RESUMO
Use of recombinant glycosidases is a promising approach for the production of minor ginsenosides, e.g., Compound K (CK) and F1, which have potential applications in the food industry. However, application of these recombinant enzymes for food-grade preparation of minor ginsenosides are limited by the lack of suitable expression hosts and low productivity. In this study, Corynebacterium glutamicum ATCC13032, a GRAS strain that has been used extensively for the industrial-grade production of additives for foodstuffs, was employed to express a novel ß-glucosidase (MT619) from Microbacterium testaceum ATCC 15829 with high ginsenoside-transforming activity. A cellulose-binding module was additionally fused to the N-terminus of MT619 for immobilization on cellulose, which is an abundant and safe material. Via one-step immobilization, the fusion protein in cell lysates was efficiently immobilized on regenerated amorphous cellulose at a high density (maximum 984 mg/g cellulose), increasing the enzyme concentration by 286-fold. The concentrated and immobilized enzyme showed strong conversion activities against protopanaxadiol- and protopanaxatriol-type ginsenosides for the production of CK and F1. Using gram-scale ginseng extracts as substrates, the immobilized enzyme produced 7.59 g/L CK and 9.42 g/L F1 in 24 h. To the best of our knowledge, these are the highest reported product concentrations of CK and F1, and this is the first time that a recombinant enzyme has been immobilized on cellulose for the preparation of minor ginsenosides. This safe, convenient, and efficient production method could also be effectively exploited in the preparation of food-processing recombinant enzymes in the pharmaceutical, functional food, and cosmetics industries.
Assuntos
Enzimas Imobilizadas/metabolismo , Ginsenosídeos/metabolismo , beta-Glucosidase/metabolismo , Actinomycetales/enzimologia , Actinomycetales/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biotransformação , Celulose/química , Clonagem Molecular , Corynebacterium glutamicum/enzimologia , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Enzimas Imobilizadas/química , Enzimas Imobilizadas/genética , Expressão Gênica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sapogeninas/metabolismo , beta-Glucosidase/química , beta-Glucosidase/genéticaRESUMO
The anthracycline antibiotic doxorubicin is commonly used antineoplastic drug in breast cancer treatment. Like most chemotherapy, doxorubicin does not selectively target tumorigenic cells with high proliferation rate and often causes serve side effects. In the present study, we demonstrated the cellular senescence and senescence associated secretory phenotype (SASP) of both breast tumor cell MDA-MB-231 and normal epithelial cell MCF-10A induced by clinical dose of doxorubicin (100 nM). Senescence was confirmed by flattened morphology, increased level of beta galactose, accumulating contents of lysosome and mitochondrial, and elevated expression of p16 and p21 proteins. Similarly, SASP was identified by highly secreted proteins IL-6, IL-8, GRO, GM-CSF, MCP-1, and MMP1 by antibody array assay. Reciprocal experiments, determined by cell proliferation and apoptosis assays and cell migration and cell invasion, indicated that SASP of MDA-MB-231 cell induces growth arrest of MCF-10A, whereas SASP of MCF-10A significantly stimulates the proliferation of MDA-MB-231. Interestingly, SASP from both cells powerfully promotes the cell migration and cell invasion of MDA-MB-231 cells. Treatment with the natural product ginsenoside Rh2 does not prevent cellular senescence or exert senolytic. However, SASP from senescent cells treated with Rh2 greatly attenuated the above-mentioned bystander effect. Altogether, Rh2 is a potential candidate to ameliorate this unwanted chemotherapy-induced senescence bystander effect.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Efeito Espectador/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Células Epiteliais/efeitos dos fármacos , Ginsenosídeos/farmacologia , Apoptose/efeitos dos fármacos , Mama/efeitos dos fármacos , Mama/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Humanos , Interleucina-6/metabolismo , Invasividade Neoplásica/patologiaRESUMO
Ginsenosides are known to have various highly pharmacological activities, such as anti-cancer and anti-inflammatory effects. However, the search for the most effective ginsenosides against the pathogenesis of atopic dermatitis (AD) and the study of the effects of ginsenosides on specific cytokines involved in AD remain unclear. In this study, ginsenoside Rh2 was shown to exert the most effective anti-inflammatory action on thymic stromal lymphopoietin (TSLP) and interleukin 8 in tumor necrosis factor-alpha and polyinosinic: polycytidylic acid induced normal human keratinocytes by inhibiting proinflammatory cytokines at both protein and transcriptional levels. Concomitantly, Rh2 also efficiently alleviated 2,4-dinitrochlorobenzene-induced AD-like skin symptoms when applied topically, including suppression of immune cell infiltration, cytokine expression, and serum immunoglobulin E levels in NC/Nga mice. In line with the in vitro results, Rh2 inhibited TSLP levels in AD mice via regulation of an underlying mechanism involving the nuclear factor κB pathways. In addition, in regard to immune cells, we showed that Rh2 suppressed not only the expression of TSLP but the differentiation of naïve CD4+ T-cells into T helper type 2 cells and their effector function in vitro. Collectively, our results indicated that Rh2 might be considered as a good therapeutic candidate for the alternative treatment of AD.
