(12) United States Patent (10) Patent N0.: US 8,653,256 B2 Yoo Et A1
US008653256B2
(12) United States Patent (10) Patent N0.: US 8,653,256 B2 Yoo et a1. (45) Date of Patent: Feb. 18, 2014
(54) METHOD OF PRODUCING TURANOSE (56) References Cited USING AMYLOSUCRASE, AND SWEETENER USING THE TURANOSE U.S. PATENT DOCUMENTS (75) Inventors: Sang-Ho Yoo, Seoul (KR); Ren Wang, 6,265,635 Bl 7/2001 Kossmann et al. Seoul (KR); Cheon-Seok Park, 6,818,421 B2 11/2004 Kossmann et al. Seongnam-si (KR) 7,456,003 B2 11/2008 Kossmann et al. (73) Assignee: Industry-Academia Cooperation FOREIGN PATENT DOCUMENTS Group of Sej ong University, Seoul JP 3182665 B2 4/2001 (KR) KR 10-0352532 B1 2/2004 KR 10-2009-0081491 A 7/2009 ( * ) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 OTHER PUBLICATIONS U.S.C. 154(b) by 0 days. Albenne et al. Molecular Basis of the Amylose-like Polymer Forma tion Catalyzed by Neisseria polysaccharea Amylosucrase. 279:726 (21) Appl. No.: 13/497,609 734, 2004* (22) PCT Filed: Jan. 31, 2011 Gabrielle Potocki De Montalk et al., “Amylosucrase from Neisseria polysaccharea: novel catalytic properties”, FEBS Letters, 2000, pp. (86) PCT N0.: PCT/KR2011/000645 219-223, vol. 471. Shigeyuki Hamada, “Role of sweeteners in the etiology and preven § 371 (0X1)’ tion of dental caries”, Pure Appl. Chem., 2002, pp. 1293-1300, vol. (2), (4) Date: Mar. 22, 2012 74, No. 7. Mary An Godshall, “The Expanding World of Nutritive and Non (87) PCT Pub. No.: WO2012/060519 Nutritive Sweeteners”, Sugar Journal, Jan. 2007, pp. 12-20.
PCT Pub. Date: May 10, 2012 * cited by examiner
(65) Prior Publication Data Primary Examiner * Wu-Cheng Winston Shen US 2012/0238744 A1 Sep. 20, 2012 Assistant Examiner * Yi-Horng Shiao (74) Attorney, Agent, or Firm * Sherr & Jiang, PLLC (30) Foreign Application Priority Data (57) ABSTRACT Nov. 2, 2010 (KR) ...... 10-2010-0108174 The present invention relates a method of producing turanose (51) Int. Cl. using amylosucrase and a sweetener including the turanose. C12P 19/18 (2006.01) This method enables production of high-purity turanose (52) US. Cl. through an enzymatic reaction occurring by treating a solu USPC ...... 536/123.13 tion including only sucrose or a solution including fructose (58) Field of Classi?cation Search and sucrose with amylosucrase. USPC ...... 536/123.13 See application ?le for complete search history. 6 Claims, 3 Drawing Sheets US. Patent Feb. 18, 2014 Sheet 1 of3 US 8,653,256 B2
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?gure 6 US 8,653,256 B2 1 2 METHOD OF PRODUCING TURANOSE present invention, Wherein the method includes acting amy USING AMYLOSUCRASE, AND SWEETENER losucrase on a sucrose solution to produce turanose, and USING THE TURANOSE purifying the reaction product. CROSS REFERENCE TO PRIOR APPLICATIONS Advantageous Effects This application is a National Stage Patent Application of PCT International Patent Application No. PCT/KR2011/ According to the present invention, the method of produc 000645 (?led on Jan. 31, 2011) under 35 U.S.C. §371,Which ing turanose using amylosucrase enables production of high claims priority to Korean Patent Application No. 10-2010 purity turanose through an enZymatic reaction occurring by 0108174 (?led on Nov. 2, 2010), Which are all hereby incor treating a solution containing only sucrose or a solution con porated by reference in their entirety. taining fructose and sucrose With amylo sucrase. Accordingly, turanose may be very useful for use as a calorie-free sWeet TECHNICAL FIELD ener, and may function as a critical sWeetener or additive in food, cosmetic, and pharmaceutic industries. The present invention relates to a method of producing turanose using amylosucrase and a sWeetener using the tura DESCRIPTION OF DRAWINGS nose. The above and other features and advantages of the present BACKGROUND ART