Solibacillus palustris sp. nov., isolated from wetland soil of ecology park.

A Gram-staining-positive, motile, aerobic and rod-shaped bacterium, designated strain MA9T was isolated from wetland soil of ecology park, in Seoul, Republic of Korea. This bacterium was characterized to determine its taxonomic position by using the polyphasic approach. Strain MA9T grew at 10-37 °C and at pH 6.0-9.5 on TSB. Menaquinone MK-7 was the predominant respiratory quinone and iso-C15 : 0, iso-C16 : 0 and C16 : 1 ω7c alcohol were the major fatty acids. The main polar lipids were phosphatidylethanolamine (PE), phosphatidylserine (PS), diphosphatidylglycerol (DPG) and phosphatidylglycerol (PG). The peptidoglycan type of the cell wall was A4α l-Lys-d-Glu. Based on 16S rRNA gene sequencing, strain MA9T clustered with species of the genus Solibacillus and appeared closely related to S. silvestris DSM 12223T (97.8 % sequence similarity), S. cecembensis DSM 21993T (97.6 %), S. isronensis DSM 21046T (97.6 %) and S. kalamii DSM 101595T (96.6 %). The G+C content of the genomic DNA was 37.0 mol%. Digital DNA-DNA hybridization between strain MA9T and type strains of S. silvestris, S. isronensis, S. cecembensis and S. kalamii resulted in values below 70 %. Strain MA9T could be differentiated genotypically and phenotypically from the recognized species of the genus Solibacillus. The isolate therefore represents a novel species, for which the name Solibacillus palustris sp. nov. is proposed, with the type strain MA9T (=KACC 22212T = LMG 32188T).

Isolation and characterization of a novel glycoside hydrolase positive bacterial strain named Terrimonas ginsenosidimutans sp. nov. with ginsenoside-converting activity.

A novel yellow-pigmented catalase- and oxidase-positive bacterial strain (designated NA20T) was isolated from wetland soil and characterized. Results of 16S rRNA and draft genome sequence analysis placed strain NA20T within the genus Terrimonas of the family Chitinophagaceae. Strain NA20T showed ≤97.1 % sequence similarity to members of the genus Terrimonas and the highest sequence similarity was found to Terrimonas lutea DYT (97.1%). The draft genome of strain NA20T 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 NA20T contained iso-C15 : 0, iso-C15 : 0 G, iso-C17 : 0 3-OH and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) 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 NA20T 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 NA20T within the genus Terrimonas, for which the name Terrimonas ginsenosidimutans sp. nov. is proposed. The type strain is NA20T (=KACC 22218T=LMG 32198T).

Isolation and Characterization of Sphingomonas telluris, Sphingomonas caseinilyticus Isolated from Wet Land Soil.

Two novel bacterial strains, designated as SM33T and NSE70-1T, were isolated from wet soil in South Korea. To get the taxonomic positions, the strains were characterized. The genomic information (both 16S rRNA gene and draft genome sequence analysis) show that both novel isolates (SM33T and NSE70-1T) belong to the genus Sphingomonas. SM33T share the highest 16s rRNA gene similarity (98.2%) with Sphingomonas sediminicola Dae20T. In addition, NSE70-1T show 96.4% 16s rRNA gene similarity with Sphingomonas flava THG-MM5T. The draft genome of strains SM33T and NSE70-1T consist of a circular chromosome of 3,033,485 and 2,778,408 base pairs with DNA G+C content of 63.9, and 62.5%, respectively. Strains SM33T and NSE70-1T possessed the ubiquinone Q-10 as the major quinone, and a fatty acid profile with C16:0, C18:1 2-OH, C16:1 ω7c/C16:1 ω6c (summed feature 3) and C18:1 ω7c/C18:1 ω6c (summed feature 8) as major fatty acids. The major polar lipids of SM33T and NSE70-1T were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, sphingoglycolipid and phosphatidylcholine, respectively. Moreover, genomic, physiological, and biochemical results allowed the phenotypic and genotypic differentiation of strains SM33T and NSE70-1T from their closest and other species of the genus Sphingomonas with validly published names. Therefore, the SM33T and NSE70-1T represent novel species of the genus Sphingomonas, for which the name Sphingomonas telluris sp. nov. (type strain SM33T = KACC 22222T = LMG 32193T), and Sphingomonas caseinilyticus (type strain NSE70-1T = KACC 22411T = LMG 32495T).

Ketonization of Ginsenoside C-K by Novel Recombinant 3-ß-Hydroxysteroid Dehydrogenases and Effect on Human Fibroblast Cells.