Assuntos
Citocinas/metabolismo , Dermatite Atópica/tratamento farmacológico , Ginsenosídeos/uso terapêutico , NF-kappa B/metabolismo , Células Th2/metabolismo , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Citocinas/análise , Dermatite Atópica/induzido quimicamente , Dermatite Atópica/patologia , Dinitroclorobenzeno/toxicidade , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Ginsenosídeos/farmacologia , Humanos , Imunoglobulina E/sangue , Masculino , Camundongos , Pele/metabolismo , Pele/patologia , Células Th2/citologia , Linfopoietina do Estroma do TimoRESUMO
Cutaneous wound healing is a well-orchestrated event in which many types of cells and growth factors are involved in restoring the barrier function of skin. In order to identify whether ginsenosides, the main active components of Panax ginseng, promote wound healing, the proliferation and migration activities of 15 different ginsenosides were tested by MTT assay and scratched wound closure assay. Among ginsenosides, gypenoside LXXV (G75) showed the most potent wound healing effects. Thus, this study aimed to investigate the effects of G75 on wound healing in vivo and characterize associated molecular changes. G75 significantly increased proliferation and migration of keratinocytes and fibroblasts, and promoted wound closure in an excision wound mouse model compared with madecassoside (MA), which has been used to treat wounds. Additionally, RNA sequencing data revealed G75-mediated significant upregulation of connective tissue growth factor (CTGF), which is known to be produced via the glucocorticoid receptor (GR) pathway. Consistently, the increase in production of CTGF was confirmed by western blot and ELISA. In addition, GR-competitive binding assay and GR translocation assay results demonstrated that G75 can be bound to GR and translocated into the nucleus. These results demonstrated that G75 is a newly identified effective component in wound healing.
Assuntos
Anti-Inflamatórios/farmacologia , Fator de Crescimento do Tecido Conjuntivo/genética , Fármacos Dermatológicos/farmacologia , Receptores de Glucocorticoides/genética , Ferida Cirúrgica/tratamento farmacológico , Cicatrização/efeitos dos fármacos , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/isolamento & purificação , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Fármacos Dermatológicos/química , Fármacos Dermatológicos/isolamento & purificação , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Ginsenosídeos/química , Ginsenosídeos/isolamento & purificação , Ginsenosídeos/farmacologia , Gynostemma/química , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Panax/química , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Transporte Proteico , Receptores de Glucocorticoides/metabolismo , Transdução de Sinais , Pele/efeitos dos fármacos , Pele/lesões , Pele/metabolismo , Ferida Cirúrgica/genética , Ferida Cirúrgica/metabolismo , Ferida Cirúrgica/patologia , Cicatrização/fisiologiaRESUMO
Minor ginsenosides, such as compound K, Rg3(S), which can be produced by deglycosylation of ginsenosides Rb1, showed strong anti-cancer effects. However, the anticancer effects of gypenoside LXXV, which is one of the deglycosylated shapes of ginsenoside Rb1, is still unknown due to the rarity of its content in plants. Here, we cloned and characterized a novel ginsenoside-transforming ß-glucosidase (BglG167b) derived from Microbacterium sp. Gsoil 167 which can efficiently hydrolyze gypenoside XVII into gypenoside LXXV, and applied it to the production of gypenoside LXXV at the gram-scale with high specificity. In addition, the anti-cancer activity of gypenoside LXXV was investigated against three cancer cell lines (HeLa, B16, and MDA-MB231) in vitro. Gypenoside LXXV significantly reduced cell viability, displaying an enhanced anti-cancer effect compared to gypenoside XVII and Rb1. Taken together, this enzymatic method would be useful in the preparation of gypenoside LXXV for the functional food and pharmaceutical industries.