invention Will become more apparent by describing in detail 20 exemplary embodiments thereof With reference to the Turanose is a reducing disaccharide naturally present in attached draWings in Which: honey and its sWeetness poWer is about half that of sucrose. It FIG. 1 shoWs a HPAEC chromatogram of a non-puri?ed is an analog of sucrose, and has a chemical structure of turanose sample; 3-O-0t-D-glucopyranosyl-D-fructose. FIGS. 2 and 4 shoW a HPLC-MS chromatogram of a puri Because turano se is not fermented by a microorganism that 25 ?ed turanose sample respectively; causes dental caries, the turanose can be used as a calorie-free FIG. 3 shoWs a HPAEC chromatogram of a puri?ed tura sWeetener. Accordingly, turanose can play an important role in food, cosmetic, and pharmaceutic industries. nose sample; Japanese publications, patent No. 1993-252974 and J. FIG. 5 shoWs the puri?ed turanoses analyZed based on Appl. Glycosci., 51, 223-227, are the only references that NMR spectrum; and disclose an enZymatic process for producing turanose by 30 FIG. 6 shoWs a TLC chromatogram of a puri?ed turanose treating an aqueous solution containing an amylaceous sub sample, stance and fructose With cyclomaltodextrin glucanotrans Wherein Lanes 1 and 7 shoW results of G1(glucose)-G7 ferase. In fact, the enZymatic process includes tWo important (maltoheptaose) reference material; Lane 2 shoWs results of a steps: transglycosylation of the amylaceous substances to sucrose reference material; Lane 3 shoWs results of a turanose fructose by cyclomaltodextrin glucanotransferase and hydro 35 reference material; Lane 4 shoWs results of a fructose refer lyZation of the ot-1,4-glucan chains of the transfer products by ence material; Lane 5 shoWs results of a non-puri?ed turanose glucoamylase. sample; and Lane 6 shoWs results of a puri?ed turanose However, a method of easily producing high-purity and sample. high-yield turano se has not been developed. Accordingly, it is very important to develop such a method of producing tura 40 BEST MODE nose so as to produce a calorie-free sWeetener for use in foods, and additives for use in cosmetic products or medical prod An aspect of the present invention provides a method of ucts. producing turanose according to an embodiment of the present invention, Wherein the method includes acting amy DISCLOSURE 45 losucrase on a sucrose solution to produce a reaction product, and purifying the reaction product to obtain a turanose. Technical Problem Amylosucrase used herein may be derived from any one of microorganism selected from the group consisting of micro In response, the inventors of the present invention studied organism Neisseriapolysaccharea, Deinococcus geotherma on hoW to produce high-purity turanose With high ef?ciency, 50 lis, and Alleromonas macleodii. For example, the amylosu and found that by acting amylosucrase as an Zymogen on a crase may be derived from Neisseriapolysaccharea, and may substrate, such as sucrose, or the like, turanose Was produced be isolated by using a method disclosed in US. Pat. No. With high e?iciency, and also, through a simple puri?cation 6,265,635. process, high-purity turanose Was able to be produced With According to an embodiment of the present invention, high-e?iciency, thereby completing the present invention. 