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Journal of Biotechnology 138 (2008) 33–41

Contents lists available at ScienceDirect

Journal of Biotechnology

journa l homepage: www.e lsev ier .com/ locate / jb io tec

ynthesis of novel fructooligosaccharides by substratend enzyme engineering

afael Beinea, Roxana Morarua, Manfred Nimtzb, Shukrallah Na’amniehc,lice Pawlowskic, Klaus Buchholza, Jürgen Seibela,b,∗

Department for Carbohydrate Technology, Technical University of Braunschweig, Hans-Sommer Str. 10, 38106 Braunschweig, GermanyDivision of Structural Biology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, GermanyX-Zyme GmbH, Merowingerplatz 1A, 40225 Düsseldorf, Germany

r t i c l e i n f o

rticle history:eceived 15 May 2008eceived in revised form 4 July 2008ccepted 30 July 2008

a b s t r a c t

Fructooligosaccharides (FOSs) and polyfructosides (PSs) have received particular attention due to its ben-eficial effects as prebiotics. Here we report the synthesis of a new class of fructooligosaccharides bysubstrate and enzyme engineering. Using an engineered levansucrase enzyme (SacB of Bacillus subtilis),and sucrose analogues (�-Xyl-1,2-�-Fru or �-Gal-1,2-�-Fru), the product profile shifted from the fruc-

eywords:estose analogueligosaccharidesevansucrasenzyme engineering

tan (levan) polymer to a range of new higher oligosaccharides (xylooligofructosides), or polysaccharides(galactopolyfructosides), of varying size. Further the enzyme was tailored by random mutagenesis, forthe synthesis of short-chain fructooligosaccharides to yield variant A5 (N242H), which is unable to pro-duce polymers. It shifts its product pattern to short-chain oligosaccharides and hydrolysis and enabledin combination with the sucrose analogue Xyl-Fru for the first time the direct synthesis of a 6-kestose

-2,6-rides

lp2

fwaC((Fldao

analogue (�-Xyl-1,2-�-Fruthat cap fructooligosaccha

. Introduction

Oligosaccharides are found on cell surfaces as glycoproteinr glycolipid conjugates and play important structural and func-ional roles in numerous biological recognition processes. Theserocesses include viral and bacterial infection, cancer metas-asis, inflammatory response, innate and adaptive immunity,nd many other receptor-mediated signalling processes (Varki,993; Wong, 2005). Oligosaccharides and polysaccharides arelso widely used in the food and cosmetics sector (Egglestonnd Cote, 2003; Seibel et al., 2006b). Although fructooligosac-harides (FOSs) presumably do not play an important role onell surfaces, they have received particular attention becausef their favourable features, being low in calories and noncar-ogenic, and acting as selective energy sources for beneficial

icroorganisms in the intestinal flora (Salminen et al., 1996).ost bacterial fructosyltransferases known are levansucrases (EC

.4.1.10) synthesizing fructan polymers composed of �(2 → 6)inked fructose units (levans) (Meng and Fütterer, 2003) and inu-

∗ Corresponding author at: Division of Structural Biology, Helmholtz Centre fornfection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany.el.: +49 531 6181 7002; fax: +49 531 6181 7099.

E-mail address: juergen.seibel@helmholtz-hzi.de (J. Seibel).

oetAAawf(S

168-1656/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.jbiotec.2008.07.1998

�-Fru). The different glycopyranosyl-residues (i.e. galactose and xylose)may alter prebiotic and biochemical properties.

© 2008 Elsevier B.V. All rights reserved.

osucrases (Inu, EC 2.4.1.9), producing �(2 → 1) linked fructanolymers (inulins) (Olivares-Illana et al., 2003; van Hijum et al.,003).

Levansucrase, SacB, of Bacillus subtilis NCIMB 11871 synthesizesrom sucrose both the high-molecular weight polysaccharide levanith a molecular mass up to 3 × 106 Da, and, in the presence of

cceptor molecules, low-molecular weight FOS (Avigad et al., 1957;heetham et al., 1989). It belongs to glycoside hydrolase family 68GH 68) according to the Carbohydrate-Active Enzymes databaseCAZy database; http://www.cazy.org) (Henrissat, 1991). Meng andütterer recently determined the crystal structures of B. subtilisevansucrase in the ligand-free form and bound to the fructosylonor substrate sucrose and very recently also raffinose (Mengnd Fütterer, 2003, 2008). It shows a fivefold �-propeller topol-gy (Meng and Fütterer, 2003, 2008). The glucopyranosyl residuef sucrose is stabilized through various hydrogen bonds into thenzymes active core. Three catalytic amino acids have been iden-ified: Asp86 is acting as a nucleophile, Glu342 is the acid andsp247 stabilizes the transition state (Meng and Fütterer, 2003).lso Ozimek et al. provided clear mutant evidence for these cat-

lytic residues (Ozimek et al., 2004). Asp86 forms a covalent bondith the fructofuranosyl residue of sucrose to yield an enzyme-

ructofuranosyl intermediate which has been identified previouslyChambert and Gonzy-Treboul, 1976a; Meng and Fütterer, 2003).imilar fructosyltransferases of Bacillus megaterium, Lactobacillus