Assuntos
Actinobacteria/química , Antineoplásicos Fitogênicos/metabolismo , Proteínas de Bactérias/metabolismo , Ginsenosídeos/metabolismo , beta-Glucosidase/metabolismo , Actinobacteria/enzimologia , Animais , Antineoplásicos Fitogênicos/isolamento & purificação , Antineoplásicos Fitogênicos/farmacologia , Proteínas de Bactérias/genética , Biotransformação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Ginsenosídeos/isolamento & purificação , Ginsenosídeos/farmacologia , Gynostemma , Células HeLa , Humanos , Melanoma Experimental/tratamento farmacológico , Camundongos , Panax/química , Extratos Vegetais/biossíntese , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , beta-Glucosidase/genéticaRESUMO
Topical use of ginsenosides, the major bioactive substances in Panax ginseng, has been used for the treatment of irritated skin complaints. However, the protective mechanisms of ginsenosides remain unclear. In the present study, we investigated the anti-inflammatory role of ginsenoside F2 (GF2) on the skin inflammation. To induce irritant dermatitis, 12-O-tetradecanoylphorbol-13-acetate (TPA) was applied on the surface of the mouse ears with or without treatments of GF2 and dexamethasone for 24 h. Protective effects of GF2 on edema and inflammation were assessed by measuring ear thickness, weights of skin punch, and inflammatory responses. In gross findings, treatments with GF2 significantly decreased skin thickness and weight compared to those of TPA-treated groups, which was comparable with the protective effects of dexamethasone. In addition, expression of inflammatory mediators was remarkably reduced in GF2-treated ears compared to that of vehicle-treated ears of mice. Interestingly, immunohistochemistry and flow cytometry analyses revealed that TPA treatment significantly increased infiltration of interleukin-17 (IL-17) producing dermal γδ T cells, while frequencies of γδ T cells was decreased by GF2 treatment, subsequently ameliorating inflammation in skin. Concomitantly, TPA-mediated skin inflammation was significantly ameliorated in IL-17A knock out mice. Furthermore, GF2 treatment inhibited infiltration and generation of reactive oxygen species (ROS) of neutrophils in damaged ears compared with vehicle-treated mice. These results clearly suggest that GF2 treatment ameliorates TPA-induced dermal inflammation by inhibiting production of IL-17 and ROS in γδ T cells and neutrophils, respectively. Therefore, as a natural compound, application of GF2 may be a novel therapeutic approach for treating skin inflammation.
Assuntos
Dermatite/prevenção & controle , Orelha Externa/efeitos dos fármacos , Edema/prevenção & controle , Ginsenosídeos/farmacologia , Administração Cutânea , Animais , Dermatite/etiologia , Dermatite/metabolismo , Orelha Externa/metabolismo , Orelha Externa/patologia , Edema/induzido quimicamente , Edema/metabolismo , Citometria de Fluxo , Expressão Gênica/efeitos dos fármacos , Ginsenosídeos/administração & dosagem , Interleucina-17/genética , Interleucina-17/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Substâncias Protetoras/administração & dosagem , Substâncias Protetoras/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Acetato de Tetradecanoilforbol/toxicidadeRESUMO
Human leukocyte antigen (HLA)-E is a non-classical major histocompatibility complex class I (Ib) molecule, which plays an important role in immunosuppression. In this study, we investigated the immunomodulating effect of HLA-E in a xenogeneic system, using human placental artery-derived endothelial (hPAE) cells expressing HLA-E in a mouse model. In vitro cell lysis analysis by primed lymphocytes in combination with siRNA transfection showed that HLA-E is necessary for inhibition of the immune response. Similarly, in vivo cell implantation analysis with siRNA-mediated down-regulation of HLA-E demonstrates that HLA-E is involved in immunosuppression. As hPAE cells efficiently transdifferentiate into myoblasts/myocytes in vitro, we transplanted the cells into mdx mice, a model of Duchenne muscular dystrophy. hPAE cells conferred dystrophin to myocytes of the 'immunocompetent' mdx mice with extremely high efficiency. These findings suggest that HLA-E-expressing cells with a myogenic potential represent a promising source for cell-based therapy of patients with muscular dystrophy.
