Enzymatic Process for High-Yield Turanose Production and Its

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Enzymatic Process for High-Yield Turanose Production and Its Potential Property as an Adipogenesis Regulator Min-Oh Park,† Byung-Hoo Lee,‡ Eunjin Lim,§ Ji Ye Lim,§ Yuri Kim,§ Cheon-Seok Park,∥ Hyeon Gyu Lee,⊥ Hee-Kwon Kang,*,† and Sang-Ho Yoo*,† †

Department of Food Science and Technology, and Carbohydrate Bioproduct Research Center, Sejong University, Gunja-Dong, Gwangjin-Gu, Seoul 143-747, Republic of Korea ‡ Department of Food Science and Biotechnology, College of BioNano Technology, Gachon University, Seongnam, Gyeonggi-do 461-701, Republic of Korea § Department of Nutritional Science and Food Management, Ewha Womans University, Seoul 120-750, Republic of Korea ∥ Graduate School of Biotechnology, and Institute of Life Science and Resources, Kyung Hee University, Seocheon, Kiheung, Yongin 446-701, Republic of Korea ⊥ Department of Food and Nutrition, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791, Republic of Korea ABSTRACT: Turanose is a sucrose isomer naturally existing in honey and a promising functional sweetener due to its low glycemic response. In this study, the extrinsic fructose effect on turanose productivity was examined in Neisseria amylosucrase reaction. Turanose was produced, by increasing the amount of extrinsic fructose as a reaction modulator, with high concentration of sucrose substrate, which resulted in 73.7% of production yield. In physiological functionality test, lipid accumulation in 3T3-L1 preadipocytes in the presence of high amounts of pure glucose was attenuated by turanose substitution in a dose-dependent manner. Turanose treatments at concentrations representing 50%, 75%, and 100% of total glucose concentration in cell media significantly reduced lipid accumulation by 18%, 35%, and 72%, respectively, as compared to controls. This result suggested that turanose had a positive role in controlling adipogenesis, and enzymatic process of turanose production has a potential to develop a functional food ingredient for controlling obesity and related chronic diseases. KEYWORDS: turanose, amylosucrase, fructose, adipogenesis, Neisseria polysaccharea

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INTRODUCTION Amylosucrase from Neisseria polysaccharea (NpAS) is a glucosyltransferase (E.C. 2.4.1.4) that primarily catalyzes the synthesis of linear α-1,4-linked glucans by transferring glucose from sucrose and releasing fructose.1,2 NpAS was cloned and expressed in Escherichia coli systems.3−5 Because amylose-like glucose polymers accumulated in the culture media of NpAS, amylosucrase, which is responsible for producing this biomacromolecule, was expected to be secreted outside cells during the growth.4 On the basis of the amino acid sequences, NpAS is classified into the glycoside hydrolase family 13, which harbors unique (β/α)8 barrels.4,5 The structural conformation of NpAS protein was determined, and some important amino acid residues in the protein were identified for glycogen binding and chain elongation.1,6−8 Furthermore, information on the donor and acceptor binding sites suggested that the proportion of resulting products from the NpAS reaction may be regulated by targeted enzyme mutagenesis.8 NpAS generated diverse products from the reaction with sucrose as a sole substrate such as turanose, trehalulose, glucose, fructose, and linear soluble/insoluble α-glucans.2 These insoluble glucans, which were produced from sucrose, were thought to be formed through chain−chain association and concomitant crystallization of elongated α-glucan chains by successive transfers of glucosyl moieties of sucrose on the acceptor molecules.9 The maltooligosaccharide disproportionation10 and chain elongation of glycogen11 suggest that diverse precursor molecules © XXXX American Chemical Society

