Food Chemistry 138 (2013) 1183–1188

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Food Chemistry

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Simple, fast, and efficient process for producing and purifying trehalulose

Yutuo Wei a,c,1, Jiayuan Liang a,c,1, Ying Huang a,c, Panxian Lei a,c, Liqin Du a,c, Ribo Huang a,b,c,⇑a State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, 100 Daxue Road, Nanning, Guangxi 530004, Chinab National Engineering Research Center for Non-Food Biorefinery, Guangxi Academy of Sciences, 98 Daling Road, Nanning, Guangxi 530007, Chinac College of Life Science and Technology, Guangxi University, Nanning 530004, China

a r t i c l e i n f o a b s t r a c t

Article history:Received 22 July 2012Received in revised form 19 October 2012Accepted 20 November 2012Available online 5 December 2012

Keywords:Trehalose synthaseThermus thermophilusTrehaluloseSucrosePurification

0308-8146/$ - see front matter � 2013 Published byhttp://dx.doi.org/10.1016/j.foodchem.2012.11.115

⇑ Corresponding author at: Guangxi Academy of ScieResearch Center, 98 Daling Road, Nanning, Guangxi2503902; fax: +86 771 2503908.

E-mail address: rbhuang@gxas.ac.cn (R. Huang).1 These authors contributed equally to this work.

A new property of recombinant trehalose synthase (GTase) from Thermus thermophilus HB-8 (ATCC27634) was found and described in this study. GTase can act on sucrose and catalyze trehalulose forma-tion without isomaltose, isomaltulose, or isomelezitose, releasing small amounts of glucose and fructoseas byproducts. Maximum trehalulose yield (approximately 81%) was obtained at an optimum tempera-ture of 65 �C and was independent of substrate concentration. A simple, fast, and efficient method of pro-ducing and purifying trehalulose is then described. In the first step, GTase catalyzed trehaluloseformation using a 20% sucrose substrate. Miscellaneous sugars were then rapidly removed, while treha-lulose was completely preserved by Saccharomyces cerevisiae cells. Finally, the cells were separated bycentrifugation, and salt ions were removed by an ion-exchange resin, subsequently obtaining a high-purity trehalulose solution. A trehalulose recovery rate of over 95% was achieved using this process. Thismethod has a simple process, fast separation efficiency, and low investment in production equipment, sogreatly to improve production efficiency and reduce production costs.

� 2013 Published by Elsevier Ltd.

1. Introduction

Trehalulose (1-O-a-D-glucosylpyranosyl-b-D-fructose) is areducing disaccharide that is naturally present in small quantitiesin honey (Low & Sporns, 1988; Nakajima, Sugitani, Tanaka, & Fujii,1990). It is a structural isomer of sucrose (a-D-glucosylpyranosyl-1,2-b-D-fructofuranoside) and has been proven to have two opticalisomers, namely, 1-O-a-D-glucosylpyranosyl-b-D-fructofuranoseand 1-O-a-D-glucopyranosyl-b-D-fructopyranose (Cookson, Cheetham,& Rathbone, 1987). The sweetness of this sugar is approxi-mately 60% that of sucrose; however, it is non-cariogenic (Ooshimaet al., 1991) and shows a slower rate of monosaccharide releaseinto the blood. Trehalulose has high solubility (Ooshima et al.,1991) and exhibits physiological functions and chemical proper-ties similar to those of isomaltulose and trehalose. It can preventdental cavities, mitigate diabetes mellitus, and help maintain bodyweight (Minami, Fujiwara, Ooshima, Nakajima, & Hamada, 1990;Ooshima et al., 1991). The growing list of disorders related to car-diovascular pathologies, several forms of cancer, diabetes, obesity,osteoporosis, and infectious dental diseases are associated withexcessive sugar intake. Therefore, the development of a healthy

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sugar substitute is urgently needed (Ravaud et al., 2007). Trehalu-lose is a promising substance that has a wide range of potentialapplications in the food, cosmetics, pharmaceutical, and otherindustries.

