Fengxie Jin

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.

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).

Thermostable α-amylase and α-galactosidase production from the thermophilic and aerobic Bacillus sp.JF strain

Extracellular thermostable α-amylase (EC 3.2.1.1) and intercellular α-galactosidase (EC 3.2.1.22) from one fermentation of a thermophilic and aerobic Bacillus sp.JF2 strain were studied and modelled. The cell growth of the thermophilic Bacillus sp.JF2 strain could be expressed by the Monod model, the μmax was 0.084/h, the activation energy for cell growth and death was Eg=7.42 and Ed=−18.8 cal/g mol K. The thermostable α-amylase production was expressed by a model combining growth-associated and non-growth-associated models. Intercellular α-galactosidase production was expressed by the growth-associated model; and the relationship equation between the substrate, cell growth, and two enzyme production was also defined, and the all constants were defined.

Hymenobacter daecheongensis sp. nov., isolated from stream sediment.

A Gram-negative, strictly aerobic, non-spore-forming, rod-shaped, red-pink bacterium, designated strain Dae14(T), was isolated from stream sediment collected near Daecheong Dam, South Korea, and its taxonomic position was investigated by using a polyphasic approach. Phylogenetic analysis of 16S rRNA gene sequences showed that strain Dae14(T) belonged to the genus Hymenobacter. Sequence similarities between strain Dae14(T) and the type strains of Hymenobacter species with validly published names ranged from 91.3 to 94.3 %. The predominant cellular fatty acids of strain Dae14(T) were iso-C(15 : 0), C(16 : 1)omega5c, summed feature 5 (iso-C(17 : 1) I and/or anteiso-C(17 : 1) B) and iso-C(16 : 0). The DNA G+C content was 62.2 mol%. Results of phylogenetic, chemotaxonomic and phenotypic characterization indicated that strain Dae14(T) can be distinguished from all known Hymenobacter species and represents a novel species, for which the name Hymenobacter daecheongensis sp. nov. is proposed, with Dae14(T) (=KCTC 22258(T)=LMG 24498(T)) as the type strain.

Preparation of minor ginsenosides C-Mc, C-Y, F2, and C-K from American ginseng PPD-ginsenoside using special ginsenosidase type-I from Aspergillus niger g.848

Background Minor ginsenosides, those having low content in ginseng, have higher pharmacological activities. To obtain minor ginsenosides, the biotransformation of American ginseng protopanaxadiol (PPD)-ginsenoside was studied using special ginsenosidase type-I from Aspergillus niger g.848. Methods DEAE (diethylaminoethyl)-cellulose and polyacrylamide gel electrophoresis were used in enzyme purification, thin-layer chromatography and high performance liquid chromatography (HPLC) were used in enzyme hydrolysis and kinetics; crude enzyme was used in minor ginsenoside preparation from PPD-ginsenoside; the products were separated with silica-gel-column, and recognized by HPLC and NMR (Nuclear Magnetic Resonance). Results The enzyme molecular weight was 75 kDa; the enzyme firstly hydrolyzed the C-20 position 20-O-β-D-Glc of ginsenoside Rb1, then the C-3 position 3-O-β-D-Glc with the pathway Rb1→Rd→F2→C-K. However, the enzyme firstly hydrolyzed C-3 position 3-O-β-D-Glc of ginsenoside Rb2 and Rc, finally hydrolyzed 20-O-L-Ara with the pathway Rb2→C-O→C-Y→C-K, and Rc→C-Mc1→C-Mc→C-K. According to enzyme kinetics, Km and Vmax of Michaelis–Menten equation, the enzyme reaction velocities on ginsenosides were Rb1 > Rb2 > Rc > Rd. However, the pure enzyme yield was only 3.1%, so crude enzyme was used for minor ginsenoside preparation. When the crude enzyme was reacted in 3% American ginseng PPD-ginsenoside (containing Rb1, Rb2, Rc, and Rd) at 45°C and pH 5.0 for 18 h, the main products were minor ginsenosides C-Mc, C-Y, F2, and C-K; average molar yields were 43.7% for C-Mc from Rc, 42.4% for C-Y from Rb2, and 69.5% for F2 and C-K from Rb1 and Rd. Conclusion Four monomer minor ginsenosides were successfully produced (at low-cost) from the PPD-ginsenosides using crude enzyme.