Assuntos
Distrofina/genética , Distrofina/metabolismo , Células Endoteliais , Antígenos de Histocompatibilidade Classe I/genética , Distrofia Muscular de Duchenne/genética , Animais , Artérias/citologia , Técnicas de Cultura de Células , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunocompetência/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos mdx , Desenvolvimento Muscular/genética , Distrofia Muscular de Duchenne/imunologia , Distrofia Muscular de Duchenne/terapia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Antígenos HLA-ERESUMO
Here, we isolated and characterized a new ginsenoside-transforming ß-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-ß-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 Vmax 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 ß-glucosidase of glycoside hydrolase family 3.
Assuntos
Bacteroidetes/enzimologia , Ginsenosídeos/metabolismo , beta-Glucosidase/metabolismo , Bacteroidetes/genética , Biotransformação , Análise por Conglomerados , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Cinética , Dados de Sequência Molecular , Peso Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNA , Homologia de Sequência , beta-Glucosidase/química , beta-Glucosidase/genéticaRESUMO
This study focused on the cloning, expression, and characterization of ginsenoside-transforming recombinant ß-glucosidase from Actinosynnema mirum KACC 20028(T) in order to biotransform ginsenosides efficiently. The gene, termed as bglAm, encoding a ß-glucosidase (BglAm) belonging to the glycoside hydrolase family 3 was cloned. bglAm consisted of 1,830 bp (609 amino acid residues) with a predicted molecular mass of 65,277 Da. This enzyme was overexpressed in Escherichia coli BL21(DE3) using a GST-fused pGEX 4T-1 vector system. The recombinant BglAm was purified with a GST·bind agarose resin and characterized. The optimum conditions of the recombinant BglAm were pH 7.0 and 37 °C. BglAm could hydrolyze the outer and inner glucose moieties at the C3 and C20 of the protopanaxadiol-type ginsenosides (i.e., Rb(1) and Rd, gypenoside XVII) to produce protopanaxadiol via gypenoside LXXV, F(2), and Rh(2)(S) with various pathways. BglAm can effectively transform the ginsenoside Rb(1) to gypenoside XVII and Rd to F(2); the K (m) values of Rb(1) and Rd were 0.69 ± 0.06 and 0.45 ± 0.02 mM, respectively, and the V (max) values were 16.13 ± 0.29 and 51.56 ± 1.35 µmol min(-1) mg(-1) of protein, respectively. Furthermore, BglAm could convert the protopanaxatriol-type ginsenoside Re and Rg(1) into Rg(2)(S) and Rh(1)(S) hydrolyzing the attached glucose moiety at the C6 and C20 positions, respectively. These various ginsenoside-hydrolyzing pathways of BglAm may assist in producing the minor ginsenosides from abundant major ginsenosides.
Assuntos
Actinomycetales/enzimologia , Ginsenosídeos/metabolismo , Proteínas Recombinantes/metabolismo , beta-Glucosidase/metabolismo , Eletroforese em Gel de Poliacrilamida , Glicosídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/genética , Temperatura , beta-Glucosidase/genéticaRESUMO
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 Km and Vmax 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.
Assuntos
Ginsenosídeos/metabolismo , Glicosídeo Hidrolases/metabolismo , Leuconostoc/enzimologia , Biotransformação , Cromatografia Líquida , Clonagem Molecular , DNA Bacteriano/química , DNA Bacteriano/genética , Estabilidade Enzimática , Escherichia coli/genética , Microbiologia de Alimentos , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/isolamento & purificação , Concentração de Íons de Hidrogênio , Cinética , Coreia (Geográfico) , Leuconostoc/genética , Leuconostoc/isolamento & purificação , Dados de Sequência Molecular , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , TemperaturaRESUMO
The gene encoding an α-L-arabinofuranosidase that could biotransform ginsenoside Rc {3-O-[ß-D-glucopyranosyl-(1-2)-ß-D-glucopyranosyl]-20-O-[α-L-arabinofuranosyl-(1-6)-ß-D-glucopyranosyl]-20(S)-protopanaxadiol} to ginsenoside Rd {3-O-[ß-D-glucopyranosyl-(1-2)-ß-D-glucopyranosyl]-20-O-ß-D-glucopyranosyl-20(S)-protopanaxadiol} was cloned from a soil bacterium, Rhodanobacter ginsenosidimutans strain Gsoil 3054(T), and the recombinant enzyme was characterized. The enzyme (AbfA) hydrolyzed the arabinofuranosyl moiety from ginsenoside Rc and was classified as a family 51 glycoside hydrolase based on amino acid sequence analysis. Recombinant AbfA expressed in Escherichia coli hydrolyzed non-reducing arabinofuranoside moieties with apparent K (m) values of 0.53 ± 0.07 and 0.30 ± 0.07 mM and V (max) values of 27.1 ± 1.7 and 49.6 ± 4.1 µmol min(-1) mg(-1) of protein for p-nitrophenyl-α-L-arabinofuranoside and ginsenoside Rc, respectively. The enzyme exhibited preferential substrate specificity of the exo-type mode of action towards polyarabinosides or oligoarabinosides. AbfA demonstrated substrate-specific activity for the bioconversion of ginsenosides, as it hydrolyzed only arabinofuranoside moieties from ginsenoside Rc and its derivatives, and not other sugar groups. These results are the first report of a glycoside hydrolase family 51 α-L-arabinofuranosidase that can transform ginsenoside Rc to Rd.
