InsP6-sensitive variants of the Gle1 mRNA export factor rescue growth and fertility defects of the ipk1 low-phytic-acid mutation in Arabidopsis.

Myo-inositol-1,2,3,4,5,6-hexakisphosphate (InsP(6)), also known as phytic acid, accumulates in large quantities in plant seeds, serving as a phosphorus reservoir, but is an animal antinutrient and an important source of water pollution. Here, we report that Gle1 (GLFG lethal 1) in conjunction with InsP(6) functions as an activator of the ATPase/RNA helicase LOS4 (low expression of osmotically responsive genes 4), which is involved in mRNA export in plants, supporting the Gle1-InsP(6)-Dbp5 (LOS4 homolog) paradigm proposed in yeast. Interestingly, plant Gle1 proteins have modifications in several key residues of the InsP(6) binding pocket, which reduce the basicity of the surface charge. Arabidopsis thaliana Gle1 variants containing mutations that increase the basic charge of the InsP(6) binding surface show increased sensitivity to InsP(6) concentrations for the stimulation of LOS4 ATPase activity in vitro. Expression of the Gle1 variants with enhanced InsP(6) sensitivity rescues the mRNA export defect of the ipk1 (inositol 1,3,4,5,6-pentakisphosphate 2-kinase) InsP(6)-deficient mutant and, furthermore, significantly improves vegetative growth, seed yield, and seed performance of the mutant. These results suggest that Gle1 is an important factor responsible for mediating InsP(6) functions in plant growth and reproduction and that Gle1 variants with increased InsP(6) sensitivity may be useful for engineering high-yielding low-phytate crops.

Evaluation of sterols as markers of fungal spoilage in red pepper powder.

Red pepper powder (RPP) made from ground dried red pepper (Capsicum annuum L.) is prone to adulteration with fungal-spoiled RPP to gain unfair profits in Korea. This study aimed to investigate the effects of fungal infection on the ergosterol and phytosterol content of RPP and evaluate the potential of the sterol content as a marker for identifying fungal-spoiled RPP. Ergosterol was detected only in fungal-spoiled RPP and not in unspoiled RPP [

Curcumol metabolized by rat liver S9 fraction and orally administered in mouse suppressed the proliferation of colon cancer in vitro and in vivo.

Following 3R (reduction, refinement, and replacement) principles, we employed the rat liver S9 fraction to mimic liver metabolism of curcumol having high in vitro IC50 on cancer cells. In HCT116 and HT29 colon cancer cells, the metabolites of curcumol by S9 fraction exerted more enhanced activity in inducing cell cycle arrest and apoptosis via regulating the expression of cyclin D1, CDK1, p21, PARP and Bcl-2 than curcumol. In addition, oral administration of curcumol at 4 mg/kg BW significantly suppressed the development of colon tumor induced by azoxymethane/dextran sulfate sodium, and induced cell cycle arrest and apoptosis in tumor tissues. In mass analysis, curcumenol and curzerene were identified as the metabolites of curcumol by S9 fraction metabolism. Taken together, curcumol metabolites showed the enhanced suppressive effect on colon cancer, suggesting that S9 fraction can be considered as simple, fast, and bio-mimicking platform for the screening of chemical libraries on different chronic diseases.

Cryopreservation of mouse spermatogonial stem cells in dimethylsulfoxide and polyethylene glycol.

Assisted reproductive techniques involving isolation, culture, and transplantation of spermatogonial stem cells (SSCs) have the potential to create transgenic livestock and to treat male infertility caused by cancer treatments such as chemotherapy or radiation. Because stem cells may need to be preserved for several years before reintroduction to the patients' testes, efficient SSC cryopreservation techniques need to be developed. SSCs can reinitiate spermatogenesis in recipient testes after freezing; however, optimal cryopreservation protocols have not been identified. The objective of this study was to develop an efficient cryopreservation method for SSCs using permeable cryoprotectant agents (PCAs) or additive cryoprotectant agents (ACAs). To identify an efficient cryopreservation method, populations of mouse testis cells enriched for SSCs were cultured in vitro and frozen using conventional freezing media containing various PCAs or ACAs for 1 wk or 1, 3, 6, 12, or 24 mo. Additionally, various molecular weights and concentrations of polyethylene glycol (PEG) were evaluated. Recovery rate, culture potential, and stem cell activity were significantly greater for cells frozen in 2.5% PEG with a molecular weight of 1000 compared to other treatment groups. These cells also retained the ability to colonize recipient testes, generate normal spermatogenesis, and contribute to viable offspring. The systematic analysis of many cryoprotectant agents indicates that 2.5% PEG (molecular weight 1000) is the most effective agent for efficient SSC cryopreservation.