34 R. Beine et al. / Journal of Biotechnology 138 (2008) 33–41

Scheme 1. Possible reaction pathway of levansucrases for the transfructosylation and formation of polyfructosides. (a) A sucrose analogue coordinates in the active site oft the fru( gue (hp

raTiatittasfbtieo(trtesc

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he enzyme. (b) The glycopyranoside is cleaved and a reactive oxocarbenium ion ofc) Covalent fructosyl–enzyme complex, substituted by C6-OH of the sucrose-analoosition) and the free enzyme active site.

euteri and Gluconacetobacter diazotrophicus have these three cat-lytic residues, too (Homann et al., 2007; Ozimek et al., 2004).he levansucrase from B. subtilis has been extensively character-zed regarding the reaction mechanism and kinetics by Chambertnd Gonzy-Treboul (1976b) and Chambert et al. (1974). The reac-ion proceeds via a ping–pong mechanism. The hydrolysis reactions slower by two orders of magnitude than that of the fructosylransfer reaction (for the reversible reaction in 1 M glucose solu-ion), highlighting the differences between glycosyltransferasesnd hydrolases (Chambert and Gonzy-Treboul, 1976b) We havehown that all reaction steps are reversible; thus the equilibrium oformation of the sucrose analogue galactofructoside (Gal-Fru) cane shifted towards higher yields by eliminating glucose as a reac-ion byproduct, and when incubating Gal-Fru with glucose sucroses formed (Baciu et al., 2005; Seibel et al., 2006c). In silico dockingxperiments have been previously used to explore the mechanismf ordered acceptor and the donor substrate binding to levansucraseSeibel et al., 2006c). The X-ray structure of levansucrase suggestshe equatorial hydroxyl group in position 2 of the glucopyranosylesidue in sucrose directs Glu342 into a productive orientationo protonate the glycosidic bond (Meng and Fütterer, 2003). Thisxplains how the levansucrase recognizes novel substrates (i.e. theucrose analogues) and interacts with them specifically at the gly-opyranoside moiety (Scheme 1).

Kinetic and docking studies support the hypothesis that theucrose derivatives bind in a mode similar to sucrose (Scheme 1)Seibel et al., 2006c).

FOSs with prebiotic properties (e.g., kestose and nystose) areructose oligosaccharides joined by �(2 → 1) or �(2 → 6) link-

2

ti

ctosyl-residue is formed and subsequently attacked by Asp86 of the levansucrase.ere as the acceptor) to form a trisaccharide. (d) The trisaccharide formed (acceptor

ges and terminated with a glucose molecule linked to fructosey an �(1 → 2) bond as seen in sucrose. FOSs and inulin transithrough the stomach and small intestine while becoming neitherbsorbed nor degraded and reach the colon. FOSs and polyfructo-ides (PSs) are fermented by resident bacterial groups and promotehe proliferation of bifidobacteria. Beside their positive effects on

icroorganism growth, nondigestible oligosaccharides like galac-ooligosaccharides (GOSs) and xylooligosaccharides (XOSs) havedditional benefits (Boehm et al., 2005; Hsu et al., 2004), suchs inhibition of bacterial adherence on hepatocytes, thus reduc-ng the adherence of enteropathogenic Escherichia coli. Our aimas to combine structural features of GOSs and XOS with FOSs

o design a new class of FOSs. Very recently we demonstrated therst examples of the synthesis of 1-kestose analogues by using aombination of sucrose analogues (substrate engineering) as novelubstrates and a highly active recombinant �-fructofuranosidaserom Aspergillus niger (Zuccaro et al., 2008). Here we aimed atxpanding our approach with levansucrase enzymes to synthe-ize 6-kestose analogues. The different glycopyranosyl-residuesi.e. galactose and xylose) that cap kestose analogues may be rec-gnized by carbohydrate binding cell receptors.

. Materials and methods

.1. Protein expression and purification

E. coli BL21(DE3) cells containing plasmid p24FTF11871 (wild-ype) (Seibel et al., 2006c) or p24FTF1187/A5 (N242H) were grownn Luria–Bertani medium (Anumula and Taylor, 1992) at 37 ◦C

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R. Beine et al. / Journal of B

ith 100 �g mL−1 ampicillin to an OD580 of 0.7; at this OD580,he expression of sacB gene was induced by the addition of 1 mMsopropyl-�-d-thiogalactoside (IPTG). Induced cells were culturedor 24 h at 28 ◦C, harvested by centrifugation, and resuspended in0 mM Na2HPO4/NaH2PO4 (pH 6.2). Cells were disrupted by soni-ation, and pelleted by centrifugation. The wild-type enzyme andariant A5 (N242H) were purified using a CM-Sepharose column asescribed previously (Biedendieck et al., 2007).

.2. Synthesis of sucrose analogues

�-d-Fructofuranosyl-�-d-mannopyranoside,-d-fructofuranosyl-�-d-galactopyranoside, �-d-fructofuranosyl--d-allopyranoside, �-d-fructofuranosyl-�-d-fucopyranosidend �-d-fructofuranosyl-�-d-xylopyranoside (Man-Fru, Gal-Fru,ll-Fru, d-Fuc-Fru, and Xyl-Fru) synthesis, purification and charac-

erisation by NMR and mass spectroscopy (MS) were performed asescribed previously (Seibel et al., 2005, 2006c).