55 amylosucrase shoWs a maximum activity of 2.4 U/ml, and Accordingly, the present invention provides a method of because an amount of turanose produced in this case is 2.38 producing high-yield and high-purity turanose in the indus mg/ml, it is con?rmed that a speci?c activity of amylosucrase trial scale and a sWeetener using the turanose. is 1.01 U/mg protein. Accordingly, in order to improve the According to an aspect of the present invention, a method yield of turanose, amylosucrase may be included in an of producing turanose includes acting amylosucrase on a 60 amount of 20 to 500 mg, for example, 40 to 150 mg, based on sucrose solution to produce turanose, and purifying the reac 100 g of sucrose. If the amount of amylosucrase is outside this tion product. range, reaction conversion ef?ciency may be decreased or a side-reaction speed may be increased. Technical Solution The microorganism Neisseria polysaccharea may be cul 65 tured in such a condition that a microorganism is groWn to An aspect of the present invention provides a method of produce amylosucrase that constitutes an enZyme, and in producing turanose according to an embodiment of the general, the microorganism Neisseria polysaccharea may be US 8,653,256 B2 3 4 isolated and cultured at a temperature of 37° C. and at a pH of During the enzymatic reaction according to the present 7.0 to 7.2. A culturing time may not be restricted as long as a invention, a reaction pH may be in a range of about 6.0 to 9.0, microorganism is suf?ciently groWn, and for example, may for example, 6.5 to 7.5. be in a range of 24 to 48 hours. After the culturing of a During the enzymatic reaction according to the present microorganism is completed, cells may be collected from the invention, the reaction time may vary according to an enzy culture through a typical solid liquid phase separation matic reaction condition. For example, When 100-400 units/ L method. For example, centrifuging may be performed of puri?ed amylosucrase is used, the reaction time may be in thereon at a temperature of 4° C. and then a supernatant a range of 24 to 120 hours. thereof is removed to obtain cells. In the reaction mixture obtained according to the present In particular, an amylosucrase gene derived from the invention Which is a saccharide composition, the yield of microorganism Neisseriapolysaccharea may be expressed as turanose may be 10% or more, for example, 50% or more, and a recombined proteinoplast in a host cell, for example, E. coli, for example, 73%. The obtained saccharide composition is bacillus, lactobacillus, or fungi. ?ltered to remove impurities therefrom and then, decolorized With activated carbon. As a cytozyme, a coenzyme extracted from a cell through Also, according to the production scale and reaction con a typical method may be used. For example, a coenzyme may ditions, various high-purity puri?cation methods may be used be obtained as folloWs: an enzyme is extracted from a cell to purify and recollect the produced turanose. For example, through a pulverization using ultrasonic Waves or homogena ion-exchange resin chromatography, column chromatogra tion and then the extract is treated With centrifuging or mem phy using activated carbon, column chromatography using brane ?ltering. A coenzyme may be used Without additional 20 silicagel, fractional crystallization, or the like may be used to treatments or may be used after puri?cation according to easily purify a product including high-purity turanose from typical methods. For example, a coenzyme extract is ?ltered the reaction mixture, and the obtained product is concentrated to remove insoluble materials therefrom, and the ?ltrate is as a syrup product. If necessary, the syrup product is dried into passed through a nickel-nitrilotriacetic acid (Ni-NTA) a?in a poWder product. ity column to purify recombined His6-tagged amylosucrase, 25 Also, another aspect of the present invention provides a and amicon ultra centrifugal ?lter devices are used at a tem sWeetener including a turanose that is produced by using the perature of 4° C. to elute the amylosucrase to obtain an method described above. electrophoretically uniform enzyme. Turanose is not fermented by a microorganism that causes A puri?ed enzyme may be directly used, or may be immo dental caries. Accordingly, turanose may be very useful for bilized on a