Anticancer Activity of Continentalic Acid in B-Cell Lymphoma.

Aralia continentalis has been used in Korea as a folk remedy for arthralgia, rheumatism, and inflammation. However, its anti-lymphoma effect remains uncharacterized. Here, we demonstrate that A. continentalis extract and its three diterpenes efficiently kill B-lymphoma cells. Our in vitro and in vivo results suggest that the cytotoxic activities of continentalic acid, a major diterpene from A. continentalis extract, are specific towards cancer cells while leaving normal murine cells and tissues unharmed. Mechanistically, continentalic acid represses the expression of pro-survival Bcl-2 family members, such as Mcl-1 and Bcl-xL. It dissociates the mitochondrial membrane potential, leading to the stimulation of effector caspase 3/7 activities and, ultimately, cell death. Intriguingly, this agent therapeutically synergizes with roflumilast, a pan-PDE4 inhibitor that has been successfully repurposed for the treatment of aggressive B-cell malignancies in recent clinical tests. Our findings unveiled that A. continentalis extract and three of the plant's diterpenes exhibit anti-cancer activities. We also demonstrate the synergistic inhibitory effect of continentalic acid on the survival of B-lymphoma cells when combined with roflumilast. Taken in conjunction, continentalic acid may hold significant potential for the treatment of B-cell lymphoma.

Salt Stress Response of Sulfolobus acidocaldarius Involves Complex Trehalose Metabolism Utilizing a Novel Trehalose-6-Phosphate Synthase (TPS)/Trehalose-6-Phosphate Phosphatase (TPP) Pathway.

The crenarchaeon Sulfolobus acidocaldarius has been described to synthesize trehalose via the maltooligosyltrehalose synthase (TreY) and maltooligosyltrehalose trehalohydrolase (TreZ) pathway, and the trehalose glycosyltransferring synthase (TreT) pathway has been predicted. Deletion mutant analysis of strains with single and double deletions of ΔtreY and ΔtreT in S. acidocaldarius revealed that in addition to these two pathways, a third, novel trehalose biosynthesis pathway is operative in vivo: the trehalose-6-phosphate (T6P) synthase/T6P phosphatase (TPS/TPP) pathway. In contrast to known TPS proteins, which belong to the GT20 family, the S. acidocaldarius TPS belongs to the GT4 family, establishing a new function within this group of enzymes. This novel GT4-like TPS was found to be present mainly in the Sulfolobales The ΔtreY ΔtreT Δtps triple mutant of S. acidocaldarius, which lacks the ability to synthesize trehalose, showed no altered phenotype under standard conditions or heat stress but was unable to grow under salt stress. Accordingly, in the wild-type strain, a significant increase of intracellular trehalose formation was observed under salt stress. Quantitative real-time PCR showed a salt stress-mediated induction of all three trehalose-synthesizing pathways. This demonstrates that in Archaea, trehalose plays an essential role for growth under high-salt conditions.IMPORTANCE The metabolism and function of trehalose as a compatible solute in Archaea was not well understood. This combined genetic and enzymatic approach at the interface of microbiology, physiology, and microbial ecology gives important insights into survival under stress, adaptation to extreme environments, and the role of compatible solutes in Archaea Here, we unraveled the complexity of trehalose metabolism, and we present a comprehensive study on trehalose function in stress response in S. acidocaldarius This sheds light on the general microbiology and the fascinating metabolic repertoire of Archaea, involving many novel biocatalysts, such as glycosyltransferases, with great potential in biotechnology.

Sulfolobus acidocaldarius Transports Pentoses via a Carbohydrate Uptake Transporter 2 (CUT2)-Type ABC Transporter and Metabolizes Them through the Aldolase-Independent Weimberg Pathway.

