Exploration and Characterization of Novel Glycoside Hydrolases from the Whole Genome of Lactobacillus ginsenosidimutans and Enriched Production of Minor Ginsenoside Rg3(S) by a Recombinant Enzymatic Process.

BACKGROUND: Several studies have reported that ginsenoside Rg3(S) is effective in treating metastatic diseases, obesity, and various cancers, however, its presence in white ginseng cannot be estimated, and only a limited amount is present in red ginseng. Therefore, the use of recombinant glycosidases from a Generally Recognized As Safe (GRAS) host strain is a promising approach to enhance production of Rg3(S), which may improve nutritional activity, human health, and quality of life. METHOD: Lactobacillus ginsenosidimutans EMML 3041T, which was isolated from Korean fermented pickle (kimchi), presents ginsenoside-converting abilities. The strain was used to enrich the production of Rg3(S) by fermenting protopanaxadiol (PPD)-mix-type major ginsenosides (Rb1, Rb2, Rc, and Rd) in four different types of food-grade media (1, MRS; 2, Basel Food-Grade medium; 3, Basel Food-Grade medium-I, and 4, Basel Food-Grade medium-II). Due to its tendency to produce Rg3(S), the presence of glycoside hydrolase in Lactobacillus ginsenosidimutans was proposed, the whole genome was sequenced, and the probable glycoside hydrolase gene for ginsenoside conversion was cloned. RESULTS: The L. ginsenosidimutans EMML 3041T strain was whole genome sequenced to identify the target genes. After genome sequencing, 12 sets of glycoside hydrolases were identified, of which seven sets (α,ß-glucosidase and α,ß-galactosidase) were cloned in Escherichia coli BL21 (DE3) using the pGEX4T-1 vector system. Among the sets of clones, only one clone (BglL.gin-952) showed ginsenoside-transforming abilities. The recombinant BglL.gin-952 comprised 952 amino acid residues and belonged to glycoside hydrolase family 3. The enzyme exhibited optimal activity at 55 °C and a pH of 7.5 and showed a promising conversion ability of major ginsenoside Rb1→Rd→Rg3(S). The recombinant enzyme (GST-BglL.gin-952) was used to mass produce Rg3(S) from major ginsenoside Rb1. Scale-up of production using 50 g of Rb1 resulted in 30 g of Rg3(S) with 74.3% chromatography purity. CONCLUSION: Our preliminary data demonstrated that this enzyme would be beneficial in the preparation of pharmacologically active minor ginsenoside Rg3(S) in the functional food and pharmaceutical industries.

Acclimation of hydrogen peroxide enhances salt tolerance by activating defense-related proteins in Panax ginseng C.A. Meyer.

The effect of exogenously applied hydrogen peroxide on salt stress tolerance was investigated in Panax ginseng. Pretreatment of ginseng seedlings with 100 µM H2O2 increased the physiological salt tolerance of the ginseng plant and was used as the optimum concentration to induce salt tolerance capacity. Treatment with exogenous H2O2 for 2 days significantly enhanced salt stress tolerance in ginseng seedlings by increasing the activities of ascorbate peroxidase, catalase and guaiacol peroxidase and by decreasing the concentrations of malondialdehyde (MDA) and endogenous H2O2 as well as the production rate of superoxide radical (O2(-)). There was a positive physiological effect on the growth and development of salt-stressed seedlings by exogenous H2O2 as measured by ginseng dry weight and both chlorophyll and carotenoid contents. Exogenous H2O2 induced changes in MDA, O2(-), antioxidant enzymes and antioxidant compounds, which are responsible for increases in salt stress tolerance. Salt treatment caused drastic declines in ginseng growth and antioxidants levels; whereas, acclimation treatment with H2O2 allowed the ginseng seedlings to recover from salt stress by up-regulation of defense-related proteins such as antioxidant enzymes and antioxidant compounds.

Stenotrophomonas panacihumi sp. nov., isolated from soil of a ginseng field.

