilized on cellulose for the preparation of minor ginsenosides. This safe, convenient, and efficient production method could also be effectively exploited in the preparation of food-processing recombinant enzymes in the pharmaceutical, functional food, and cosmetics industries.

Neuroblastoma (NB) is the most common childhood cancer, with a very poor prognosis. More than 60% of children with NB die within five years; therefore, a more effective therapy for NB is required. Although ginsenoside has been shown to significantly inhibit the growth of various cancers, the effect of ginsenoside Rk1 on neuroblastoma has not been known yet. Hence, we examined the anticancer effects of highly pure Rk1 on neuroblastoma cell lines. The apoptotic effects of Rk1 on neuroblastoma cells were examined using cell viability assay, flow cytometry and cell staining assay, and the change in gene expression levels were analysed using RT-PCR, western blots, and immunohistochemistry. The metastatic effect of Rk1 was monitored by wound healing assay, invasion and migration with Matrigels. Rk1 inhibited neuroblastoma cell viability dose-dependently. Rk1-induced apoptosis was investigated through nuclear condensation and mitochondrial membrane potential loss, and it showed that Rk1 can induce cell cycle arrest at the G0/G1 phase but also inhibit the metastatic ability of neuroblastoma cells. Moreover, Rk1 (30 mg/kg) injections markedly inhibited xenograft tumor growth. These findings demonstrate that Rk1 might be valuable in the development of anti-cancer agents for neuroblastoma treatment.

Ginsenoside Rh 2 , a protopanaxadiol saponin from ginseng, has been reported to have strong anti-inflammatory activity. However, the concentration of ginsenoside Rh 2 is very low (>0.001%) in the total ginseng extracted, which is not enough for production despite its high pharmacological effects. Thus, in this study, we evaluated the anti-inflammatory effect of ginsenoside Rh 2 -mix (GRh 2 -mix) on lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophage cells. From the high-performance liquid chromatography analysis, it was confirmed that the GRh 2 -mix was mainly composed of 20(S)-Rh 2 , 20(R)-Rh 2 , Rk 2 , and Rh 3 . The LPS-stimulated RAW 264.7 cells were treated with different concentrations of GRh 2 -mix (100, 200, 400, 500 μg/mL). The cell counting kit-8 assay showed that the GRh 2 -mix treatment increased cell proliferation in LPS-stimulated RAW 264.7 murine macrophage cells. The GRh 2 -mix inhibited nitric oxide production in a dose-dependent manner, suggesting an anti-inflammatory effect. Furthermore, reverse transcription polymerase chain reaction and Western blot results also indicated that the GRh 2 -mix suppressed inflammatory genes such as iNOS, TNF-α, COX-2, IL-1β, IL-6, and NF-κB. In summary, these results suggest that the GRh 2 -mix exhibits anti-inflammatory activity via the downregulation of the NF-κB pathway and has high efficiency with a simple production procedure.

Biotic elicitation is an important biotechnological strategy for triggering the accumulation of secondary metabolites in adventitious root cultures. These biotic elicitors can be obtained from safe, economically important strains of bacteria found in the rhizosphere and fermented foods. Here, we assayed the effects of filtered cultures of five nitrogen-fixing bacteria and four types of fermentation bacteria on mutant adventitious Panax ginseng root cultures induced in a previous study by colchicine treatment. The biomass, pH, and electrical conductivity (EC) of the culture medium were altered at 5 days after treatment with bacteria. The saponin content was highest in root cultures treated with Mesorhizobium amorphae (GS3037), with a concentration of 105.58 mg g -1 dry weight saponin present in these cultures versus 74.48 mg g -1 dry weight in untreated root cultures. The accumulation of the ginsenosides Rb 2 and Rb 3 dramatically increased (19.4- and 4.4-fold, and 18.8- and 4.8-fold) 5 days after treatment with M. amorphae (GS3037) and Mesorhizobium amorphae (GS336), respectively. Compound K production increased 1.7-fold after treatment with M. amorphae (GS3037) compared with untreated root cultures. These results suggest that treating mutant adventitious root cultures with biotic elicitors represents an effective strategy for increasing ginsenoside production in Panax ginseng.

