Production of ginsenosides Rg1 and Rh1 by hydrolyzing the outer glycoside at the C-6 position in protopanaxatriol-type ginsenosides using β-glucosidase from Pyrococcus furiosus

The specific activity of a recombinant β-glucosidase from Pyrococcus furiosus for protopanaxatriol (PPT)-type ginsenosides followed the order Rf > R₁ > Re > R₂ > Rg₂, which were converted to Rh₁, Rg₁, Rg₁, Rh₁, and Rh₁, respectively. No activity was observed with Rg₁ and Rh₁. Thus, P. furiosus β-glucosidase hydrolyzed the outer glycoside at the C-6 position in PPT-type ginsenosides whereas the enzyme did not hydrolyze the inner glucoside at the C-6 position and the glucoside at the C-20 position. The activity for Rf was optimal at 95 °C, pH 5.5, 5 mM ginsenoside, and 32 U enzyme l^?1. Under these conditions, P. furiosus β-glucosidase completely converted from R₁ to Rg₁ after 10 h, with a productivity of 0.4 g l^?1 h^?1 and completely converted Rf to Rh₁ after 1.2 h, with a productivity of 2.74 g l^?1 h^?1.     

Production of rare ginsenosides (compound Mc, compound Y and aglycon protopanaxadiol) by β-glucosidase from Dictyoglomus turgidum that hydrolyzes β-linked, but not α-linked, sugars in ginsenosides

Optimal hydrolytic activity of β-glucosidase from Dictyoglomus turgidum for the ginsenoside Rd was at pH 5.5 and 80?°C, with a half-life of ~11?h. The enzyme hydrolysed β-linked, but not α-linked, sugar moieties of ginsenosides. It produced the rare ginsenosides, aglycon protopanaxadiol (APPD), compounds Y, and Mc, via three unique transformation pathways: Rb(1)?→?Rd?→?F(2)?→?compound K?→?APPD, Rb(2)?→?compound Y, and Rc?→?compound Mc. The enzyme converted 0.5?mM Rb(2) and 0.5?mM Rc to 0.5?mM compound Y and 0.5?mM compound Mc after 3?h, respectively, with molar conversion yields of 100?%.     

Highly selective hydrolysis for the outer glucose at the C-20 position in ginsenosides by β-glucosidase from Thermus thermophilus and its application to the production of ginsenoside F2 from gypenoside XVII

β-Glucosidase from Thermus thermophilus has specific hydrolytic activity for the outer glucose at the C-20 position in protopanaxadiol-type ginsenosides without hydrolysis of the inner glucose. The hydrolytic activity of the enzyme for gypenoside XVII was optimal at pH 6.5 and 90 °C, with a half-life of 1 h with 3 g enzyme l^?1 and 4 g gypenoside XVII l^?1. Under the optimized conditions, the enzyme converted the substrate gypenoside XVII to ginsenoside F₂ with a molar yield of 100 % and a productivity of 4 g l^?1 h^?1. The conversion yield and productivity of ginsenoside F₂ are the highest reported thus far among enzymatic transformations.     

Hydrolysis of the outer β-(1,2)-d-glucose linkage at the C-3 position of ginsenosides by a commercial β-galactosidase and its use in the production of minor ginsenosides

Commercial β-galactosidase from Aspergillus oryzae (SUMILACT L^TM) was used for the bioconversion of the ginsenosides Rb1, Rb2, Rc, Rd, and Rg3 to gypenoside-XVII, compound-O, compound-MC1, F2, and Rh2, respectively. The optimal conditions were pH 4.5, 50?°C, 60?U·mL^?1 enzyme, and 8.0?mM substrate. Interestingly, the enzyme hydrolyzed only the outer β-(1,2)-d-glucose linkage at the C-3 position of ginsenosides. Under optimum conditions, the enzyme completely converted Rb1, Rb2, Rc, Rd, and Rg3 to gypenoside-XVII, compound-O, compound-MC1, F2, and Rh2, respectively, with the highest productivity.     