The study isolated a Gram-negative, rod-shaped, non-motile bacterium from the soil of a ginseng field in Daejeon, South Korea and characterized it to determine its taxonomic position. Phylogenetic analysis, based on the 16S rRNA gene sequence, revealed that strain MK06(T) belongs to the family Xanthomonadacea, and showed the highest degree of sequence similarity to Stenotrophomonas rhizophila e-p10(T) (98.6%), Xanthomonas campestris LMG 568T (98.0%), Stenotrophomonas maltophilia ATCC 1d3637(T) (97.3%), and Stenotrophomonas humi R-32729(T) (96.9%). Chemotaxonomic data revealed that strain MK06(T) possesses ubiquinone Q-8 as the predominant respiratory lipoquinone, which is common in the genus Stenotrophomonas, and that the predominant fatty acids were 15:0 iso (41.1%), 15:0 anteiso (12.6%), and 17:1 iso omega9c (8.6%). The results of physiological and biochemical tests clearly demonstrated that strain MK06(T) represents a distinct species and supported its affiliation with the genus Stenotrophomonas. Based on these data, MK06(T) (KCTC, 22893(T); JCM, 16536(T); KEMB, 9004-002(T)) should be classified as the type strain for a novel species, for which we propose the name Stenotrophomonas panacihumi sp. nov.

Microbacterium ginsengiterrae sp. nov., a beta-glucosidase-producing bacterium isolated from soil of a ginseng field.

Strain DCY37(T) was isolated from a soil sample of a ginseng field in the Republic of Korea and characterized in order to determine its taxonomic position. Cells were Gram-staining-positive, heterotrophic, strictly aerobic, non-motile short rods. 16S rRNA gene sequence analysis revealed that strain DCY37(T) belongs to the genus Microbacterium. According to 16S rRNA gene sequence analysis, it is closely related to Microbacterium aerolatum DSM 14217(T) (98.8 %), Microbacterium hydrocarbonoxydans DSM 16089(T) (98.5 %), Microbacterium natoriense JCM 12611(T) (98.5 %), Microbacterium foliorum (98.4 %) and Microbacterium phyllosphaerae (98.3 %). However, DNA-DNA hybridization studies showed reassociation values of less than 70 % between representative strains and DCY37(T). The DNA G+C content was 64.5 mol%. Strain DCY37(T) possessed chemotaxonomic markers that were consistent with classification in the genus Microbacterium, i.e. MK-12 and MK-13 as the major menaquinones and anteiso-C(15 : 0), anteiso-C(17 : 0) and iso-C(16 : 0) as the predominant cellular fatty acids. The major cell wall sugars were ribose, xylose and galactose. The diamino acid in cell-wall hydrolysates of strain DCY37(T) was ornithine and major cell-wall amino acids were alanine, glycine, d-glutamic acid and serine. The major polar lipids were glycolipid, phosphatidylglycerol, diphosphatidylglycerol and unknown aminolipids. Based on these data, DCY37(T) (=KCTC 19526(T) =JCM 15516(T)) should be classified as the type strain of a novel species of the genus Microbacterium, for which the name Microbacterium ginsengiterrae sp. nov. is proposed.

Lysobacter soli sp. nov., isolated from soil of a ginseng field.