can be applied as acceptor molecules by NpAS. The production of sucrose isomers, turanose and trehalulose, indicated that fructose might also be utilized as an acceptor.2 Sucrose is widely utilized as a sweetener in the food industry. However, excessive intakes of sucrose may cause tooth decay, obesity, and diabetes mellitus, which increases the necessity for unfermentable low-calorie alternative sweeteners. Turanose, 3-O-α-D-glucosyl-D-fructose, is a sucrose isomer that naturally exists in honey, and provides the sweetness as well as slowly hydrolyzable property as compared to sucrose, which can be applied as a potential low glycemic sweetener similar to isomaltulose.12−14 This novel disaccharide displayed one-half of the sweetness as compared to sucrose,15 and cannot be utilized by Streptococcus mutans.16 Additionally, its hydrolysis rate by a rat intestinal enzyme mixture was as low as 54% of sucrose and 6% of maltose at a 5 mM concentration level.16 Currently, obesity is considered to be an important public health problem and a major risk factor for many chronic diseases such as diabetes mellitus, coronary heart disease, stroke, dyslipidemia, and certain types of cancer.17 In addition, adipose tissue has been found to play a pivotal role in regulating energy metabolism. For adipocyte differentiation, induction of adipogenic Received: January 21, 2016 Revised: May 23, 2016 Accepted: May 25, 2016

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DOI: 10.1021/acs.jafc.5b05849 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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and 0.75 M) concentrations, different volumes of sucrose and fructose stock solutions and 50 mM Tris−HCl buffer (pH 7.0) were premixed in 50 mL tubes. After the tube was preheated at 35 °C for 10 min, 400 U of enzyme per liter was added to prepare 10 mL of the total reaction volume. The enzymatic reaction then was initiated and performed in a water bath with a constant shaking speed of 120 rpm. At preassigned time intervals, 0.5 mL of the reaction mixture was collected and heated in a boiling water bath for 10 min to inactivate the enzyme. All experiments were performed in triplicate. Carbohydrate Analysis by HPAEC-PAD. The analysis of NpAS reaction products including glucose, fructose, sucrose, turanose, trehalulose, and maltooligosaccharides was performed using highperformance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD), using an ED40 electrochemical detector (Dionex, Sunnyvale, CA). The CarboPac PA1 analytical column (4 × 250 mm, Dionex) and its guard column (4 × 50 mm, Dionex) were maintained at 30 °C. Before injection, the reaction mixture was, first, centrifuged at 9000g for 10 min to remove the denatured enzyme protein and any insoluble linear α-(1,4)-glucan products. The reaction mixture then was diluted with distilled water and was filtered through a syringe membrane filter (0.45 μm). The injection volume was 25 μL, and the elution of individual sugars was achieved using 150 mM NaOH solution at a flow rate of 1.0 mL/min. A gradient of sodium acetate (from 0 to 600 mM in 100 min) in 150 mM NaOH was applied at a flow rate of 1.0 mL/min. The amount of sucrose, fructose, and turanose was determined by comparison with their corresponding standard curves. Cell Culture and Differentiation of 3T3-L1 Cells. The preadipocyte cell line, 3T3-L1 (American Type Culture Collection, Rockville, MD), was maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen, Carlsbad, CA) without glucose with 10% calf serum (Invitrogen) and 1% penicillin-streptomycin (100 U/mL and 100 μg/mL; Invitrogen). To differentiate the 3T3-L1 cells into adipocytes, the 3T3-L1 cells were allowed to reach confluence before the medium was changed to DMEM containing 10% fetal bovine serum (FBS) (Hyclone, Logan, UT), 10 μg/mL insulin, 1 μM dexamethasone, and 500 μM 3-isobutyl-1-methylxanthine. The cells then were treated with 25 mM glucose or various doses of turanose. For these experiments, 50%, 75%, and 100% of the total glucose concentration was replaced by turanose (T 50, T 75, and T 100, respectively). After 2 days, the medium was changed to DMEM containing 10% FBS, 1% penicillin-streptomycin, and 10 μg/mL insulin, and the cells were grown for an additional 3 days. Cell Viability Assays. Cell viability was assessed using a colorimetric MTT assay. Briefly, 3T3-L1 cells were seeded in 96-well plates and were treated with 25 mM glucose or various doses of turanose where 50%, 75%, and 100% of the total glucose concentration was replaced by turanose (T 50, T 75, and T 100, respectively). After 24 and 48 h, each set of cells was washed with PBS and incubated with a 0.5 mg/mL MTT stock solution (200 μL). After 4 h, the supernatants were aspirated and DMSO (200 μL) was added to each well to dissolve the formazan crystals present. Absorbance values at 540 nm were measured with a microplate reader (Molecular Devices, Sunnyvale, CA). The results are expressed as the percentage of viable cells with the absorbance values for the control group representing 100% viability as compared to the control cells. Oil Red O Staining. To determine whether the 3T3-L1 cells had undergone differentiation, they were grown in 6-well plates and were stained with an Oil red O solution (Sigma, St. Louis, MO). After 20 min of staining, the cells were washed twice with PBS and were then fixed in 10% formalin in PBS. After 1 h, cell morphology was examined with an ECLIPSE Ti−S inverted microscope (Nikon Instrument Co. Ltd., Tokyo, Japan). The cells were subsequently washed with distilled water and were incubated with 1 mL of isopropanol for 10 min. Lipid accumulation was measured with a microplate reader (Molecular Devices, Sunnyvale, CA) that detected absorbance values at 492 nm. Quantitative Real-Time PCR. Total RNA was isolated from differentiated 3T3-L1 cells using a TRIzol reagent (Invitrogen), and cDNA was prepared by reverse transcription using a RevertAid First Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania).