.3. General description of the fructosylation reaction usingucrose and sucrose analogues

Reactions were performed with purified wild-type levansu-rase and A5 variant. Wild-type levansucrase (13 mg L−1) or variant5 (N242H; 28 mg L−1) was added to a reaction mixture con-

aining 90 g L−1 sucrose or sucrose analogues (All-Fru, Gal-Fru,-Fuc-Fru, Xyl-Fru), as substrates in 50 mM phosphate buffer (pH) and 50 mg L−1 CaCl2. Man-Fru (90 g L−1) was incubated withild-type levansucrase (328 mg L−1) or variant A5 (696 mg L−1).

roduct formation was investigated by discontinuous analysisf aliquots from the reaction mixture at suitable time intervalsp to 24 h at 37 ◦C. The enzyme was inactivated by boiling theamples in a water bath for 10 min. After cooling, the inacti-ated samples were filtered through a 0.22-�m nitrocelluloseembrane filter (Millipore, Germany). Accurate values for Km

nd kcat were determined from initial rate data at seven toen substrate concentrations by a direct fit of the data to the

ichaelis–Menten-equation using the computer program Origin.0 (OriginLab Corporation). One unit of total enzyme activityhydrolysis and transfructosylation) is defined as the release of�mol of glycopyranoside per minute at 37 ◦C and substrate satura-

ion. Analysis of the samples was carried out using several differenthromatographic systems as described below. The amount of gly-opyranoside was determined by thin layer chromatography andPLC.

.4. HPLC analysis

Enzymatic reactions were analyzed by high-performanceiquid chromatography (HPLC). HPLC was performed with

RCM monosaccharide Ca2+ column (300 mm × 7.8 mm,henomenex®, Germany) operated at 80 ◦C and an ion chro-atograph (IC) (Metrohm, Germany) with a refractive index

etector (ERC-7512, Erma, Germany) using distilled water asluent at 0.8 mL min−1. Standard sugar solutions were pre-ared in the range of 0.1–10 g L−1 (fructose, galactose, glucose,ylose, sucrose, melibiose, raffinose, 1-kestose, nystose). Theonosaccharides fructose, galactose, glucose, xylose, the dis-

ccharides sucrose and melibiose, the trisaccharides raffinosend 1-kestose, and the tetrasaccharide nystose were used asxternal standards for peak identification and quantification.he relative standard deviation of this system is approximately%.

ro(wa

nology 138 (2008) 33–41 35

.5. Densitometry analysis

Aliquots from levansucrase reactions were analyzed using thinayer chromatography (TLC) at room temperature (rt). The solventystem ethylacetate/isopropanol/water was used in a ratio of 6/3/1v/v/v) for Gal-Fru and Man-Fru reactions. For Xyl-Fru and Fuc-ru reactions, acetonitrile/water in a ratio of 4/1 (v/v) was useds mobile phase. Reaction samples (final concentration between.05 and 1.0 g L−1) were applied using “end-to-end pipettes” (3 �Lolume, HIRSCHMANN® minicaps, Hirschmann, Germany) on sil-ca thin-layer plates (TLC aluminium sheets 20 × 20 cm, silicael 60 F254 with concentrating zone 20 × 2.5 cm, MERCK, Ger-any). The carbohydrates were separated using one to five ascents

2–5 × 90 min ethylacetate/isopropanol/water or 1 × 45 min ace-onitrile/water). Spots were detected by dipping the plates into theetecting reagent (0.3% (w/v) of N-(1-naphtyl)-ethylenediamine,luka, Germany) and 5% (v/v) concentrated sulfuric acid inethanol using a CAMAG Chromatogram Immersion Device III

speed 2, time 4, Camag, Switzerland), followed by heating in anven at 110 ◦C for 30 min. The sugars were visualized as dark spotsn a pale background. The intensity of spots was quantitated (inrange of 150–3000 ng sugar) by densitometric scanning the TLClate using a Bio-Rad Imaging Densitometer using Quantity One®

oftware (Version 4.2).

.6. Preparative chromatography

Separations of oligosaccharides derived from the sucrose ana-ogue reaction were carried out on a 9 cm × 46 cm column of BioGel2 (fine) that was eluted at rt with distilled water at a flow rate of8 mL h−1. The carbohydrate content of each fraction was analyzedy HPLC.

.7. High-performance anion-exchange chromatography (HPAEC)

Analytical HPAEC was performed on a Dionex DX 300 chro-atography system equipped with either a CarboPac PA1 column

r a CarboPac PA-100 column. Samples were eluted from the Car-oPac PA1 column (4 mm × 250 mm, Dionex, USA) at 1 mL min−1

sing a linear gradient of aqueous sodium acetate (1 M, pH 6.0)inearly from 1 to 8% over 25 min, and then from 8 to 15% over0 min. Samples were eluted from the CarboPac PA-100 column4 mm × 50 mm, Dionex) with sodium acetate (1 M) in aqueousodium hydroxide (0.1 M) linearly from 20 to 40% over 65 min.lution of the oligosaccharides was monitored with pulsed amper-metric detection, including a post-column derivatization at pH 6.0sing 1.5 M sodium hydroxide.