Sulfolobus spp. possess a great metabolic versatility and grow heterotrophically on various carbon sources, such as different sugars and peptides. Known sugar transporters in Archaea predominantly belong to ABC transport systems. Although several ABC transporters for sugar uptake have been characterized in the crenarchaeon Sulfolobus solfataricus, only one homologue of these transporters, the maltose/maltooligomer transporter, could be identified in the closely related Sulfolobus acidocaldarius Comparison of the transcriptome of S. acidocaldarius MW001 grown on peptides alone and peptides in the presence of d-xylose allowed for the identification of the ABC transporter for d-xylose and l-arabinose transport and the gaining of deeper insights into pentose catabolism under the respective growth conditions. The d-xylose/l-arabinose substrate binding protein (SBP) (Saci_2122) of the ABC transporter is unique in Archaea and shares more similarity to bacterial SBPs of the carbohydrate uptake transporter-2 (CUT2) family than to any characterized archaeal one. The identified pentose transporter is the first CUT2 family ABC transporter analyzed in the domain of Archaea Single-gene deletion mutants of the ABC transporter subunits exemplified the importance of the transport system for d-xylose and l-arabinose uptake. Next to the transporter operon, enzymes of the aldolase-independent pentose catabolism branch were found to be upregulated in N-Z-Amine and d-xylose medium. The α-ketoglutarate semialdehyde dehydrogenase (KGSADH; Saci_1938) seemed not to be essential for growth on pentoses. However, the deletion mutant of the 2-keto-3-deoxyarabinoate/xylonate dehydratase (KDXD [also known as KDAD]; Saci_1939) was no longer able to catabolize d-xylose or l-arabinose, suggesting the absence of the aldolase-dependent branch in S. acidocaldariusIMPORTANCE Thermoacidophilic microorganisms are emerging model organisms for biotechnological applications, as their optimal growth conditions resemble conditions used in certain biotechnologies such as industrial plant waste degradation. Because of its high genome stability, Sulfolobus acidocaldarius is especially suited as a platform organism for such applications. For use in (ligno)cellulose degradation, it was important to understand pentose uptake and metabolism in S. acidocaldarius This study revealed that only the aldolase-independent Weimberg pathway is required for growth of S. acidocaldarius MW001 on d-xylose and l-arabinose. Moreover, S. acidocaldarius employs a CUT2 ABC transporter for pentose uptake, which is more similar to bacterial than to archaeal ABC transporters. The identification of pentose-inducible promoters will expedite the metabolic engineering of S. acidocaldarius for its development into a platform organism for (ligno)cellulose degradation.

Structural basis for the transglycosylase activity of a GH57-type glycogen branching enzyme from Pyrococcus horikoshii.

Glycogen branching enzyme (GBE) catalyzes the formation of α-1,6-branching points during glycogenesis by cleaving α-1,4 bonds and making new α-1,6 bonds. Most GBEs belong to the glycoside hydrolase 13 family (GH13), but new GBEs in the GH57 family have been isolated from Archaea. Here, we determined the crystal structure of a GH57 GBE from the hyperthermophilic archaeon Pyrococcus horikoshii (PhGBE) at a resolution of 2.3 Å. PhGBE exhibits both α-1,6-branching activity and endo-α-1,4 hydrolytic activity. PhGBE has a central (ß/α)7-barrel domain that contains an embedded helix domain and an α-helix-rich C-terminal domain. The active-site cleft is located at the interface of the central and C-terminal domains. Amino acid substitution at Trp22, which is separate from the catalytic nucleophilic residue, abolished both enzymatic activities, indicating that Trp22 might be responsible for substrate recognition. We also observed that shortening of the flexible loop near the catalytic residue changed branched chain lengths of the reaction products with increased hydrolytic activity. Taken together, our findings propose a molecular mechanism for how GH57 GBEs exhibit the two activities and where the substrate binds the enzyme.

Crystal structure of ß-N-acetylglucosaminidase CbsA from Thermotoga neapolitana.

CbsA from the thermophilic marine bacteria Thermotoga neapolitana is a chitinolyitc enzyme that can cleave a glycosidic bond of the polymer N-acetylglucosamine at the non-reducing end. This enzyme has particularly high activity on di-N-acetylchitobiose. CbsA consists of a family of 3 glycoside hydrolase (GH3)-type catalytic domains and a unique C-terminal domain. The C-terminal domain distinguishes CbsA from other GH3-type enzymes. Sequence analyses have suggested that CbsA has the Asp-His dyad as a general acid/base with the NagZ of Bacillus subtilis and the Salmonella enterica serovar Typhimurium. Here, we determined the crystal structure of CbsA from T. neapolitana at a resolution of 2.0 Å using the Zn-SAD method, revealing a unique homodimeric assembly facilitated by the C-terminal domains in the dimer. We observed that CbsA is strongly inhibited by ZnCl2, and two zinc ions were consistently bound in the active site. Our results can explain the zinc ion's inhibition mechanism in the subfamily of GH3 enzymes, and provide information on the structural diversity and substrate specificity of this hydrolase family.