Strain DCY21(T), a Gram-negative, gliding and rod-shaped aerobic bacterium was isolated from soil of a ginseng field in the Republic of Korea and characterized using a polyphasic approach in order to determine its taxonomic position. Comparative 16S rRNA gene sequence analysis revealed that strain DCY21(T) clustered with the species of the genus Lysobacter. It was closely related to Lysobacter gummosus LMG 8763(T) (97.9 %), Lysobacter capsici YC5194(T) (97.6 %), Lysobacter antibioticus DSM 2044(T) (97.5 %), Lysobacter niastensis DSM 18481(T) (97.2 %) and Lysobacter enzymogenes DSM 2043(T) (96.9 %). The major cellular fatty acids of strain DCY21(T) were iso-C(15 : 0) (34.3 %), iso-C(17 : 1)omega9c (19.5 %) and iso-C(17 : 0) (17.2 %) and the major isoprenoid quinone was Q-8. The major polar lipids of strain DCY21(T) were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and phosphatidyl-N-methylethanolamine. The G+C content of the total DNA was 65.4 mol%. The DNA-DNA relatedness values, and biochemical and physiological characteristics strongly supported the genotypic and phenotypic differentiation of strain DCY21(T) from species of the genus Lysobacter. Strain DCY21(T) therefore represents a novel species, for which the name Lysobacter soli sp. nov. is proposed. The type strain is DCY21(T) (=KCTC 22011(T) =LMG 24126(T)).

Stenotrophomonas ginsengisoli sp. nov., isolated from a ginseng field.

A Gram-negative, non-spore-forming, rod-shaped bacterium, designated strain DCY01(T), was isolated from soil from a ginseng field in South Korea and was characterized in order to determine its taxonomic position. 16S rRNA gene sequence analysis revealed that strain DCY01(T) belonged to the Gammaproteobacteria and was most closely related to Stenotrophomonas koreensis KCTC 12211(T) (98.4 % similarity), Stenotrophomonas humi R-32729(T) (97.2 %), Stenotrophomonas terrae R-32768 (97.1 %), Stenotrophomonas maltophilia DSM 50170(T) (96.9 %) and Stenotrophomonas nitritireducens DSM 12575(T) (96.8 %). Chemotaxonomic analyses revealed that strain DCY01(T) possessed a quinone system with Q-8 as the predominant compound, and iso-C(15 : 0) (28.2 %), C(16 : 0) 10-methyl (13.2 %), iso-C(15 : 1) F (10.8 %) and C(15 : 0) (7.5 %) as major fatty acids, corroborating assignment of strain DCY01(T) to the genus Stenotrophomonas. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The results of DNA-DNA hybridization and physiological and biochemical tests clearly demonstrated that strain DCY01(T) represents a species distinct from recognized Stenotrophomonas species. Based on these data, DCY01(T) (=KCTC 12539(T)=NBRC 101154(T)) should be classified as the type strain of a novel species of the genus Stenotrophomonas, for which the name Stenotrophomonas ginsengisoli sp. nov. is proposed.

Sphingopyxis panaciterrulae sp. nov., isolated from soil of a ginseng field.

A Gram-negative, rod-shaped, motile bacterium was isolated from the soil of a ginseng field in Daejeon, South Korea, and characterized in order to determine its taxonomic position. Phylogenetic analysis based on 16S rRNA gene sequence analysis revealed that strain DCY34(T) belonged to the family Sphingomonadaceae, and the highest degree of sequence similarity was found with Sphingopyxis witflariensis W-50(T) (97.1 %), Sphingopyxis ginsengisoli Gsoil 250(T) (97.0 %), Sphingopyxis chilensis S37(T) (96.9 %), Sphingopyxis macrogoltabida IFO 15033(T) (96.8 %), Sphingopyxis alaskensis RB2256(T) (96.7 %) and Sphingopyxis taejonensis JSS54(T) (96.7 %). Chemotaxonomic data revealed that strain DCY34(T) possessed ubiquinone Q-10 as the predominant respiratory lipoquinone, which is common to members of the genus Sphingopyxis. The predominant fatty acids were C18:1ω7c (27.5 %), summed feature 4 (C16:1ω7c and/or C15 :0 iso 2-OH; 18.6 %), C16:0 (15.6 %) and summed feature 8 (C19:1ω6c and/or unknown 18.864; 15.4 %). The major polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, sphingoglycolipid and an unknown polar lipid. The results of physiological and biochemical tests clearly demonstrated that strain DCY34(T) represented a separate species and supported its affiliation to the genus Sphingopyxis. Based on these data, the new isolate represents a novel species, for which the name Sphingopyxis panaciterrulae sp. nov. is proposed. The type strain is DCY34(T) (=KCTC 22112(T)=JCM 14844(T)).

