Hyung-Gwan Lee

Identification and Characterization of a Novel Terrabacter ginsenosidimutans sp. nov. β-Glucosidase That Transforms Ginsenoside Rb1 into the Rare Gypenosides XVII and LXXV

ABSTRACT A new β-glucosidase from a novel strain of Terrabacter ginsenosidimutans (Gsoil 3082T) 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-[β-d-glucopyranosyl-(1-2)-β-d-glucopyranosyl]-20-O-[β-d-glucopyranosyl-(1-6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol} to the more pharmacologically active rare ginsenosides gypenoside XVII {3-O-β-d-glucopyranosyl-20-O-[β-d-glucopyranosyl-(1-6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol}, gypenoside LXXV {20-O-[β-d-glucopyranosyl-(1-6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol}, and C-K [20-O-(β-d-glucopyranosyl)-20(S)-protopanaxadiol]. A BLAST search of the bgpA sequence revealed significant homology to family 3 glycoside hydrolases. Expressed in E. coli, β-glucosidase had apparent Km values of 4.2 ± 0.8 and 0.14 ± 0.05 mM and Vmax values of 100.6 ± 17.1 and 329 ± 31 μmol·min−1·mg of protein−1 against p-nitrophenyl-β-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°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 β-glucosidase of the glycoside hydrolase family 3.

Bioconversion of ginsenosides Rb1, Rb2, Rc and Rd by novel β-glucosidase hydrolyzing outer 3-O glycoside from Sphingomonas sp. 2F2: Cloning, expression, and enzyme characterization

A new β-glucosidase gene (bglSp) was cloned from the ginsenoside converting Sphingomonas sp. strain 2F2 isolated from the ginseng cultivating filed. The bglSp consisted of 1344 bp (447 amino acid residues) with a predicted molecular mass of 49,399 Da. A BLAST search using the bglSp sequence revealed significant homology to that of glycoside hydrolase superfamily 1. This enzyme was overexpressed in Escherichia coli BL21 (DE3) using a pET21-MBP (TEV) vector system. Overexpressed recombinant enzymes which could convert the ginsenosides Rb(1), Rb(2), Rc and Rd to the more pharmacological active rare ginsenosides gypenoside XVII, ginsenoside C-O, ginsenoside C-Mc(1) and ginsenoside F(2), respectively, were purified by two steps with Amylose-affinity and DEAE-Cellulose chromatography and characterized. The kinetic parameters for β-glucosidase showed the apparent K(m) and V(max) values of 2.9±0.3 mM and 515.4±38.3 μmol min(-1)mg of protein(-1) against p-nitrophenyl-β-d-glucopyranoside. The enzyme could hydrolyze the outer C3 glucose moieties of ginsenosides Rb(1), Rb(2), Rc and Rd into the rare ginsenosides Gyp XVII, C-O, C-Mc(1) and F(2) quickly at optimal conditions of pH 5.0 and 37°C. A little ginsenoside F(2) production from ginsenosides Gyp XVII, C-O, and C-Mc(1) was observed for the lengthy enzyme reaction caused by the side ability of the enzyme.

Rhodanobacter ginsenosidimutans sp. nov., isolated from soil of a ginseng field in South Korea

A novel gammaproteobacterium, designated Gsoil 3054(T), was isolated from soil of a ginseng field in Pocheon province, South Korea, and was characterized using a polyphasic approach to determine its taxonomic position. The strain was Gram-negative, aerobic, non-motile, non-spore-forming and rod-shaped. Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain belonged to the genus Rhodanobacter; it was most closely related to Rhodanobacter fulvus Jip2(T) and Rhodanobacter thiooxydans LCS2(T) (97.9 and 97.2 % 16S rRNA gene sequence similarity, respectively). Chemotaxonomic data, i.e. Q-8 as the predominant ubiquinone and iso-C(15 : 0), 10-methyl C(16 : 0) and iso-C(17 : 0) as major fatty acids, also supported classification of strain Gsoil 3054(T) in the genus Rhodanobacter. However, DNA-DNA hybridization values of Gsoil 3054(T) with R. fulvus Jip2(T) and R. thiooxydans LCS2(T) were 45 and 31 %, respectively. Moreover, physiological and biochemical tests enabled strain Gsoil 3054(T) to be differentiated phenotypically from other established species of Rhodanobacter. Therefore, the isolate represents a novel species, for which the name Rhodanobacter ginsenosidimutans sp. nov. is proposed; the type strain is Gsoil 3054(T) (=KACC 12822(T) =DSM 21013(T) =KCTC 22231(T) =LMG 24457(T)).