Membrane-bound amylopullulanase is essential for starch metabolism of Sulfolobus acidocaldarius DSM639.

Sulfolobus acidocaldarius DSM639 produced an acid-resistant membrane-bound amylopullulanase (Apu) during growth on starch as a sole carbon and energy source. The physiological role of Apu in starch metabolism was investigated by the growth and starch degradation pattern of apu disruption mutant as well as biochemical properties of recombinant Apu. The Δapu mutant lost the ability to grow in minimal medium in the presence of starch, and the amylolytic activity observed in the membrane fraction of the wild-type strain was not detected in the Δapu mutant when the cells were grown in YT medium. The purified membrane-bound Apu initially hydrolyzed starch, amylopectin, and pullulan into various sizes of maltooligosaccharides, and then produced glucose, maltose, and maltotriose in the end, indicating Apu is a typical endo-acting glycoside hydrolase family 57 (GH57) amylopullulanase. The maltose and maltotriose observed in the culture medium during the exponential and stationary phase growth indicates that Apu is the essential enzyme to initially hydrolyze the starch into small maltooligosaccharides to be transported into the cell.

Glycoconjugates synthesized via transglycosylation by a thermostable α-glucosidase from Thermoplasma acidophilum and its glycosynthase mutant.

A new method is presented for synthesizing arbutin glycosides using α-glucosidase (AglA) from Thermoplasma acidophilum and its glycosynthase mutant. An α-glycosynthase was constructed by substituting the catalytic nucleophile with the non-nucleophile glycine. Enzyme activity was then recovered using an external nucleophile. The transglycosylation reaction of AglA using maltose as a donor and arbutin as an acceptor produced arbutin coupled with a glucose moiety. The products were isolated and further analysed using preparative recycling HPLC. Arbutin glycosides linked to C-3, C-4, and C-6 were identified using NMR. The transglycosylation products of AglA were used as substrates for the enzyme reaction, which were hydrolyzed back again and reduced final yields. The glycosynthase mutant produced one main arbutin glycoside linked to C-4 with a yield of 38 % without further observed hydrolysis.

Taxonomic Variations of Bacterial and Fungal Communities depending on Fermentation Temperature in Traditional Korean Fermented Soybean Food, Doenjang.

Meju, a fermented soybean brick, is a key component in soybean foods like doenjang and ganjang, harboring a variety of microorganisms, including bacteria and fungi. These microorganisms significantly contribute to the nutritional and sensory characteristics of doenjang and ganjang. Amplicon-based next-generation sequencing was applied to investigate how the microbial communities of meju fermented at low and high temperatures differ and how this variation affects the microbial communities of doenjang, a subsequently fermented soybean food. Our metagenomic data showed distinct patterns depending on the fermentation temperature. The microbial abundance in the bacterial community was increased under both temperatures during the fermentation of meju and doenjang. Weissella was the most abundant genus before the fermentation of meju, however, it was replaced by Bacillus at high temperature-fermented meju and lactic acid bacteria such as Weissella and Latilactobacillus at low temperature-fermented meju. Leuconostoc, Logiolactobacillus, and Tetragenococcus gradually took over the dominant role during the fermentation process of doenjang, replacing the previous dominant microorganisms. Mucor was dominant in the fungal community before and after meju fermentation, whereas Debaryomyces was dominant under both temperatures during doenjang fermentation. The dominant fungal genus of doenjang was not affected regardless of the fermentation temperature of meju. Strong correlations were shown for specific bacteria and fungi linked to specific fermentation temperatures. This study helps our understanding of meju fermentation process at different fermentation temperatures and highlights different bacteria and fungi associated with specific fermentation periods which may influence the nutritional and organoleptic properties of the final fermented soybean foods doenjang.