Effects of Protein Hydrolysate from Silkworm (Bombyx mori) pupae on the C2C12 Myogenic Differentiation.

This study investigated the effects and active compounds of silkworm pupae, an edible insect, on C2C12 muscle differentiation. The protein of silkworm pupae was extracted using sonication after defatting with hexane. Subsequently, the extract was rehydrated using Alcalase to obtain a protein hydrolysate. The silkworm pupae protein hydrolysate effectively promoted C2C12 myogenic differentiation without cytotoxicity. Subsequently, the hydrolysate was fractionated into four subfractions using preparative high-performance liquid chromatography (Prep-HPLC). Subfraction 1 was the most effective in promoting C2C12 myogenic differentiation and significantly upregulated the expression of myoblast transcription factors, 1.5-fold of myoblast determination protein 1 (MyoD), 2-fold of myogenin, and 3-fold of myosin heavy chain (MyHC). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and multivariate statistical analysis were used to identify the active peptides in silkworm pupae responsible for the observed effects; then, dipeptides and essential amino acids, such as isoleucine (Ile), valine (Val), and methionine (Met), were identified. In addition, Val, Ile, and two dipeptides underwent quantification to determine the potential bioactive peptides that enhanced C2C12 myogenic differentiation. This study suggests that the peptides from silkworm pupae could be used as a nutraceutical to enhance muscle growth.

Selective Detection of Escherichia coli K12 and Staphylococcus aureus in Mixed Bacterial Communities Using a Single-Walled Carbon Nanotube (SWCNT)-Functionalized Electrochemical Immunosensor with Dielectrophoretic Concentration.

An electrochemical immunosensor has been developed for the rapid detection and identification of potentially harmful bacteria in food and environmental samples. This study aimed to fabricate a microwire-based electrochemical immunosensor (MEI sensor) for selective detection of Escherichia coli and Staphylococcus aureus in microbial cocktail samples using dielectrophoresis (DEP)-based cell concentration. A gold-coated tungsten microwire was functionalized by coating polyethylenimine, single-walled carbon nanotube (SWCNT) suspension, streptavidin, biotinylated antibodies, and then bovine serum albumin (BSA) solutions. Double-layered SWCNTs and 5% BSA solution were found to be optimized for enhanced signal enhancement and nonspecific binding barrier. The selective capture of E. coli K12 or S. aureus cells was achieved when the electric field in the bacterial sample solution was generated at a frequency of 3 MHz and 20 Vpp. A linear trend of the change in the electron transfer resistance was observed as E. coli concentrations increased from 5.32 × 102 to 1.30 × 108 CFU/mL (R2 = 0.976). The S. aureus MEI sensor fabricated with the anti-S. aureus antibodies also showed an increase in resistance with concentrations of S. aureus (8.90 × 102-3.45 × 107 CFU/mL) with a correlation of R2 = 0.983. Salmonella typhimurium and Listeria monocytogenes were used to evaluate the specificity of the MEI sensors. The functionalization process developed for the MEI sensor is expected to contribute to the sensitive and selective detection of other harmful microorganisms in food and environmental industries.

Natural Occurrence of Alternaria Toxins in Agricultural Products and Processed Foods Marketed in South Korea by LC-MS/MS.

Alternaria mycotoxins including alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT), altertoxin-I (ATX-I), tentoxin (TEN), and tenuazonic acid (TeA), are ubiquitous contaminants in agricultural products. A method for the simultaneous determination of these six toxins by ultrahigh performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) with solid phase extraction (SPE) was validated in rice, sesame, tomato, and apple juice matrices. The performance of the method was evaluated in terms of linearity (R2 > 0.999), the limit of detection (0.04-1.67 µg/kg), the limit of quantification (0.12-5.06 µg/kg), recovery (80.0-114.7%), and precision (<17.7%). The validated method was applied to monitor 152 marketed food samples in South Korea, as well as to investigate the co-occurrence and correlation between Alternaria toxins. The mean occurrence levels were 2.77 µg/kg for AOH, 4.36 µg/kg for AME, 0.14 µg/kg for ALT, 0.11 µg/kg for ATX-I, 0.43 µg/kg for TEN, and 104.56 µg/kg for TeA. Mean and extreme (95th percentile) daily dietary exposures of South Koreans to Alternaria toxins were estimated to be 22.93 ng/kg b.w./day and 86.07 ng/kg b.w./day, respectively.