Assuntos
Ginsenosídeos/metabolismo , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Xanthomonadaceae/enzimologia , Xanthomonadaceae/genética , Clonagem Molecular , Escherichia coli/enzimologia , Escherichia coli/genética , Glicosídeo Hidrolases/química , Hidrólise , Cinética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Microbiologia do Solo , Especificidade por SubstratoRESUMO
Cascade reaction systems, such as protein fusion and synthetic protein scaffold systems, can both spatially control the metabolic flux and boost the productivity of multistep enzymatic reactions. Despite many efforts to generate fusion proteins, this task remains challenging due to the limited expression of complex enzymes. Therefore, we developed a novel fusion system that bypasses the limited expression of complex enzymes via a post-translational linkage. Here, we report a split intein-mediated cascade system wherein orthogonal split inteins serve as adapters for protein ligation. A genetically programmable, self-assembled, and traceless split intein was utilized to generate a biocatalytic cascade to produce the ginsenoside compound K (CK) with various pharmacological activities, including anticarcinogenic, anti-inflammatory, and antidiabetic effects. We used two types of split inteins, consensus atypical (Cat) and Rma DnaB, to form a covalent scaffold with the three enzymes involved in the CK conversion pathway. The multienzymatic complex with a size greater than 240 kDa was successfully assembled in a soluble form and exhibited specific activity toward ginsenoside conversion. Furthermore, our split intein cascade system significantly increased the CK conversion rate and reduced the production time by more than 2-fold. Our multienzymatic cascade system that uses split inteins can be utilized as a platform for regulating multimeric bioconversion pathways and boosting the production of various high-value substances.
Assuntos
Ginsenosídeos , Inteínas , Inteínas/genética , Processamento de Proteína , Proteínas/metabolismoRESUMO
A mixed natural preservative composed of ε-polylysine (ε-PL), chestnut 70% ethanol extract (NE), and cinnamon hydrothermal extract (CW), was investigated for the reduction of Staphylococcus aureus. The minimum inhibitory concentration (MIC) and minimum bacterial concentration (MBC) of seven natural extracts were investigated against a cocktail of three strains of S. aureus (ATCC 25923, ATCC 33591, and ATCC 33594). Three important factors (ε-PL, NE, and CW) were selected by using the Plackett-Burman (PB) design for the response surface model (P < 0.001). Following a central composite design, S. aureus were treated with mixtures of natural preservatives that included ε-PL, NE, and CW. The MIC and MBC of ε-PL and the natural extracts and ranged from 1 to 16 mg/mL (R2 = 0.9857). The mixed natural preservative presented a synergistic antibacterial effect, at the optimum point. These results suggest that mixed natural preservatives of ε-PL, NE, and CW can lower the economic cost of food processing.
RESUMO
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) (94.6â%). The phylogenetic distances from other described species with validly published names within the order Bacillales were greater than 9.0â%. 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(15â:â0) and anteiso-C(15â:â0). 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) (â=âKCTC 13942(T) â=âDSM 18389(T)).