Characterization of a recombinant cellobiose 2-epimerase from Dictyoglomus turgidum that epimerizes and isomerizes β-1,4- and α-1,4-gluco-oligosaccharides

A recombinant putative N-acyl-d-glucosamine 2-epimerase from Dictyoglomus turgidum was identified as a cellobiose 2-epimerase by evaluating its substrate specificity. The purified enzyme was a 46?kDa monomer with a specific activity of 16.8?μmol?min^?1?mg^?1 for cellobiose. The epimerization activity was maximal at pH 7.0 and 70?°C with a half-life of 55?h. The isomerization of the glucose at the reducing end of β-1,4- and α-1,4-linked gluco-oligosaccharides to a fructose moiety by the enzyme took place after the epimerization of the glucose to a mannose moiety. The enzyme converted cellobiose to 12.8?% 4-O-β-d-glucopyranosyl-d-mannose and 54.6?% 4-O-β-d-glucopyranosyl-d-fructose as an equilibrium and converted lactose to 12.8?% epilactose and 54.3?% lactulose.     

Production of fructooligosaccharides in high yield using a mixed enzyme system of β-fructofuranosidase and glucose oxidase

A mixed enzyme system, with -fructofuranosidase (obtained from Aspergillus japonicus) and commercial glucose oxidase (Gluzyme, Novo Nordisk), produced fructooligosaccharides (FOS) in high yield from sucrose. The reaction was performed in an aerated stirred tank reactor controlled at pH 5.5 by a slurry of CaCO₃. Glucose, an inhibitor of -fructofuranosidase, produced in the reaction was converted by glucose oxidase to gluconic acid, which was then precipitated to calcium gluconate in solution. The system produced more than 90% (w/w) FOS on a dry weight basis, the remainder was glucose, sucrose and a small amount of calcium gluconate. Most of the FOS and sucrose was hydrolyzed to fructose in the mixed enzyme system with glucose oxidase and -fructofuranosidase from Asp. niger.     

Agrobacterium-mediated transformation of ginseng (Panax ginseng) and mitotic stability of the inserted β-glucuronidase gene in regenerants from isolated protoplasts

Cotyledonary expiants of ginseng zygotic embryos were cocultured with Agrobacterium tumefadens strain LBA4404 harboring the binary vector pBI121 for 48 h and transferred onto MS medium supplemented with 1 mgl^–1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1 mgl^–1 kinetin, and 100 mgl^–1 kanamycin. After 8 weeks of culture, kanamycin-resistant calli formed on the cut surfaces of cotyledonary expiants and subsequently they gave rise to numerous somatic embryos. Eight weeks after transfer onto medium containing 1 mgl^–1 each of 6-benzyladenine (BA) and gibberellic acid, most of them developed into plantlets. Southern analysis confirmed that the -glucuronidase (GUS) gene was incorporated into the genomic DNA of regenerants. Protoplasts were enzymatically isolated from transformed somatic embryo segments and cultured in liquid medium containing 60 gl^–1 myo-inositol, 1 mgl^–1 2,4-D, 0.5 mgl^–1 BA, and 0.5 mgl^–1 kinetin. Plants were regenerated from protoplasts via somatic embryogenesis. The polymerase chain reaction method revealed that 92% of the regenerants retained the GUS gene. When treated with X-glucuronide, 78% of the regenerants showed a GUS-positive response. The overall results indicate that the transgene is stably transmitted during somatic ontogeny and stably expressed in most the regenerants, whereas it may be deleted or impaired in some portion of them.Abbreviations BA 6-benzyladenine; 2,4-D,2,4-dichlorophenoxyacetic acid - DIG digoxigenine - GA₃ gibberellic acid - X-gluc X-glucuronide - GUS -glucuronidase - MS Murashige and Skoog (1962)     

Production of β-fructofuranosidases by Aspergillus niveus using agroindustrial residues as carbon sources: Characterization of an intracellular enzyme accumulated in the presence of glucose