Protective effect of a mixture of marigold and rosemary extracts on UV-induced photoaging in mice.

UV irradiation exposure may induce photoaging of the skin tissue. Various plant extracts have been recognized as effective protectants against UV-induced damage. Here, a mixture of marigold and rosemary extracts was evaluated for its anti-photoaging effects as a potential nutraceutical product for skin health. Hexane extract of marigold and ethanolic extract of rosemary were prepared, and the formulated mixture was investigated. A UV-induced photoaged mouse model was prepared, and the protective effects of the extract mixture were compared with those of hyaluronic acid (positive control). Expression of various photoaging-related biomarkers such as matrix metalloproteinases (MMPs), interleukins, tumor necrosis factor-alpha, procollagen type I, 8-hydroxy-deoxyguanosine, superoxide dismutase, glutathione peroxidase, and catalase were determined. UV irradiation significantly enhanced the expression of these biomarkers through an inflammatory response, however, the mixture of marigold and rosemary extracts exerted inhibitory effects and protected from UV-induced damage. Suppression of inflammatory response were the mechanisms underlying this protective function of the mixture of marigold and rosemary extracts. Histological evaluation also supported these protective effects against photoaging.

Authentication of pomegranate juice using multidimensional analysis of its metabolites.

With the growing popularity and demand of pomegranate juice, its adulteration has also steadily increased. In this study, to authenticate pure or adulterated juice, the major components of pomegranate juice were compared with those of grape, peach, and apple juices (which are common adulterants in pomegranate juice) using liquid chromatography-electrospray-tandem mass spectrometry, ion chromatography, and inductively coupled plasma spectrometry. The various parameters evaluated were as follows: the ratio of malic acid to citric acid content, presence of tartaric acid, and levels of glucose, fructose, and mannitol, and sucrose. Potassium was the most abundant mineral in pomegranate juice, and the content ratio of other minerals/potassium did not exceed 0.1. The reliability of this method was confirmed in blind tests and monitoring experiments with commercial pomegranate juice. In conclusion, a simple and effective method was developed to detect adulteration in pomegranate juice.

Garcinone C suppresses colon tumorigenesis through the Gli1-dependent hedgehog signaling pathway.

BACKGROUND: Although garcinone C, a natural xanthone derivative identified in the pericarp of Garcinia mangostana, has been demonstrated to exert different health beneficial activities in oxidative stress and ß-amyloid aggregation, the role of garcinone C in colon tumorigenesis has not been investigated. In addition, aberrant Hedgehog (Hh) signaling activation is associated with tumorigenesis including colon cancer. Here, we hypothesized that garcinone C can prevent colon tumorigenesis through regulating the Hh signaling pathway. METHOD: Colony formation assay and flow cytometry were used to evaluate the effect of garcinone C on the proliferation and cell cycle progression of colon cancer cells. Protein expression of cell cycle related markers and Hh/Gli1 signaling mediators were determined. The regulatory effect of orally administered garcinone C on the Hh/Gli1 signaling pathway and colon tumorigenesis was evaluated in an azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced colon cancer animal model. RESULTS: Garcinone C suppressed the proliferation of colon cancer cells, induced G0/G1 cell cycle arrest, as well as regulated the expression of cell cycle-related markers such as cyclin D1, cyclin E, CDK6, and p21. Garcinone C inhibited the expression of Gli1, a key mediator of Hedgehog signaling, and protein kinase B (AKT) phosphorylation in Smo-independent colon cancer cells. In the AOM/DSS-induced colon tumorigenesis model, garcinone C significantly inhibited tumor development, regulated the expression of cell cycle markers and Gli1, and reduced AKT phosphorylation in colon tumor tissues, which is consistent with our in vitro results. CONCLUSION: Garcinone C can suppress colon tumorigenesis in vitro and in vivo through Gli1-dependent non-canonical Hedgehog signaling, suggesting that it may serve as a potent chemopreventive agent against colon tumorigenesis.

Amylose-Lipid Complex as a Fat Replacement in the Preparation of Low-Fat White Pan Bread.