Assuntos
Bacilos Gram-Positivos Formadores de Endosporo/classificação , Panax/microbiologia , Filogenia , Microbiologia do Solo , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , DNA Ribossômico/genética , Ácidos Graxos/química , Bacilos Gram-Positivos Formadores de Endosporo/genética , Bacilos Gram-Positivos Formadores de Endosporo/isolamento & purificação , Dados de Sequência Molecular , RNA Ribossômico 16S/genética , República da Coreia , Análise de Sequência de DNA , Vitamina K 2/análogos & derivados , Vitamina K 2/químicaRESUMO
Duchenne muscular dystrophy is an X-linked recessive genetic disease characterized by severe skeletal muscular degeneration. The placenta is considered to be a promising candidate cell source for cellular therapeutics because it contains a large number of cells and heterogenous cell populations with myogenic potentials. We analyzed the myogenic potential of cells obtained from six parts of the placenta, that is, umbilical cord, amniotic epithelium, amniotic mesoderm, chorionic plate, villous chorion, and decidua basalis. In vitro cells derived from amniotic mesoderm, chorionic plate, and villous chorion efficiently transdifferentiate into myotubes. In addition, in vivo implantation of placenta-derived cells into dystrophic muscles of immunodeficient mdx mice restored sarcolemmal expression of human dystrophin. Differential contribution to myogenesis in this study may be attributed to placental portion-dependent default cell state. Molecular taxonomic characterization of placenta-derived maternal and fetal cells in vitro will help determine the feasibility of cell-based therapy.
Assuntos
Distrofina/metabolismo , Membranas Extraembrionárias/citologia , Membranas Extraembrionárias/transplante , Mesoderma/citologia , Mesoderma/transplante , Distrofia Muscular de Duchenne/terapia , Animais , Biomarcadores/metabolismo , Membrana Celular/metabolismo , Forma Celular , Transplante de Células , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos mdx , Camundongos SCID , Desenvolvimento Muscular , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Placenta/citologia , GravidezRESUMO
A new beta-glucosidase from a novel strain of Terrabacter ginsenosidimutans (Gsoil 3082(T)) 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-[beta-D-glucopyranosyl-(1-2)-beta-D-glucopyranosyl]-20-O-[beta-D-glucopyranosyl-(1-6)-beta-D-glucopyranosyl]-20(S)-protopanaxadiol} to the more pharmacologically active rare ginsenosides gypenoside XVII {3-O-beta-D-glucopyranosyl-20-O-[beta-D-glucopyranosyl-(1-6)-beta-D-glucopyranosyl]-20(S)-protopanaxadiol}, gypenoside LXXV {20-O-[beta-v-glucopyranosyl-(1-6)-beta-D-glucopyranosyl]-20(S)-protopanaxadiol}, and C-K [20-O-(beta-D-glucopyranosyl)-20(S)-protopanaxadiol]. A BLAST search of the bgpA sequence revealed significant homology to family 3 glycoside hydrolases. Expressed in E. coli, beta-glucosidase had apparent K(m) values of 4.2 +/- 0.8 and 0.14 +/- 0.05 mM and V(max) values of 100.6 +/- 17.1 and 329 +/- 31 micromol x min(-1) x mg of protein(-1) against p-nitrophenyl-beta-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 degrees 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 beta-glucosidase of the glycoside hydrolase family 3.
Assuntos
Actinomycetales/enzimologia , Actinomycetales/isolamento & purificação , Ginsenosídeos/metabolismo , beta-Glucosidase/genética , beta-Glucosidase/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biotransformação , Clonagem Molecular , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli/genética , Gynostemma/metabolismo , Cinética , Proteínas de Membrana , Dados de Sequência Molecular , Estrutura Molecular , Panax , Filogenia , Extratos Vegetais/metabolismo , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Microbiologia do Solo , Especificidade por Substrato , Transferases (Outros Grupos de Fosfato Substituídos)RESUMO
Duchenne muscular dystrophy (DMD), the most common lethal genetic disorder in children, is an X-linked recessive muscle disease characterized by the absence of dystrophin at the sarcolemma of muscle fibers. We examined a putative endometrial progenitor obtained from endometrial tissue samples to determine whether these cells repair muscular degeneration in a murine mdx model of DMD. Implanted cells conferred human dystrophin in degenerated muscle of immunodeficient mdx mice. We then examined menstrual blood-derived cells to determine whether primarily cultured nontransformed cells also repair dystrophied muscle. In vivo transfer of menstrual blood-derived cells into dystrophic muscles of immunodeficient mdx mice restored sarcolemmal expression of dystrophin. Labeling of implanted cells with enhanced green fluorescent protein and differential staining of human and murine nuclei suggest that human dystrophin expression is due to cell fusion between host myocytes and implanted cells. In vitro analysis revealed that endometrial progenitor cells and menstrual blood-derived cells can efficiently transdifferentiate into myoblasts/myocytes, fuse to C2C12 murine myoblasts by in vitro coculturing, and start to express dystrophin after fusion. These results demonstrate that the endometrial progenitor cells and menstrual blood-derived cells can transfer dystrophin into dystrophied myocytes through cell fusion and transdifferentiation in vitro and in vivo.