Pedobacter composti sp. nov., isolated from compost

A Gram-negative, aerobic, rod-shaped, non-motile, non-spore-forming bacterial strain, designated TR6-06(T), was isolated from a compost sample in South Korea and characterized taxonomically by using a polyphasic approach. The organism grew optimally at 30 degrees C and pH 6.5-7.0. The isolate was positive for catalase and oxidase tests, but negative for gelatinase and urease and for indole and H(2)S production. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain TR6-06(T) was most closely affiliated with members of the genus Pedobacter of the family Sphingobacteriaceae. Strain TR6-06(T) exhibited 16S rRNA gene sequence similarity values of 89.9-93.5 % to the type strains of species of the genus Pedobacter. The G+C content of the genomic DNA of strain TR6-06(T) was 41.9 mol%. The predominant respiratory quinone was MK-7. The major fatty acids were iso-C(15 : 0), iso-C(17 : 0) 3-OH, C(16 : 1)omega7c and anteiso-C(15 : 0). These chemotaxonomic data support the affiliation of strain TR6-06(T) to the genus Pedobacter. However, on the basis of its phenotypic properties and phylogenetic distinctiveness, strain TR6-06(T) (=KCTC 12638(T)=LMG 23490(T)) should be classified as the type strain of a novel species, for which the name Pedobacter composti sp. nov. is proposed.

Status, Alert System, and Prediction of Cyanobacterial Bloom in South Korea

Bloom-forming freshwater cyanobacterial genera pose a major ecological problem due to their ability to produce toxins and other bioactive compounds, which can have important implications in illnesses of humans and livestock. Cyanobacteria such as Microcystis, Anabaena, Oscillatoria, Phormidium, and Aphanizomenon species producing microcystins and anatoxin-a have been predominantly documented from most South Korean lakes and reservoirs. With the increase in frequency of such blooms, various monitoring approaches, treatment processes, and prediction models have been developed in due course. In this paper we review the field studies and current knowledge on toxin producing cyanobacterial species and ecological variables that regulate toxin production and bloom formation in major rivers (Han, Geum, Nakdong, and Yeongsan) and reservoirs in South Korea. In addition, development of new, fast, and high-throughput techniques for effective monitoring is also discussed with cyanobacterial bloom advisory practices, current management strategies, and their implications in South Korean freshwater bodies.

Paenibacillus sediminis sp nov., a xylanolytic bacterium isolated from a tidal flat

A Gram-positive, rod-shaped, xylanolytic, spore-forming bacterium, strain GTH-3(T), was isolated from a tidal flat adjacent to Ganghwa Island, Republic of Korea, and was characterized to determine its taxonomic position. On the basis of 16S rRNA gene sequence similarity, strain GTH-3(T) was shown to belong to the family Paenibacillaceae, being most closely related to the type strains of Paenibacillus ginsengisoli (94.9 %), Paenibacillus anaericanus (94.8 %), Paenibacillus urinalis (94.4 %), Paenibacillus cookii (94.2 %), Paenibacillus alvei (94.1 %) and Paenibacillus chibensis (94.0 %). The G+C content of the genomic DNA of strain GTH-3(T) was 45.9±0.2 mol% (mean±sd). The major menaquinone was MK-7. The major fatty acids were anteiso-C(15:0) and iso-C(16:0). The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Phenotypic and chemotaxonomic data supported the affiliation of strain GTH-3(T) to the genus Paenibacillus. The results of physiological and biochemical tests allowed strain GTH-3(T) to be distinguished genotypically and phenotypically from recognized species of the genus Paenibacillus. Strain GTH-3(T) is therefore considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus sediminis sp. nov. is proposed. The type strain is GTH-3(T) ( = DSM 23491(T) = LMG 25635(T)).

Novosphingobium sediminicola sp. nov. isolated from freshwater sediment.

A yellow-pigmented, Gram-negative, short rod-shaped, non-motile and non-spore-forming bacterial strain, designated HU1-AH51(T), was isolated from freshwater sediment and was characterized using a polyphasic approach, in order to determine its taxonomic position. On the basis of 16S rRNA gene sequence similarity, strain HU1-AH51(T) was shown to belong to the genus Novosphingobium, showing the highest level of sequence similarity with respect to Novosphingobium resinovorum NCIMB 8767(T) (96.0 %), Novosphingobium naphthalenivorans TUT562(T) (96.0 %) and Novosphingobium panipatense SM16(T) (96.0 %). Strain HU1-AH51(T) had a genomic DNA G+C content of 62.6 mol% and Q-10 as the predominant respiratory quinone. Furthermore, the major polyamine component (spermidine) in the cytoplasm and the presence of sphingoglycolipids suggested that strain HU1-AH51(T) belongs to the family Sphingomonadaceae. On the basis of its phenotypic properties and phylogenetic distinctiveness, strain HU1-AH51(T) represents a novel species of the genus Novosphingobium, for which the name Novosphingobium sediminicola sp. nov. is proposed. The type strain is HU1-AH51(T) ( = LMG 24320(T)  = KCTC 22311(T)).