Amylose-lipid complex (ALC) was prepared with corn starch and stearic acid and used as a shortening replacement in white pan bread preparation. ALCs were prepared using various concentrations of stearic acid to corn starch (1%, 3%, 5%, and 7%) under different temperatures (55, 65, and 75 °C) and for different durations of time (30, 60, and 120 min); then, their complexing properties were assessed using iodine reagent and X-ray diffraction. The complexing reaction at 75 °C for 60 min showed the highest complexing index of the tested conditions; the in vitro digestibility of ALC was lower than that of corn starch. White pan bread was prepared with ALCs and their characteristics, including appearance, loaf volume, and starch retrogradation during storage at room temperature for four days, were compared with those of control bread. With increasing ALC replacement concentrations, loaf volume and shape were significantly affected; however, starch retrogradation was significantly retarded and energy value decreased by ALC replacement. Overall, 50% replacement of shortening by ALC appeared to be a reasonable level for retaining the basic characteristics of the bread while retarding the staling process. These results indicate that ALCs may be potentially useful in the bakery industry for preparing low calorie and low-fat products.

Propolis Suppresses UV-Induced Photoaging in Human Skin through Directly Targeting Phosphoinositide 3-Kinase.

Propolis is a resinous substance generated by bees using materials from various plant sources. It has been known to exhibit diverse bioactivities including anti-oxidative, anti-microbial, anti-inflammatory, and anti-cancer effects. However, the direct molecular target of propolis and its therapeutic potential against skin aging in humans is not fully understood. Herein, we investigated the effect of propolis on ultraviolet (UV)-mediated skin aging and its underlying molecular mechanism. Propolis suppressed UV-induced matrix metalloproteinase (MMP)-1 production in human dermal fibroblasts. More importantly, propolis treatment reduced UV-induced MMP-1 expression and blocked collagen degradation in human skin tissues, suggesting that the anti-skin-aging activity of propolis can be recapitulated in clinically relevant conditions. While propolis treatment did not display any noticeable effects against extracellular signal-regulated kinase (ERK), p38, and c-jun N-terminal kinase (JNK) pathways, propolis exerted significant inhibitory activity specifically against phosphorylations of phosphoinositide-dependent protein kinase-1 (PDK1) and protein kinase B (Akt). Kinase assay results demonstrated that propolis can directly suppress phosphoinositide 3-kinase (PI3K) activity, with preferential selectivity towards PI3K with p110α and p110δ catalytic subunits over other kinases. The content of active compounds was quantified, and among the compounds identified from the propolis extract, caffeic acid phenethyl ester, quercetin, and apigenin were shown to attenuate PI3K activity. These results demonstrate that propolis shows anti-skin-aging effects through direct inhibition of PI3K activity.

Effect of Equilibration Time and Temperature on Murine Spermatogonial Stem Cell Cryopreservation.

Cryopreservation of spermatogonial stem cells (SSCs) is essential for preservation of valuable livestock and clinical applications. Although optimal equilibration of cryoprotectants has emerged as a promising approach to improve the cryopreservation efficiency, standard equilibration protocols have not yet been considered in cryopreservation of SSCs. This study aimed to establish a standard equilibration protocol to improve the cryopreservation efficiency of murine germ cells enriched for SSCs. After time- and temperature-dependent equilibration, the germ cells were cryopreserved with 10% dimethyl sulfoxide (DMSO) and 200 mM trehalose. To investigate cryopreservation efficiency at different equilibration conditions, the survival and proliferation rates were assessed after thawing, and then, cytotoxicity and intracellular trehalose quantification were analyzed. Protein (PLZF, GFRα1, VASA, and c-Kit) and gene (Bcl6b, Erm, Dazl, and Sycp1) expression was determined using immunofluorescence and real-time quantitative polymerase chain reaction (RT-qPCR), respectively. The proliferation rate increased significantly following equilibration for 20 minutes at room temperature (RT; 163.7% ± 24.6%) or 4°C (269.0% ± 18.2%). Cytotoxicity was reduced in 10% DMSO with 200 mM trehalose compared with that of 10% DMSO alone. Also, intracellular trehalose was observed after equilibration. The immunofluorescence and RT-qPCR data revealed that the murine germ cells enriched for SSCs retained their self-renewal ability after cryopreservation following equilibration. The most effective protocol was equilibration with 10% DMSO and 200 mM trehalose for 20 minutes at RT or 4°C, which is due to synergistic effects of intracellular and extracellular trehalose. This improved methodology will contribute toward the development of a standardized freezing protocol for murine germ cells enriched for SSCs and thereby expand their application in various fields.

High-phytate/low-calcium diet is a risk factor for crystal nephropathies, renal phosphate wasting, and bone loss.