support material by using a typical method and 30 use as a calorie-free sWeetener, and may function as a critical then used. For example, a binding method With respect to an sWeetener or additive in food, cosmetic, and pharmaceutic ion exchanger, a covalent binding or adsorption method With industries. respect to a resin and a membrane may be used. Through this MODE FOR INVENTION immobilization, an enzyme as a synthetic catalyst may be 35 easily recollected and may be reused several times. In gen Hereinafter, the present invention is described in detail eral, an enzyme puri?cation process requires long process With reference to examples beloW. HoWever, the present times and high costs. Accordingly, the immobilization and invention is not limited to the examples. reuse of an enzyme may contribute to a substantial co st reduc tion. 40 Example 1 An activity of puri?ed amylosucrase according to the present invention may be measured by using the folloWing Enzyme Preparation and Puri?cation method. That is, 100 pl of an enzyme solution is added to 900 pl of 50 mM Tris-HCl buffer solution (pH 7.0) including 1. Preparation of Stock Culture of Recombinant E. coli 0.1M sucrose as a substrate, and the mixture is reacted at a 45 BL21 temperature of 3 5 ° C. for 30 minutes, and the reaction mixture A gene corresponding to NpAS Was obtained from Neis is heated at a temperature of 100° C. for 10 minutes to stop the seria polysaccharea ATCC 43768 through polymerized enzymatic reaction. An amount of fructose liberated into the chain reaction (PCR) using 2 primers based on a Neisseria reaction mixture is analyzed through a dinitrosalicylic acid polysaccharea amylosucrase nucleotide sequence. That is, (DNS) method using fructose as a reference material. One 50 the PCR Was carried out using N. polysaccharea genome unit of amylosucrase activity is de?ned as an amount of DNA as a template and tWo oligonucleotides (SEQ ID NO: 1: enzyme that promotes production of l umol fructose per NPASl 5'-GGA TCC GAT GTT GAC CCC CAC GCA GCA minute under analysis conditions. A-3'; and SEQ ID NO:2: NPAS2 5'-GGC AAG CTT CAG As a substrate used in the present invention, only sucrose GCG ATT TCG AGC CAC AT-3'). may be used, or sucrose supplemented With fructose may be 55 The result PCR product Was cloned in T-easy vector used. Concentrations of the used sucrose and fructose may (Promega, Madison, Wis., USA), and any mistake that may not be particularly restricted. For example, even When acted occur during PCR Was veri?ed through DNA sequencing. An on a 0.25M or 0.75M fructose-supplemented 1.0M or 2.5M insert Was cleaved by using BamHI and Hind III, and then the sucrose solution Which constitutes a substrate, the enzyme obtained fragment Was inserted into a pRSET-B vector may act to produce turanose. Also, a solution including very 60 treated With the same enzyme to obtain pRSET-NpAS that high-concentration substrate or insoluble substrate may be constitutes NpAS expression vector. For the purpose of el? used. cient gene expression, E. coli BL21 Was transformed using During the enzymatic reaction according to the present the pRSET-NpAS. invention, a reaction temperature may not be limited as long 2. Enzyme Preparation as the enzyme is not deactivated. For example, the reaction 65 A pH of a liquid nutrient culture medium including 1% temperature may be about 40° C. or less, or may be in a range (W/v) Bactotryptone, 0.5% (W/v) yeast extract, 0.5% (W/v) of 20 to 40° C. sodium chloride, and Water Was controlled to be 7.0. 