Song-Gun Kim

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.

A novel denitrifying bacterial isolate that degrades trimethylamine both aerobically and anaerobically via two different pathways

Abstract. The aerobic and anaerobic degradation of trimethylamine by a newly isolated denitrifying bacterium from an enrichment culture with trimethylamine inoculated with activated sludge was studied. Based on 16S rDNA analysis, this strain was identified as a Paracoccus sp. The isolate, strain T231, aerobically degraded trimethylamine, dimethylamine and methylamine and released a stoichiometric amount of ammonium ion into the culture fluid as a metabolic product, indicating that these methylated amines were completely degraded to formaldehyde and ammonia. The strain degraded trimethylamine also under denitrifying conditions and consumed a stoichiometric amount of nitrate, demonstrating that complete degradation of trimethylamine was coupled with nitrate reduction. Cell-free extract prepared from cells grown aerobically on trimethylamine exhibited activities of trimethylamine mono-oxygenase, trimethylamine N-oxide demethylase, dimethylamine mono-oxygenase, and methylamine mono-oxygenase. Cell-free extract from cells grown anaerobically on trimethylamine and nitrate exhibited activities of trimethylamine dehydrogenase and dimethylamine dehydrogenase. These results indicate that strain T231 had two different pathways for aerobic and anaerobic degradation of trimethylamine. This is a new feature for trimethylamine metabolism in denitrifying bacteria.

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.

Annual variation of Microcystis genotypes and their potential toxicity in water and sediment from a eutrophic reservoir

The relative genetic diversity of microcystin-producing Microcystis in the water and sediment of the Daechung Reservoir, Korea, was investigated over an entire year, including the cyanobacterial bloom season. The cells of potentially toxic Microcystis strains containing mcyJ genotypes and cells containing the genus-specific cpcBA gene were quantified by a real-time PCR. The ratio of cells with mcyJ genotypes to the total Microcystis population in the water body was the highest (68.3%) in August when the cyanobacterial bloom reached its peak and the microcystin concentration in the water began to increase. A denaturing gradient gel electrophoresis profile analysis of the mcyJ genotypes performed to monitor any changes in the toxic Microcystis population showed the appearance of new genotypes and the disappearance of existing genotypes in the reservoir water collected during the summer months, when compared with the profile for the samples collected in spring and autumn. However, very little change was observed over the course of the year as regards the population diversity of the sediment samples.

Determination of Cyanobacterial Diversity during Algal Blooms in Daechung Reservoir, Korea, on the Basis of cpcBA Intergenic Spacer Region Analysis

ABSTRACT The detection and prevention of cyanobacterial blooms are important issues in water quality management. As such, the diversity and community dynamics of cyanobacteria during cyanobacterial bloom in the Daechung Reservoir, Korea, were studied by analyzing the intergenic spacer (IGS) region between phycocyanin subunit genes cpcB and cpcA (cpcBA IGS). To amplify the cpcBA IGS from environmental samples, new PCR primers that could cover a wider range of cyanobacteria than previously known primers were designed. In the samples taken around the bloom peak (2 September 2003), seven groups of cpcBA IGS sequences were detected, and none of the amplified cpcBA IGSs was closely related to the cpcBA IGS from chloroplasts. Apart from the Microcystis-, Aphanizomenon (Anabaena)-, Pseudanabaena-, and Planktothrix (Oscillatoria)-like groups, the three other groups of cpcBA IGS sequences were only distantly related to previously reported sequences (<85% similarity to their closest relatives). The most prominent changes during the bloom were the gradual decrease and eventual disappearance of the Aphanizomenon (Anabaena)-like group before the bloom peak and the gradual increase and sudden disappearance of Planktothrix (Oscillatoria)-like groups right after the bloom peak. The community succession profile obtained based on the cpcBA IGS analysis was also supported by a PCR-denaturing gradient gel electrophoresis analysis of the 16S rRNA genes.

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.

Phylogenetic analysis of Antrodia and related taxa based on partial mitochondrial SSU rDNA sequences

Sequences of mitochondrial SSU rDNA were obtained from six species of Antrodia and related fungal taxa to reveal their phylogenetic relationships. Phylogenetic analysis showed that the species of Antrodia did not cluster into a single clade. Brown rot fungi were separated into two main groups through which Antrodia was dispersed. Antrodia sinuosa, A. serialis, A. heteromorpha and A. malicola clustered with Perenniporia, Fomitopsis, Piptoporus, Daedalea and Melanoporia within one group of brown rot fungi, while A. carbonica and A. vaillantii clustered with Oligoporus, Gloeophyllum and Auriporia within the other group of brown rot fungi, indicating that Antrodia is a heterogeneous genus and that brown rot fungi have evolved convergently.

Identification and Characterization of a Mucilaginibacter sp. Strain QM49 β-Glucosidase and Its Use in the Production of the Pharmaceutically Active Minor Ginsenosides (S)-Rh1 and (S)-Rg2

ABSTRACT 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 V max 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.

Isolation and characterization of novel halotolerant and/or halophilic denitrifying bacteria with versatile metabolic pathways for the degradation of trimethylamine

Four denitrifying bacteria capable of degrading trimethylamine under both aerobic and denitrifying conditions were newly isolated from coastal sediments and wastewater contaminated by marine water. All strains were in alpha-Proteobacteria. Strain GP43 was classified as a member of genus Paracoccus, and strain PH32, PH34 and GRP21 were novel organisms with remote phylogenetic position from other genus alpha-Proteobacteria. Among these four strains were the halophilic strains PH32, PH34 and GRP21, which did not grow in the absence of sodium chloride in culture medium. Cells grown under denitrifying conditions possessed trimethylamine dehydrogenase while cells grown aerobically possessed two different enzymes for oxidation of trimethylamine, trimethylamine dehydrogenase and trimethylamine monooxygenase. The newly isolated strain PH32, PH34 and GRP21 may be the first halophilic bacteria to degrade trimethylamine under denitrifying conditions.

Pedobacter daechungensis sp. nov., from freshwater lake sediment in South Korea

A novel bacterial strain, designated Dae 13T, was isolated from sediment from a freshwater lake in Daejeon, South Korea, and was characterized taxonomically by using a polyphasic approach. The isolate was Gram-negative, aerobic, non-motile, non-spore-forming and rod-shaped. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate belonged to the genus Pedobacter in the family Sphingobacteriaceae but was clearly separate from established species of this genus. The 16S rRNA gene sequence similarities between strain Dae 13(T) and type strains of Pedobacter species with validly published names ranged from 91.6 to 97.5 %. The G+C content of the genomic DNA was 33.8 mol%. Chemotaxonomic data, i.e. the presence of MK-7 as the major menaquinone and iso-C(15 : 0), C(16 : 0) and summed feature 3 (iso-C(15 : 0) 2-OH and/or C(16 : 1)omega7c) as the major fatty acids, supported the affiliation of strain Dae 13T to the genus Pedobacter. However, the results of physiological and biochemical tests allowed phenotypic differentiation of the isolate with respect to Pedobacter species with validly published names. Therefore, strain Dae 13T represents a novel species within the genus Pedobacter, for which the name Pedobacter daechungensis sp. nov. is proposed. The type strain is Dae 13T (=KCTC 12637T=LMG 23489T).