The production of β-fructofuranosidases by Aspergillus niveus, cultivated under submerged fermentation using agroindustrial residues, was investigated. The highest productivity of β-fructofuranosidases was obtained in Khanna medium supplemented with sugar cane bagasse as carbon source. Glucose enhanced the production of the intracellular enzyme, whereas that of the extracellular one was decreased. The intracellular β-fructofuranosidase was a trimeric protein of approximately 141 kDa (gel filtration) with 53.5% carbohydrate content, composed of 57 kDa monomers (SDS-PAGE). The optimum temperature and optimum pH were 60 °C and 4.5, respectively. The purified enzyme showed good thermal stability and exhibited a half-life of 53 min at 60 °C. β-Fructofuranosidase activity was slightly activated by Cu²⁺, Mn²⁺, Mg²⁺, and Na⁺ at 1 mM concentration. The enzyme hydrolyzed sucrose, raffinose, and inulin, with K_d values of 5.78 mM, 5.74 mM, and 1.74 mM, respectively.     

Isolation and Characterization of Δ6-Desaturase,an ELO-Like Enzyme and Δ5-Desaturase from the Liverwort Marchantia Polymorpha and Production of Arachidonic and Eicosapentaenoic Acids in the Methylotrophic Yeast Pichia Pastoris

The liverwort Marchantia polymorpha contains high proportions of arachidonic and eicosapentaenoic acids. In general, these C20 polyunsaturated fatty acids (PUFA) are synthesized from linoleic and alpha -linolenic acids, respectively, by a series of reactions catalyzed by Delta(6)-desaturase, an ELO-like enzyme involved in Delta(6) elongation and Delta(5)-desaturase. Here we report the isolation and characterization of the cDNAs, MpDES6, MpELO1 and MpDES5, coding for the respective enzymes from M. polymorpha. Co-expression of the MpDES6, MpELO1 and MpDES5 cDNAs resulted in the accumulation of arachidonic and eicosapentaenoic acids in the methylotrophic yeast Pichia pastoris. Interestingly, Delta(6) desaturation by the expression of the MpDES6 cDNA appears to occur both in glycerolipids and the acyl-CoA pool, although other lower-plant Delta(6)-desaturases are known to have a strong preference for glycerolipids.     

The effects of detergents DDM and β-OG on the singlet excited state lifetime of the chlorophyll a in cytochrome b_6f complex from spinach chloroplasts

The singlet excited state lifetime of the chlorophyll a (Chl a) in cytochrome b6f (Cyt b6f) complex was reported to be shorter than that of free Chl a in methanol, but the value was different for Cyt b6f com-plexes from different sources (~200 and ~600 ps are the two measured results). The present study demonstrated that the singlet excited state lifetime is associated with the detergents n-dodecyl-β-D- maltoside (DDM) and n-octyl-β-D-glucopyranoside (β-OG), but has nothing to do with the different sources of Cyt b6f complexes. Compared with the Cyt b6f dissolved in β-OG, the Cyt b6f in DDM had a lower fluorescence yield, a lower photodegradation rate of Chl a, and a shorter lifetime of Chl a excited state. In short, the singlet excited state lifetime, ~200 ps, of the Chl a in Cyt b6f complex in DDM is closer to the true in vivo.     

Physical location of the carotenoid biosynthesis genes Psy and β-Lcy in Capsicum annuum (Solanaceae) using heterologous probes from Citrus sinensis (Rutaceae)

Carotenoids are responsible for a range of fruit colors in different hot pepper (Capsicum) varieties, from white to deep red. Color traits are genetically determined by three loci, Y, C1, and C2, which are associated with carotenogenic genes. Although such genes have been localized on genetic maps of Capsicum and anchored in Lycopersicon and Solanum, physical mapping in Capsicum has been restricted to only a few clusters of some multiple copy genes. Heterologous probes from single copy genes have been rarely used. Fluorescent in situ hybridization was performed in Capsicum annuum varieties with different fruit colors, using heterologous probes of Psy and β-Lcy genes obtained from a BAC library of the sweet orange (Citrus sinensis). The probes hybridized in the terminal portion of a chromosome pair, confirming the location of these genes in genetic maps. The hybridized segments showed variation in size in both chromosomes.     