Correlation between monosaccharide, oligosaccharide, and microbial community profile changes in traditional soybean brick (meju) fermentation.

Meju is essential for making diverse traditional fermented soybean foods in Korea. To understand the changes in carbohydrates during fermentation, we aimed to identify autochthonous microorganisms from spontaneously fermented meju and compare the alterations in monosaccharides and oligosaccharides throughout the fermentation process. Microbial diversity was determined using a metabarcoding approach, and monosaccharide and oligosaccharide profiles were obtained by HPLC-Q-TOF MS and HPLC-MS/MS analyses, respectively. The dominant bacterial genera were Weissella, Lactobacillus, and Leuconostoc, while Mucor was highly abundant in the fungal community. The total monosaccharide content increased from Day 0 to Day 50, with the highest amount being 4.37 mg/g. Oligosaccharide profiling revealed the degradation of soybean dietary fiber during fermentation, and novel oligosaccharide structures were also discovered. Correlation analysis revealed that the fungus Mucor was positively related to pentose-containing oligosaccharides, galactose, and galacturonic acid, indicating that Mucor may degrade soybean dietary fibers such as xylogalacturonan, arabinogalactan, and rhamnogalacturonan. The negative relationships between the abundances of Weissella and oligo- and monosaccharides suggested that the bacteria may utilize saccharides for fermentation. These findings provide insights into the mechanisms underlying carbohydrate degradation and utilization; the key components involved in saccharide transformation that contribute to the characteristics of traditional meju were subsequently identified.

Identification and characterization of MalA in the maltose/maltodextrin operon of Sulfolobus acidocaldarius DSM639.

A putative maltose/maltodextrin operon was found in the Sulfolobus acidocaldarius DSM639 genome. The gene cluster consisted of 7 genes (malA, trmB, amyA, malG, malF, malE, and malK). Here, we report the identification of MalA, which is responsible for the hydrolysis of maltose or maltodextrin to glucose in S. acidocaldarius. The transcription level of malA was increased 3-fold upon the addition of maltose or starch to the medium. Moreover, the α-glucosidase activity for maltose as a substrate in cell extracts of S. acidocaldarius DSM639 was also 11- and 10-fold higher during growth in YT medium (Brock's mineral salts, 0.1% [wt/vol] tryptone, and 0.005% [wt/vol] yeast extract) containing maltose or starch, respectively, than during growth on other sugars. The gene encoding MalA was cloned and expressed in S. acidocaldarius. The enzyme purified from the organism was a dodecamer in its active state and showed strong maltose-hydrolyzing activity at 100°C and pH 5.0. MalA was remarkably thermostable, with half-lives of 33.8 h, 10.6 h, and 1.8 h at 95°C, 100°C, and 105°C, respectively. Substrate specificity and kinetic studies of MalA with maltooligosaccharides indicated that MalA efficiently hydrolyzed maltose to maltopentaose, which is a typical characteristic of GH31-type α-glucosidases. However, glycogen or starch was not hydrolyzed. Reverse transcription-PCR, sugar uptake, and growth studies of the wild-type DSM639 and ΔmalEFG mutant on different sugars demonstrated that MalA located in the mal operon gene cluster is involved in maltose and starch metabolism in S. acidocaldarius.

Structural and functional basis for substrate specificity and catalysis of levan fructotransferase.

Levan is ß-2,6-linked polymeric fructose and serves as reserve carbohydrate in some plants and microorganisms. Mobilization of fructose is usually mediated by enzymes such as glycoside hydrolase (GH), typically releasing a monosaccharide as a product. The enzyme levan fructotransferase (LFTase) of the GH32 family catalyzes an intramolecular fructosyl transfer reaction and results in production of cyclic difructose dianhydride, thus exhibiting a novel substrate specificity. The mechanism by which LFTase carries out these functions via the structural fold conserved in the GH32 family is unknown. Here, we report the crystal structure of LFTase from Arthrobacter ureafaciens in apo form, as well as in complexes with sucrose and levanbiose, a difructosacchride with a ß-2,6-glycosidic linkage. Despite the similarity of its two-domain structure to members of the GH32 family, LFTase contains an active site that accommodates a difructosaccharide using the -1 and -2 subsites. This feature is unique among GH32 proteins and is facilitated by small side chain residues in the loop region of a catalytic ß-propeller N-domain, which is conserved in the LFTase family. An additional oligosaccharide-binding site was also characterized in the ß-sandwich C-domain, supporting its role in carbohydrate recognition. Together with functional analysis, our data provide a molecular basis for the catalytic mechanism of LFTase and suggest functional variations from other GH32 family proteins, notwithstanding the conserved structural elements.