gene expression occurs in early stages of differentiation and continues until the terminal differentiation stage when lipid droplets accumulate.18 It has been reported that high levels of glucose promote adipose-derived stem cell differentiation. Furthermore, mRNA levels of sterol regulatory element-binding protein-1C (SREBP-1C) and peroxisome proliferator-activated receptor γ (PPARγ) have been found to be up-regulated under high glucose conditions as compared to low glucose conditions during lipid accumulation.19 There are other naturally occurring sugars (e.g., trehaloase, leucrose, and isomaltulose) that display properties that make them potential alternative sweeteners, but the lack of low-cost mass production processes makes their commercialization challenging. In a previous effort to establish an industrially feasible turanose production process, a dual enzyme treatment using cyclomaltodextrin glucanotransferase and glucoamylase was adopted.15 In the above-mentioned report, the production yield of turanose from α-cyclodextrin and D-fructose mixture was approximately 45% (w/v) after 24 h of reaction. Another study by Albenne et al. (2004) found that turanose could be produced with 14% yield from the NpAS reaction system in the presence of 100 mM sucrose substrate.7 In our previous study, it was also shown that turanose was maximally produced with a 56.2% conversion yield under the reaction condition of 2 M sucrose level at 35 °C.20 It is well-known that either the addition of extra acceptor materials or the increase of acceptor to donor ratio enhances the efficiency of the enzymatic transglycosylating reaction.21,22 Thus, we added extrinsic fructose and used sucrose as a possible acceptor molecule in the NpAS reaction to facilitate turanose production. The result of this research can be used to optimize conditions for producing greater yields of turanose, which can then be applied as a novel sweetener to regulate lipid accumulation.

