Biotechnology Techniques13: 43–48, 1999.© 1999Kluwer Academic Publishers. Printed in the Netherlands.

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Rapid method for trehalulose production and its purification bypreparative high-performance liquid chromatography

Thierry Veronese1, Alain Bouchu2 & Patrice Perlot1,∗1Eridania-Beghin-Say, Institut National des Sciences Appliqu´ees, Hall Gilbert Durand, Avenue de Rangueil, 31077Toulouse Cedex, France2Eridania-Beghin-Say, CEI, BP 2132, 27, boulevard du 11 novembre, 69603 Villeurbanne Cedex, France∗Author for correspondence

Received 26 October 1998; Accepted 24 November 1998

Key words:HPLC, trehalulose, isomaltulose, sucrose-isomerase,Erwinia rhapontici

Abstract

Trehalulose was produced with a good yield by enzymatic conversion of sucrose and easily purified by preparativeHPLC using a single Ca2+-based column. In addition, the structure of this sugar was confirmed by13C and1Hn.m.r studies.

Introduction

Trehalulose (1-O-α-D-glucopyranosyl-D-fructose) is astructural isomer of sucrose which is naturally presentin honey (Nakajimaet al.1990). The sweetness of thissugar is approximately 60% of that of sucrose but itis non-cariogenic (Ooshimaet al. 1991) and showsa slower rate of release of monosaccharides into theblood (Yamadaet al. 1985). Several studies have fo-cused on finding possibilities to produce trehalulosein order to use it as a food ingredient. The synthesisof this disaccharide was first detected after the actionof α-glucosidases from yeast strains (Avigad 1959).However, the largest production was obtained by usingmicro-organisms that produce isomaltulose (6-O-α-D-glucopyranosyl-D-fructose) and trehalulose when theyare cultivated or incubated (immobilized or disrupted)with sucrose. Strains which were isolated in the firststudies, such asProtaminobacter rubrum(Weiden-hagen & Lorenz 1957),Serratia plymuthica(Fujiiet al. 1983) orErwinia rhapontici(Cheetham 1983),were described as producing mainly isomaltulose (80–90%), but during the past ten years attention has beenpaid to screening strains which convert sucrose with abetter selectivity for trehalulose than for isomaltulose.As a result, micro-organisms belonging to the genusKlebsiellawhich yield 60–75% trehalulose were iso-

lated (Tsuyukiet al. 1992), but better yields wereobtained withPseudomonas mesoacidophilaMX-45(Miyata et al. 1992) andAgrobacterium radiobacterMX-232 (Nagai-Miyataet al. 1993) since these bac-teria could produce about 90% trehalulose. In mostcases, the ability of these micro-organisms to iso-merise sucrose was demonstrated to be due to the ac-tion of anα-glucosyltransferase (a sucrose-isomerase)which converts sucrose simultaneously into isomal-tulose and trehalulose (Matteset al.1995, Nagaiet al.1994, Idenoet al.1991, Parket al.1996, Véronèse &Perlot 1998).

These properties have allowed the industrial pro-duction of isomaltulose (Palatinose or Lylose) byenzymatic conversion of sucrose. In this case, isoma-ltulose is easily separated from trehalulose and otherby-products by crystallization (Sarkkiet al. 1997).However, even if such a process can be use for trehalu-lose synthesis, its recovery is more difficult since thisdisacharide is extremely soluble. As a result, mainlytrehalulose rich syrups have been produced at a largescale and used in confectionery and other sweetenedfood in Japan (Nishimotoet al.1997). However, somepatents mention the separation of trehalulose fromisomaltulose by ionic chromatography using severalcolumn in series (Munir 1982) and a recent studyalso mentions the possibility to separate these two iso-

44

Reaction time (h)

0 5 10 15 20 25

Con

cent

ratio

ns (

g/l)

0

100

200

300

400

500

600

Fig. 1. Time course of sucrose conversion in the batch reactor.Sucrose (#), trehalulose (�), isomaltulose (�).

mers by simulated moving bed (Kishiharaet al.1989).Nevertheless, these processes have a low productivity.