Naturally occurring ginsenoside F1 (20-O-β-D-glucopyranosyl-20(S)-protopanaxatriol) is rare. Here, we produced gram-scale quantities of ginsenoside F1 from a crude protopanaxatriol saponin mixture comprised mainly of Re and Rg1 through enzyme-mediated biotransformation using recombinant β-glucosidase (BgpA) cloned from a soil bacterium, Terrabacter ginsenosidimutans Gsoil 3082 T . In a systematic step-by-step process, the concentrations of substrate, enzyme, and NaCl were determined for maximal production of F1. At an optimized NaCl concentration of 200 mM, the protopanaxatriol saponin mixture (25 mg/ml) was incubated with recombinant BgpA (20 mg/ml) for 3 days in a 2.4 L reaction. Following octadecylsilyl silica gel column chromatography, 9.6 g of F1 was obtained from 60 g of substrate mixture at 95% purity, as assessed by chromatography. These results represent the first report of gramscale F1 production via recombinant enzyme-mediated biotransformation.

A Gram-stain-negative, oxidase- and catalase-positive, strictly aerobic, non-gliding bacterial strain, designated as NPT, was isolated from lake water and subjected to a taxonomic study using a polyphasic approach. Colonies of strain NP5T were light-yellow-coloured, circular with regular margins, and opaque. Cells were rods, 0.2-0.5 µm wide and 1.2-3.0 µm long. Strain NP5T possessed β-glycosidase activity, which was responsible for its ability to convert ginsenosides Rb1, Rc and Rd (three main active components of ginseng) to ginsenoside F2. Phylogenetic study based on the 16S rRNA gene sequence put strain NP5T in a distinct lineage in the family Flavobacteriaceae, sharing less than 96.0 % sequence similarity with members of the closely related genera Chryseobacterium, Bergeyella, Epilithonimonas and 'Candidatus Amoebinatus'. The novel isolate showed the highest sequence similarity with the genus Chryseobacterium. Strain NP5T contained MK-6 as predominant quinone, and iso-C15 : 0, iso-C17 : 0 3-OH, iso-C16 : 0 3-OH, C18 : 0 3-OH, anteiso-C15 : 0 and C16 : 1ω7c and/or C16 : 1ω6c (summed feature 3) as major fatty acids. The DNA G+C content was 44.8 mol%. The main polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The physiological, biochemical and taxonomical characteristics allowed the phenotypic differentiation of strain NP5T from its closest phylogenetic neighbours. On the basis of the evidence of this polyphasic study, isolate NP5T represents a novel genus and species in the family Flavobacteriaceae for which the name Daejeonia ginsenosidivorans gen. nov., sp. nov. is proposed. The type strain is NP5T (=KACC 18626T=LMG 29198T).

The ginsenoside Rh2 has strong anti-cancer, anti-inflammatory, and anti-diabetic effects. However, the application of ginsenoside Rh2 is restricted because of the small amounts found in Korean white and red ginsengs. To enhance the production of ginsenoside Rh2-MIX (comprising 20( S )-Rh2, 20( R )-Rh2, Rk2, and Rh3 as a 10-g unit) with high specificity, yield, and purity, a new combination of enzymatic conversion using the commercial enzyme Viscozyme L followed by acid treatment was developed. Viscozyme L treatment at pH 5.0 and 50°C was used initially to transform the major ginsenosides Rb1, Rb2, Rc, and Rd into ginsenoside F2, followed by acid-heat treatment using citric acid 2% (w/v) at pH 2.0 and 121°C for 15 min. Scale-up production in a 10-L jar fermenter, using 60 g of the protopanaxadiol-type ginsenoside mixture from ginseng roots, produced 24 g of ginsenoside Rh2-MIX. Using 2 g of Rh2-MIX, 131 mg of 20( S )-Rh2, 58 mg of 20( R )-Rh2, 47 mg of Rk2, and 26 mg of Rh3 were obtained at over 98% chromatographic purity. Then, the anti-cancer effect of the four purified ginsenosides was investigated on B16F10, MDA-MB-231, and HuH-7 cell lines. As a result, these four rare ginsenosides markedly inhibited the growth of the cancer cell lines. These results suggested that rare ginsenoside Rh2-MIX could be exploited to prepare an anti-cancer supplement in the functional food and pharmaceutical industries.

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 β-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., Competing Interests: The authors declare no competing financial interest.

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 β-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&#95;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&#95;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.