.8. Kinetic analysis

The total activity minus the hydrolytic activity (fructose release)eflected the transglycosylation enzyme activity (polymer and fruc-ooligosaccharide formation). The reaction products synthesized byevansucrase from B. subtilis with sucrose analogues as substrates

ere mixed with the reaction products derived from sucrose toighlight novel and identical products.

.9. Analysis by electrospray ionization MS

Aliquots (1–3 �L) corresponding to 2–20 pmol of oligosaccha-

ides were applied to a nanospray gold-coated glass capillary placedrthogonally in front of the entrance hole of a QTOF-II instrumentMicromass, UK). Then 1000 V was applied to the capillary and ionsere separated by the time-of-flight (TOF) analyzer. For MS/MS

nalysis, parent ions were selected by the quadrupole mass filter

36 R. Beine et al. / Journal of Biotechnology 138 (2008) 33–41

F antly ah with A

ai

2

b1tG

2

u21mmutf(mT

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pom

ig. 1. (a) Wild-type levansucrase (right) formed from the substrate sucrose dominydrolysis; (b) view of the active centre of the wild-type levansucrase from B. subtilis

nd subjected to collision-induced dissociation. Resulting daughterons were further separated by the TOF-analyzer.

.10. GC–MS analysis

The intact polysaccharides were permethylated as describedy Anumula and Taylor (1992), followed by hydrolysis (4 N TFA,10 ◦C, 2 h), reduction (NaBD4), and acetylation. The resulting par-ially methylated alditol acetates were analyzed on a ThermoQuestCQ ion trap GC/MS system (MS: EI mode, GC: 30 m DB 5 column).

.11. Random mutagenesis of the levansucrase gene sacB

The E. coli strain BL21 (DE3) (Novagen, Madison, WI, USA) wassed for library construction. Plasmid p24FTF1187 (Seibel et al.,006c) carrying the wild-type sacB gene from B. subtilis NCIMB1871 was used as template in mutagenic PCR. DNA-isolation, -anipulation and -transformation were performed with standardethods (Sambrook et al. 1989). Error prone PCR was carried out

sing primers 5′-GATATAAACATATGAACATCAAAAAGTTTGCA-3′ for

he forward primer and 5′-CCAACTCGAGTTTGTTAACGTTTAATTG 3′

or the reverse primer with restriction sites for NdeI and XhoIunderlined). Mutagenic PCR was performed in a 100-�l reaction

ixture containing 16 mM (NH4)2SO4, 67 mM Tris pH 8.8, 0.01%ween 20, 2.5 mM MgCl2, 40 �M MnCl2, 0.2 mM (dATP/dGTP),

21m(2

polyfructoside while variant A5 (left) synthesized the trisaccharide 6-kestose andsn242 (wt). The substitution with His (A5) abrogated the polysaccharide synthesis.

mM (dCTP/dTTP), 2 fmol plasmid DNA as template, 40 pmol ofach primer and 5 U Tac polymerase (AppliChem, Darmstadt, Ger-any). Thermal cycling parameters were 97 ◦C for 7 min (1 cycle),

5 ◦C for 1 min, 54 ◦C for 1 min, 72 ◦C for 2 min (25 cycles), and 72 ◦Cor 10 min. Purified restricted PCR products were ligated with NdeI-hoI digested expression vector pET24a + and transformed intoompetent BL21 (DE3) cells using standard methods (Sambrookt al., 1989). Mutated sacB PCR products were cloned into plas-id pET24a + (Novagen, Madison, WI, USA). Error rate of mutagenic

CR was verified by sequencing 10 DNA samples isolated fromecombinant clones (Sequiserve, Vaterstetten, Germany). Sequencenalysis revealed an error rate of ∼1–2 base substitutions perene.

.12. Library screening

Transformed cells were plated on Lauria–Bertani (LB) agarlates supplemented with 50 �g mL−1 kanamycine and grownvernight at 37 ◦C. Single colonies were picked into 364-wellicrotiter plates containing 100 �L medium and incubated for

4 h at 37 ◦C. The master plates were duplicated by transferring a�L aliquot to a 96-well microtiter plate containing 100 �l LB-Kmedium + 1 mM IPTG and grown for 16 h at 30 ◦C. Sucrose solution

50 �L, 100 gL−1) was added and the reaction analysed by TLC after4 h.

R. Beine et al. / Journal of Biotechnology 138 (2008) 33–41 37

-(1,2)-

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Scheme 2. Structures of sucrose analogues with glycosidic bonds �

.13. ˇ-d-Fructofuranosyl-(2 → 6)-ˇ-d-fructofuranosyl-(2 → 1)--d-xylopyranoside (Xyl-(Fru)2)

White solid, m.p. 235 ◦C: Rf = 0.168 (6:3:1 EtOAc–isopropanol–ater, 3 ascends in 90 min); [˛]D + 4.83 (c 0.725, H2O); 1H NMR