The chemical synthesis of trehalulose is highly difficult, and itsindustrial production proceeds exclusively from sucrose usingimmobilized microorganisms. Several organisms or enzymes(including trehalulose synthase, isomaltulose synthase, and su-crose isomerase) can convert sucrose into trehalulose, or a mixtureof isomaltulose and trehalulose to produce glucose and fructose inresidual amounts through sucrose hydrolysis. Depending on theenzyme, the composition of enzyme products varies from mainlyisomaltulose (66–91%) (Cheetham, 1984; Véronèsea & Perlot,1999; Wu & Birch, 2005; Zhang, Li, & Zhang, 2002) to predomi-nantly trehalulose (�90%) (Nagai, Sugitani, & Tsuyuki, 1994). Inparticular, sucrose isomerase from whiteflies converts sucrose intomainly trehalulose, without isomaltulose (Salvucci, 2003). How-ever, the paper did not report trehalulose yield. Hamerli and Birch(2011) developed sugarcane plants to produce trehalulose throughthe expression of a vacuole-targeted trehalulose synthase modifiedfrom a gene of ‘‘Pseudomonas mesoacidophila MX-45’’. The trehalu-lose concentration in the juice increased with internode maturity,reaching approximately 600 mM and a near-complete sucrose con-version in the most mature internodes. The plants remained vigor-ous, and trehalulose production in selected lines was retained overmultiple vegetative generations under glasshouse and fieldconditions.

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Complex products are generated during trehalulose production,including a considerable number of impurities. This condition, cou-pled with high solubility, renders the separation, purification, andcrystallization of trehalulose difficult. Moreover, currently avail-able reports on separation and purification methods are inade-quate. Véronèse, Bouchu, and Perlot (1999) and Kishihara et al.(1989) obtained high-purity trehalulose via preparative chroma-tography and through the use of a simulated moving-bed adsorber(Kishihara et al., 1989; Véronèse et al., 1999). Cookson et al. (1987)purified trehalulose from a complex mixture using four reversed-phase preparative-scale HPLC columns, with distilled water asthe eluent, achieving a final purity of 98%. The mixture, whichwas produced by immobilized microbial cells, consisted of a trisac-charide, sucrose, isomaltulose, glucose, and fructose. However, thehigh cost and low yield of the aforementioned separation andpurification methods make them unsuitable for commercialproduction.

Zdziebło and Synowiecki (2006) and Wang et al. (2012), re-ported the properties of a trehalose synthase (GTase or TST) fromThermus thermophilus HB-8 (ATCC 27634). The enzyme catalyzesthe conversion of maltose into trehalose at an optimum tempera-ture of 65 �C. In the current paper, a new property of GTase is de-scribed. The sucrose products under the catalysis of GTase wereconfirmed via high-performance liquid chromatography (HPLC).Given the substrate availability and higher product yield, this en-zyme is suitable for the industrial production of trehalulose fromsucrose. Thus, a simple, fast, and efficient process of producingand purifying trehalulose is proposed.

2. Materials and methods

2.1. Bacterial strains, plasmids and reagents

Saccharomyces cerevisiae CICC1001 obtained from CICC (China)and T. thermophilus HB8 (ATCC27634) were used in the currentstudy. Escherichia coli JM109 (Promega, USA) was used for routinecloning, whereas E. coli BL21 was used for gene expression. PlasmidpSE380 (Invitrogen, USA) was used as a cloning and expressionvector. E. coli and recombinant E. coli strains were routinely grownat 37 �C and 200 rpm either in a Luria–Bertani medium alone orwith 100 lg/ml ampicillin. The restriction enzymes, ligase, andLA Taq DNA polymerase were all obtained from TaKaRa (Shiga,Japan). The 732 cation exchange resin and 717 anion exchange re-sin (Product Nos. 10024260 and 10024160) were obtained fromSinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

Trehalulose was obtained from Wako (Japan). All other saccha-rides were purchased from Sigma (St. Louis, MO, USA). All other re-agents were of analytical grade.

2.2. Construction of recombinant expression vectors

All experimental methods were in accordance with previouslydescribed techniques (Wang et al., 2012). The expression vectorswere linked with 6� His-tag at the C terminus for purification ina nickel–nitriloacetic acid (Ni–NTA) column (Qiagen, Hilden,Germany). The recombinant plasmid (pSE380-GTase) was trans-formed into E. coli BL21 for GTase expression.