A Gram-reaction-negative, catalase- and oxidase-positive, aerobic, non-motile, light yellow and rod-shaped bacterium (designated Gsoil 809T) isolated from soil of ginseng field, was characterized by a polyphasic approach to clarify its taxonomic position. Strain Gsoil 809T was observed to grow optimally at 30 °C and at pH 7.0 on nutrient agar medium. Strain Gsoil 809T possessed β-glucosidase activity, which was responsible for its ability to transform protopanaxatriol-type ginsenoside Rg1 to ginsenoside Rh1. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain Gsoil 809T belongs to the genus Arachidicoccus of the family Chitinophagaceae and was most closely related to Arachidicoccusrhizosphaerae Vu-144T (98.1 % 16S rRNA gene sequence similarity). The DNA G+C content was 39.4 mol%. The DNA-DNA hybridization value between strain Gsoil 809T and A.rhizosphaerae Vu-144T was 41.27±1.03 %. The major polar lipids were phosphatidylethanolamine and an unknown polar lipid. The predominant quinone was MK-7. The major fatty acids were iso-C15 : 0, iso-C15 : 1 G, iso-C17 : 0 3-OH and summed feature 3, which supported the affiliation of Gsoil 809T to the genus Arachidicoccus. Strain Gsoil 809T contained homospermidineas the major polyamine. Moreover, the physiological and biochemical test results and low DNA-DNA relatedness value allowed the phenotypic and genotypic differentiation of strain Gsoil 809T from recognized species of the genus Arachidicoccus. Therefore, strain Gsoil 809T represents a novel species of the genus Arachidicoccus, for which the name Arachidicoccus ginsenosidivorans sp. nov. is proposed. The type strain is Gsoil 809T (=KCTC 22820T=JCM 30984T).

A Gram-stain-negative, strictly aerobic, non-motile, light yellow, short-rod bacterium (designated GJ30-7T) isolated from compost, was characterized using a polyphasic approach, in order to clarify its taxonomic position. Strain GJ30-7T was observed to grow optimally at 30 °C and at pH 7.0 on R2A agar medium. Strain Gsoil GJ30-7T possessed β-glucosidase activity, which was responsible for its ability to transform ginsenosides Rb1 and Rc (the two main active components of ginseng) to ginsenoside F2. Phylogenetic analysis, based on 16S rRNA gene sequence similarities, indicated that GJ30-7T belongs to the genus Sphingobacterium of the family Sphingobacteriaceae and was most closely related to Sphingobacterium yanglingense JCM 30166T (92.6 %), Sphingobacterium psychroaquaticum KACC 18188T (92.6 %), and Sphingobacterium thermophilum KCTC 23708T (92.0 %). The DNA G+C content was 43 mol% and MK-7 was the major isoprenoid quinone. The main polar lipids were phosphatidylethanolamine, one unidentified phospolipid and one unknown polar lipid. In contrast to standard and reference strains, unidentified sphingolipid was also present. The predominant fatty acids of strain GJ30-7T were iso-C15 : 0, iso-C17 : 0 3-OH, C16 : 1ω7c and/or C16 : 1ω6c (summed feature 3) and iso-C17 : 1ω9c and/or C16 : 0 10-methyl (summed feature 9), supporting the affiliation of strain GJ30-7T to the genus Sphingobacterium. However, strain GJ30-7T could be distinguished genotypically and phenotypically from species of the genus Sphingobacterium with validly published names. The novel isolate therefore represents a novel species, for which the name Sphingobacterium jejuense sp. nov. is proposed, with the type strain GJ30-7T (=KACC 18625T=JCM 30948T).

A ginsenoside-transforming bacterium, designated Gsoil 1550T, was isolated from soil of a ginseng field and subjected to a polyphasic taxonomic analysis. Colonies of strain Gsoil 1550T were yellow, of low convexity and with regular margin. Cells were long rods, 0.5-1.2 µm wide and 1.6-3 µm long. The isolate grew at 10-37 °C and at pH 5-9 on R2A agar medium; maximum growth occurred at 30 °C and pH 6-7. Phylogenetic study based on the 16S rRNA gene sequence positioned Gsoil 1550T in a distinct lineage in the family Chitinophagaceae, sharing 92.5-92.8 % sequence similarity with members of the closely related genera Terrimonas, Parafilimonas, Sediminibacterium and Parasegetibacter. Strain Gsoil 1550T contained menaquinone MK-7 as the predominant quinone, and iso-C15 : 0, iso-C17 : 0 3-OH, C16 : 0 and iso-C15 : 1 G as major fatty acids. The DNA G+C content was 44.6 mol%. Strain Gsoil 1550T could be distinguished from other members of the family Chitinophagaceae by a number of chemotaxonomic and phenotypic characteristics. The major polar lipid of strain Gsoil 1550T was phosphatidylethanolamine. Based on this polyphasic taxonomic analysis, strain Gsoil 1150T represents a novel species within a new genus, for which the name Panacibacter ginsenosidivorans gen. nov., sp. nov. is proposed. The type strain of Panacibacter ginsenosidivorans is Gsoil 1550T (=KCTC 12658T=JCM 31452T).