500 ml US 8,653,256 B2 5 6 of the culture medium Was loaded into a 1000 ml Erlenmeyer heating at a temperature of 100° C. for 10 minutes. Thereafter, ?ask that hadbeen sterilized at a temperature of 120° C. for 15 a saccharide composition of each of the samples Was assayed minutes, followed by cooling, inoculation With the previously using HPAEC in a manner similar to that used as described prepared stock culture of the recombined E. coli BL21, and above, and results thereof are shoWn in Table 2. incubation in the presence of 0.01% (W/v) ampicillin at a temperature of 37° C. When an optical density of cells Was TABLE 2 about 0.6, 1 ml of 0. 1 M isopropyl-[3-D-thiogalactopyranoside Reaction Was added to the culture to induce gene expression. After 12 time (hour) Saccharide compo ition (%) hours of incubation at a temperature of 16° C., the culture Was centrifuged at a temperature of 4° C. for 20 minutes at reaction Other time (hour) Glucose Fructose Sucrose Turanose saccharides 5,500>
TABLE 4 TABLE 6
Temperature (0 C_) Turanose yield (%) Fructose (M)
Turanose yield (%) 30 35 40 5 turanose yield (%) 0 0.25 050 0_75
Sucrose 10 33 9 I 01 317 I 0 3 29 6 I 0 0 Sucrose 1.0 30.9 1 0.8 45.8 1 0.1 57.2 1 0.2 62.8 11.1 concentration (M) concentration (M) Sucros? 15 387+04 361+05 347+00 sucrose 1.5 36.5 10.6 49.8101 58.6103 65.4104
Concentration (M) I I _ I I _ I I _ ' concentration (M) 20 342 02 511 00 387 11 sucrose 2.0 50.9101 60.0101 67.1104 73.1106 sucrose . . 1 . . 1 . . 1 . concentration (M) concentration (M)
As shoWn in Table 6, the higher the fructose concentration As shown in Table 4, When acted on 2.0M sucrose as a is, ?nally, more turanose Was produced. When 0.75M fruc substrate at a temperature of 35° C. for 72 hours, the puri?ed tose Was added to the respective reaction media, the enzyme amylosucrase according to the present invention produced produced turanose With a high yield of about 60%. about 50% turanose. Example 7 Example 5 Preparation of Puri?ed Turanose from Sucrose 20 Effect of Sucrose Concentration on Turanose Yield 1. Enzyme Preparation and Puri?cation A stock culture of recombined E. coli BL21 Was inoculated 400 units/L of the puri?ed amylosucrase according to the on 5 L of sterilized liquid nutrient culture medium including present invention Was added to a 1.0, 1.5, 2.0 or 2.5M sucrose 25 1% (W/v) Bactotryptone, 0.5% (W/v) yeast extract, 0.5% containing solution, and the respective enzymatic reactions (W/v) sodium chloride, and Water, folloWed by incubation in Were performed at a temperature of 35° C. Samples Were the presence of 0.01% (W/v) ampicillin at a temperature of collected at predetermined time intervals, and the remaining 37° C. for 4 hours. Thereafter, 10 ml of 0.1M isopropyl-[3-D enzyme Was deactivated by heating at a temperature of 100° thiogalactopyranoside Was added to the culture. After 16 C. for 10 minutes. Thereafter, a saccharide composition of 30 hours of incubation at a temperature of 16° C., centrifuging each of the samples Was assayed using HPAEC, and turanose Was performed thereon at a temperature of 4° C. for 30 min yields of the respective samples at various sucrose concen utes at 5,500> Reaction 24 30.7 1 0.2 26.5 1 0.2 25.1 1 0.1 17.9 1 0.0 40 (N i-NTA) al?nity column, and concentrated by using an ami time 72 31.7 1 0.3 36.11 0.5 51.11 0.0 41.0 1 0.8 con ultra centrifugal ?lter devices (Millipore Corporation, (hour) 120 31.8 1 0.5 37.4 1 0.3 52.0 1 0.9 56.2 1 0.3 Billerica, USA). About 100 ml of concentrated enzyme solu tion shoWed, ?nally, an activity of about 2.4 units/ml. 2. Preparation of Puri?ed Turanose from Sucrose As shoWn in Table 5, the higher the sucrose concentration 45 A solution including sucrose having a ?nal concentration is, the higher ?nal concentration the turanose is. When 2.5M of 2.5M Was prepared, and 400 units/L of the puri?ed amy sucrose solution Was used, after 120 hours of reaction, the losucrase Was added to the solution, and then an enzymatic enzyme produced turanose at a high yield of about 55%. reaction Was performed thereon at a temperature of 35° C. for 120 hours. Thereafter, the reaction mixture Was