At the laboratory scale, pure trehalulose can be ob-tained using preparative HPLC. Cooksonet al. (1987)have proposed a purification by passing solutions threetimes through four reversed-phase preparative-scaleHPLC C18-columns in series and Muniret al. (1987)have purified trehalulose with a procedure combiningCa2+ and cellulose chromatographies, and methanolextractions. However, both these complex processeshave low productivities. More recently, trehalulosehas been purified from the excrement of the sweetpotato whitefly by HPLC techniques involving twocalcium-based columns in series (Bateset al.1990).

Trehalulose structure has been first determinedby 13C n.m.r (Cooksonet al. 1987) and con-firmed later with additional1H n.m.r studies (Bateset al. 1990) and these results have shown theexistence of two major tautomers in water, 1-O-α-D-glucopyranosyl-D-fructofuranose and 1-O-α-D-glucopyranosyl-D-fructopyranose (major form). Fur-ther studies have investigated the influence of temper-ature on the variation of these two forms in water andDMSO (Lichtenthaler & Rönninger 1990).

In this paper, we propose a simple manner to pro-duce trehalulose with a good yield using the potentialof Erwinia rhaponticiNCPPB 1578 and a rapid andeasier method to purify this sugar by HPLC using asingle column. The purity and the structure of tre-

halulose was confirmed by mass spectrometry andby comparing our n.m.r data with those available inlitterature.

Material and methods

Sucrose-isomerase production. Erwinia rhaponticiNCPPB 1578 was cultivated in a 2-l fermenter con-taining 1.5 l of medium at 30◦C, pH 6.8, for 20 h withan aeration rate of 1 vvm at 600 rpm. The mediumhad the following composition (g/l): sucrose, 50.0;KH2PO4, 5.0; mgSO4, 0.4; (NH4)2SO4, 1.0; yeastextract, 7.5. The culture medium was centrifuged andthe pellet obtained was washed three times with phos-phate buffer 50 mM, pH 6.5 and concentrated aboutten times in the same buffer. This cell suspension canbe used directly as catalyst for isomerisation reaction.

Standard assay of sucrose-isomerase activities.En-zyme activities were measured by incubating 1 mlcell suspension at 5 g/l with 9 ml sucrose solutionat 292 mM, in 50 mM phosphate buffer (pH 6.2) at35◦C. Several samplings of the reaction mixture weredone, and the activity was calculated by plotting prod-ucts formation versus reaction time. One unit activity(U) is defined as the amount of enzyme that can re-lease oneµmol product (isomaltulose or trehalulose)in 1 min at the initial stage of the reaction under thestandard assay conditions. Sucrose conversion intoisomaltulose is the main enzyme activity. Thus, theinternational unit activity (UI) is defined as the amountof enzyme that can release oneµmol isomaltuloseper minute at the inital stage under the standard assayconditions.

Sugars analysis. Samples were delivered by an ICS1708 autosampler with 10µl fixed loop to a DionexDX40 chromatography system (Dionex). Separationwas carried out in a 4× 250 mm Dionex Carbo-packPA100 column preceeded by a 3× 25 mm DionexCarbo-pack PA100 Guard column. Mobile phase waspropelled by a Dionex GP40 quaternary pump at a1 ml/min. It was composed of a two eluent mixcontaining nanopure water and 0.15 M NaOH. Botheluents were degassed by helium sparging in a Dionexdegassing module. Detection was done by a DionexED40 module with a gold working electrode and apH Ag/AgCl reference. Sensitivity was set at 10µC.Potentials for amperometric detection were applied asfollow: delay time t1= 200 ms, potential E1= 50 mV;

45

2 4 6 8 10 12 14 16

29500

30000

30500

31000

31500

Time (min.)

fructoseglucose

trehalulose

isomaltulose

2 4 6 8 10 12 14 16

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

3.6e4

3.7e4

3.8e4

Time (min.)

trehalulose

(A) (B)

Fig. 2. Analysis of the sugar solution obtained after sucrose conversion before purification (A) and after purification on preparative HPLC (B).

electrode oxidation by the sample, t2= 200 ms, E2=50 mV; electrode cleaning t3= 200 ms, E3= 750 mV;electrode reduction t4= 400 ms, E4= −150 mV.