Structure of a novel α-amylase AmyB from Thermotoga neapolitana that produces maltose from the nonreducing end of polysaccharides.

An intracellular α-amylase, AmyB, has been cloned from the hyperthermophilic bacterium Thermotoga neapolitana. AmyB belongs to glycoside hydrolase family 13 and liberates maltose from diverse substrates, including starch, amylose, amylopectin and glycogen. The final product of AmyB is similar to that of typical maltogenic amylases, but AmyB cleaves maltose units from the nonreducing end, which is a unique property of this α-amylase. In this study, the crystal structure of AmyB from T. neapolitana has been determined at 2.4 Šresolution, revealing that the monomeric AmyB comprises domains A, B and C like other α-amylases, but with structural variations. In the structure, a wider active site and a putative extra sugar-binding site at the top of the active site were found. Subsequent biochemical results suggest that the extra sugar-binding site is suitable for recognizing the nonreducing end of the substrates, explaining the unique activity of this enzyme. These findings provide a structural basis for the ability of an α-amylase that has the common α-amylase structure to show a diverse substrate specificity.

Identification of antigenic Edwardsiella tarda surface proteins and their role in pathogenesis.

Edwardsiella tarda causes an infectious fish disease called edwardsiellosis. Several outer membrane proteins (OMPs) are associated with virulence factors and are attractive as vaccine candidates. In this study, 4 immuno-reactive OMPs of E. tarda were detected using anti-sera from flounder infected with E. tarda. Using matrix-assisted laser desorption/ionization mass spectrometry analyses, 2 of the 4 OMPs were identified as OmpA and murein lipoprotein (Lpp), which are highly conserved surface proteins in gram-negative bacteria. For further characterization of these surface proteins, we generated ompA- and lpp-inactivated mutants by insertion of a kanamycin cassette in the corresponding genes, and named these mutants E. tarda CK99 and CK164, respectively. As expected, immuno-reactive OmpA and Lpp proteins were absent in E. tarda CK99 and CK164, respectively, confirming that OmpA and Lpp are antigenic surface proteins. Interestingly, the LD(50) value of E. tarda CK164 in fish (2.0 × 10(8) colony-forming unit [CFU]/fish) was greater than that of the parental strain (3.0 × 10(7) CFU/fish). The LD(50) of E. tarda CK99 did not differ from that of its parental strain. After administering attenuated E. tarda CK164 to fish, we monitored the E. tarda-specific immune response profile. We observed that the E. tarda-specific serum IgM titer increased in a time-dependent manner, and was much higher than the value observed after the administration of a heat-killed E. tarda control. Moreover, fish vaccinated with E. tarda CK164 were 100% protected when challenged by CK41, a pathogenic strain. Our results suggest that E. tarda CK164 can potentially be used for developing an effective live attenuated vaccine for edwardsiellosis that can be applied in the aquaculture industry.

Crystallization and preliminary X-ray crystallographic analysis of the ß-N-acetylglucosaminidase CbsA from Thermotoga neapolitana.

The ß-N-acetylglucosaminidase CbsA was cloned from the thermophilic Gram-negative bacterium Thermotoga neapolitana. Although CbsA contains a family 3 glycoside hydrolase-type (GH3-type) catalytic domain, it can be distinguished from other GH3-type ß-N-acetylglucosaminidases by its high activity towards chitobiose. The homodimeric CbsA contains a unique domain at the C-terminus for which the three-dimensional structure is not yet known. In this study, CbsA was overexpressed and the recombinant protein was purified using Ni-NTA affinity and gel-filtration chromatography. The purified CbsA protein was crystallized using the vapour-diffusion method. A diffraction data set was collected to a resolution of 2.0 Å at 100 K. The crystal belonged to space group R32. To obtain initial phases, the crystallization of selenomethionyl-substituted protein and the production of heavy-atom derivative crystals are in progress.