heated at a Example 6 50 temperature of 100° C. for 10 minutes, cooled, ?ltered through a 0.45 pm syringe ?lter to remove insoluble materials therefrom, and puri?ed by preparative high-speed liquid Fructose Addition Effect on Turanose Yield chromatography (model LC-9104; JAI Ltd., Tokyo, Japan) sequentially including tWo hydrophilic preparative columns Solutions including various concentrations of sucrose (1.0, 55 of JAIGEL W-251 and W-252 (20 mm><500 mm). Samples Were eluted With deionized Water at a How rate of 3 mL/min. 1.5, or 2.0M) and fructose (0.25, 0.50, or 0.75M) Were pre A turanose fraction Was obtained, concentrated, and lyo pared, and then 400 units/L of the puri?ed amylosucrase philized to produce turanose-rich saccharide poWder With a according to the present invention Was added to the respective purity of about 96% and a yield of about 65%. solutions, and an enzymatic reaction Was performed thereon 60 3. Sugar Composition Assay at a temperature of 35° C. After 96 hours of reaction, the Sugar composition as say Was performed on the hi gh-purity samples Were heated at a temperature of 100° C. for 10 saccharide poWder including turanose. minutes to deactivate the remaining enzyme, and saccharide 1) HPAEC compositions of the respective samples Were assayed using The high-purity saccharide poWder Was assayed by using a HPAEC. Turanose yields of the respective samples at various 65 DX-300 series HPAEC system (Dionex, USA) including a sucrose and fructose concentrations are shoWn in Table 6 pulse current detector (Dionex, USA), and in this case, com beloW. mercially available glucose, fructose, sucrose, maltose and US 8,653,256 B2 1 0 turanose Were used as reference materials. As a result, as Example 8 shown in FIG. 3, it Was con?rmed that turanose Was formed from sucrose. Preparation of Puri?ed Turanose from Sucrose 2) HPLC-MS The mass spectrum of the puri?ed turanose Was obtained 5 A solution including sucrose With a ?nal concentration of on Agilent 1100 series MSD (Agilent Technologies, Palo 2.0M and fructose With a ?nal concentration of 0.75M Was Alto, Calif., USA) by using a cation electrospray mode. The prepared, and 400 units/L of the puri?ed amylosucrase Was sample solution Was delivered at a How rate of 0.8 ml/min to added to the solution, and an enZymatic reaction Was per an electrosprayer and a loading amount Was 10 pl. While a formed at a temperature of 3 5 ° C. for 96 hours. Thereafter, the capillary voltage Was maintained at 4 kV, a fragmentor volt reaction mixture Was heated at a temperature of 1000 C. for 10 age Was ?xed at 150 V. The temperature of the electro sprayer minutes to deactivate the remaining enZyme, folloWed by Was maintained at 3500 C., and a detection scan range Was in cooling to room temperature. The obtained reaction mixture a range ofm/Z 100 to 1400. included about 73% of turanose (see FIG. 1). To isolate tura As a result, as shoWn in FIG. 4, the high-purity saccharide nose from other components, the reaction mixture Was ?l poWder shoWed a peak corresponding to an ion (M+Na)+ at tered through a 0.45 pm syringe ?lter to remove insoluble MS m/Z 365.3 and thus, it Was con?rmed that the saccharide materials therefrom, and then subjected to a preparative high Was disaccharide. speed liquid chromatography (model LC-9104; JAI Ltd., 3) NMR Tokyo, Japan) sequentially including tWo hydrophilic pre 20 NMR assay Was performed using a 600 MHZ NMR system parative columns of JAIGEL W-251 and W-252 (20 mm><500 (Rheinstetten, Germany), and in this case, the puri?ed tura mm). Samples Were eluted With deioniZed Water at a How rate nose Was used as being dissolved in pure Water D20. A of 3 mUmin. A turanose fraction (fraction C of FIG. 2) Were heteronuclear single quantum correlation (HSQC), 13 C and obtained, concentrated, and lyophiliZed to produce