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

Two novel strains of Gram-negative, non-sporulating, short rod-shaped, motile bacteria, designated DCY25T and DCY28, were isolated from soil of a ginseng field in South Korea and characterized in order to determine their taxonomic position. 16S rRNA gene sequence analysis showed that strains DCY25T and DCY28 belonged to the Betaproteobacteria, the highest sequence similarities being found with Pusillimonas noertemannii BN9T (96.9%), Bordetella trematum DSM 11334(T) (95.9%), Achromobacter denitrificans DSM 30026T (95.9%), Achromobacter insolitus LMG 6003T (95.8%) and Pigmentiphaga kullae K24T (95.5%). Chemotaxonomic data revealed that both strains DCY25T and DCY28 possessed ubiquinone Q-8. Fatty acid analysis of strain DCY25T demonstrated the presence of 19:0 cyclo omega8c (22.8%) and 16:0 (16.6%). The polar lipid profiles of strains DCY25T and DCY28 included phosphatidylethanolamine, phosphatidylglycerol, two unknown aminolipids and diphosphatidylglycerol. The G+C contents of strains DCY25T and DCY28 were 57.3 and 57.2 mol%, respectively. DNA-DNA relatedness values, biochemical and physiological characteristics strongly supported the genotypic and phenotypic differentiation of strains DCY25T and DCY28 from Pusillimonas noertemannii. Therefore, strains DCY25T and DCY28 should be classified in a novel species, for which the name Pusillimonas ginsengisoli sp. nov. is proposed. The type strain is DCY25T (=KCTC 22046T =JCM 14767T); strain DCY28 (=KCTC 22047=JCM 14768) is a reference strain.

Microbacterium soli sp. nov., an alpha-glucosidase-producing bacterium isolated from soil of a ginseng field.

Five Gram-type-positive, aerobic, rod-shaped, non-motile strains of Microbacterium (DCY 17(T), Ms1, Ms2, Ms3 and Ms4) were isolated from soil from a ginseng field in Daejeon, South Korea. On the basis of 16S rRNA gene sequence similarity, these strains were shown to be related to Microbacterium esteraromaticum DSM 8609(T) (96.1 %), M. xylanilyticum DSM 16914(T) (96.0 %), M. aquimaris JS54-2(T) (95.6 %), M. insulae DS-66(T) (95.5 %), M. ketosireducens IFO 14548(T) (95.5 %) and M. arabinogalactanolyticum DSM 8611(T) (95.4 %). Chemotaxonomic data revealed that the type strain, DCY 17(T), possesses menaquinones MK-12, MK-11 and MK-13 and the predominant fatty acids C(15 : 0) anteiso (32.5 %), C(15 : 0) iso (27.5 %), C(16 : 0 ) iso (17.0 %), C(17 : 0) anteiso (13.2 %), C(17 : 0) iso (6.1 %) and C(14 : 0) iso (2.1 %). The DNA G+C content of strain DCY 17(T) is 70.2 mol% and those of strains Ms1 to Ms4 are in the range 68.9-73.5 mol%. The physiological and biochemical tests suggested that these strains represent a novel species. Based on these data, DCY 17(T) (=KCTC 19237(T) =LMG 24010(T)) is classified as the type strain of a novel Microbacterium species, for which the name Microbacterium soli sp. nov. is proposed.

Polygalacturonase inhibiting protein: isolation, developmental regulation and pathogen related expression in Panax ginseng C.A. Meyer.