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MATERIALS AND METHODS

Materials. Fructose, sucrose, glucose (G1), maltose (G2), maltotriose (G3), maltotetraose (G4), maltopentaose (G5), maltohexaose (G6), and maltoheptaose (G7) were purchased from Sigma−Aldrich Chemical Co. (St. Louis, MO). Turanose was obtained from TCI (Tokyo, Japan), and trehalulose was purchased from Mitsui Sugar Co., Ltd. (Tokyo, Japan). Other chemicals used were of analytical grade. Preparation of NpAS. The NpAS preparation was performed as previously described.23 Neisseria polysaccharea (ATCC 43768) was purchased from the American Type Culture Collection (ATCC, Manassas, VA), and Escherichia coli TOP10 and BL21 were used as cloning and expression host, respectively. Gene cloning, expression, enzyme extraction, and purification were carried out as previously described.23 Nickel-nitrilotriacetic acid (Ni-NTA) affinity column chromatography (Qiagen, Hilden, Germany) was used to purify the 6× his-tagged recombinant NpAS. Purified protein content of the purified NpAS that was used in this study was 4.38 mg/mL, which was determined using the Bradford method with bovine serum albumin as a standard.24 Determination of Enzyme Activity. To test the enzyme activity of amylosucrase, an assay was performed in 50 mM Tris−HCl buffer (pH 7.0) with 0.1 M sucrose as a substrate at 35 °C for 30 min. One unit (U) of NpAS activity corresponds to the amount of enzyme that catalyzes the release of 1 μmol of reducing sugars per minute under the assay conditions.25 The amount of reducing sugars released in the reaction was determined using the dinitrosalicylic acid (DNS) method with fructose as a standard.26 Turanose Production from Sucrose by NpAS. Sucrose and fructose stock solutions were prepared in 50 mM Tris−HCl buffer (pH 7.0) with concentrations of 4.0 and 3.0 M, respectively. To achieve the intended sucrose (1.0, 1.5, and 2.0 M) and fructose (0.25, 0.50, B

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Journal of Agricultural and Food Chemistry To analyze mRNA levels, the qPCR for each gene was performed using Rotor-Gene SYBR Green PCR Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Real-time PCR was performed by using a Rotor-Gene Q instrument (Qiagen, Austin, TX) that was programmed as follows: 95 °C for 15 min, followed by 40 cycles of 94 °C for 15 s, 55 °C for 30 s, and 70 °C for 30 s. Each reaction consisted of 2 μL of cDNA, 5 μL of SYBR Green PCR master mix, 1 μL of forward primer, 1 μL of reverse primer, and 1 μL of RNase-free water. All data were normalized to the housekeeping gene, glyceraldeyde 3-phosphate dehydrogenase (GAPDH), which was detected as an internal control. The primers used for PCR included the following: mouse SREBP-1C, 5′-TAG AGC ATA TCC CCC AGG TG-′3 (forward) and 5′-GGT ACG GGC CAC AAG AAG TA-′3 (reverse); PPARγ, 5′-CGA GAA GGA GAA GCT GTT GG-′3 (forward) and 5′-TCA GCG GGA AGG ACT TTA TGT ATG-′3 (reverse); FAS, 5′-CTT CGC CAA CTC TAC CAT GG-′3 (forward) and 5′-TTC CAC ACC CAT GAG CGA GT-′3 (reverse); GAPDH, 5′-GCC TTC CGT GTT CCT ACC C-′3 (forward) and 5′-TGC CTG CTT CAC CAC CTT C-′3 (reverse). Statistical Analysis. The results presented are the mean ± standard error of the mean (SEM) for each group. Comparisons were made using one-way analysis of variance (ANOVA), followed by Newman−Keuls multiple comparison test in the Graph-Pad Prism 3.0 software (Graph-Pad Software, Inc., San Diego, CA). A P-value less than 0.05 was considered statistically significant. At least three independent experiments were performed for each statistical analysis.

