Chang-Hao Cui

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.

Enzymatic Biotransformation of Ginsenoside Rb1 and Gypenoside XVII into Ginsenosides Rd and F2 by Recombinant β-glucosidase from Flavobacterium johnsoniae.

This study focused on the enzymatic biotransformation of the major ginsenoside Rb1 into Rd for the mass production of minor ginsenosides using a novel recombinant β-glucosidase from Flavobacterium johnsoniae. The gene (bglF3) consisting of 2,235 bp (744 amino acid residues) was cloned and the recombinant enzyme overexpressed in Escherichia coli BL21(DE3) was characterized. This enzyme could transform ginsenoside Rb1 and gypenoside XVII to the ginsenosides Rd and F2, respectively. The glutathione S-transferase (GST) fused BglF3 was purified with GST-bind agarose resin and characterized. The kinetic parameters for β-glucosidase had apparent Km values of 0.91±0.02 and 2.84±0.05 mM and Vmax values of 5.75±0.12 and 0.71±0.01 μmol·min-1·mg of protein-1 against p-nitrophenyl-β-D-glucopyranoside and Rb1, respectively. At optimal conditions of pH 6.0 and 37℃, BglF3 could only hydrolyze the outer glucose moiety of ginsenoside Rb1 and gypenoside XVII at the C-20 position of aglycon into ginsenosides Rd and F2, respectively. These results indicate that the recombinant BglF3 could be useful for the mass production of ginsenosides Rd and F2 in the pharmaceutical or cosmetic industry.

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.

Mucilaginibacter composti sp. nov., with ginsenoside converting activity, isolated from compost

The Gram-negative, strictly aerobic, non-motile, non-spore-forming, rod shaped bacterial strain designated TR6-03T was isolated from compost, and its taxonomic position was investigated by using a polyphasic approach. Strain TR6-03T grew at 4–42°C and at pH 6.0–8.0 on R2A and nutrient agar without NaCl supplement. Strain TR6-03T had β-glucosidase activity, which was responsible for its ability to transform ginsenoside Re (one of the dominant active components of ginseng) to Rg2. On the basis of 16S rRNA gene sequence similarity, strain TR6-03T was shown to belong to the family Sphingobacteriaceae and to be related to Mucilaginibacter lappiensis ANJLI2T (96.3% sequence similarity), M. dorajii FR-f4T (96.1%), and M. rigui WPCB133T (94.1%). The G+C content of the genomic DNA was 45.6%. The predominant respiratory quinone was MK-7 and the major fatty acids were summed feature 3 (comprising C16:1 ω7c and/or iso-C15:0 20H), iso-C16:0 and iso-C17:0 3OH. DNA and chemotaxonomic data supported the affiliation of strain TR6-03T to the genus Mucilaginibacter. Strain TR6-03T 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 composti sp. nov. is proposed, with the type strain TR6-03T (=KACC 14956T = KCTC 12642T =LMG 23497T).

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.

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.

Tumebacillus ginsengisoli sp. nov., isolated from soil of a ginseng field

A gram-reaction-positive, rod-shaped, spore-forming bacterium, designated Gsoil 1105(T), was isolated from soil of a ginseng field in Pocheon Province in South Korea and characterized in order to determine its taxonomic position. Comparative analysis of the 16S rRNA gene sequence showed that the isolate belongs to the order Bacillales, showing the highest level of sequence similarity with respect to Tumebacillus permanentifrigoris Eur1 9.5(T) (94.6 %). The phylogenetic distances from other described species with validly published names within the order Bacillales were greater than 9.0 %. Strain Gsoil 1105(T) had a genomic DNA G+C content of 55.6 mol% and menaquinone 7 (MK-7) as the major respiratory quinone. The major fatty acids were iso-C(15 : 0) and anteiso-C(15 : 0). On the basis of its phenotypic properties and phylogenetic distinctiveness, strain Gsoil 1105(T) represents a novel species of the genus Tumebacillus, for which the name Tumebacillus ginsengisoli sp. nov. is proposed. The type strain is Gsoil 1105(T) ( = KCTC 13942(T)  = DSM 18389(T)).

Characterization of a Ginsenoside-Transforming β-glucosidase from Paenibacillus mucilaginosus and Its Application for Enhanced Production of Minor Ginsenoside F2

A novel β-glucosidase (BglPm) was identified from Paenibacillus mucilaginosus KCTC 3870T 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.

Identification and Characterization of a Ginsenoside-Transforming β-Glucosidase from Pseudonocardia sp. Gsoil 1536 and Its Application for Enhanced Production of Minor Ginsenoside Rg2(S)

The ginsenoside Rg2(S), which is one of the pharmaceutical components of ginseng, is known to have neuroprotective, anti-inflammation, and anti-diabetic effects. However, the usage of ginsenoside Rg2(S) is restricted owing to the small amounts found in white and red ginseng. To enhance the production of ginsenoside Rg2(S) as a 100 gram unit with high specificity, yield, and purity, an enzymatic bioconversion method was developed to adopt the recombinant glycoside hydrolase (BglPC28), which is a ginsenoside-transforming recombinant β-glucosidase from Pseudonocardia sp. strain Gsoil 1536. The gene, termed bglPC28, encoding β-glucosidase (BglPC28) belonging to the glycoside hydrolase family 3 was cloned. bglPC28 consists of 2,232 bp (743 amino acid residues) with a predicted molecular mass of 78,975 Da. This enzyme was overexpressed in Escherichia coli BL21(DE3) using a GST-fused pGEX 4T-1 vector system. The optimum conditions of the recombinant BglPC28 were pH 7.0 and 37°C. BglPC28 can effectively transform the ginsenoside Re to Rg2(S); the K m values of PNPG and Re were 6.36±1.10 and 1.42±0.13 mM, respectively, and the V max values were 40.0±2.55 and 5.62±0.21 µmol min−1 mg−1 of protein, respectively. A scaled-up biotransformation reaction was performed in a 10 L jar fermenter at pH 7.0 and 30°C for 12 hours with a concentration of 20 mg/ml of ginsenoside Re from American ginseng roots. Finally, 113 g of Rg2(S) was produced from 150 g of Re with 84.0±1.1% chromatographic purity. These results suggest that this enzymatic method could be usefully exploited in the preparation of ginsenoside Rg2(S) in the cosmetics, functional food, and pharmaceutical industries.

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.