This study aimed to produce a pharmacologically active minor ginsenoside F2 from the major ginsenosides Rb1 and Rd by using a recombinant Lactococcus lactis strain expressing a heterologous β-glucosidase gene. The nucleotide sequence of the gene (BglPm) was derived from Paenibacillus mucilaginosus and synthesized after codon optimization, and the two genes (unoptimized and optimized) were expressed in L. lactis NZ9000. Codon optimization resulted in reduction of unfavorable codons by 50% and a considerable increase in the expression levels (total activities) of β-glucosidases (0.002 unit/mL, unoptimized; 0.022 unit/mL, optimized). The molecular weight of the enzyme was 52 kDa, and the purified forms of the enzymes could successfully convert Rb1 and Rd into F2. The permeabilized L. lactis expressing BglPm resulted in a high conversion yield (74%) of F2 from the ginseng extract. Utilization of this microbial cell to produce F2 may provide an alternative method to increase the health benefits of Panax ginseng.

A Gram-reaction-negative, aerobic, non-motile and rod-shaped bacterial strain designated Gsoil 636T was isolated from soil of a ginseng cultivation field in Pocheon Province, South Korea and its taxonomic position was investigated using a polyphasic approach. Gsoil 636T grew at 18-30 °C and at pH 6.0-8.0 on R2A medium. Gsoil 636T possessed β-glucosidase activity, which was responsible for its ability to transform ginsenoside Rb1 (ones of the dominant active components of ginseng) to F2. On the basis of 16S rRNA gene sequence similarity, Gsoil 636T was shown to belong to the family Chitinophagaceae and to be related to Flavisolibacter ginsengiterrae Gsoil 492T (96.7 % sequence similarity), Flavisolibacter ginsengisoli Gsoil 643T (96.6 %) and Flavisolibacter rigui 02SUJ3T (96.6 %). The G+C content of the genomic DNA was 48.9 %. The predominant respiratory quinone was MK-7 and the major fatty acids were iso-C15 : 0, summed feature 3 (comprising C16 : 1ω6c and/or C16 : 1ω7c) and iso-C17 : 0 3-OH. DNA and chemotaxonomic data supported the affiliation of Gsoil 636T to the genus Flavisolibacter. Gsoil 636T could be differentiated genotypically and phenotypically from the species of the genus Flavisolibacter with validly published names. The isolate therefore represents a novel species, for which the name Flavisolibacter ginsenosidimutans sp. nov. is proposed, with the type strain Gsoil 636T (KCTC 22818T = JCM 18197T = KACC 14277T).

The Gram-reaction-negative, strictly aerobic, non-motile, nonspore-forming, and rod-shaped bacterial strain designated BS11(T) was isolated from the compost and its taxonomic position was investigated by using a polyphasic approach. Strain BS11(T) grew optimally at 30-37°C and at pH 7.0 in the absence of NaCl on nutrient agar. Strain BS11(T) displayed β-glucosidase activity that was responsible for its ability to transform ginsenoside Rb1 (one of the dominant active components of ginseng) to Rd. On the basis of 16S rRNA gene sequence similarity, strain BS11(T) was shown to belong to the family Sphingomonadaceae and was related to Sphingosinicella vermicomposti YC7378(T) (96.3% sequence similarity), S. xenopeptidilytica 3-2W4(T) (96.2%), S. microcystinivorans Y2(T) (96.1%), and S. soli KSL-125(T) (95.9%). The G+C content of the genomic DNA was 64.9%. The major menaquinone was Q-10 and the major fatty acids were summed feature 7 (comprising C18:1 ω7c/ω9t/ω12t; 40.6%), C16:0 (22.5%), C17:1 ω6c (13.7%) and C17:0 (9.1%). DNA and chemotaxonomic data supported the affiliation of strain BS11(T) to the genus Sphingosinicella. Strain BS11(T) could be differentiated genotypically and phenotypically from the recognized species of the genus Sphingosinicella. The novel isolate therefore represents a novel species, for which the name Sphingosinicella ginsenosidimutans sp. nov. is proposed, with the type strain BS11(T) (=KACC 16619T =JCM 18201(T)).