system was more effective in improving the turanose production (Table 1). Fructose has been recognized as a competitive inhibitor against NpAS2 as well as amylosucrases from other Neisseria species.27,28 However, the action mode of fructose on NpAS reaction patterns has not been carefully addressed. In this study, the addition of extrinsic fructose neither attenuated vtur by NpAS nor accelerated transglucosylation onto the fructose molecule as an acceptor. If the extrinsic or released fructose from sucrose acts as an acceptor in the transglucosylating reaction of NpAS, the production rate of sucrose isomers increases with the initial fructose concentration ([fructose]ini). However, the production rate of sucrose isomers including turanose remained constant, regardless of [fructose]ini (Table 1). This independence of the production rate of sucrose isomers on [fructose]ini in the NpAS reaction implies that instead of using extrinsic fructose or released fructose, the fructose moiety derived from sucrose was directly utilized as an acceptor to produce turanose without being released from the enzyme. The biocatalytic conversion process of sucrose by NpAS can be divided into two unique reaction patterns: linear α-glucan production and sucrose isomers production. When the [sucrose]ini increased from 1.0 to 2.0 M, the consumption rate of sucrose decreased from 47.0 to 36.4 mM h−1, possibly resulting from the substrate inhibition effect (Table 1). Furthermore, the addition of extrinsic fructose up to 0.75 M clearly showed a decrease in sucrose consumption velocity within the tested range of [sucrose]ini. Meanwhile, the production rate of linear α-glucan decreased along with increasing [fructose]ext (Figure 2). Regardless of [fructose]ext in this reaction mixture, the discrepancies between the rates of sucrose consumption (vsuc) and vfru were almost identical within the same [sucrose]ini and were 1.84, 2.06, and 2.19 mM h−1 at 1.0, 1.5, and 2.0 M [sucrose]ini, respectively. This observation indicated that the rate discrepancy became larger when [sucrose]ini increased. Furthermore, it was clear that higher [sucrose]ini increased the production rate of sucrose isomers more effectively than the increase of [fructose]ext in the NpAS reaction. However, there was an obvious decrease in vsuc that was observed along with the increase in [fructose]ext, which resulted in a linear α-glucan production rate decrease (Table 1). Although both vsuc and vfru decreased along with [fructose]ext, the production rate of turanose (vtur), which was the major sucrose isomer product in this reaction system, slightly increased.

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RESULTS AND DISCUSSION Development of High-Yield Bioprocess for Turanose Production. As shown in Table 1, the rate of turanose production at 1.0 M sucrose was 15.7 mM h−1, and the yield of turanose was 31.0% (w/v). At 2.0 M sucrose, 51.1% turanose (w/v) was produced at a rate of 21.9 mM h−1. The increase in [sucrose]ini contributed to improving turanose yield as well as acceleration of vtur. By adding 0.75 M fructose, the turanose yield increased from 31.0% to 65.4% at 1 M sucrose and from 51.1% to 73.7% at 2.0 M sucrose (Table 1). At 1.0 and 1.5 M sucrose, the vtur clearly increased, and a lower increase in the vtur was observed at 2.0 M sucrose (Figure 1). The positive effect of extrinsic fructose on enhancing turanose yield was much greater than that of initial sucrose. The turanose yield after adding 0.75 M extrinsic fructose substantially increased by 34.4% and 22.6% at 1.0 and 2.0 M [sucrose]ini, respectively. As compared to the yield enhancement of 20.1% by increasing [sucrose]ini from 1.0 to 2.0 M, the 0.75 M [fructose]ext in NpAS reaction

Table 1. Reaction Velocity Parameters of NpAS and Turanose Production Yield reaction velocity (×10−2 M/h)a vfru

reaction condition 1.0 M suc +0.25 +0.50 +0.75 1.5 M suc +0.25 +0.50 +0.75 2.0 M suc +0.25 +0.50 +0.75

M fruc M fruc M fruc M fruc M fruc M fruc M fruc M fruc M fruc

2.80 1.29 0.57 0.33 1.90 1.04 0.60 0.35 1.34 0.80 0.42 0.27

± ± ± ± ± ± ± ± ± ± ± ±

Δ(vsuc − vfru)

vsuc 0.12 0.10 0.05 0.01 0.02 0.02 0.01 0.01 0.02 0.02 0.02 0.01

4.70 3.05 2.37 2.21 3.90 3.11 2.75 2.35 3.64 2.99 2.64 2.32

± ± ± ± ± ± ± ± ± ± ± ±

0.08 0.02 0.02 0.04 0.05 0.09 0.01 0.02 0.07 0.12 0.06 0.30

1.90 1.76 1.80 1.88 2.00 2.07 2.15 2.00 2.30 2.19 2.22 2.05

turanose yield (%)b

vtur 1.57 1.62 1.66 1.67 1.84 1.94 2.04 1.97 2.19 2.24 2.21 2.16

± ± ± ± ± ± ± ± ± ± ± ±

0.02 0.02 0.02 0.02 0.03 0.01 0.01 0.02 0.01 0.05 0.02 0.05

31.0 47.8 58.4 65.4 37.3 51.7 59.3 66.7 51.1 60.1 67.5 73.7

± ± ± ± ± ± ± ± ± ± ± ±

1.0 0.2 0.7 0.2 0.7 0.4 0.5 0.5 0.3 0.1 0.6 0.5

a vfru = the production rate of fructose, vsuc = the consumption rate of sucrose, Δ(vsuc − vfru) = the difference between the rates of sucrose consumption and fructose production, and vtur = the production rate of turanose. bTuranose yield (%) = (the produced amount of turanose)/ (the initial amount of sucrose) × 100. suc, sucrose; fru, fructose.