Vomifoliol 9-O-α-arabinofuranosyl (1→6)-β-D-glucopyranoside from the leaves of Diospyros Kaki stimulates the glucose uptake in HepG2 and 3T3-L1 cells

A novel α-glucosidase inhibitor, vomifoliol 9-O-α-arabinofuranosyl (1→6)-β-D-glucopyranoside, was isolated for the first time from leaves of Diospyros Kaki and its bioactivity analyzed. This inhibitor exhibited strong anti-α-glucosidase activity with an IC50 value of 170.62nM and stimulated a dose-dependent increase in the uptake of a fluorescent d-glucose analog, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG), in HepG2 cells at a rate higher than that of insulin controls. It was also found to be associated with adipocyte differentiation and moderate increases in 2-NBDG uptake by 3T3-L1 cells. These findings suggest that vomifoliol 9-O-α-arabinofuranosyl (1→6)-β-D-glucopyranoside could augment peripheral glucose as an insulin-sensitizing agent against Type 2 diabetes mellitus.     

Characterization of a cDNA encoding a novel DNA-binding protein, SPF1, that recognizes SP8 sequences in the 5′ upstream regions of genes coding for sporamin and β-amylase from sweet potato

We isolated a cDNA encoding a DNA-binding protein, SPF1, of sweet potato that binds to the SP8a (ACTGTGTA) and SP8b (TACTATT) sequences present in the 5 upstream regions of three different genes coding for sporamin and -amylase of tuberous roots. SPF1 comprises 549 amino acids and is enriched in both basic and acidic residues. The amino acid sequence of SPF1 shows no significant homology to any known protein sequences, suggesting that it may represent a new class of DNA-binding protein. Binding studies with ³⁵S-labeled SPF1, synthesized in vitro, and synthetic DNA fragments indicated that, although SPF1 binds to both the SP8a and SP8b sequences, it binds much more strongly to SP8a than to SP8b. SPF1 bound to the SP8a sequence as a monomer. The DNA-binding domain of SPF1 was localized within the C-terminal half of this protein, and a 162-amino acid fragment of SPF1 (Met³¹⁰-Arg⁴⁷²) showed DNA-binding activity with no change in target sequence specificity. This fragment contains a region enriched in basic amino acids adjacent to a highly acidic stretch. A sequence which is highly homologous to a 40-amino acid sequence in the basic region of the DNA-binding domain is duplicated in the N-terminal part of SPF1. The gene coding for SPF1 is present in one or a few copies per haploid genome and the SPF1 mRNA was detected in leaves, stems and tuberous roots of the sweet potato, in addition to petioles. The level of SPF1 mRNA in the petioles decreased when leaf-petiole cuttings were treated with sucrose to induce accumulation of sporamin and -amylase mRNAs.     

Variable Deposition of Amyloid β-Protein (Aβ) with the Carboxy-Terminus that Ends at Residue Valine40 (Aβ40) in the Cerebral Cortex of Patients with Alzheimer's Disease: A Double-Labeling Immunohistochemical Study with Antibodies Specific for Aβ40 and the Aβ that Ends at Residues Alanine42/Threonine43 (Aβ42)

Amyloid -protein (A) deposits in the cerebral cortices of patients with Alzheimer's disease (AD) were investigated immunohistochemically to determine their carboxy terminal sequences. Antibodies specific for A terminating at residue valine₄₀ (A40) and at residues alanine₄₂/threonine₄₃ (A42) were used. Virtually all parenchymal A deposits were positive for A42. Many of these deposits were also partially or completely labeled for A40. The degree of A40 labeling varied from area to area within a given brain and from AD case to AD case. In contrast to parenchymal deposits, A40 labeled essentially all the vascular deposits which constitute amyloid angiopathy (AA), with A42 occurring variably in some of these deposits. Occasional AA was found, however, in which A42 predominated or was exclusively deposited. Such a diversity of A species, both in brain parenchyma and in AA, suggests that multiple C-terminal processing mechanisms occur in the cell types responsible for these deposits.