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-ß-hydroxydammar-3-one-20(S)-O-ß-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.

Ramlibacter paludis sp. nov., isolated from wetland.

A Gram-stain-negative, non-motile, rod-shaped, aerobic and white-coloured bacterium (designated XY19T) was isolated from a soil sample of wetland from Godeok Ecological Park, Gangdong-gu, Seoul, Republic of Korea. On the basis of 16S rRNA gene sequencing, strain XY19T clustered with species of the genus Ramlibacter and appeared closely related to R. ginsenosidimutans DSM 23480T (98.42 %), R. alkalitolerans JCM 32081T (97.68 %) and R. monticola JCM 31918T (97.66 %). The average nucleotide identity between strain XY19T and three strains (R. ginsenosidimutans DSM 23480T, R. alkalitolerans JCM 32081T and R. monticola JCM 31918T) were 80.7, 81.1 and 81.4 %. And the digital DNA-DNA hybridization (dDDH) calculated between strain XY19T and each of the three strains (R. ginsenosidimutans DSM 23480T, R. alkalitolerans JCM 32081T and R. monticola JCM 31918T) were 24.1, 24.4 and 24.5 %. ANI value and dDDH results were a novel species of the genus Ramlibacter. Growth occurs at 10-37 °C on R2A medium in the pressence of 0-1 % NaCl (w/v) and at pH 6.0-8.5. The DNA G+C content of the genomic DNA was 68.7 mol%, and ubiquinone-8 (Q-8) was the major respiratory quinone. The major cellular fatty acids (>5 %) were C16:1 ω7c and/or C16:1 ω6c (summed feature 3), C16 : 0, C17 : 0 cyclo and C18:1 ω7c and/or C18:1 ω6c (summed feature 8). The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, three unidentified lipids and unidentified aminophospholipid. Physiological and biochemical characteristics indicated that strain XY19T represents a novel species of the genus Ramlibacter, for which the name Ramlibacter paludis sp. nov. is proposed. The type strain is XY19T (= KACC 22220T = LMG 32190T).

Characterization of four novel bacterial species of the genus Sphingomonas, Sphingomonas anseongensis, Sphingomonas alba, Sphingomonas brevis and Sphingomonas hankyongi sp.nov., isolated from wet land.

Four novel bacterial strains, designated as RG327T, SE158T, RB56-2T and SE220T, were isolated from wet soil in the Republic of Korea. To determine their taxonomic positions, the strains were fully characterized. On the basis of genomic information (16S rRNA gene and draft genome sequences), all four isolates represent members of the genus Sphingomonas. The draft genomes of RG327T, SE158T, RB56-2T and SE220T consisted of circular chromosomes of 2 226 119, 2 507 338, 2 593 639 and 2 548 888 base pairs with DNA G+C contents of 64.6, 63.6, 63.0 and 63.1 %, respectively. All the isolates contained ubiquinone Q-10 as the predominant quinone compound and a fatty acid profile with C16 : 0, C17 : 1ω6c, C18 : 1 2-OH, summed feature 3 (C16 : 1ω7c/C16 : 1ω6c) and summed feature 8 (C18 : 1ω7c/C18 : 1ω6c) as the major fatty acids, supporting the affiliation of strains RG327T, SE158T, RB56-2T and SE220T to the genus Sphingomonas. The major identified polar lipids in all four novel isolates were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, sphingoglycolipid and phosphatidylcholine. Moreover, the physiological, biochemical results and low level of DNA-DNA relatedness and average nucleotide identity values allowed the phenotypic and genotypic differentiation of RG327T, SE158T, RB56-2T and SE220T from other species of the genus Sphingomonas with validly published names and indicated that they represented novel species of the genus Sphingomonas, for which the names Sphingomonas anseongensis sp. nov. (RG327T = KACC 22409T = LMG 32497T), Sphingomonas alba sp. nov. (SE158T = KACC 224408T = LMG 324498T), Sphingomonas brevis (RB56-2T = KACC 22410T = LMG 32496T) and Sphingomonas hankyongi sp. nov., (SE220T = KACC 22406T = LMG 32499T) are proposed.

Sphingomonas cremea sp. nov., isolated from ginseng soil.