Phosphate overload contributes to mineral bone disorders that are associated with crystal nephropathies. Phytate, the major form of phosphorus in plant seeds, is known as an indigestible and of negligible nutritional value in humans. However, the mechanism and adverse effects of high-phytate intake on Ca2+ and phosphate absorption and homeostasis are unknown. Here, we show that excessive intake of phytate along with a low-Ca2+ diet fed to rats contributed to the development of crystal nephropathies, renal phosphate wasting, and bone loss through tubular dysfunction secondary to dysregulation of intestinal calcium and phosphate absorption. Moreover, Ca2+ supplementation alleviated the detrimental effects of excess dietary phytate on bone and kidney through excretion of undigested Ca2+-phytate, which prevented a vicious cycle of intestinal phosphate overload and renal phosphate wasting while improving intestinal Ca2+ bioavailability. Thus, we demonstrate that phytate is digestible without a high-Ca2+ diet and is a risk factor for phosphate overloading and for the development of crystal nephropathies and bone disease.

Classification of fermented soybean paste during fermentation by 1H nuclear magnetic resonance spectroscopy and principal component analysis.

Fermented soybean paste (doenjang, FSP) is a traditionally fermented Korean food produced by fermentation with various microorganisms that is known to exhibit various beneficial bioactivities. To investigate the changes in nonvolatile metabolites of FSP during fermentation, samples produced with six fermentation times were analyzed using an (1)H nuclear magnetic resonance spectroscopy (NMR)-based metabolomics technique. This revealed clear separation of 50% methanol extracts of the FSP samples with different fermentation times in the principal component plots by combining PC1 and PC2, which cumulatively accounted for 94.2% of the variance. Major compounds contributing to the separation of 50% methanol extracts of FSP with various fermentation times were isoleucine/leucine, lactate, alanine, acetic acid, glutamine, choline, tyrosine, and phenylalanine. In addition, the (1)H NMR spectra of chloroform extracts were separated mainly by a combination of PC1 and PC3, which accounted for 72.6% of the variance. The present study suggests the usefulness of a (1)H NMR-based metabolomics approach to discriminate FSP samples subjected to different fermentation times, and this is the first report regarding metabolomic profiling of FSP.

Effect of amylose content in corn starch modification by Thermus aquatiqus 4-alpha-glucanotransferase.

Corn starches with different amylose contents were enzymatically modified using Thermus aquaticus 4-alpha-glucanotransferase (TAalphaGTase). On treating, chain length distribution of the product became broader (degree of polymerization; DP 3-40) after isoamylolysis when compared to the untreated corn starch. In addition, a variety of different size cycloamyloses were formed by glucanotransferring activity of TAalphaGTase. Cycloamyloses (CAs) with DP 5-40 were detectable in all of the TAalphaGTase-treated corn starches. The amount of CAs produced by the enzyme treatment seems to increase as amylose content of starch increased when judged by high-performance anion exchange chromatography (HPAEC) and high-performance size exclusion chromatography (HPSEC) analyses. Thus, it was suggested that the extent of modification on starch molecules was enhanced in proportion to amylose content of starch by transferring activity of TAalphaGTase. In turn, it could be useful for developing an efficient process of CA production using this enzyme.

Metabolomic Screening of Anti-Inflammatory Compounds from the Leaves of Actinidia arguta (Hardy Kiwi).

The metabolomic screening of potential anti-inflammatory compounds in the leaves of Actinidia arguta was performed by using LC-MS/MS. Ethanol extracts were prepared, and the anti-inflammatory effects were investigated based on nitric oxide (NO) synthesis and inducible nitric oxide synthase expression in lipopolysaccharide-induced RAW 264.7 macrophages. The 75% ethanol extract showed the highest inhibitory effect on nitric oxide (NO) production, and it was further separated by in vitro bioassay-guided fractionation using preparative LC with reversed-phase column separation. Through multiple steps of fractionation, sub-fraction 1-3 was finally purified, and caffeic acid derivatives, such as caffeoylthreonic acid and danshensu (salvianic acid A), were successfully identified as key anti-inflammatory compounds by LC-MS/MS and metabolomics analyses. This is the first study identifying anti-inflammatory compounds in A. arguta (Actinidia arguta) leaves through bioassay-guided fractionation and metabolomics screening. Results of this study would be useful for the application of A. arguta leaves as a nutraceutical.

Comparative Characterization of Protein Hydrolysates from Three Edible Insects: Mealworm Larvae, Adult Crickets, and Silkworm Pupae.