Geodermatophilus soli sp. nov. and Geodermatophilus terrae sp. nov., two actinobacteria isolated from grass soil.

Two strains, PB34(T) and PB261(T), were isolated from grass soil sampled in Daejeon, Republic of Korea. Comparative 16S rRNA gene sequence studies showed the two bacteria to be clearly affiliated with the phylum Actinobacteria and most closely related to the genus Geodermatophilus, showing 16S rRNA gene sequence similarities to the type strains of species of the genus Geodermatophilus of 95.0-96.3 % and sharing 98.5 % similarity between the two strains. The two strains were Gram-stain-positive, aerobic, motile and rod-shaped bacteria. The peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. The predominant menaquinones were MK-9(H4) and MK-9(H0). The major fatty acids were iso-C15 : 0, iso-C16 : 0, iso-C17 : 0 and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) for strain PB34(T) and iso-C14 : 0, iso-C15 : 0, iso-C16 : 0 and C16 : 0 for strain PB261(T). The G+C contents of the genomic DNA of strains PB34(T) and PB261(T) were 73.2 mol% and 74.1 mol%, respectively. Thus, based on the evidence of a polyphasic study, it is proposed that strains PB34(T) and PB261(T) represent two novel species, for which the names Geodermatophilus soli sp. nov. (type strain PB34(T) = KCTC 19880(T) = JCM 17785(T)) and Geodermatophilus terrae sp. nov. (type strain PB261(T) = KCTC 19881(T) = JCM 17786(T)) are proposed.

Olivibacter soli sp. nov., Olivibacter ginsengisoli sp. nov. and Olivibacter terrae sp. nov., from soil of a ginseng field and compost in South Korea.

Three novel strains, designated Gsoil 034T, Gsoil 060T and Jip13T, isolated from soil from a ginseng field and compost in South Korea, were characterized using a polyphasic approach to clarify their taxonomic positions. These isolates were found to be Gram-negative, aerobic and heterotrophic, non-spore-forming, non-motile and rod-shaped. Phylogenetic analysis based on 16S rRNA gene sequences indicated that these three isolates formed a cluster with the monospecific genus Olivibacter within the family Sphingobacteriaceae but were clearly separated from Olivibacter sitiensis. The 16S rRNA gene sequence similarities between these isolates and the type strain of O. sitiensis were in the range 88.5-90.1 %. Phenotypic and chemotaxonomic data (MK-7 as the major isoprenoid quinone and iso-C15 : 0 2-OH and/or C16 : 1omega7c, iso-C15 : 0 and iso-C17 : 0 3-OH as the major fatty acids) supported the affiliation of these strains to the genus Olivibacter. However, the results of physiological and biochemical tests allowed phenotypic differentiation of the isolates from Olivibacter species with validly published names. Therefore strains Gsoil 034T, Gsoil 060T and Jip13T represent three novel species of the genus Olivibacter, for which the names Olivibacter soli sp. nov. (type strain Gsoil 034T =KCTC 12645T =LMG 23492T), Olivibacter ginsengisoli sp. nov. (type strain Gsoil 060T =KCTC 12646T =LMG 23491T) and Olivibacter terrae sp. nov. (type strain Jip13T =KCTC 12644T =LMG 23494T) are proposed.

Streptomyces panacagri sp. nov., isolated from soil of a ginseng field

A Gram-positive, spore-forming, aerobic actinomycete, strain Gsoil 519T, was isolated from soil of a ginseng field of Pocheon province in South Korea. The closest phylogenetic relatives were Streptomyces marinus Sp080513GE-26T (97.94 % 16S rRNA gene sequence similarity), Streptomyces albiaxialis NRRL B-24327T (97.84 %), Streptomyces albus subsp. albus DSM 40313T (97.84 %), Streptomyces almquistii NBRC 13015T (97.81 %), Streptomyces gibsonii NBRC 15415T (97.81 %), Streptomyces rangoonensis NBRC 13078T (97.81 %), Streptomyces sodiiphilus YIM 80305T (97.77 %) and Streptomyces flocculus NBRC 13041T (97.67 %). The G+C content of the genomic DNA was 71.8 mol%. The chemotaxonomic data [MK-9(H6) and MK-9(H8) as the major menaquinones; ll-diaminopimelic acid as a component of the cell-wall peptidoglycan; ribose, xylose, mannose and glucose as the major cell-wall sugars; and anteiso-C15:0, iso-C15:0, iso-C17:0, anteiso-C17:0 and C16:0 as the major fatty acids] supported the affiliation of strain Gsoil 519T to the genus Streptomyces. The physiological and biochemical characteristics and the low level of DNA-DNA relatedness differentiated the isolate genotypically and phenotypically from recognized members of the genus Streptomyces. The isolate, therefore, represents a novel species, for which the name Streptomyces panacagri sp. nov. is proposed, with Gsoil 519T (=KCTC 19139T=DSM 41871T) as the type strain.