Min-Ho Yoon

Bioconversion of major ginsenosides Rg1 to minor ginsenoside F1 using novel recombinant ginsenoside hydrolyzing glycosidase cloned from Sanguibacter keddieii and enzyme characterization

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.

Sphingosinicella ginsenosidimutans sp. nov., with ginsenoside converting activity

The Gram-reaction-negative, strictly aerobic, non-motile, nonspore-forming, and rod-shaped bacterial strain designated BS11T was isolated from the compost and its taxonomic position was investigated by using a polyphasic approach. Strain BS11T grew optimally at 30–37°C and at pH 7.0 in the absence of NaCl on nutrient agar. Strain BS11T 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 BS11T was shown to belong to the family Sphingomonadaceae and was related to Sphingosinicella vermicomposti YC7378T (96.3% sequence similarity), S. xenopeptidilytica 3–2W4T (96.2%), S. microcystinivorans Y2T (96.1%), and S. soli KSL-125T (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 C18:1ω7c/ω9t/ω12t; 40.6%), C16:0 (22.5%), C17:1ω6c (13.7%) and C17:0 (9.1%). DNA and chemotaxonomic data supported the affiliation of strain BS11T to the genus Sphingosinicella. Strain BS11T 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 BS11T (=KACC 16619T =JCM 18201T).

Mass production of the ginsenoside Rg3(S) through the combinative use of two glycoside hydrolases.

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.

Methanobrevibacter boviskoreani sp. nov., isolated from the rumen of Korean native cattle.

Three strictly anaerobic, methanogenic strains JH1(T), JH4 and JH8 were isolated from rumen of the Korean native cattle (HanWoo; Bos taurus coreanae) in South Korea. The colonies were circular, opaque, and slightly yellowish. Phylogenetic analyses of 16S rRNA gene and mcrA (encoding α subunit of methyl-coenzyme M reductase) sequences confirmed the affiliation of the novel strains with the Methanobacteriales, and Methanobrevibacter wolinii SH(T) was the most closely related species. The 16S rRNA gene and mcrA sequence similarities between strains JH1(T), JH4 and JH8 and M. wolinii SH(T) were 96.2 and 89.0 % respectively, and DNA-DNA hybridization of the isolates and M. wolinii DSM 11976(T) showed a 20 % reassociation. Strain JH1(T) exhibited 92 % DNA-DNA relatedness with strains JH4 and JH8, and their 16S rRNA gene and mcrA sequences were identical. Cells stained Gram-positive and were non-motile rods, 1.5-1.8 µm long and 0.6 µm wide. The strains were able to use H2/CO2 and formate. The optimum temperature and pH ranges for growth were 37-40 °C and pH 6.5-7.0. The DNA G+C content of strain JH1(T) was 28 mol%. Based on data from this study using a polyphasic approach, the three strains represent a novel species of genus Methanobrevibacter, for which the name Methanobrevibacter boviskoreani sp. nov. is proposed. The type strain is JH1(T) ( = KCTC 4102(T) = JCM 18376(T)).

Genome sequence of Methanobrevibacter sp. strain jh1, isolated from rumen of Korean native cattle.

ABSTRACT The Methanobrevibacter sp. strain JH1 was isolated from the rumen of Korean native cattle (HanWoo; Bos taurus coreanae). Here, we provide a 2.06-Mb draft genome sequence of strain JH1 that might provide more information about the lifestyle of rumen methanogens and about the genes and proteins that can be targeted to curb methane emissions.