600 MHz, D2O) ı = 5.27 (d, J1,2 3.8 Hz, 1H, 1-H), 4.17–4.16 (d, J3′ ,4′.9 Hz, 1H, 3′-H), 4.06–4.03 (t, J4′ ,3′ = J4′ ,5′ 8.9 Hz, 1H, 4′-H), 3.92–3.88dt, J5′ ,4′ = 8.9, J5′ ,6′ 2.8 Hz, 1H, 5′-H), 3.77–3.74 (2d, J6a′ ′ ,5′ ′ = J6b′ ′ ,5′ ′.8 Hz, 2H, 6a′ ′ -H, 6b′ ′ -H), 3.84-3.80 (m, 2H, 3-H, 5-H), 3.63 (s, 2H,′-H2), 3.70 (s, 2H, 1′ ′-H2), 3.61–3.59 (m, 1H, 4-H), 3.50–3.48 (dd,

ig. 2. Reaction and product formation over a 24-h period using the substrates Gal-ru (a) and Man-Fru (b), both with wild-type levansucrase. Fructan formation isbserved with Gal-Fru (a) as the substrate, but not using Man-Fru (a) Gal-Fru: �,al: �, d-Fru: �, polyfructoside: �; (b) Man-Fru: �, Man: �, d-Fru: �].

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�-fructoside that were synthesized and used for substrate studies.

2,3 9.8, J2,1 3.6 Hz, 1H, 2-H), 4.13–4.11 (d, J3′ ,4′ 8.9 Hz, 1H, 3′ ′-H).04–4.02 (t, J4′ ′ ,3′ ′ = J4′ ′5′ ′ 8.9 Hz, 1H, 4′ ′-H), 3.81–3.75 (dt, J5′ ′ ,4′ ′ = 8.9,

5′ ′6′ ′ 2.8 Hz, 1H, 5′ ′-H) 13C NMR (600 MHz, D2O) ı = 106.75 (C-2′ ′),06.54 (C-2′), 95.20 (C-1), 83.96 (C-5′ ′), 83.13 (C-5′), 79.25 (C-3′ ′),8.77 (C-3′), 77.40 (C-4′ ′), 77.31 (C-4′), 75.57 (C-3), 73.91 (C-2), 72.10C-4), 65.66 (C-6′ ′), 65.33 (C-6′), 64.60 (C-5), 63.40 (C-1′ ′), 62.50C-1′). ESIMS: m/z 497.0 100% [M + Na+].

. Results

.1. Enzyme engineering of levansucrase SacB from B. subtilisCIMB 11871 for FOS synthesis

Oligofructans like kestose and nystose are well known forheir health benefits in human nutrition (Hiramaya et al., 1993).

e envisage that the substitution of glucose-residue to anotherlycopyranosyl-residue like xylose will effect the structural andiochemical properties of the new molecule. In contrast to shorthain FOSs the glycopyranosyl-residue in polysaccharides mayot influence its structure and properties tremendously. Thus,ur first aim was to generate a levansucrase that does not pro-uce polysaccharides but instead short-chain FOSs. To increase theligofructoside formation and to reduce the levan production ofhe levansucrase from B. subtilis NCIMB 11871, random mutagene-is was applied to the entire levansucrase gene sacB by error-proneCR with Taq-polymerase. Adjusting the concentration of Mn2+ andhe ratio of deoxyribonucleoside triphosphates during PCR led ton error rate of ∼1–2 base substitutions per gene, as was obvi-us from sequence analysis. Recombinant clones of the mutantibrary were transferred in 96-well microtiter plates and the reac-ion plates were tested with sucrose on polymer formation by TLC.fter screening of 200 mutants of the library (15,000 clones) oneariant (A5) was identified, that catalyzed the increased forma-ion of oligofructosides, hydrolysis and in contrast to the wild-typenzyme no levan production was detected (Fig. 1a). Sequence anal-sis of the variant A5 revealed the presence of two mutations inhe gene, V106A and N242H (Fig. 1b). We used these novel insightsor a functional characterization of a novel levansucrase from B.egaterium (74% identity with SacB from B. subtilis) (Homann et

l., 2007). There we found that V115A (Val106 B. subtilis num-ering) mutation has no effect on levansucrases (Homann et al.,007).

.2. Kinetic parameters of levansucraseSacB wild-type and

ariant A5 with sucrose analogues

The convenient synthetic routes using levansucrases andlucansucrases are limited, however, to the transfer of fruc-ose and glucose with sucrose as substrate. Sucrose ana-

38 R. Beine et al. / Journal of Biotechnology 138 (2008) 33–41

Table 1Kinetic parameters determined for wild-type levansucrase from B. subtilis and A5 with sucrose and sucrose analogues

Substrate Km (mM)a,b kcat (s−1)a,b kcat/Km (mM−1 s−1) ��G (kcal mol−1)

Sucrose (wild-type) c14c 85.5 (3.2) 6.1 0Man-Fru (wild-type) 30.8 (7.9) 0.4 (0.1) 0.01 3.8All-Fru (wild-type) – – – –Gal-Fru (wild-type) 20.3 (4.1) 36.7 (2.8) 1.8 0.7D-Fuc-Fru (wild-type) 40.5 (5.7) 70.6 (4.9) 1.7 0.8Xyl-Fru (wild-type) 28.5 (4.3) 72.6 (3.1) 2.6 0.5Sucrose (variant A5) 222 (44.5) 40.4 (4.2) 0.2 –