A novel Gram-stain-negative, aerobic, rod-shaped, non-motile, cream-coloured strain (G124T) was isolated from ginseng soil collected in Yeongju, Republic of Korea. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain G124T belongs to a distinct lineage within the genus Sphingomonas (family Sphingomonadaceae, order Sphingomonadales and class Alphaproteobacteria). Strain G124T was closely related to Sphingomonas rhizophila THG-T61T (98.5 % 16S rRNA gene sequence similarity), Sphingomonas mesophila SYSUP0001T (98.3 %), Sphingomonas edaphi DAC4T (97.6 %) and Sphingomonas jaspsi TDMA-16T (97.6 %). The strain contained ubiquinone 10 as the major respiratory quinone. The major polar lipid profile of strain G124T comprised phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine and sphingoglycolipids. The predominant cellular fatty acids of strain G124T were summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c; 33.4 %), summed feature 3 (C16 : 1 ω6c/C16 : 1 ω7c; 27.2 %) and C16 : 0 (18.3 %). The genome size of strain G124T was 2 549 305 bp. The genomic DNA G+C content is 62.0 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between strain G124T and other Sphingomonas species were in the range of 71.2-75.9 % and 18.7-19.9 %, respectively. Based on the polyphasic analysis such as biochemical, phylogenetic and chemotaxonomic characteristics, strain G124T represents a novel species of the genus Sphingomonas, for which the name Sphingomonas cremea sp. nov. is proposed. The type strain is G124T (=KACC 21691T=LMG 31729T).

Arthrobacter hankyongi sp. nov., Isolated From Wet Land.

A Gram-staining-positive, catalase positive and oxidase negative, non-motile, non-flagellated, and oval-shaped bacterium, was designated as I2-34 T, isolated from wetland in Soul South Korea. Colonies were round, entire, raised, and cream colored after two days of incubation on R2A agar plates at 25 °C. Based on genomes (both 16S rRNA gene and draft genome) sequence analysis, strain I2-34 T belongs to the genus Arthrobacter and was most closely related to Arthrobacter deserti YIM CS25T (98.0%). The strain I2-34 T had a circular genome with length of 5,186,447 base pairs (67 contigs) and 4830 total genes. Out of 4696 were protein-coding genes, 54 tRNA and 4 rRNA genes. The chemotaxonomic analysis indicates iso-C16:0, anteiso-C15:0, and anteiso-C17:0 as major fatty acids, phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), and two unidentified glycolipids (GL1, GL2) as major polar lipids. The predominant quinone was MK-8(H2). The peptidoglycan type was A3α with an. L-Lys-L-Ala interpeptide bridge. Thus, the experimental data demonstrated here show that the novel isolate shares the similar major fatty acids, major polar lipid PG, DPG, and GLs, major and major quinone MK8-(H2) with the described members of the genus Arthrobacter. However, the low 16S rRNA gene sequence (98.0%), and some physiological and biochemical characteristics differentiate the I2-34 T from its closest phylogenetic neighbors. As a result, the isolate represents a novel species in within the genus Arthrobacter and family Micrococcaceae for which the name Arthrobacter hankyongi sp. nov. is proposed. The type strain is I2-34 T (= KACC 22217 T, LMG 32197 T).

Repressing effect of transformed ginsenoside Rg3-mix against LPS-induced inflammation in RAW264.7 macrophage cells.

BACKGROUND: Rg3-ginsenoside, a protopanaxadiol saponin, is a well-known adaptogen used for the prevention of cancer and inflammation. However, despite its distinct biological activity, the concentration of Rg3 in the total ginseng extract is insufficient for therapeutic applications. This study aims to convert PPD-class of major ginsenosides into a mixture of minor ginsenoside, to analyze its immune-regulatory role in macrophage cells. RESULTS: Using heat and organic acid treatment, three major ginsenosides, Rc, Rd, and Rb1, were converted into a mixture of minor ginsenosides, GRg3-mix [Rg3(S), Rg3(R), Rg5, and Rk1]. Purity and content analysis of the transformed compound were performed using thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), compared with their standards. Preceding with the anti-inflammatory activity of GRg3-mix, lipopolysaccharide (LPS)-stimulated murine RAW264.7 macrophage cells were treated with various concentrations of GRg3-mix (6.25, 12.5, 25, and 50 µg/mL). The cell viability assay revealed that the level of cell proliferation was increased, while the nitric oxide (NO) assay showed that NO production decreased dose-dependently in activated RAW264.7 cells. The obtained results were compared to those of pure Rg3(S) ≥ 98% (6.25, 12.5, and 25 µg/mL). Preliminary analysis of the CCK-8 and NO assay demonstrated that GRg3-mix can be used as an anti-inflammatory mediator, but mRNA and protein expression levels were evaluated for further confirmation. The doses of GRg3-mix significantly suppressed the initially upregulated mRNA and protein expression of inflammation-related enzymes and cytokines, namely inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear transcription factor kappa B (NF-κB), tumor necrosis factor (TNF-α), and interleukins (IL-6 and IL1B), as measured by reverse transcription-polymerase chain reaction and western blotting. CONCLUSIONS: Our pilot data confirmed that the mixture of minor ginsenosides, namely GRg3-mix, has high anti-inflammatory activity and has an easy production procedure.