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., (© The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The ginsenoside Rg2(S), which is one of the pharmaceutical components of ginseng, is known to have neuroprotective, anti-inflammation, and anti-diabetic effects. However, the usage of ginsenoside Rg2(S) is restricted owing to the small amounts found in white and red ginseng. To enhance the production of ginsenoside Rg2(S) as a 100 gram unit with high specificity, yield, and purity, an enzymatic bioconversion method was developed to adopt the recombinant glycoside hydrolase (BglPC28), which is a ginsenoside-transforming recombinant β-glucosidase from Pseudonocardia sp. strain Gsoil 1536. The gene, termed bglPC28, encoding β-glucosidase (BglPC28) belonging to the glycoside hydrolase family 3 was cloned. bglPC28 consists of 2,232 bp (743 amino acid residues) with a predicted molecular mass of 78,975 Da. This enzyme was overexpressed in Escherichia coli BL21(DE3) using a GST-fused pGEX 4T-1 vector system. The optimum conditions of the recombinant BglPC28 were pH 7.0 and 37 °C. BglPC28 can effectively transform the ginsenoside Re to Rg2(S); the Km values of PNPG and Re were 6.36 ± 1.10 and 1.42 ± 0.13 mM, respectively, and the Vmax values were 40.0 ± 2.55 and 5.62 ± 0.21 µmol min-1 mg-1 of protein, respectively. A scaled-up biotransformation reaction was performed in a 10 L jar fermenter at pH 7.0 and 30°C for 12 hours with a concentration of 20 mg/ml of ginsenoside Re from American ginseng roots. Finally, 113 g of Rg2(S) was produced from 150 g of Re with 84.0 ± 1.1% chromatographic purity. These results suggest that this enzymatic method could be usefully exploited in the preparation of ginsenoside Rg2(S) in the cosmetics, functional food, and pharmaceutical industries.

The focus of this study was the cloning, expression, and characterization of recombinant ginsenoside hydrolyzing β-glucosidase from Arthrobacter chlorophenolicus with an ultimate objective to more efficiently bio-transform ginsenosides. The gene bglAch, consisting of 1,260 bp (419 amino acid residues) was cloned and the recombinant enzyme, overexpressed in Escherichia coli BL21 (DE3), was characterized. The GST-fused BglAch was purified using GST·Bind agarose resin and characterized. Under optimal conditions (pH 6.0 and 37°C) BglAch hydrolyzed the outer glucose and arabinopyranose moieties of ginsenosides Rb1 and Rb2 at the C20 position of the aglycone into ginsenoside Rd. This was followed by hydrolysis into F2 of the outer glucose moiety of ginsenoside Rd at the C3 position of the aglycone. Additionally, BglAch more slowly transformed Rc to F2 via C-Mc1 (compared to hydrolysis of Rb1 or Rb2). These results indicate that the recombinant BglAch could be useful for the production of ginsenoside F2 for use in the pharmaceutical and cosmetic industries.

A novel β-glucosidase (BglPm) was identified from Paenibacillus mucilaginosus KCTC 3870(T) which has ginsenoside converting activity. The gene, termed bglPm, consists of 1,260 bp and belongs to glycoside hydrolase family 1 (GH1). After being overexpressed and purified from Escherichia coli, the enzymatic properties of BglPm were investigated. The enzyme exhibited an optimal activity at 45°C and pH 7.5 and showed high bioconversion ability for major ginsenoside Rb1 and Rd into ginsenoside F2. Thus, it was used for mass production of relatively high pure F2 from relatively abundant protopanaxadiol type ginsenosides mixture (PPDGM) with combined usage of ginsenoside Rc-hydrolyzing enzyme. Scale-up of production using 250 g of the PPDGM resulted in 152 g of F2 with 80.1% chromatography purity and 95.7% recovery. These results suggest that this enzyme would be useful in the preparation of pharmacologically active ginsenoside F2 in the functional food and pharmaceutical industries.

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Here, we isolated and characterized a new ginsenoside-transforming β-glucosidase (BglQM) from Mucilaginibacter sp. strain QM49 that shows biotransformation activity for various major ginsenosides. The gene responsible for this activity, bglQM, consists of 2,346 bp and is predicted to encode 781 amino acid residues. This enzyme has a molecular mass of 85.6 kDa. Sequence analysis of BglQM revealed that it could be classified into glycoside hydrolase family 3. The enzyme was overexpressed in Escherichia coli BL21(DE3) using a maltose binding protein (MBP)-fused pMAL-c2x vector system containing the tobacco etch virus (TEV) proteolytic cleavage site. Overexpressed recombinant BglQM could efficiently transform the protopanaxatriol-type ginsenosides Re and Rg1 into (S)-Rg2 and (S)-Rh1, respectively, by hydrolyzing one glucose moiety attached to the C-20 position at pH 8.0 and 30°C. The Km values for p-nitrophenyl-β-d-glucopyranoside, Re, and Rg1 were 37.0 ± 0.4 μM and 3.22 ± 0.15 and 1.48 ± 0.09 mM, respectively, and the Vmax values were 33.4 ± 0.6 μmol min(-1) mg(-1) of protein and 19.2 ± 0.2 and 28.8 ± 0.27 nmol min(-1) mg(-1) of protein, respectively. A crude protopanaxatriol-type ginsenoside mixture (PPTGM) was treated with BglQM, followed by silica column purification, to produce (S)-Rh1 and (S)-Rg2 at chromatographic purities of 98% ± 0.5% and 97% ± 1.2%, respectively. This is the first report of gram-scale production of (S)-Rh1 and (S)-Rg2 from PPTGM using a novel ginsenoside-transforming β-glucosidase of glycoside hydrolase family 3.