Transcriptome Analysis of Halotolerant Staphylococcus saprophyticus Isolated from Korean Fermented Shrimp.

Saeu-jeotgal, a Korean fermented shrimp food, is commonly used as an ingredient for making kimchi and other side dishes. The high salinity of the jeotgal contributes to its flavor and inhibits the growth of food spoilage microorganisms. Interestingly, Staphylococcus saprophyticus was discovered to be capable of growth even after treatment with 20% NaCl. To elucidate the tolerance mechanism, a genome-wide gene expression of S. saprophyticus against 0%, 10%, and 20% NaCl was investigated by RNA sequencing. A total of 831, 1314, and 1028 differentially expressed genes (DEGs) were identified in the 0% vs. 10%, 0% vs. 20%, and 10% vs. 20% NaCl comparisons, respectively. The Clusters of Orthologous Groups analysis revealed that the DEGs were involved in amino acid transport and metabolism, transcription, and inorganic ion transport and metabolism. The functional enrichment analysis showed that the expression of the genes encoding mechanosensitive ion channels, sodium/proton antiporters, and betaine/carnitine/choline transporter family proteins was downregulated, whereas the expression of the genes encoding universal stress proteins and enzymes for glutamate, glycine, and alanine synthesis was upregulated. Therefore, these findings suggest that the S. saprophyticus isolated from the saeu-jeotgal utilizes different molecular strategies for halotolerance, with glutamate as the key molecule.

Engineering of Sulfolobus acidocaldarius for Hemicellulosic Biomass Utilization.

The saccharification of cellulose and hemicellulose is essential for utilizing lignocellulosic biomass as a biofuel. While cellulose is composed of glucose only, hemicelluloses are composed of diverse sugars such as xylose, arabinose, glucose, and galactose. Sulfolobus acidocaldarius is a good potential candidate for biofuel production using hemicellulose as this archaeon simultaneously utilizes various sugars. However, S. acidocaldarius has to be manipulated because the enzyme that breaks down hemicellulose is not present in this species. Here, we engineered S. acidocaldarius to utilize xylan as a carbon source by introducing xylanase and ß-xylosidase. Heterologous expression of ß-xylosidase enhanced the organism's degradability and utilization of xylooligosaccharides (XOS), but the mutant still failed to grow when xylan was provided as a carbon source. S. acidocaldarius exhibited the ability to degrade xylan into XOS when xylanase was introduced, but no further degradation proceeded after this sole reaction. Following cell growth and enzyme reaction, S. acidocaldarius successfully utilized xylan in the synergy between xylanase and ß-xylosidase.

Identification of the antibacterial action mechanism of diterpenoids through transcriptome profiling.

Effective antibacterial substances of Aralia continentalis have anti-biofilm and bactericidal activity to the oral pathogen Streptococcus mutans. In this study, three compounds extracted from A. continentalis were identified as acanthoic acid, continentalic acid, and kaurenoic acid by NMR and were further investigated how these diterpenoids affect the physiology of the S. mutans. When S. mutans was exposed to individual or mixed fraction of diterpenoids, severe growth defects and unique morphology were observed. The proportion of unsaturated fatty acids in the cell membrane was increased compared to that of saturated fatty acids in the presence of diterpenoids. Genome-wide gene expression profiles with RNA-seq were compared to reveal the mode of action of diterpenoids. Streptococcus mutans commonly enhanced the expression of 176 genes in the presence of the individual diterpenoids, whereas the expression of 232 genes was considerably reduced. The diterpenoid treatment modulated the expression of genes or operon(s) involved in cell membrane synthesis, cell division, and carbohydrate metabolism of S. mutans. Collectively, these findings provide novel insights into the antibacterial effect of diterpenoids to control S. mutans infection, which causes human dental caries.

Discovery of novel inhibitors for human intestinal maltase: virtual screening in a WISDOM environment and in vitro evaluation.