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Figure 2. HPAEC analysis of the linear α-glucan production pattern of NpAS depending on different concentrations of extrinsic fructose. NpAS reacted with 1.0 (A), 1.5 (B), and 2.0 M (C) sucrose and 0 M (a), 0.25 M (b), 0.50 M (c), and 0.75 M (d) of extrinsic fructose at each sucrose concentration. Standard materials at the bottom of each panel are as follows: 1, glucose; 2, maltose; 3, maltotriose; 4, maltotetraose; 5, maltopentaose; 6, maltohexaose; 7, maltoheptaose.

Figure 1. Effect of extrinsic fructose on the turanose production rate of NpAS. NpAS reacted with 1.0 (A), 1.5 (B), and 2.0 M (C) sucrose and 0 (○), 0.25 (□), 0.50 (△), and 0.75 M (◇) of extrinsic fructose at each sucrose concentration.

Thus, it was suggested that the noticeable decrease in vsuc might directly decrease the linear α-glucan synthesis without disturbing turanose production. Turanose Suppresses Lipid Accumulation in Adipocytes by Down-Regulating mRNA Expression of SREBP1C, PPARγ, and FAS. Following the differentiation of 3T3-L1 cells, cell viability was evaluated using MTT assays. No significant difference was observed among the tested groups (Figure 3A and B). However, Oil red O staining of the differentiated cell groups showed that in the presence of 25 mM glucose, lipid accumulation in adipocyte cells was significantly increased (Figure 3C). In contrast, treatment with turanose significantly reduced lipid accumulation in a dose-dependent manner (Figure 3D). Treatments with turanose at concentrations

representing 50%, 75%, and 100% of the total glucose concentration (25 mM) in cell media significantly reduced lipid accumulation by 18%, 35%, and 72%, respectively, as compared to the control cells, which contained 100% glucose at a concentration of 25 mM in cell media (p < 0.001 for all). An increased adipocyte differentiation under high glucose level conditions suggests that cross-talk occurs between signal transduction pathways that mediate obesity and diabetes signaling. Additionally, this cross-talk provides positive feedback to enhance metabolic dysfunction in a diabetic state. Moreover, the results of the present study are consistent with those involving another analogue, palatinose (isomaltulose), D

DOI: 10.1021/acs.jafc.5b05849 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 3. Turanose suppresses lipid accumulation in 3T3-L1 adipocytes. Cell viability of differentiated 3T3-L1 cells was analyzed in MTT assays after 24 h (A) or 48 h (B) incubation (A,B). Lipid accumulation was examined and quantified after the 3T3-L1 cells were stained with Oil red O in 6-well plates. a, Ctrl; b, Glu; c, T 50; d, T 75; and e, T 100. The stained cells were imaged at 100× magnification (C,D). All data were analyzed using one-way ANOVA and Newman−Keuls multiple comparison test. Different letters for given bars indicate the values that significantly differ from each other (p < 0.001). Glu, treatment with 25 mM glucose in the cell media; T 50, T 75, and T 100, treatments where 50%, 75%, or 100% of the total glucose concentration (25 mM) was replaced by turanose, respectively, in the cell media.