Overall activation energy (��G) was calculated from relative values of kcat/Km using the kinetic constants of sucrose and an analogue in the equation ��G =RT ln

(kcat(suc)/Km(suc)

kcat(analogue)/Km(analogue)

).

a Parameters determined as described in Section 2. Reaction times were 60 min, and enzyme concentrations used for each substrate were as follows: Gal-Fru 0.2–5 U mL−1,Man-Fru 7.4–148 U mL−1, Xyl-Fru 0.1 U to 1.0 mL−1, d-Fuc-Fru 0.3–0.6 U mL−1. Enzyme concentrations used for each substrate were added as estimated to convert about 10%o

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fractionated into five groups using a BioGel P2 gel permeationcolumn chromatography. The unit length was determined by MSanalysis. The ESI-MS analysis results agree with the structuralassignments proposed from HPAEC and TLC analysis. As exem-

Table 2Endpoint reactions of different substrates with wild-type levansucrase and variantA5

Substrate Hydrolysis (%) Transglycosylation products (%)

Sucrose (wild-type) 46 54Sucrose (A5) 62 38Man-Fru (wild-type) 95 5All-Fru (wild-type)a <1 <1Gal-Fru (wild-type) 52 48Gal-Fru (A5) 97 3D-Fuc-Fru (wild-type) 51 49Xyl-Fru (wild-type) 47 53Xyl-Fru (A5) 83 17

f the different initial sugar concentrations within 60 min.b Values in parentheses represent the error limits (*) on the reported number.c Data correspond to literature data (Mäntsälä and Puntala, 1982).

ogues containing the same high energy �-glycosidic bondike sucrose, such as, �-d-fructofuranosyl-�-d-mannopyranoside,-d-fructofuranosyl-�-d-galactopyranoside, �-d-fructofuranosyl--d-allopyranoside, �-d-fructofuranosyl-�-d-fucopyranoside and-d-fructofuranosyl-�-d-xylopyranoside (Man-Fru, Gal-Fru, All-ru, d-Fuc-Fru, and Xyl-Fru) have been synthesized (Scheme 2)Baciu et al., 2005; Seibel et al., 2005, 2006c).

They have been evaluated as donor substrates for bacterial fruc-osyltransferases (Biedendieck et al., 2007; Seibel et al., 2006a). Weave determined the kinetic parameters for wild-type levansucrase

rom B. subtilis and variant A5 with sucrose and sucrose analoguesTable 1). The kcat/Km value reflects the first step, most likely theormation of the fructosyl-enzyme intermediate. Essentially a weakeduction in kcat/Km was seen for most sucrose-analogues as com-ared to sucrose. The kcat/Km of Xyl-Fru was decreased threefold,hat of d-Fuc-Fru and Gal-Fru were decreased fourfold, and that of

an-Fru was reduced by 610-fold relative to sucrose. There was noeasurable reaction with All-Fru; less than 1% was transformed,ost due to hydrolysis. The kcat value for Man-Fru was lowered

30-fold. The Km value includes the affinity of substrate for enzyme.he Km value for Man-Fru remaining very similar to that of sucroseould suggest that the equatorial position of the hydroxyl group

n position 2 is crucial for catalysis. The kcat value for sucrose withariant A5 decreased twofold and that of Km increased 16-fold com-ared with the wild-type enzyme, leading to a 32-fold reduction ofcat/Km.

.3. FOS and PS synthesis using substrate analogues and theevansucrase variant

Sucrose analogues (90 gL−1) were incubated with purified wild-ype levansucrase and levansucrase variant A5 (1.48 U mL−1 for

ost analogues, 37.1 U mL−1 for Man-Fru) at 37 ◦C and the reactionrogress was followed by TLC and HPAEC (Fig. 2). All substratesxcept All-Fru were consumed within 24 h. The wild-type enzymeonverted 54% of the sucrose into transglycosylation products,6% to levan and 18% to FOSs. Variant A5 converted only 38%o transfructosylation products, nearly exclusively to 6-kestosend 6-nystose. The wild-type enzyme converted 52% of the Gal-ru into transglycosylation products, almost exclusively levan-typeolysaccharide, and 48% of Gal-Fru was hydrolysed. Reaction

ith Gal-Fru and variant A5, which is unable to produce levan,

ielded only 3% FOS, the dominant reaction was hydrolysis. Xyl-Fruielded 53% and d-Fuc-Fru 49% transglycosylation products withild-type levansucrase. Variant A5 yielded 17% transglycosylationroducts from Xyl-Fru, the rest was hydrolyzed as well. Man-Fru

Rt53

as predominantly hydrolyzed (95%) by the wild-type enzyme.nly 5% transglycosylation products were synthesized during the

eaction with Man-Fru. These were also hydrolyzed when the reac-ion time was extended. The levan formed in the reaction maye a better substrate for the levansucrase than Man-Fru itselfTable 2).