Isomaltulose and trehalulose production.Our kinet-ics studies on the sucrose-isomerase fromErwiniarhaponticiNCPPB 1578 showed that the relative tre-halulose production increases when temperature de-creases (15% of the total activity at 30◦C, 60% at1◦C). Moreover, other by-products such as glucose,fructose or isomaltose were not detected at low tem-peratures. In order to enhance the trehalulose yield,the conversion reaction was thus done at 1◦C, and theloss of activity due to this condition was compensatedby increasing the enzyme amount. A 500 ml reactorcontaining 400 ml of a 50% (w/v) sucrose solutionin 0.1 M phosphate buffer pH 6.2 was inoculated by50 ml of the enzymatic solution The temperature wasmaintained at 1◦C by a chiller.

Cleaning of the sugar solution.After total sucroseconversion, cells were removed by centrifugationand the sugar solution was filtered through a cellu-lose/kieselgurs deep filter (cut-off range 0.5–0.8µm,KS 50 Seitz) in a 43 mm diameter filtration cell. Thesolution to be filtered was propelled by a peristalticpump at 5 ml/min. A following microfiltration wasperformed with the same apparatus but with a 0.2µmcut-off filter. A last step of cleaning was done by ul-trafiltration of the sugar solution in a 50 ml stirred

Fig. 3. Elution profile of isomaltulose and trehalulose obtained onthe preparative HPLC system.

cell through a 10 000 daltons molecular weight cut offmembrane. This solution was finally concentrated to70% (w/w) with a rotative evaporator.

Preparative HPLC purification. The preparativeHPLC apparatus was composed of an HPLC pump,connected to an injector with a 30µl injection loop.Sugars were detected by a refractive-index detector.Separation was performed with a PL-Hi-Plex Ca 300×25 mm preparative column at room temperature withultrapure water as solvent at 1 ml/min. The purifica-

46

tion process was automatisated using an electric timerconnected to the injector, to a peristaltic pump to fillthe injection loop with the 70% (w/w) solution (about275 mg for each injection), and to a collector to collectthe trehalulose peak. This system allowed successiveinjections which enhanced the productivity.

Trehalulose storage. After purification, the trehalu-lose solution was concentrated to 10% (w/w) with arotative evaporator.

Trehalulose structure. A Brucker AM-500 instru-ment was used for n.m.r studies.1H n.m.r was car-ried out at 500 MHz with sodium 4,4-dimethyl-4-silapentanesulfonate atδ 0 as reference, and13C n.m.rwas done at 125 MHz using acetone (31.07 ppm) asreference. Two-dimensional n.m.r spectra (1H-1H and1H-13C COSY) were performed to determine n.m.rshifts and coupling constants.

Mass spectrometry analysis.Fast atom bombard-ment spectra were recorded on a quadrupolar NermagR 1010C spectrometer. Accelerated atoms (Xenon)were produced by a M−scan (Wallis) gun using a 9 kVacceleration tension.

Results and discussion

Enzyme production. The maximum sucrose iso-merase activity was obtained after 20 h of culture. Werecovered a 100 ml cell suspension with an activity of240 UI/ml.

Sucrose conversion.Figure 1 shows the time courseof sucrose conversion in the batch reactor. The cal-culation of each activity at the initial rate of thereaction shows that 60% of the activity is dedicatedto trehalulose formation and 40% to isomaltulose.

Therefore, after total sucrose conversion, the re-moval of the cells, the cleaning procedure and thefollowing concentration to a 70% (w/w) syrup, we ob-tained the sugar composition shown in Figure 2A andTable 1. Our sugar solution contained 63% of trehalu-lose but if desired, this percentage can be increasedby concentrating the syrup in a rotary evaporator andby recrystallizing isomaltulose by cooling the solutionand seeding it with crystalline isomaltulose. The crys-tals can be removed by centrifugation to yield a motherliquor which is richer in trehalulose.

Table 1. Composition of the sugar solution obtainedafter sucrose conversion in the batch reactor.