Assuntos
Distrofina/genética , Menstruação/sangue , Distrofia Muscular Animal/terapia , Animais , Diferenciação Celular , Fusão Celular , Linhagem Celular , Endométrio/citologia , Feminino , Técnicas de Transferência de Genes , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos NOD , Camundongos Endogâmicos mdx , Camundongos Knockout , Camundongos SCID , Desenvolvimento Muscular , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapiaRESUMO
BACKGROUND: The separation of isomeric compounds from a mixture is a recurring problem in chemistry and phytochemistry research. The purification of pharmacologically active ginsenoside Rb3 from ginseng extracts is limited by the co-existence of its isomer Rb2. The aim of the present study was to develop an enzymatic elimination-combined purification method to obtain pure Rb3 from a mixture of isomers. METHODS: To isolate Rb3 from the isomeric mixture, a simple enzymatic selective elimination method was used. A ginsenoside-transforming glycoside hydrolase (Bgp2) was employed to selectively hydrolyze Rb2 into ginsenoside Rd. Ginsenoside Rb3 was then efficiently separated from the mixture using a traditional chromatographic method. RESULTS: Chromatographic purification of Rb3 was achieved using this novel enzymatic elimination-combined method, with 58.6-times higher yield and 13.1% less time than those of the traditional chromatographic method, with a lower minimum column length for purification. The novelty of this study was the use of a recombinant glycosidase for the selective elimination of the isomer. The isolated ginsenoside Rb3 can be used in further pharmaceutical studies. CONCLUSIONS: Herein, we demonstrated a novel enzymatic elimination-combined purification method for the chromatographic purification of ginsenoside Rb3. This method can also be applied to purify other isomeric glycoconjugates in mixtures.
RESUMO
BACKGROUND: Ginsenosides, triterpene saponins of Panax species, are considered the main active ingredients responsible for various pharmacological activities. Herein, a new protopanaxatriol-type ginsenoside called "ginsenoside MT1" is described; it was accidentally found among the enzymatic conversion products of ginsenoside Re. METHOD: We analyzed the conversion mechanism and found that recombinant ß-glucosidase (MT619) transglycosylated the outer rhamnopyranoside of Re at the C-6 position to glucopyranoside at C-20. The production of MT1 by trans-rhamnosylation was optimized and pure MT1 was obtained through various chromatographic processes. RESULTS: The structure of MT1 was elucidated based on spectral data: (20S)-3ß,6α,12ß,20-tetrahydroxydammarene-20-O-[α-L-rhamnopyranosyl(1â2)-ß-D-glucopyranoside]. This dammarane-type triterpene saponin was confirmed as a novel compound. CONCLUSION: Based on the functions of ginsenosides with similar structures, we believe that this ginsenoside MT1 may have great potential in the development of nutraceutical, pharmaceutical or cosmeceutical products.
Assuntos
Enzimas/metabolismo , Ginsenosídeos/biossíntese , Ginsenosídeos/química , Ramnose/metabolismo , BiotransformaçãoRESUMO
Ginseng has been shown to produce a cognitive improvement effect. The key molecular components in ginseng that produce pharmacological effects are ginsenosides. Previous studies reported a memory improvement effect of a few major ginsenosides. However, the identity of specific minor ginsenosides mediating such function remains unknown. Here, we report that a minor ginsenoside F1 improves memory function in APPswe/PSEN1dE9 (APP/PS1) double-transgenic Alzheimer's disease (AD) model mice. After 8-wk oral administration of F1 jelly, we observed that spatial working memory, but not context-dependent fear memory, was restored in AD mice. To search for a possible underlying molecular and cellular mechanism, we investigated the effect of F1 on Aß plaque. We observed F1 administration reduced the Aß plaque area and density in the cortex, but not in the hippocampus of AD mice. Next, we tested for the effect of F1 on the expression level of key molecules involved in learning and memory. Results from Western blot assay revealed that an abnormally reduced level of a phosphorylated form of CREB in the hippocampus of AD mice was restored to a normal level by F1 administration. Moreover, in the same animals, BDNF level was augmented in the cortex. Our results, therefore, suggest that minor ginsenoside F1 constitutes a promising target to develop therapeutic agents for AD.