Wan-Taek Im

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.

A Gram-stain negative, strictly aerobic, non-motile, non-spore-forming, and rod-shaped bacterial strain designated FW-3^T was isolated from fresh water and its taxonomic position was investigated by using a polyphasic approach. Strain FW-3^T was found to grow at 10–37 °C and at pH 7.0 in the absence of NaCl on nutrient agar. On the basis of 16S rRNA gene sequence similarity, strain FW-3^T was shown to belong to the family Acetobacteraceae and to be related to Roseomonas lacus TH-G33^T (97.2 % sequence similarity) and Roseomonas terrae DS-48^T (96.4 %). The G+C content of the genomic DNA was determined to be 68.0 %. The major menaquinone was determined to be Q-10 and the major fatty acids were identified as summed feature 7 (comprising C_18:1 ω9c/ω12t/ω7c as defined by the MIDI system; 55.4 %), and C_18:1 2OH (29.8 %). DNA and chemotaxonomic data supported the affiliation of strain FW-3^T to the genus Roseomonas. Strain FW-3^T could be differentiated genotypically and phenotypically from the recognized species of the genus Roseomonas. The novel isolate therefore represents a novel species, for which the name Roseomonas sediminicola sp. nov. is proposed, with the type strain FW-3^T (=KACC 16616^T = JCM 18210^T).

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 Rg ₁ into ( S )-Rg ₂ and ( S )-Rh ₁ , respectively, by hydrolyzing one glucose moiety attached to the C-20 position at pH 8.0 and 30°C. The K _m values for p -nitrophenyl-β- d -glucopyranoside, Re, and Rg ₁ 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 )-Rh ₁ and ( S )-Rg ₂ at chromatographic purities of 98% ± 0.5% and 97% ± 1.2%, respectively. This is the first report of gram-scale production of ( S )-Rh ₁ and ( S )-Rg ₂ from PPTGM using a novel ginsenoside-transforming β-glucosidase of glycoside hydrolase family 3.

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 C_16:1 ω6c and/or C_16:1 ω7c), iso-C_15:0 and C_16:0. 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 Gram-negative, strictly aerobic, non-motile, non-spore-forming and rod-shaped bacterial strain designated KHI67^T was isolated from sediment of the Gapcheon River in South Korea and its taxonomic position was investigated by using a polyphasic approach. Strain KHI67^T was observed to grow optimally at 25–30 °C and at pH 7.0 on nutrient and R2A agar. On the basis of 16S rRNA gene sequence similarity, strain KHI67^T was shown to belong to the family Sphingomonadaceae and was related to Sphingomonas faeni MA-olki^T (97.6 % sequence similarity), Sphingomonas aerolata NW12^T (97.5 %) and Sphingomonas aurantiaca MA101b^T (97.3 %). The G + C content of the genomic DNA was determined to be 65.6 %. The major ubiquinone was found to be Q-10, the major polyamine was identified as homospermidine and the major fatty acids identified were summed feature 8 (comprising C_18:1 ω7c/ω6c; 37.0 %), C_16:0 (13.0 %), summed feature 3 (comprising C_16:1 ω7c/C_16:1 ω6c; 12.8 %) and C_14:0 2OH (9.3 %). DNA and chemotaxonomic data supported the affiliation of strain KHI67^T to the genus Sphingomonas. The DNA–DNA relatedness values between strain KHI67^T and its closest phylogenetic neighbours were below 15 %. Strain KHI67^T could be differentiated genotypically and phenotypically from the recognised species of the genus Sphingomonas. The isolate therefore represents a novel species, for which the name Sphingomonas ginsenosidivorax sp. nov. is proposed, with the type strain KHI67^T (=KACC 14951^T = JCM 17076^T = LMG 25801^T).

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-C_16:0, iso-C_16:1 H, iso-C_14:0, iso-C_15:0 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).

Two novel bacteria, designated strains Gsoil 634^T and Dae 20^T, were isolated in South Korea from soil of a ginseng field and freshwater sediment, respectively and were characterized by a polyphasic approach to clarify their taxonomic positions. Phylogenetic analysis based on 16S rRNA gene sequences indicated that, although they probably represented two distinct species (indicated by a sequence similarity of 96.6 %), both strain Gsoil 634^T and strain Dae 20^T belonged to the genus Sphingomonas and were most closely related to ‘Sphingomonas humi’ PB323 (97.8 % and 96.7 % sequence similarity, respectively), Sphingomonas kaistensis PB56^T (96.8 % and 96.7 %), Sphingomonas astaxanthinifaciens TDMA-17^T (96.6 % and 95.4 %) and Sphingomonas jaspsi TDMA-16^T (95.6 % and 95.8 %). For both novel strains, the major ubiquinone was Q-10, the major polyamine was homospermidine, the major cellular fatty acids included summed feature 7 (C_18 : 1ω7c, C_18 : 1ω9t and/or C_18 : 1ω12t), C_17 : 1ω6c and C_16 : 0, and the polar lipids included sphingoglycolipid. These chemotaxonomic data supported the affiliation of both strains to the genus Sphingomonas . However, the DNA–DNA relatedness value between strain Gsoil 634^T and ‘Sphingomonas humi’ PB323^T was 31 %. Moreover, the results of physiological and biochemical tests allowed the phenotypic differentiation of strains Gsoil 634^T and Dae 20^T from established members of the genus Sphingomonas . Based on these data, the two isolates represent two novel species in the genus Sphingomonas , for which the names Sphingomonas ginsengisoli sp. nov. (type strain Gsoil 634^T = KCTC 12630^T = DSM 18094^T = LMG 23739^T) and Sphingomonas sediminicola sp. nov. (type strain Dae 20^T = KCTC 12629^T = DSM 18106^T = LMG 23592^T) are proposed.

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.

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 K_m and V_max 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.