Human intestinal maltase (HMA) is an α-glucosidase that hydrolyses α-1,4-linkages from the non-reducing end of malto-oligosaccharides. HMA is an important target to discover of new drugs for the treatment of type 2 diabetes. In this study, 308,307 compounds were virtually screened with HMA using Autodock 3.0.5 in a WISDOM production environment to discover novel inhibitors. The 42 top-scoring free binding energy compounds, representing 17 groups containing potential hydrogen bonding with key residues in the active site pocket of HMA, were tested in vitro for their inhibitory activities against recombinant HMA expressed from Pichia pastoris. Compounds 17 and 18 were competitive inhibitors exclusively for HMA without any in vitro inhibition for human pancreatic α-amylase. The K(i) values were 20 µM for both compound 17 and 18.

Enzymatic Synthesis of Resveratrol α-Glucoside by Amylosucrase of Deinococcus geothermalis.

Glycosylation of resveratrol was carried out by using the amylosucrase of Deinococcus geothermalis, and the glycosylated products were tested for their solubility, chemical stability, and biological activities. We synthesized and identified these two major glycosylated products as resveratrol-4'-O-α-glucoside and resveratrol-3-O-α-glucoside by nuclear magnetic resonance analysis with a ratio of 5:1. The water solubilities of the two resveratrol-α-glucoside isomers (α-piceid isomers) were approximately 3.6 and 13.5 times higher than that of ß-piceid and resveratrol, respectively, and they were also highly stable in buffered solutions. The antioxidant activity of the α-piceid isomers, examined by radical scavenging capability, showed it to be initially lower than that of resveratrol, but as time passed, the α-piceid isomers' activity reached a level similar to that of resveratrol. The α-piceid isomers also showed better inhibitory activity against tyrosinase and melanin synthesis in B16F10 melanoma cells than ß-piceid. The cellular uptake of the α-piceid isomers, which was assessed by ultra-performance liquid chromatography (UPLC) analysis of the cell-free extracts of B16F10 melanoma cells, demonstrated that the glycosylated form of resveratrol was gradually converted to resveratrol inside the cells. These results indicate that the enzymatic glycosylation of resveratrol could be a useful method for enhancing the bioavailability of resveratrol.

Identification of the Genes Related to the Glycogen Metabolism in Hyperthermophilic Archaeon, Sulfolobus acidocaldarius.

Glycogen is a polysaccharide that comprises α-1,4-linked glucose backbone and α-1,6-linked glucose polymers at the branching points. It is widely found in organisms ranging from bacteria to eukaryotes. The physiological role of glycogen is not confined to being an energy reservoir and carbon source but varies depending on organisms. Sulfolobus acidocaldarius, a thermoacidophilic archaeon, was observed to accumulate granular glycogen in the cell. However, the role of glycogen and genes that are responsible for glycogen metabolism in S. acidocaldarius has not been identified clearly. The objective of this study is to identify the gene cluster, which is composed of enzymes that are predicted to be involved in the glycogen metabolism, and confirm the role of each of these genes by constructing deletion mutants. This study also compares the glycogen content of mutant and wild type and elucidates the role of glycogen in this archaeon. The glycogen content of S. acidocaldarius MR31, which is used as a parent strain for constructing the deletion mutant in this study, was increased in the early and middle exponential growth phases and decreased during the late exponential and stationary growth phases. The pattern of the accumulated glycogen was independent to the type of supplemented sugar. In the comparison of the glycogen content between the gene deletion mutant and MR31, glycogen synthase (GlgA) and α-amylase (AmyA) were shown to be responsible for the synthesis of glycogen, whereas glycogen debranching enzyme (GlgX) and glucoamylase (Gaa) appeared to affect the degradation of glycogen. The expressions of glgC-gaa-glgX and amyA-glgA were detected by the promoter assay. This result suggests that the gradual decrease of glycogen content in the late exponential and stationary phases occurs due to the increase in the gene expression of glgC-gaa-glgX. When the death rate in nutrient limited condition was compared among the wild type strain, the glycogen deficient strain and the strain with increased glycogen content, the death rate of the glycogen deficient strain was found to be higher than any other strain, thereby suggesting that the glycogen in S. acidocaldarius supports cell maintenance in harsh conditions.