Figure 4. Turanose down-regulates the mRNA expression of SREBP-1C, PPARγ, and FAS in 3T3-L1 cells (A−C). The levels of SREBP-1C, PPARγ, and FAS mRNA were measured using quantitative real time PCR, and levels of GAPDH were used as a loading control. All data were analyzed using one-way ANOVA and Newman−Keuls multiple comparison test. Different letters for given bars indicate the values that significantly differ from each other (p < 0.05). Glu, treatment with 25 mM glucose in the cell media; T 50, T 75, and T 100, treatments where 50%, 75%, or 100% of the total glucose concentration (25 mM) was replaced by turanose, respectively, in the cell media. E

DOI: 10.1021/acs.jafc.5b05849 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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(3) Remaud-Simeon, M.; Albaret, F.; Canard, B.; Varlet, I.; Colonna, P.; Willemot, R. M.; Monsan, P. Studies on a recombinant amylosucrase. In Progress in Biotechnology; Steffen, B., Petersen, B. S., Sven, P., Eds.; Elsevier: New York, 1995; Vol. 10, pp 313−320. (4) Buttcher, V.; Welsh, T.; Willmitzer, L.; Kossmann, J. Cloning and characterization of the gene for amylosucrase from Neisseria polysaccharea: production of a linear alpha-1,4-glucan. J. Bacteriol. 1997, 179, 3324−3330. (5) Potocki de Montalk, G.; Remaud-Simeon, M.; Willemot, R. M.; Planchot, V.; Monsan, P. Sequence analysis of the gene encoding amylosucrase from Neisseria polysaccharea and characterization of the recombinant enzyme. J. Bacteriol. 1999, 181, 375−381. (6) Sarcabal, P.; Remaud-Simeon, M.; Willemot, R.; Potocki de Montalk, G.; Svensson, B.; Monsan, P. Identification of key amino acid residues in Neisseria polysaccharea amylosucrase. FEBS Lett. 2000, 474, 33−37. (7) Albenne, C.; Skov, L. K.; Mirza, O.; Gajhede, M.; Feller, G.; D’Amico, S.; Andre, G.; Potocki-Veronese, G.; van der Veen, B. A.; Monsan, P.; Remaud-Simeon, M. Molecular basis of the amylose-like polymer formation catalyzed by Neisseria polysaccharea amylosucrase. J. Biol. Chem. 2004, 279, 726−734. (8) Albenne, C.; Potocki de Montalk, G.; Monsan, P.; Skov, L. K.; Mirza, O.; Gajhede, M.; Remaud-Simeon, M. Site-directed mutagenesis of key amino acids in the active site of amylosucrase from Neisseria polysaccharea. Biol. Brat. 2002, 57, 119−128. (9) Kim, B. S.; Kim, H. S.; Hong, J. S.; Huber, K. C.; Shim, J. H.; Yoo, S.-H. Effects of amylosucrase treatment on molecular structure and digestion resistance of pre-gelatinised rice and barley starches. Food Chem. 2013, 138, 966−975. (10) Albenne, C.; Skov, L. K.; Mirza, O.; Gajhede, M.; PotockiVeronese, G.; Monsan, P.; Remaud-Simeon, M. Maltooligosaccharide disproportionation reaction: an intrinsic property of amylosucrase from Neisseria polysaccharea. FEBS Lett. 2002, 527, 67−70. (11) Potocki de Montalk, G.; Remaud-Simeon, M.; Willemot, R. M.; Monsan, P. Characterisation of the activator effect of glycogen on amylosucrase from Neisseria polysaccharea. FEMS Microbiol. Lett. 2000, 186, 103−108. (12) White, J. W., Jr; Hoban, N. Composition of honey. IV. Identification of the disaccharides. Arch. Biochem. Biophys. 1959, 80, 386−392. (13) Takewaki, S.-i.; Kimura, A.; Kubota, M.; Chiba, S. Substrate specificity and subsite affinities of honeybee α-glucosidase II. Biosci., Biotechnol., Biochem. 1993, 57, 1508−1513. (14) Dahlqvist, A. Characterization of hog intestinal invertase as a glucosido-invertase. III. Specificity of purified invertase. Acta Chem. Scand. 1960, 14, 63−71. (15) Shibuya, T.; Mandai, T.; Kubota, M.; Fukuda, S.; Kurimoto, M.; Tsujisaka, Y. Production of turanose by cyclomaltodextrin glucanotransferase from Bacillus stearothermophilus. J. Appl. Glycosci. 2004, 51, 223−227. (16) Hodoniczky, J.; Morris, C. A.; Rae, A. L. Oral and intestinal digestion of oligosaccharides as potential sweeteners: A systematic evaluation. Food Chem. 2012, 132, 1951−1958. (17) Larsson, B.; Björntorp, P.; Tibblin, G. The health consequences of moderate obesity. Int. J. Obes. 1981, 5, 97−116. (18) Gregoire, F. M.; Smas, C. M.; Sul, H. S. Understanding adipocyte differentiation. Physiol. Rev. 1998, 78, 783−809. (19) Aguiari, P.; Leo, S.; Zavan, B.; Vindigni, V.; Rimessi, A.; Bianchi, K.; Franzin, C.; Cortivo, R.; Rossato, M.; Vettor, R.; Abatangelo, G.; Pozzan, T.; Pinton, P.; Rizzuto, R. High glucose induces adipogenic differentiation of muscle-derived stem cells. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 1226−1231. (20) Wang, R.; Bae, J. S.; Kim, J. H.; Kim, B. S.; Yoon, S. H.; Park, C. S.; Yoo, S.-H. Development of an efficient bioprocess for turanose production by sucrose isomerisation reaction of amylosucrase. Food Chem. 2012, 132, 773−779. (21) Jaitak, V.; Kaul, V. K.; Kumar, N.; Singh, B.; Savergave, L.; Jogdand, V.; Nene, S. Simple and efficient enzymatic transglycosylation