A more detailed analysis of the transfructosylation productsighlighted large differences between the products formed fromucrose analogues. Although very small amounts of FOSs wereroduced from the substrates Gal-Fru and d-Fuc-Fru by wild-type

evansucrase, new xylo-fructooligosaccharides (XFOSs), includingyl-(Fru)n-Fru (n = 1–26), were formed from Xyl-Fru with the wild-ype enzyme. The decrease of the lower molecular weight XFOSsuring extended reaction times correlated with the appearance ofigher molecular weight XFOSs (Fig. 3).

Wild-type levansucrase produces a wide range of XFOS withyl-Fru, but variant A5 does not. The main transfructosylation prod-ct with Xyl-Fru is Xyl-Fru2 (Fig. 4) and a high rate of hydrolysis

s detected. The 17% of transglycosylation products consist of 15%yl-Fru2 and only 2% other products, mainly Xyl-Fru3.

.4. Oligosaccharide analysis by ESI-MS

The reaction products from Xyl-Fru and wild-type enzyme were

eaction conditions as described in Section 2. Reaction times and enzyme concen-rations used for each substrate were as follows: 1345 min Gal-Fru and Man-Fru,10 min others; 90 g L−1 initial substrate concentration, pH 6, 37 ◦C, 0.05 g L−1 CaCl2,7.1 U mL−1 for Man-Fru, 1.48 U mL−1 for others.a Reaction with All-Fru is very slow compared with sucrose.

R. Beine et al. / Journal of Biotechnology 138 (2008) 33–41 39

F , (b) 41 of loa

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ig. 3. HPAEC of the XFOS products of Xyl-Fru (90 g L−1) at 37 ◦C after (a) 10 min.48 U mL−1. The chromatographic analysis shows a significant shift of the formationnd d) with increasing reaction time.

lified in the ESI spectrum, oligosaccharides produced by thisevansucrase from Xyl-Fru yielded a major molecular ion series cor-esponding to FOSs [Fru1–10 + Na]+ at m/z 203, 365, 527, 689, etc. Theorresponding derivatives bearing one additional xylose residue[Xyl-Fru1–9 + Na]+ appeared at m/z 335, 497, 659, etc. The xyloseontaining FOS ([Xyl-Fru3 + Na]+ at m/z 659 was further subjectedo tandem mass analysis (MS/MS) (data not shown). The spectrahowed a characteristic fragment ion at m/z 527 for [Fru3 + Na]+,hich provided evidence for the sequence Xyl-Fru3. These resultsere confirmed by an analogous analysis of the permethylatederivatives (data not shown).

The trisaccharide obtained from the reaction of variant A5 andyl-Fru was isolated by BioGel P2 gel permeation column chro-atography. The ESI-MS analysis showed the adduct ion at m/z

97 [M + Na]+ which characterize a xylofructooligosaccharide withhe molecular formula C17H30O15 (Xyl-Fru2). Further structuralvidence of the �-d-fructofuranosyl-(2 → 6)-�-d-fructofuranosyl-2 → 1)-�-d-xylopyranoside (Xyl-Fru2, a 6-kestose analogue) waserified by 1H NMR and 13C NMR spectroscopy.

.5. Methylation analysis

It was expected that the xylofructooligosaccharides composed

f �(2 → 6) linked fructose units (levan). To unequivocally iden-ify the linkage pattern of the monosaccharide units of the XFOSshe polysaccharides were permethylated (Anumula and Taylor,992), followed by hydrolysis with trifluoro acetic acid (TFA). Theonosaccharide mixture of fructose and xylose units were reduced

(le(G

5 min, (c) 510 min and (d) 1345 min incubation using wild-type levansucrase atw molecular weight (a and b) towards higher molecular weight oligosaccharides (c

y NaBD4 to glucitol and mannitol derivatives (originating fromhe fructose residues) and xylitol (from xylose residue) and per-cetylated. The resulting partially methylated alditol acetates werenalyzed by GC/MS (Table 3). The derivatives characteristic for 2,6-ubstituted fructose (1,3,4-Tri-O-methyl glucitol, mannitol) wereetected, corresponding to the inner fructose residues of the fruc-ooligosaccharide chains. In contrast, trace amounts of Fru(2 → 1)inkages were observed. The terminal position of the xylosyl residueas verified as 2,3,4-Tri-O-methyl-xylitol derivative.

. Discussion

Levansucrase essentially converts sucrose into a large levanolymer, only few intermediate oligosaccharides can be detectednd polysaccharides of high molecular weight are obtained fromhe start of the reaction (Ozimek et al., 2006b). Here we testeducrose analogues as alternative substrates for B. subtilis levansu-rase. Our sucrose analogues Man-Fru, Gal-Fru, Xyl-Fru, and Fuc-Fruere accepted as substrates by B. subtilis levansucrase. With theseew substrates we have obtained novel oligosaccharide productshat may be of scientific and/or practical interest.

The fructan synthesis with sucrose-analogue Gal-Fru by levan-ucrase proceeded in the same manner like sucrose. Gal-(Fru)n-Fru

n > 12) was the dominate polymer from Gal-Fru with simi-ar transfructosylation efficiency. In contrast the use of Xyl-Fruffected the formation of the oligosaccharide XFOS series Xyl-Fru)n-Fru (n = 1–8). This was not observed with sucrose oral-Fru. Thus, the substrate analogues influence the lengths

40 R. Beine et al. / Journal of Biotech

Fig. 4. HPAEC of the XFOS products using (a) 90 g L−1 Xyl-Fru with 2.00 U mL−1

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The data obtained from these studies could be subsequently

TT

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ild-type levansucrase at 37 ◦C after 115 min incubation, and using (b) 90 g L−1 Xyl-ru with 0.78 U mL−1 variant A5 at 40 ◦C after 120 min incubation. The selectiveormation of the trisaccharide by the variant A5 is obvious.

f the products, the contribution and the efficiency of thenzymatic reaction. What could be a reasonable explana-ion?