Rhodanobacter panaciterrae sp. nov., a bacterium with ginsenoside-converting activity isolated from soil of a ginseng field

A novel gammaproteobacterium, designated LnR5-47(T), was isolated from soil of a ginseng field in Liaoning province, China. The isolate was a Gram-negative, aerobic, non-motile, non-spore-forming rod. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain LnR5-47(T) belonged to the genus Rhodanobacter. The isolate was most closely related to Rhodanobacter ginsengisoli GR17-7(T), Rhodanobacter terrae GP18-1(T), Dyella ginsengisoli Gsoil 3046(T), Rhodanobacter soli DCY45(T), Dyella soli JS12-10(T) and Dyella japonica IAM 15069(T) (98.0, 97.9, 97.7, 97.3, 97.2 and 97.1% 16S rRNA gene sequence similarity, respectively). Chemotaxonomic data (Q-8 as the predominant ubiquinone, and iso-C(16:0), iso-C(17:1)ω9c and iso-C(15:0) as the major fatty acids) also supported the affiliation of strain LnR5-47(T) with the genus Rhodanobacter. However, DNA-DNA relatedness between strain LnR5-47(T) and its closest phylogenetic neighbours was <25.8%. Moreover, physiological and biochemical tests phenotypically differentiated the isolate from other members of the genus Rhodanobacter. Therefore, strain LnR5-47(T) represents a novel species, for which the name Rhodanobacter panaciterrae sp. nov. is proposed; the type strain is LnR5-47(T) (=KACC 12826(T)=KCTC 22232(T)=LMG 24460(T)).

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.

Lysobacter terrae sp. nov. isolated from Aglaia odorata rhizosphere soil.

A Gram-stain negative, facultatively anaerobic, non-motile, rod-shaped bacterium, designated strain THG-A13(T), was isolated from Aglaia odorata rhizosphere soil in Gyeonggi-do, Republic of Korea. Based on 16S rRNA gene sequence comparisons, strain THG-A13(T) had close similarity with Lysobacter niabensis GH34-4(T) (98.5 %), Lysobacter oryzae YC6269(T) (97.9 %) and Lysobacter yangpyeongensis GH19-3(T) (97.3 %). Chemotaxonomic data revealed that strain THG-A13(T) possesses ubiquinone-8 (Q8) as the predominant isoprenoid quinone and iso-C15 : 0, iso-C16 : 0 and iso-C17 : 1ω9c as the major fatty acids. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol) and diphosphatidylglycerol. The G+C content was 66.3 mol%. The DNA-DNA relatedness values between strain THG-A13(T) and its closest phylogenetic neighbours were below 18.0 %. These data corroborated the affiliation of strain THG-A13(T) to the genus Lysobacter. These data suggest that the isolate represents a novel species for which the name Lysobacter terrae sp. nov. is proposed, with THG-A13(T) as the type strain ( = KACC 17646(T) = JCM 19613(T)).

Phycicoccus ginsenosidimutans sp. nov., isolated from soil of a ginseng field

A Gram-positive, non-motile, non-spore-forming, aerobic, coccoid-shaped bacterium, designated BXN5-13(T), was isolated from the soil of a ginseng field from Baekdu Mountain in Jilin district, China. Strain BXN5-13(T) grew optimally at 30 °C and pH 6.5-7.5 with 0-2  % (w/v) NaCl. Strain BXN5-13(T) had β-glucosidase activity that was connected with ginsenoside-converting ability, so that it was able to convert ginsenoside Rb(1) to ginsenoside F2. On the basis of 16S rRNA gene sequence analysis, the closest phylogenetic relatives of strain BXN5-13(T) were Phycicoccus aerophilus 5516T-20(T) (98.4  % 16S rRNA gene sequence similarity), P. bigeumensis MSL-03(T) (98.3  %), P. dokdonensis DS-8(T) (97.9  %) and P. jejuensis KSW2-15(T) (96.9  %). Lower sequence similarity (<97.0  %) was found with the type strains of other recognized species of the family Intrasporangiaceae. The predominant quinone was MK-8(H₄). The major fatty acids (>10  %) were iso-C₁₅:₀, C₁₇:₀, anteiso-C₁₅:₀ and iso-C₁₆:₀. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and phosphatidylinositol. The cell-wall peptidoglycan contained meso-diaminopimelic acid. The chemotaxonomic data and the high genomic DNA G+C content of strain BXN5-13(T) (70.8l %) supported its affiliation with the genus Phycicoccus. DNA-DNA relatedness between strain BXN5-13(T) and its closest phylogenetic neighbours was below 16  %. Strain BXN5-13(T) represents a novel species within the genus Phycicoccus, for which the name Phycicoccus ginsenosidimutans sp. nov. is proposed. The type strain is BXN5-13(T) (=KCTC 19419(T)=DSM 21006(T)=LMG 24462(T)).