A comparative characterization of proteins from three edible insects-Tenebrio molitor (mealworm) larvae, Gryllus bimaculatus (cricket), and Bombyx mori (silkworm) pupae-was performed in this study. Proteins were extracted from edible insects and their hydrolysates were prepared through enzymatic hydrolysis with commercial enzymes (Flavourzyme: 12%; Alcalase: 3%). Solubility was significantly higher following enzymatic hydrolysis, while foamability was lower compared to those of the protein control. Angiotensin-converting enzyme was significantly inhibited after enzymatic hydrolysis, especially following Alcalase treatment, with IC50 values of 0.047, 0.066, and 0.065 mg/mL for G. bimaculatus, T. molitor larvae, and B. mori pupae, respectively. Moreover, the Alcalase-treated group of B. mori pupae and the T. molitor larvae group treated with a mixture of enzymes showed the effective inhibition of α-glucosidase activity. The anti-inflammatory activity of the insect hydrolysates was assessed via nitric oxide production from macrophages, and B. mori pupae samples exhibited significant activity regardless of the method of hydrolysis. These results indicate the functional properties of protein and hydrolysates from three species of edible insects, which may be useful in their future exploitation.

ß-Thujaplicin inhibits basal-like mammary tumor growth by regulating glycogen synthase kinase-3ß/ß-catenin signaling.

ß-Thujaplicin, a natural monoterpenoid, has been demonstrated to exert health beneficial activities in chronic diseases. However, it has not been studied in regulating estrogen receptor (ER) negative breast cancer. Here, we investigated the effect of ß-thujaplicin on inhibiting ER-negative basal-like breast cancer and the underlying mechanism of action using an in vitro and in vivo xenograft animal model. ß-Thujaplicin induced G0/G1 phase cell cycle arrest and regulated cell cycle mediators, cyclin D1, cyclin E, and cyclin-dependent kinase 4 (CDK 4), leading to the inhibition of the proliferation of ER-negative basal-like MCF10DCIS.com human breast cancer cells. It also modulated the phosphorylation of protein kinase B (AKT) and glycogen synthase kinase (GSK-3ß) and the protein level of ß-catenin. In an MCF10DCIS.com xenograft animal model, ß-thujaplicin significantly inhibited tumor growth, reduced tumor weight, and regulated the expression of cell cycle proteins, phosphorylation of AKT and GSK-3ß, and protein level of ß-catenin in the tumor tissues. These results demonstrate that ß-thujaplicin can suppress basal-like mammary tumor growth by regulating GSK-3ß/ß-catenin signaling, suggesting that ß-thujaplicin may be a potent chemopreventive agent against the basal-like subtype of breast cancer.

Enzymatic synthesis of dimaltosyl-beta-cyclodextrin via a transglycosylation reaction using TreX, a Sulfolobus solfataricus P2 debranching enzyme.

Di-O-alpha-maltosyl-beta-cyclodextrin ((G2)(2)-beta-CD) was synthesized from 6-O-alpha-maltosyl-beta-cyclodextrin (G2-beta-CD) via a transglycosylation reaction catalyzed by TreX, a debranching enzyme from Sulfolobus solfataricus P2. TreX showed no activity toward glucosyl-beta-CD, but a transfer product (1) was detected when the enzyme was incubated with maltosyl-beta-CD, indicating specificity for a branched glucosyl chain bigger than DP2. Analysis of the structure of the transfer product (1) using MALDI-TOF/MS and isoamylase or glucoamylase treatment revealed it to be dimaltosyl-beta-CD, suggesting that TreX transferred the maltosyl residue of a G2-beta-CD to another molecule of G2-beta-CD by forming an alpha-1,6-glucosidic linkage. When [(14)C]-maltose and maltosyl-beta-CD were reacted with the enzyme, the radiogram showed no labeled dimaltosyl-beta-CD; no condensation product between the two substrates was detected, indicating that the synthesis of dimaltosyl-beta-CD occurred exclusively via transglycosylation of an alpha-1,6-glucosidic linkage. Based on the HPLC elution profile, the transfer product (1) was identified to be isomers of 6(1),6(3)- and 6(1),6(4)-dimaltosyl-beta-CD. Inhibition studies with beta-CD on the transglycosylation activity revealed that beta-CD was a mixed-type inhibitor, with a K(i) value of 55.6 micromol/mL. Thus, dimaltosyl-beta-CD can be more efficiently synthesized by a transglycosylation reaction with TreX in the absence of beta-CD. Our findings suggest that the high yield of (G2)(2)-beta-CD from G2-beta-CD was based on both the transglycosylation action mode and elimination of the inhibitory effect of beta-CD.