Microlunatus panaciterrae sp. nov., a β-glucosidase-producing bacterium isolated from soil in a ginseng field

A novel bacterial strain, designated Gsoil 954(T), showing beta-glucosidase activity was isolated from a soil sample from a ginseng field in Pocheon Province, South Korea. The isolate was a Gram-positive, aerobic, motile, coccus-shaped, non-endospore-forming bacterium. Comparative analysis of 16S rRNA gene sequences revealed that strain Gsoil 954(T) belonged to the family Propionibacteriaceae, the highest levels of sequence similarity being found with the type strains of Microlunatus ginsengisoli (96.4 %) and Microlunatus phosphovorus (96.2 %). The strain showed <95.9 % similarity with respect to other species within the family Propionibacteriaceae. In addition, the presence of ll-2,6-diaminopimelic acid in the cell-wall peptidoglycan, MK-9(H(4)) as the major menaquinone and anteiso-C(15 : 0) and iso-C(15 : 0) as the major cellular fatty acids suggested its affiliation with the genus Microlunatus. The G+C content of the genomic DNA was 65.1 mol%. On the basis of its phenotypic characteristics and phylogenetic distinctiveness, strain Gsoil 954(T) represents a novel species within the genus Microlunatus, for which the name Microlunatus panaciterrae sp. nov. is proposed. The type strain is Gsoil 954(T) (=KCTC 13058(T)=DSM 18662(T)).

Cellulomonas chitinilytica sp. nov., a chitinolytic bacterium isolated from cattle-farm compost

A bacterial strain, designated X.bu-b T, with chitin-, xylan-, cellulose- and starch-degrading activities, was isolated from compost at a cattle farm near Daejeon, Republic of Korea. The strain comprised Gram-positive, aerobic or facultatively anaerobic, non-motile, rod-shaped bacteria. On the basis of an analysis of 16S rRNA gene sequences, the phylogenetic position of X.bu-b T was within the genus Cellulomonas, and the strain exhibited relatively high sequence similarities with respect to Cellulomonas biazotea DSM 20112T (98.1 %), C. cellasea DSM 20118T (98.1 %), C. fimi DSM 20113T (98.0 %), C. terrae DB5T (97.9 %), C. humilata ATCC 25174T (97.7 %), C. xylanilytica XIL11 T (97.5 %), C. uda DSM 20107T (97.4 %), C. gelida DSM 20111 T (97.3 %), C. iranensis OT (97.3 %) and C. flavigena DSM 20109T (97.0 %). The phylogenetic distance from other Cellulomonas species with validly published names was greater than 3 % (i.e. less than 97.0 % sequence similarity). Chemotaxonomic data also supported the classification of strain X.bu-b T within the genus Cellulomonas: L-ornithine was the cell-wall diamino acid, anteiso-C15:0 and anteiso-C17:0 were the major fatty acids, rhamnose, galactose, xylose and ribose were the cell-wall sugars, MK-9(H4) was the predominant menaquinone and diphosphatidylglycerol and phosphatidylglycerol were present in the polar lipids. The G+C content of the genomic DNA was 73.6 mol%. DNA-DNA hybridization experiments showed that the values for DNA-DNA relatedness between strain X.bu-b T and the phylogenetically closest neighbours were below 23 %. On the basis of its phenotypic properties and phylogenetic distinctiveness, strain X.bu-b T represents a novel species of the genus Cellulomonas, for which the name Cellulomonas chitinilytica sp. nov. is proposed. The type strain is X.bu-b T (=KCTC 19133T =DSM 17922T).

Mucilaginibacter ginsenosidivorax sp. nov., with ginsenoside converting activity isolated from sediment

A Gram-reaction-negative, strictly aerobic, non-motile, non-spore-forming, and rod-shaped bacterial strain designated KHI28T was isolated from sediment in Gapcheon (river) and its taxonomic position was investigated using a polyphasic approach. Strain KHI28T grew at 10–42°C and at pH 5.5–8.5 on R2A and nutrient agar without additional NaCl as a supplement. Strain KHI28T 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 KHI28T was shown to belong to the family Sphingobacteriaceae and to be related to Mucilaginibacter dorajii DR-f4T (97.9% sequence similarity), M. polysacchareus DRP28T (97.3%), and M. lappiensis ANJLI2T (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 C16:1ω6c and/or C16:1ω7c), iso-C15:0 and C16:0. DNA and chemotaxonomic data supported the affiliation of strain KHI28T to the genus Mucilaginibacter. Strain KHI28T 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 KHI28T (=KACC 14955T =LMG 25804T).