which is composed of glucose and fructose. In a study by Sato et al., ingestion of palatinose by Zucker fatty rats led to a reduced accumulation of fat and a lower respiratory quotient.29 To investigate the molecular changes that mediate the effects of turanose on adipogenesis in differentiated 3T3-L1 cells, mRNA levels of SREBP-1C, PPARγ, and FAS were analyzed. All three levels were higher in the presence of 25 mM glucose than in the control (p < 0.001 for all). Conversely, all three mRNA levels were significantly lower in the presence in a dosedependent manner (p < 0.05 for all) (Figure 4). These three genes represent early adipogenic markers, and they have been shown to play important roles in the differentiation of adipocytes and lipogenesis. SREBP-1C, an adipogenic transcription factor, is highly expressed in adipocytes and hepatocytes and contributes to fatty acid synthesis by controlling other lipogenic genes, including FAS and acetyl CoA carboxylase (ACC).30,31 With increased expression of FAS, mature adipocytes largely accumulate lipid content via de novo long chain fatty acid synthesis and triacylglycerol esterification in response to lipogenic hormones.32 Several dietary carbohydrates have been tested for their effects on fat accumulation. Recently, it has been reported that D-xylose suppresses adipogenesis and is able to regulate lipid metabolism genes, including SREBP-1C, CCAAT/enhancer-binding protein α (CEBP/α), FAS, and PPARγ in obese mice.33 Moreover, D-psicose, a C-3 epimer of D-fructose, has been found to reduce visceral fat mass in high-fat diet-induced obese rats.34 The results of the present study clearly demonstrated that 20 mM turanose had the capacity to suppress adipogenesis by downregulating mRNA expression of SREBP-1C, PPARγ, and FAS. Thus, turanose at cellular levels may potentially mediate beneficial health effects by suppressing preadipocyte differentiation to prevent obesity and related metabolic diseases. However, further studies are needed to confirm the involved signal transduction pathways, and clinical studies are needed to explore the potential applications in humans.

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AUTHOR INFORMATION

Corresponding Authors

*Tel.: +82-2-3408-3911. Fax: +82-2-3408-3899. E-mail: [email protected]. *Tel.: +82-2-3408-3221. Fax: +82-2-3408-4319. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (no. 2015R1A2A1A15056119). This research was also supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2013R1A1A1011762).

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REFERENCES

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DOI: 10.1021/acs.jafc.5b05849 J. Agric. Food Chem. XXXX, XXX, XXX−XXX