Sugars Concentration

(g/l)

Trehalulose 298

Isomaltulose 193

Isomaltose 0.65

Glucose 1.0

Fructose 1.1

Preparative HPLC purification. Figure 3 shows anexample of the elution profile of isomaltulose and tre-halulose obtained with our preparative HPLC system.The sample applied to the column was 30µl of the70% (w/w) sugar syrup, which is thus equivalent to17.2 mg of trehalulose and 7.4 mg of isomaltulose.The two peaks were very well separated, so the totalityof the trehalulose injected can be collected after 90min with a 99.9% purity as shown on Figure 2B. Thisproductivity of about 11 mg of trehalulose per hour hasbeen increased by successive injections every 40 minwhich allows us to reach a productivity of 26 mg perhour (620 mg a day).

13C and 1H n.m.r studies. Table 2 gives the n.m.rshifts and coupling constants obtained. These resultsare in accordance with those reported in previous stud-ies but, we have also determined the additional valuesof the 1H n.m.r of theβ-fructofuranose form. As aresult, we confirm the existence of the two major tau-tomeric forms of trehalulose A and B (Figure 4) witha ratio of approximately 20:4. Onlyβ-anomers havebeen detected.

Mass spectrometry studies.Pseudo-molecular ionm/z = 343 [M+H]+ is observed, mainly associatedwith several peaks corresponding to the adducts of onemolecule of trehalulose with one, two or three mole-cules of glycerol (matrix) (m/z= 435 [M+gly+H]+;527 [M+2gly+H]+ and 619 [M+3gly+H]+).

Conclusion

Obtention of pure trehalulose at the laboratory scaleis important for testing the properties of this sugarin different chemical or biological investigations. Interms of trehalulose production, a good yield has been

47

OHO

HO

O

OH

OH

O

OH

OH

OH

HO

6f

1f

β-p

OO

HOHO

O

OH

OH

HO

OH

OH

6f

1f

β-f

(A) (B)Fig. 4. Structures of the two major trehalulose forms predicted from n.m.r studies. 1-O-α-D-glucopyranosyl-D-fructopyranose (A) and1-O-α-D-glucopyranosyl-D-fructofuranose (B).

Table 2. N.m.r shifts (ppm) and coupling constants (Hz, in parentheses).

Carbon A B

number13C 1H 13C 1H

Glc-1 99.4 4.89 d (3.7) 4.92 (4)

Glc-2 72.3 3.49 dd (3.7, 9.8)

Glc-3 73.8 3.69 t (9.8)

Glc-4 70.3 3.35 t (9.8)

Glc-5 72.7 3.63 dd (9.8, 2.5)

Glc-6 61.3 3.68 dd (12, 9.8)

3.79 dd (12, 2.5)

Fru-1 69.9 3.37 d (10.4) 3.46 d (10.8)

3.85 d (10.4) 69.3 3.72 d (10.8)

Fru-2 98.6 – – 101.7 – –

Fru-3 68.7 3.76 d (10.8) 77.1 4.06 d (7.8)

Fru-4 70.4 3.83 dd (10.8, 3.8) 75.3 4.04 d (11.6, 7.8)

Fru-5 69.9 3.93 m – 81.5 3.78 m –

Fru-6 64.4 3.63 d large (12.2) 63.1 3.61 dd (11.2, 7.0)

3.97 d large (12.2) 3.73 dd (11.2,3.8)

obtained by usingErwinia rhapontici strain as cat-alyst. Low temperature is required to increase theratio trehalulose to isomaltulose, but also to avoid thesynthesis of other by-products (glucose, fructose, iso-maltose and isomelezitose). Such phenomena existsfor other isomaltulose producing micro-organisms andthus,Serratia plymuthica(Véronèse & Perlot 1998),Protaminobacter rubrum(Fujii et al. 1983) or strainsfrom the genusKlebsiella (Tsuyuki et al. 1992) canalso be used in the same conditions. However, theirability to produce more trehalulose at low temperatureis more or less significant from case to case.

Our paper also shows that trehalulose can be eas-ily purified by using a more simple procedure thanthose described previously which were performed oncomplex apparatus (Cooksonet al.1987, Muniret al.1987, Bateset al. 1990). Moreover, the use of acalcium-based column allows the elution with waterand thereby avoids the use of toxic eluants such as ace-tonitrile. Finally, our work shows that, if trehaluloseis desired for laboratory studies, it can be obtained insignificant amounts in no more than two weeks.

In addition to these results, our work has alsoallowed us to confirm the structure of trehalulose.

48

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