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 β-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)).

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 β-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)).

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 Km and Vmax values of 0.95 ± 0.02 μM and 1.2 ± 0.1 μmol min(-1) mg of protein(-1) against p-nitrophenyl-α-L-arabinofuranoside, respectively. Using a purified MBP-Abf22-3 (10 μg/ml), 0.1 % of ginsenoside Rc was completely converted to ginsenoside Rd within 20 min.

A Gram-negative, strictly aerobic, non-motile, non-sporeforming, and rod-shaped bacterial strain designated FW-6T was isolated from a freshwater sample and its taxonomic position was investigated by using a polyphasic approach. Strain FW-6T grew optimally at 10-42 degrees C and at pH 7.0 on nutrient and R2A agar. Strain FW-6T displayed beta- glucosidase activity that was responsible for its ability to transform ginsenoside Rb1 (one of the dominant active components of ginseng) to Rd. On the basis of 16S rRNA gene sequence similarity, strain FW-6T was shown to belong to the family Sphingomonadaceae and was related to Novosphingobium aromaticivorans DSM 12444T (98.1% sequence similarity) and N. subterraneum IFO 16086T (98.0%). The G+C content of the genomic DNA was 64.4%. The major menaquinone was Q-10 and the major fatty acids were summed feature 7 (comprising C18:1 omega9c/ omega12t/omega7c), summed feature 4 (comprising C16:1 omega7c/iso- C15:0 2OH), C16:0, and C14:0 2OH. DNA and chemotaxonomic data supported the affiliation of strain FW-6T to the genus Novosphingobium. Strain FW-6T could be differentiated genotypically and phenotypically from the recognized species of the genus Novosphingobium. The isolate that has ginsenoside converting ability therefore represents a novel species, for which the name Novosphingobium ginsenosidimutans sp. nov. is proposed, with the type strain FW-6T (= KACC 16615T = JCM 18202T).

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.

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., 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 μmol 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.

This study focuses on the cloning, expression, and characterization of recombinant ginsenoside hydrolyzing glycosidase from Sanguibacter keddieii in order to biotransform ginsenosides efficiently. The gene, termed bglSk, consists of 1857 bp and revealed significant homology to that of glycoside hydrolase family 3. The enzyme was over-expressed in Escherichia coli BL21 (DE3) using a GST-fused pGEX 4T-1 vector system. The over-expressed recombinant enzymes could convert six major ginsenosides Rb(1), Rb(2), Rc, Rd, Re and Rg(1) into more pharmacologically active rare ginsenosides such as C-Y, C-Mc, C-K, Rg(2)(S), and F(1). Especially, BglSk could completely convert the Rg(1) into F(1). The GST-fused BglSk was purified with GST·bind agarose resin and then characterized. The kinetic parameters for β-glucosidase had apparent K(m) values of 0.456±0.009 and 0.167±0.003 mM and V(max) values of 30.2±0.7 and 4.1±0.1 μmol min(-1) mg of protein(-1) against p-nitrophenyl-β-d-glucopyranoside and Rb(1), respectively., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The gene encoding an α-L-arabinofuranosidase that could biotransform ginsenoside Rc {3-O-[β-D-glucopyranosyl-(1-2)-β-D-glucopyranosyl]-20-O-[α-L-arabinofuranosyl-(1-6)-β-D-glucopyranosyl]-20(S)-protopanaxadiol} to ginsenoside Rd {3-O-[β-D-glucopyranosyl-(1-2)-β-D-glucopyranosyl]-20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol} was cloned from a soil bacterium, Rhodanobacter ginsenosidimutans strain Gsoil 3054(T), and the recombinant enzyme was characterized. The enzyme (AbfA) hydrolyzed the arabinofuranosyl moiety from ginsenoside Rc and was classified as a family 51 glycoside hydrolase based on amino acid sequence analysis. Recombinant AbfA expressed in Escherichia coli hydrolyzed non-reducing arabinofuranoside moieties with apparent K (m) values of 0.53 ± 0.07 and 0.30 ± 0.07 mM and V (max) values of 27.1 ± 1.7 and 49.6 ± 4.1 μmol min(-1) mg(-1) of protein for p-nitrophenyl-α-L-arabinofuranoside and ginsenoside Rc, respectively. The enzyme exhibited preferential substrate specificity of the exo-type mode of action towards polyarabinosides or oligoarabinosides. AbfA demonstrated substrate-specific activity for the bioconversion of ginsenosides, as it hydrolyzed only arabinofuranoside moieties from ginsenoside Rc and its derivatives, and not other sugar groups. These results are the first report of a glycoside hydrolase family 51 α-L-arabinofuranosidase that can transform ginsenoside Rc to Rd.