Humibacter ginsenosidimutans sp. nov., with ginsenoside-converting activity isolated from activated sludge.

A novel Gram-stain-positive, thin rod-shaped, non-motile, aerobic and creamy-white pigmented bacterium (designated strain WJ7-1T) 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 20986T. Strain WJ7-1T 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-C17 : 0, iso-C16 : 0 and anteiso-C15 : 0. The peptidoglycan of strain WJ7-1T 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 WJ7-1T possessed ginsenoside-converting (ß-glucosidase) activity, which enabled it to transform ginsenoside protopanaxadiol-type Rb1 (one of the dominant active components of ginseng) to compound K. The genome size of strain WJ7-1T 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 WJ7-1T and the closely related strain were 79.8, 36.1 and 23.5 %, respectively, indicating that strain WJ7-1T represents a novel species of the genus Humibacter. Strain WJ7-1T 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 WJ7-1T (=KACC 19729T=LMG 30802T) represents a novel species within the genus Humibacter, for which the name Humibacter ginsenosidimutans sp. nov. is proposed.

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

A bacterial strain designated as G188T was isolated from ginseng field soil in the Republic of Korea. Phylogenetic analysis of 16S rRNA gene sequences showed that strain G188T formed a distinct lineage within the genus Nocardioides, family Nocardioidaceae, order Propionibacteriales. Sequence similarity revealed that strain G188T was most closely related to Nocardioides iriomotensis IR27-S3T (97.7 % 16S rRNA similarity). The genome size of strain G188T was 4 901 775 bp, and the genomic DNA G+C content was 72.3 mol%. The average nucleotide identity and DNA-DNA hybridization values with other Nocardioides species were less than 75.6 and 20.1 %, respectively. The main fatty acids of strain G188T were C17 : 0, C17 : 1 ω8c and iso-C16 : 0. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylinositol, and the major respiratory quinone was menaquinone 8, supporting that strain G188T was affiliated with the genus Nocardioides. Based on biochemical, chemotaxonomic and phylogenetic analyses, the novel species Nocardioides panacis G188T (KACC 21695T=LMG 31733T) is proposed.

Production of Prosaikogenin F, Prosaikogenin G, Saikogenin F and Saikogenin G by the Recombinant Enzymatic Hydrolysis of Saikosaponin and their Anti-Cancer Effect.

The saponins of Bupleurum falcatum L., saikosaponins, are the major components responsible for its pharmacological and biological activities. However, the anti-cancer effects of prosaikogenin and saikogenin, which are glycoside hydrolyzed saikosaponins, are still unknown due to its rarity in plants. In this study, we applied two recombinant glycoside hydrolases that exhibit glycoside cleavage activity with saikosaponins. The two enzymes, BglPm and BglLk, were cloned from Paenibacillus mucilaginosus and Lactobacillus koreensis, and exhibited good activity between 30-37 °C and pH 6.5-7.0. Saikosaponin A and D were purified and obtained from the crude B. falcatum L. extract using preparative high performance liquid chromatography technique. Saikosaponin A and D were converted into saikogenin F via prosaikogenin F, and saikogenin G via prosaikogenin G using enzyme transformation with high ß-glycosidase activity. The two saikogenin and two prosaikogenin compounds were purified using a silica column to obtain 78.1, 62.4, 8.3, and 7.5 mg of prosaikogenin F, prosaikogenin G, saikogenin F, and saikogenin G, respectively, each with 98% purity. The anti-cancer effect of the six highly purified saikosaponins was investigated in the human colon cancer cell line HCT 116. The results suggested that saikosaponins and prosaikogenins markedly inhibit the growth of the cancer cell line. Thus, this enzymatic technology could significantly improve the production of saponin metabolites of B. falcatum L.

Paludibacterium denitrificans sp. nov., a Novel Denitrifying Bacterium Isolated from Activated Sludge.