2.3. GTase expression and purification

After the cell density (A600) of the incubated recombinant cellsreached approximately 0.6, isopropyl-beta-D-thiogalactopyrano-side (IPTG) was added to a final concentration of 1 mM to inducegene expression. The cells were harvested via centrifugation at8000g for 10 min, washed twice with 50 mM potassium phosphate

buffer (pH 7.0), resuspended in the same buffer, and then disruptedby sonication on ice for 10 min after 10 h induction at 37 �C. Celldebris were removed through centrifugation at 12,000g for20 min. The recombinant protein was purified using Ni–NTA(Qiagen, Hilden, Germany) according to the instructions of themanufacturer. Enzyme protein content was determined using theBradford method (Bradford, 1976), with BSA as standard.

2.4. Preparation of S. cerevisiae cells

S. cerevisiae was grown in a yeast extract–peptone–glucose(YPD) medium (pH 7.0) containing 2% peptone and 1% yeast extractat 30 �C and 180 rpm. After the A600 of the incubated S. cerevisiaecells reached approximately 3.0, the cells were harvested by centri-fugation at 8000g for 10 min and washed twice with distilledwater.

2.5. Enzyme assay

The GTase reactions in a final volume of 500 ll involved incuba-tion at 65 �C for 2 h in a reaction mixture containing 0.5 mg of puri-fied enzyme, 2% sucrose, and 50 mM phosphate buffer (pH 7.0). Avolume of distilled water equal to that of the purified enzymewas added to the reaction mixture as a control. The identity ofthe sucrose products obtained under GTase catalysis was con-firmed via HPLC, which was performed at 30 �C and a pressure of76 bar using an Agilent 1100 series column (Alltima Amino 100A5u, 250 mm � 4.6 mm) and an Alltech 2000ES evaporative lightscattering detector. Acetonitrile/water (76:24) was used as solventat a flow rate of 1 ml min�1. Enzyme activity was assayed by mea-suring the amount of consumed substrate via HPLC, using the con-trol as the original substrate content. One unit of enzyme activitywas defined as the amount of enzyme that consumed 1 lmol ofsubstrate per minute.

Kinetic analysis was performed at an optimum temperature of65 �C and a pH of 7.0. The experiment was conducted for 1 h in a50 mM phosphate buffer containing the sucrose substrate at vari-ous concentrations. The Km and Vmax values were obtained usinga Lineweaver–Burk plot.

2.6. Simple process sequence of trehalulose production andpurification

A simple process sequence is outlined in Fig. 1. First step: Tre-halulose production involved incubation of a reaction mixture con-taining 90 mg of GTase protein, 20 g of sucrose, and 50 mM ofphosphate buffer (pH 7.0) at 65 �C. The generated trehalulosewas monitored by HPLC. The reaction solution was then boiledfor 10 min, and the enzyme was removed by centrifugation at12,000g for 10 min to obtain a coarse trehalulose solution. Secondstep: Harvested S. cerevisiae cells were resuspended in the coarsetrehalulose solution (w/v = 1 g/10 ml), and then transferred to a500 ml culture bottle for culturing at 30 �C and 180 rpm. Changesin fructose, glucose, and sucrose amounts were monitored inreal-time via HPLC. S. cerevisiae cells were immediately removedby centrifugation at 12,000g for 10 min when fructose, glucose,and sucrose were completely digested. The obtained solution con-sisted of a pure trehalulose solution; however, a small amount ofsalt ions and impurities were also present. Third step: Cation andanion exchange resin were used to separate and purify trehalulosefrom salt ions and impurities. The pretreatment using the ion-ex-change resin was in accordance with the instructions of the man-ufacturer. The processed filler was filled into a 2 cm � 1.5 mchromatography column. Distilled water was used as the mobilephase at a flow rate of 1 ml/min for approximately 2 h for chroma-tography column equilibration. Under the same conditions, the

Harvest S. cerevisiae cells by centrifugation at 12000g for 10 min

Separate and purify trehalulose from salt ions and impurities by cation and anion exchange resin

Add

Remove S. cerevisiae cells by centrifugation at 12000g for 10 min

Culture at 30 °C and 180 rpm for appropriate time

Boil for 10 min and centrifuge at 12000g for 10 min to obtaina coarse trehalulose solution

Incubate at 65 °C for some time

Add 90 mg of GTase protein to 100 ml of 20% sucrose solution

First step

Third step

Second step

Fig. 1. A simple process sequence for the production and purification of trehalulose. ( ) Sucrose, ( ) trehalulose, ( ) fructose, ( ) glucose, ( ) saltions, ( ) ionexchange resin.