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-β-D-(1-->2)-glucopyranosyl of Rb1 to gypenoside XVII, and then to further hydrolyze the 3-O-β-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.

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)).

A novel gammaproteobacterium, designated LnR5-47(T), was isolated from soil of a ginseng field in Liaoning province, China. The isolate was a Gram-negative, aerobic, non-motile, non-spore-forming rod. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain LnR5-47(T) belonged to the genus Rhodanobacter. The isolate was most closely related to Rhodanobacter ginsengisoli GR17-7(T), Rhodanobacter terrae GP18-1(T), Dyella ginsengisoli Gsoil 3046(T), Rhodanobacter soli DCY45(T), Dyella soli JS12-10(T) and Dyella japonica IAM 15069(T) (98.0, 97.9, 97.7, 97.3, 97.2 and 97.1% 16S rRNA gene sequence similarity, respectively). Chemotaxonomic data (Q-8 as the predominant ubiquinone, and iso-C(16:0), iso-C(17:1)ω9c and iso-C(15:0) as the major fatty acids) also supported the affiliation of strain LnR5-47(T) with the genus Rhodanobacter. However, DNA-DNA relatedness between strain LnR5-47(T) and its closest phylogenetic neighbours was <25.8%. Moreover, physiological and biochemical tests phenotypically differentiated the isolate from other members of the genus Rhodanobacter. Therefore, strain LnR5-47(T) represents a novel species, for which the name Rhodanobacter panaciterrae sp. nov. is proposed; the type strain is LnR5-47(T) (=KACC 12826(T)=KCTC 22232(T)=LMG 24460(T)).

A Gram-reaction-negative, strictly aerobic, non-motile, non-spore-forming, rod-shaped bacterial strain, designated THG 15(T), was isolated from soil of a field cultivated with Rhus vernicifera in Okcheon province, South Korea, and its taxonomic position was investigated by using a polyphasic approach. Strain THG 15(T) grew optimally at 25-30 °C and at pH 7 in the absence of NaCl on nutrient agar. Strain THG 15(T) displayed β-glucosidase (aesculinase) activity that was responsible for its ability to transform ginsenoside Rb(1) (one of the dominant active components of ginseng) into compound K via Rd and F(2). On the basis of 16S rRNA gene sequence similarities, strain THG 15(T) was shown to belong to the family Flavobacteriaceae and was most closely related to Chryseobacterium soldanellicola PSD1-4(T) (97.7 % sequence similarity), Chryseobacterium soli JS6-6(T) (97.5 %) and Chryseobacterium indoltheticum LMG 4025(T) (97.3 %). The G+C content of the genomic DNA was 35.7 mol%. The major menaquinone was MK-6 and the major fatty acids were iso-C(15 : 0) (50.3 %), iso-C(17 : 0) 3-OH (21.9 %), summed feature 4 (comprising C(16 : 1)ω7c and/or iso-C(15 : 0) 2-OH; 9.5 %) and iso-C(17 : 1)ω9c (9.3 %). DNA sequence analysis and chemotaxonomic data supported the affiliation of strain THG 15(T) to the genus Chryseobacterium. DNA-DNA relatedness values between strain THG 15(T) and its closest phylogenetic neighbours were <15 %. Strain THG 15(T) could be differentiated genotypically and phenotypically from recognized species of the genus Chryseobacterium. The isolate therefore represents a novel species, for which the name Chryseobacterium ginsenosidimutans sp. nov. is proposed. The type strain is THG 15(T) ( = KACC 14527(T)  = JCM 16719(T)).