turanose 1H NMR spectra thereof Were recoded according to a stan 25 rich saccharide poWder With a purity of about 96% and a yield dard experimental method. Data process Was performed of about 65. using TopSpin® 2.0 softWare (Bruker BioSpin, Rheinstetten, According the present invention, a solution that includes Germany). only sucrose or a solution that includes fructose and sucrose The puri?ed turanose Was analyZed based on 13C NMR is treated With amylosucrase so that high-purity turanose is spectrum thereof, and as shoWn in Table 7 beloW and FIG. 5, 30 easily produced and isolated from a substrate, such as it Was con?rmed that the puri?ed turanose Was a turanose sucrose, and the produced turanose has a high degree of tautomer, such as 3—O-ot-D-glucopyranosyl-ot-D-fructofura purity. Accordingly, the turanose may be useful for use as a nose, 3—O-ot-D-glucopyranosyl-[3-D-fructofuranose, or 3-0 critical calorie-free sWeetener or additive in food, cosmetic, ot-D-glucopyranosyl-[3-D-fructopyranose. and pharmaceutic industries. TABLE 7 Tautorner Chemical shift (ppm) 331?‘; Q] ill ot-D-glucopyranosyl unit —fructose unit C-3 C-4 C-5 3-O-ot-D 97.1 71.5 73.1 69.6 74.8 61.3 104.5 85.0 72.5 81.8 glucopyranosyl ot-D fructo?iranose 3-O-ot-D 71.7 72.9 69.6 74.6 62.5 101.9 80.8 72.6 81.1 glucopyranosyl [5-D fructo?iranose 3-O-ot-D 101.2 72.3 69.6 73.0 98.0 76.9 70.5 69.3 glucopyranosyl [5-D fructopyranos 4) TLC It should be understood that the exemplary embodiments TLC assay Was carried out by using a Whatman K6 silica described herein should be considered in a descriptive sense gel plate, and in this case, acetonitrile, ethylacetate, 1-pro only and not for purposes of limitation. Descriptions of fea panol, and Water (85:20:20115 v/v) Were used as a solvent 60 tures or aspects Within each embodiment should typically be system. 0.3% (W/v) N-(l -naphthyl)-ethylenediamine and 5% considered as available for other similar features or aspects in (v/v) sulfuric acid dissolved in methanol Were sprayed into other embodiments. the plate and then the plate Was heated at a temperature of Sequence List Text 1100 C. for 10 minutes to coloriZe the plate. 65 As a result, as illustrated in FIG. 6, it Was con?rmed that the SEQ ID NO: 1 is a sequence of NPASl primer, and saccharide Was turanose. SEQ ID NO 2 is a sequence of NPAS2 primer. US 8,653,256 B2 11 12 SEQUENCE LISTING SEQ ID NO 1 LENGTH: 28 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: NPASl <400> SEQUENCE: l ggatccgatg ttgaccccca cgcagcaa 28 SEQ ID NO 2 LENGTH: 29 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: NPAS2 <400> SEQUENCE: 2 ggcaagcttc aggcgatttc gagccacat 29 What is claimed is: 3. The method of claim 1, Wherein a reaction temperature is 1. A method of producing turanose, the method compris in a range of30 to 400 C. 4. The method of claim 1, Wherein a reaction pH is in a ing: adding an amylosucrase to a mixed solution having a 30 range of 6.5 to 7.5. sucrose and a fructose to produce a reaction product, and 5. The method of claim 1, Wherein When 100-400 units/L of purifying the reaction product to obtain a turanose, Wherein a the amylosucrase is added, a reaction time is in a range of 24 concentration of the sucrose contained in the mixed solution to 120 hours. is in a range of l .0 to 2.5M and a concentration of the fructose 6. The method of claim 1, Wherein the purifying step is contained in the mixed solution is in a range of 0.25 to 0.75M. performed by a process selected from the group consisting of 35 2. The method of claim 1, Wherein the amylosucrase is ion-exchange resin chromatography, column chromatogra derived from a microorganism selected from the group con phy using activated carbon, and column chromatography sisting of Neisseria polysaccharea, Deinococcus geotherma using silica gel. lis, and Alleromonas macleodii.