Y. Wei et al. / Food Chemistry 138 (2013) 1183–1188 1185

pure trehalulose solution obtained from the second step was thenapplied to the equilibrated chromatography column. Sample frac-tions of 10 ml each were then collected. Repeating the third steptwo or three times yielded better results.

3. Results

3.1. Construction, expression, and purification of recombinant enzyme

The inserted segment of the expression vector was sequenced.The recombinant enzyme was expressed, purified, and then ana-lyzed by SDS–PAGE (10% minigel), showing a clear protein band.According to a previous report (Wang et al., 2012), the 6� His-tag at the C terminus domain did not affect the GTase activity.Therefore, the purified enzyme protein was used in the subsequentstudies.

3.2. New properties of GTase

In previous papers (Wang et al., 2012; Zdziebło & Synowiecki,2006), the substrate specificity of GTase was not reported (includ-ing lactose, sucrose, cellobiose, isomaltose, isomaltulose, trehalu-lose, maltotriose, maltotetrose, sorbitol, and dextrin assubstrates). GTase acted on sucrose and catalyzed the trehaluloseformation, releasing small amounts of glucose and fructose asbyproducts (Fig. 2B). Other sugars were not catalyzed (data notshown). The products obtained when sucrose was used as a sub-strate are only trehalulose and small amounts of glucose and fruc-tose; isomaltose, isomaltulose, or isomelezitose were not

produced. This result can be used to improve the product yieldand reduce the production and purification costs.

The results of the kinetics analysis on GTase are shown in Ta-ble 1. The Km and Vmax for sucrose were 168.00 ± 2.1 mmol/l and1.3390 ± 0.01 mmol/min/mg, respectively. The obtained Km valuesfor maltose and trehalose were approximately twofold higher thanthat of sucrose (Wang et al., 2012), which indicated that GTase pre-ferred maltose and trehalose over sucrose as its substrate.

3.3. Range of trehalulose yield

The trehalulose yield varied for different incubation times. Fig. 3shows that the maximum trehalulose yield was approximately 81%at glucose and fructose contents of 6.2% and 6.3%, respectively;using reaction mixtures containing 2% sucrose under optimumreaction conditions. The results were the same using different sub-strate concentrations (from 10% to 80%), but not under the samereaction time (data not shown). These findings suggest that theconversion rate of sucrose to trehalulose is independent of sub-strate concentration.

3.4. Production and purification of trehalulose

In the first step described in Section 2.6, the trehalulose yieldreached its maximum value of 81% (Fig. 2B). After the enzymewas removed, small amounts of sucrose, fructose, and glucosewere also found in the coarse trehalulose solution. The smallamounts of miscellaneous sugars significantly reduced the diffi-culty of purification. Some enzymes can catalyze trehalulose

Fig. 2. High-performance liquid chromatography (HPLC) results for the production and purification process of trehalulose. The sample concentration was diluted toapproximately 0.1%, and 20 ll was loaded onto the column. (A) Standards for fructose (7.535), glucose (8.323), sucrose (11.144), and trehalulose (13.344), shown as peaks. (B)Peaks of the reacted sample and of the products [fructose (7.536), glucose (8.323), sucrose (11.168), and trehalulose (13.346)]. (C) Purified trehalulose (13.346).

Table 1Kinetics analysis for GTase.

Substrate Km (mmol/l) Vmax (mmol/min/mg) Product Byproduct(s)

Maltoseb 97.4 ± 5.9 – Trehalose GlucoseTrehaloseb 82.2 ± 3.2 – Maltose GlucoseSucrose 168.00 ± 2.1 1.3390 ± 0.02a Trehalulose Glucose and fructose

– Not listed. The data are represented as the mean values and standard deviations of three independent experiments.a Amount of substrate.b Data from the literature (Wang et al., 2012).