Meng and Fütterer reported recently about the raffinose-boundevansucrase, where they observed that the galactosyl-residue ofaffinose make water mediated H-bonds of the 6′′-hydroxyl withsn242 and Tyr237, whereas the 2′′-, 3′′- and 4′′-hydroxyl groupsoint into solvent (Meng and Fütterer, 2008). Thus, Asn242 locatedt subsite +2 may contribute to acceptor binding and the coor-

ination of the growing fructan chain. In the substrate Gal-Fru,he galactosyl residue may bind as acceptor over 6-hydroxyl ofhe galactosyl residue in the same way observed for raffinose. As

consequence the polymer can be formed. The xylosyl-residue

ustp

able 3he fructooligosaccharide mixtures were permethylated, and then hydrolyzed (TFA), follo

eracetylated derivative of Linkage type

ylitol2,3,4-Tri-O-methyl- ta-d-Xylp

lucitol1,2,3,4,5-Penta-O-methyl-(2-d)- 6-Linked d-Fruf1,3,4,6-Tetra-O-methyl- ta-d-Fruf2,3,4,6-Tetra-O-methyl ta-d-Glcp1,3,4-Tri-O-methyl (26) d-Fruf3,4,6-Tri-O-methyl (21) d-Fruf

annitol1,2,3,4,5-Penta-O-methyl-(2-d)- 6-Linked d-Fruf1,3,4,6-Tetra-O-methyl- ta-d-Fruf1,3,4-Tri-O-methyl (26) d-Fruf3,4,6-Tri-O-methyl (21) d-Fruf

he resulting partially methylated alditol acetatesa were analyzed by GC/MS.a t, terminal.

nology 138 (2008) 33–41

f Xyl-Fru has no 6-hydroxyl group and cannot be coordinateds an acceptor in the same manner like Gal-Fru or sucrose. Thisay explain the appearance of xylofructooligosaccharides. With d-

uc-Fru as substrate higher amounts of oligofructosides comparedo the substrate sucrose was observed, but also polymer forma-ion took place. This indicates that the 6-hydroxyl group of thelycopyranosyl-residue determines not exclusively between poly-er or oligosaccharide formation, but at least it contributes to the

cceptor binding.Further insights about the +2 subsite result from the variant

5. Mutant enzyme A5 (N242H) is unable to produce polymersnd shifts its product pattern to short chain oligosaccharides andydrolysis. So far most site directed mutagenesis has been donen residues directly involved in binding sucrose in the active site.utations at subsite +1 led to 1-kestose formation (Chambert and

etit-Glatron, 1991), while mutations at subsite-1 in FOS producingnzymes led to a shift to polymer formation (Ozimek et al., 2006a).ut Asn242His mutation in variant A5 located at the +2 sugar bind-

ng subsite plays an import role in polymer versus oligosaccharideynthesis as recently demonstrated (based on this study) with theomologue B. megaterium levansucrase (Homann et al., 2007) andupported by a recent study of Meng and Fütterer (2008). Due tots position at the +2 subsite, it might stabilise the third fructosylnit of the growing oligosaccharide chain and direct it as an accep-or substrate to an optimal position for further transfructosylationScheme 1) (Homann et al., 2007). The substitution of asparaginegainst histidine may effect that the coordination of this positionith the third sugar residue is abolished. This effect was supported

y a mutation of Asn252 to aspartate (corresponding to Asn 242 in. subtilis) where the polymerase activity was preserved (Homannt al., 2007). The exact mode of the acceptor binding is still unclear.

These previous discussed effects combined with Xyl-Fru as sub-trate and levansucrase variant A5 resulted in the production ofne main product, a 6-kestose analogue (�Xyl-(1,2)-�-Fru-(2,6)--Fru). As 6-kestose and XOFs have strong prebiotic activity, the

mpact of this novel product on the probiotic properties should beested in mammalian gut system. However, the efficiency and theransfer-rate of the enzyme need to be improved in future as theigh hydrolysis rate may limit the scope of oligosaccharide synthe-is.

sed to create tailor-made enzymes, producing novel fructans withpecific sizes and containing specific glycopyranosyl-residues forhe preparative synthesis of interesting and promising hetero-FOSs,olymers and glycoconjugates as biomaterials.

wed by reduction (NaBD4), and acetylation

FOS-mixture 1-Kestose Nystose

0.3 – –

0.7 – –1.0 0.3 0.3– 1.0 0.950.9 – –– 0.9 1.0

0.8 – –0.5 0.2 0.20.6 – –– 0.9 1.0

iotech

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cknowledgement

Financial support by the German Research Foundation via Son-erforschungsbereich 578 “From Gene to Product” is gratefullycknowledged.

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