Polygalacturonase inhibiting proteins (PGIPs) are the major defense proteins which play an important role in resistance to infection of pathogens. A putative novel gene encoding PGIP was isolated from Panax ginseng C.A. Meyer, which shows 70.3 and 68.4% homology with chick pea and Arabidopsis PGIPs. The RACE PCR was preformed to isolate the full-length PGIP cDNA from Panax ginseng. Sequence analysis revealed that the cDNA of PgPGIP is of 1,275 bp in length and that it's containing ORF encodes for a polypeptide of 366 amino acids. Domain analysis revealed that the deduced amino acid sequences of PgPGIP have a typical PGIP topology. The transcription level of PgPGIP was up-regulated in ginseng in response to wounding and infection with phytopathogenic fungi i.e., Rhizoctonia solani, Fusarium oxysporum, Phythium ultimum, Botrytis cinerea, Colletotrichum gloeosporioides and Cylindrocarpon destructans, which causes drastic damage in ginseng plants. The constitutive PgPGIP expression of 4 years old plant, showed elevated transcript level, especially roots, showed maximum then buds, stems and leaves, indicating that the gene is developmentally regulated. The crude PGIP extracts derived from the fungal infected plants directly reduces the aggressive potential of PGs from diverse group of fungi. Like other PGIPs, PgPGIP also possess board spectrum of inhibitory activity. Thus, the presence of PgPGIP gene and their active role in defense mechanism was proved. The structural model of PgPGIP was predicted based on the alignment generated by EBI-Align, the program "MOODELLER" and the predicted structure showed 10 ß-strands and 10 α-helixes region.

Castellaniella ginsengisoli sp. nov., a beta-glucosidase-producing bacterium.

A Gram-negative, motile bacterium, designated DCY36T, was isolated from soil of a ginseng field in South Korea and was characterized using a polyphasic taxonomic approach. Comparative 16S rRNA gene sequence analysis showed that strain DCY36T belongs to genus Castellaniella in the family Alcaligenaceae of the class Betaproteobacteria. The 16S rRNA gene sequence similarities between strain DCY36T and the three recognized representatives of the genus, Castellaniella caeni Ho-11T, Castellaniella defragrans 54PinT and Castellaniella denitrificans NKNTAUT, were 98.4, 97.5 and 98.1%, respectively. Strain DCY36T exhibited relatively low levels of DNA-DNA relatedness with respect to these three species. The G+C content of the genomic DNA was 63.7 mol%. Strain DCY36T contained ubiquinone Q-8. The major fatty acids were C16:0 (27.4%), C18:1omega7c (16.9%) and summed feature 4 (C16:1omega7c and C15:0 iso 2-OH, 32.5%). On the basis of phenotypic and genotypic properties and phylogenetic distinctiveness, strain DCY36T (=KCTC 22398T=JCM 15515T) should be classified in the genus Castellaniella as the type strain of a novel species, for which the name Castellaniella ginsengisoli sp. nov. is proposed.

Nocardioides humi sp. nov., a beta-glucosidase-producing bacterium isolated from soil of a ginseng field.

Strain DCY24(T), a Gram-reaction-positive, aerobic, rod-shaped, motile bacterium, was isolated from soil of a ginseng field in South Korea. According to 16S rRNA gene sequence analysis, it was closely related to Nocardioides aromaticivorans DSM 15131(T) (95.1 % similarity), Nocardioides simplex KCTC 9106(T) (95.0 %), Nocardioides nitrophenolicus DSM 15529(T) (94.8 %) and Nocardioides kongjuensis DSM 19082(T) (94.7 %). Chemotaxonomic data revealed that strain DCY24(T) possessed MK-8(H(4)) as the predominant menaquinone, ll-2,6-diaminopimelic acid as the diagnostic diamino acid, phosphatidylglycerol and diphosphatidylglycerol as predominant polar lipids and iso-C(16 : 0), iso-C(17 : 0) and C(18 : 1)omega9c as predominant fatty acids. The DNA G+C content was 71.0 mol%. Based on evidence from this polyphasic study, strain DCY24(T) (=KCTC 19265(T) =LMG 24128(T)) should be classified as the type strain of a novel Nocardioides species, for which the name Nocardioides humi sp. nov. is proposed.