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formation, but also tend to produce nonquantitative isomaltulose(from approximately 10–90%). In addition, trehalulose and iso-maltulose have similar properties, which lead to a low trehaluloseyield and difficult separation. However, the higher yield and fewer

byproducts produced when GTase was used completely overcamethese shortcomings. In the previously described second step, themiscellaneous sugars were rapidly removed using S. cerevisiae,which can efficiently assimilate sucrose, fructose, and glucose.

0 5 10 15 20 250

20

40

60

80

100

Reaction time (h)

Car

bohy

drat

es c

onte

nt (%

)

Fig. 3. Effect of reaction time at 65 �C on the trehalulose (j), fructose (s), andglucose (d) formation in 2% solution of sucrose (N) in phosphate buffer (pH 7.0).The percentages of the substrate and products in the total sugars were monitoredvia HPLC at different incubation times. All data are expressed as the average valuesof at least three replicates.

0.0 0.5 1.0 2.0 3.0 4.0 6.0 20.00

2

4

6

8

10

12

14

16

18

20Glucose Sucrose TrehaluloseFructose

Assimilation time (h)

Car

bohy

drat

es c

onte

nt (g

)

Fig. 4. Assimilation of the coarse trehalulose solution (100 ml) obtained from thefirst step by Saccharomyces cerevisiae. The content of various sugars were detectedby HPLC.

Y. Wei et al. / Food Chemistry 138 (2013) 1183–1188 1187

Sucrose, fructose, and glucose were completely removed, leavingonly trehalulose, when the mixture containing 10 g of S. cerevisiaecells were cultured with 100 ml of coarse trehalulose solution forapproximately 2 h at 30 �C and 180 rpm (Figs. 2C and 4). S. cerevi-siae is called baker’s yeast or budding yeast and is widely used inmaking bread and other food as well as in brewing wine. S. cerevi-siae can be used in food production and is safe for humans. Fig. 4shows that S. cerevisiae cells first consumed sucrose, followed byglucose and fructose; however, trehalulose was hardly used. Inthe third step, the cations and anions contained in the solutionwere exchanged and then adsorbed by the fillers, whereas trehalu-lose flowed out along with the mobile phase, when the trehalulosesolution from the second step was flowed through the cation andanion exchange resin. A high-purity trehalulose solution was thusobtained. After generating trehalulose from the first step, therewas almost no reduction in trehalulose content by microbial

assimilation and deionized processing programs. The changes oftrehalulose content over the whole process were monitored byHPLC. Therefore, the recovery rate of trehalulose could reach 95%or more. Trehalulose has high water solubility; however, severalattempts to obtain crystals were unsuccessful.

4. Conclusions

Trehalulose is a promising substance that has a wide range ofpotential applications in the food, cosmetics, pharmaceutical, andother industries. In this study, a new property of recombinantGTase was found and described. GTase is suitable for producingtrehalulose, using sucrose as a substrate. The conversion rate,product simplicity, and reaction temperature of the experimentalreaction are factors that meet the requirements for industrial pro-duction. Through the incorporation of microbial assimilation in theprocedures, miscellaneous sugars were quickly removed, whereastrehalulose was completely preserved. Finally, a high-purity treha-lulose solution was easily obtained through the removal of saltions using an ion exchange resin. The recovery rate for trehaluloseexceeded 95%. The method described herein belongs to the field ofbiological engineering, related to the separation and purification oftrehalulose syrup. Specifically, it cleverly utilized the differentassimilation capacity of S. cerevisiae cells to different sugars andseparated cells from liquid by centrifugation, finally removing su-crose, glucose, and fructose from the reaction liquid of trehalulose.Compared with conventional gel resins and ion exchange resinseparation methods, it is a simple, fast, and efficient process forproducing and purifying trehalulose. It is conducive for improvingproduction efficiency and reducing the production cost.

Acknowledgements

This work was supported by the National Natural Science Foun-dation of China (Grant No. 31160311) and the Natural ScienceFoundation of Guangxi (Contract No. 2012GXNSFAA053051).

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