A Gram-reaction-negative, facultatively aerobic, motile, non-spore-forming, rod-shaped, and denitrifying bacterium, designated dN18-1T, was isolated from activated sludge, Republic of Korea. This bacterium was investigated via a polyphasic approach to reveal its taxonomic position. Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain dN18-1T belongs to the genus Paludibacterium and is most closely related to P. purpuratum KCTC 42852T (96.2% sequence similarity), P. yongneupense KACC 11601T (96.1%), and P. paludis BCRC 80514T (95.2%). The average nucleotide identity values and digital DNA-DNA hybridization values calculated between strain dN18-1T and the closely related strains were 72.5-73.1% and 19.0-19.6%. The genome comprises of 3,347,996 bp with a G + C content of 57.3 mol%. Strain dN18-1T possesses ubiquinone Q-8 as a predominant respiratory quinone, and summed feature 3 (C16:1 ω6c and/or C16:1 ω7c), summed feature 8 (C18:1 ω6c/C18:1 ω7c), C16:0 and C12:0, as its major fatty acids (>5%). The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two unidentified phospholipids and four unidentified aminophospholipids. The results of ANI calculation, digital DNA-DNA hybridization, physiological and biochemical tests allowed phenotypic differentiation of strain dN18-1T from rephrase other genus Paludibacterium species with validly published names. Therefore, this isolate represents a novel species, for which the name Paludibacterium denitrificans sp. nov. (type strain dN18-1T = KACC 19537T = CGMCC 1.16961T) is proposed.

Hanamia caeni gen. nov., sp. nov., a Member of the Family Chitinophagaceae Isolated from Activated Sludge in Korea.

A novel Gram-stain-negative, aerobic, yellowish-pigmented, non-motile, rod-shaped bacterial strain, designated strain BO-59T, was isolated from the activated sludge of a wastewater treatment plant in Hanam City, South Korea. Phylogenetic study based on the 16S rRNA gene sequence positioned BO-59T in a distinct lineage in the family Chitinophagaceae, sharing less than 92.8% sequence similarity with members of the closely related genera Ferruginibacter, Flavitalea, Pseudoflavitalea, Flavisolibacter, Niastella, and Terrimonas. Phylogenomic- and genomic relatedness analyses revealed that strain BO-59T is clearly distinguished from other genera in the family Chitinophagaceae by average nucleotide identity < 66.9%) and the genome-to-genome distance (< 29.5%) values. The strain BO-59T contained MK-7 as the predominant quinone, and iso-C15:0, iso-C17:0 3OH, and iso-C15:1 G as major fatty acids (> 10%). The DNA G + C content was 39.1 mol% based on genome sequence analysis. The polar lipids of strain BO-59T were phosphatidylethanolamine, an unidentified aminophospholipid and three unidentified polar lipids. 16S rRNA gene sequence similarity, physiological, and biochemical characteristics indicated that strain BO-59T represents a novel species of a new genus, for which the name Hanamia caeni gen. nov., sp. nov. is proposed. The type strain is BO-59T (= KACC 19646T = LMG 30865 T).

Chryseobacterium panacisoli sp. nov., isolated from ginseng-cultivation soil with ginsenoside-converting activity.

A Gram-stain-negative, non-motile, non-spore-forming, aerobic, rod-shaped and yellow-pigmented bacterium, designated strain Gsoil 183T, 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 183T grew at 10-37 °C and at pH 5.0-9.0 on tryptic soy agar. Strain Gsoil 183T had ß-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 183T clustered with species of the genus Chryseobacterium and appeared to be closely related to Chryseobacterium sediminis LMG 28695T (99.1 % sequence similarity), Chryseobacterium lactis NCTC 11390T (98.6%), Chryseobacterium rhizoplanae LMG 28481T (98.6%), Chryseobacterium oncorhynchi CCUG 60105T (98.5%), Chryseobacterium viscerum CCUG 60103T (98.4%) and Chryseobacterium joostei DSM 16927T (98.3%). Menaquinone MK-6 was the predominant respiratory quinone and the major fatty acids were iso-C15 : 0, iso-C17 : 0-3OH and summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c). 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 183T 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 183T 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 183T (=KACC 15033T=LMG 23397T).

Enhanced production of ginsenoside Rh2(S) from PPD-type major ginsenosides using BglSk cloned from Saccharibacillus kuerlensis together with two glycosidase in series.