The Gram-negative, strictly aerobic, non-motile, non-spore-forming, rod shaped bacterial strain designated TR6-03(T) was isolated from compost, and its taxonomic position was investigated by using a polyphasic approach. Strain TR6-03(T) grew at 4-42°C and at pH 6.0-8.0 on R2A and nutrient agar without NaCl supplement. Strain TR6-03(T) had β-glucosidase activity, which was responsible for its ability to transform ginsenoside Re (one of the dominant active components of ginseng) to Rg(2). On the basis of 16S rRNA gene sequence similarity, strain TR6-03(T) was shown to belong to the family Sphingobacteriaceae and to be related to Mucilaginibacter lappiensis ANJLI2(T) (96.3% sequence similarity), M. dorajii FR-f4(T) (96.1%), and M. rigui WPCB133(T) (94.1%). The G+C content of the genomic DNA was 45.6%. The predominant respiratory quinone was MK-7 and the major fatty acids were summed feature 3 (comprising C(16:1) ω7c and/or iso-C(15:0) 20H), iso-C(16:0) and iso-C(17:0) 3OH. DNA and chemotaxonomic data supported the affiliation of strain TR6-03(T) to the genus Mucilaginibacter. Strain TR6-03(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 composti sp. nov. is proposed, with the type strain TR6-03(T) (=KACC 14956(T) = KCTC 12642(T) =LMG 23497(T)).

A Gram-negative, aerobic, non-motile, non-spore-forming, yellow-pigmented, rod-shaped bacterium, designated strain THG 01(T), was isolated from the soil of a ginseng field in Pocheon province, South Korea, and its taxonomic position was investigated by using a polyphasic approach. Strain THG 01(T) grew well at 25-37 °C and pH 6.0-7.5 in the absence of NaCl on nutrient agar. On the basis of 16S rRNA gene sequence similarity data, strain THG 01(T) was shown to belong to the family Flavobacteriaceae and was related to Flavobacterium anhuiense D3(T) (97.5 % similarity), Flavobacterium johnsoniae UW101(T) (96.8 %) and Flavobacterium denitrificans ED5(T) (96.7 %). 16S rRNA gene sequence similarities between the novel strain and members of other recognized species within the family Flavobacteriaceae were less than 96.7 %. The G+C content of the genomic DNA of strain THG 01(T) was 32.1 mol%. Phenotypic and chemotaxonomic data (major menaquinone was MK-6 and major fatty acids were iso-C(15 : 0), iso-C(15 : 0) 3-OH and C(16 : 1)ω7c and/or C(16 : 1)ω6c ) supported the affiliation of strain THG 01(T) to the genus Flavobacterium. DNA-DNA hybridization experiments showed that DNA-DNA relatedness values between strain THG 01(T) and its closest phylogenetic neighbours were below 11 %. The results of physiological and biochemical tests enabled strain THG 01(T) to be differentiated genotypically and phenotypically from recognized species of the genus Flavobacterium. The isolate therefore represents a novel species, for which the name Flavobacterium ginsenosidimutans sp. nov. is proposed, with THG 01(T) ( = KACC 14525(T) = JCM 16720(T)) as the type strain.

The Gram-reaction-negative, strictly aerobic, non-motile, non-spore-forming, and rod-shaped bacterial strain designated Gsoil 1429(T) was isolated from the soil of ginseng cultivating field of Pocheon province in South Korea. This bacterium was characterized in order to determine its taxonomic position by using the polyphasic approach. Strain Gsoil 1429(T) grew well at 25-37°C and at pH 7.0 on R2A and nutrient agar without NaCl supplement. Strain Gsoil 1429(T) had -glucosidase activity, which was responsible for its ability to transform ginsenoside Rb1 (one of the dominant active components of ginseng) to F(2) via gypenoside XVII. On the basis of 16S rRNA gene sequence similarity, strain Gsoil 1429(T) was shown to belong to the family Sphingomonadaceae and to be related to Sphingomonas yunnanensis YIM 003(T) (98.2% sequence similarity), S. phyllosphaerae FA2(T) (97.5%), S. koreensis JSS26(T) (97.3%), and S. asaccharolytica IFO 15499(T) (97.1%). The G+C content of the genomic DNA was 65.6%. The major respiratory quinone was Q-10 and the major fatty acids were summed feature 8 (comprising C(18:1) 7c/ωt/12t), C(16:0) and C(14:0)2OH. DNA and chemotaxonomic data supported the affiliation of strain Gsoil 1429T to the genus Sphingomonas. The DNA-DNA relatedness values between strain Gsoil 1429(T) and its closest phylogenetically neighbours were below 28%. Strain Gsoil 1429(T) could be differentiated genotypically and phenotypically from the recognized species of the genus Sphingomonas. The isolate therefore represents a novel species, for which the name Sphingomonas ginsenosidimutans sp. nov. is proposed, with the type strain Gsoil 1429(T) (=KACC 14949(T) =JCM 17074(T) =LMG 25799(T)).

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.