BACKGROUND: Ginsenoside Rh2(S) is a promising compound for the prevention of various kinds of cancers, inflammation, and diabetes. However, due to its low concentration (<0.02%), researchers are still trying to find an efficient glycoside hydrolase for the scaled-up production of Rh2(S). METHOD: Three glycoside hydrolases (BglBX10, Abf22-3, and BglSk) were cloned in Escherichia coli BL21 (DE3) and the expressed recombinant enzyme was used for the scaled-up production of Rh2(S) through the conversion of PPD-type (protopanaxadiol) major ginsenosides (Rb1, Rc, and Rd, except Rb2) extracted from Korean red ginseng. Specific and specialized bioconversion pathways were designed that evolved the initial bioconversion of PPD-mix â†’ Rg3(S) â†’ Rh2(S). The reaction was started with 50 mg/mL of PPD-mix, 20 mg/mL of BglBX10, Abf22-3, and BglSk in series, respectively. The process was completed in a 10 L jar fermenter with a 5 L working volume at 37 °C for 48 hrs. RESULTS: The designed bioconversion pathways show that Abf22-3 and BglBX10 were responsible for the conversion of Rb1, Rc and Rd â†’ Rg3(S), and then Rg3(S) was completely transformed to Rh2(S) by BglSk. As a result, 15.1 g of ginsenoside Rh2(S) with 98.0 ± 0.2% purity was obtained after strict purification using the Prep-HPLC system with a 100 φ diameter column. Additionally, BglSk was also investigated for its production activity with seven different kinds of PPD-mix type ginsenosides. CONCLUSION: Our pilot data demonstrate that BglSk is a suitable enzyme for the gram unit production of ginsenoside Rh2(S) at the industrial level.

Phnomibacter ginsenosidimutans gen. nov., sp. nov., a novel glycoside hydrolase positive bacterial strain with ginsenoside hydrolysing activity.

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-02T) was identified and subjected to a polyphasic taxonomic analysis. Based on genome analysis, strain SB-02T 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-02T 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-02T 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-C15 : 0, iso-C15 : 1 G and iso-C17 : 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-02T 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-02T (=KACC 21266T=LMG 31707T). The genome annotation of SB-02T 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.

Isolation, characterisation and genome analysis of a novel ginsenosides hydrolysing bacterium Ginsengibacter hankyongi gen. nov., sp. nov. isolated from soil.

A novel yellow-pigmented bacterial strain (designated BR5-29T), was isolated and its taxonomy was studied. Phylogenetic study based on the 16S rRNA and draft genome sequence placed the strain BR5-29T 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 BR5-29T 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 BR5-29T showed 225 carbohydrate genes which may be responsible for the conversion of major ginsenosides to minor ginsenosides. Strain BR5-29T contained MK-7 as a predominant quinone, and iso-C15:0, iso-C15:0 G, iso-C17:0 3-OH, and C16:1 ω7c and/or C16:1 ω6c (summed feature 3) as major fatty acids. The polar lipids found in the strain BR5-29T 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 C12:0, iso-C17:0 2-OH and iso-C15:1 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 BR5-29T 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 BR5-29T (= KACC 19446T = LMG 30462T). Thus, we predict that this novel strain may prove useful for the future research analysis (target gene cloning) and mass production of Rg3.

Novel enzymatic elimination method for the chromatographic purification of ginsenoside Rb3 in an isomeric mixture.

BACKGROUND: The separation of isomeric compounds from a mixture is a recurring problem in chemistry and phytochemistry research. The purification of pharmacologically active ginsenoside Rb3 from ginseng extracts is limited by the co-existence of its isomer Rb2. The aim of the present study was to develop an enzymatic elimination-combined purification method to obtain pure Rb3 from a mixture of isomers. METHODS: To isolate Rb3 from the isomeric mixture, a simple enzymatic selective elimination method was used. A ginsenoside-transforming glycoside hydrolase (Bgp2) was employed to selectively hydrolyze Rb2 into ginsenoside Rd. Ginsenoside Rb3 was then efficiently separated from the mixture using a traditional chromatographic method. RESULTS: Chromatographic purification of Rb3 was achieved using this novel enzymatic elimination-combined method, with 58.6-times higher yield and 13.1% less time than those of the traditional chromatographic method, with a lower minimum column length for purification. The novelty of this study was the use of a recombinant glycosidase for the selective elimination of the isomer. The isolated ginsenoside Rb3 can be used in further pharmaceutical studies. CONCLUSIONS: Herein, we demonstrated a novel enzymatic elimination-combined purification method for the chromatographic purification of ginsenoside Rb3. This method can also be applied to purify other isomeric glycoconjugates in mixtures.