Journal of Biotechnology 138 (2008) 3341

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Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec

Synthesis of novel fructooligosaccharides by substrate and enzyme engineering

Rafael Beine a , Roxana Moraru a , Manfred Nimtz b , Shukrallah Naamnieh c , Alice Pawlowski c , Klaus Buchholz a , Jrgen Seibel a,b,
a b c

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

a r t i c l e

i n f o

a b s t r a c t
Fructooligosaccharides (FOSs) and polyfructosides (PSs) have received particular attention due to its benecial effects as prebiotics. Here we report the synthesis of a new class of fructooligosaccharides by substrate 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 prole shifted from the fructan (levan) polymer to a range of new higher oligosaccharides (xylooligofructosides), or polysaccharides (galactopolyfructosides), of varying size. Further the enzyme was tailored by random mutagenesis, for the synthesis of short-chain fructooligosaccharides to yield variant A5 (N242H), which is unable to produce polymers. It shifts its product pattern to short-chain oligosaccharides and hydrolysis and enabled in combination with the sucrose analogue Xyl-Fru for the rst time the direct synthesis of a 6-kestose analogue (-Xyl-1,2--Fru-2,6--Fru). The different glycopyranosyl-residues (i.e. galactose and xylose) that cap fructooligosaccharides may alter prebiotic and biochemical properties. 2008 Elsevier B.V. All rights reserved.

Article history: Received 15 May 2008 Received in revised form 4 July 2008 Accepted 30 July 2008 Keywords: Kestose analogue Oligosaccharides Levansucrase Enzyme engineering

1. Introduction Oligosaccharides are found on cell surfaces as glycoprotein or glycolipid conjugates and play important structural and functional roles in numerous biological recognition processes. These processes include viral and bacterial infection, cancer metastasis, inammatory response, innate and adaptive immunity, and many other receptor-mediated signalling processes (Varki, 1993; Wong, 2005). Oligosaccharides and polysaccharides are also widely used in the food and cosmetics sector (Eggleston and Cote, 2003; Seibel et al., 2006b). Although fructooligosaccharides (FOSs) presumably do not play an important role on cell surfaces, they have received particular attention because of their favourable features, being low in calories and noncariogenic, and acting as selective energy sources for benecial microorganisms in the intestinal ora (Salminen et al., 1996). Most bacterial fructosyltransferases known are levansucrases (EC 2.4.1.10) synthesizing fructan polymers composed of (2 6) linked fructose units (levans) (Meng and Ftterer, 2003) and inu-

Corresponding author at: Division of Structural Biology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany. Tel.: +49 531 6181 7002; fax: +49 531 6181 7099. E-mail address: juergen.seibel@helmholtz-hzi.de (J. Seibel). 0168-1656/$ see front matter 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2008.07.1998

losucrases (Inu, EC 2.4.1.9), producing (2 1) linked fructan polymers (inulins) (Olivares-Illana et al., 2003; van Hijum et al., 2003). Levansucrase, SacB, of Bacillus subtilis NCIMB 11871 synthesizes from sucrose both the high-molecular weight polysaccharide levan with a molecular mass up to 3 106 Da, and, in the presence of acceptor molecules, low-molecular weight FOS (Avigad et al., 1957; Cheetham et al., 1989). It belongs to glycoside hydrolase family 68 (GH 68) according to the Carbohydrate-Active Enzymes database (CAZy database; http://www.cazy.org) (Henrissat, 1991). Meng and Ftterer recently determined the crystal structures of B. subtilis levansucrase in the ligand-free form and bound to the fructosyl donor substrate sucrose and very recently also rafnose (Meng and Ftterer, 2003, 2008). It shows a vefold -propeller topology (Meng and Ftterer, 2003, 2008). The glucopyranosyl residue of sucrose is stabilized through various hydrogen bonds into the enzymes active core. Three catalytic amino acids have been identied: Asp86 is acting as a nucleophile, Glu342 is the acid and Asp247 stabilizes the transition state (Meng and Ftterer, 2003). Also Ozimek et al. provided clear mutant evidence for these catalytic residues (Ozimek et al., 2004). Asp86 forms a covalent bond with the fructofuranosyl residue of sucrose to yield an enzymefructofuranosyl intermediate which has been identied previously (Chambert and Gonzy-Treboul, 1976a; Meng and Ftterer, 2003). Similar fructosyltransferases of Bacillus megaterium, Lactobacillus

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Scheme 1. Possible reaction pathway of levansucrases for the transfructosylation and formation of polyfructosides. (a) A sucrose analogue coordinates in the active site of the enzyme. (b) The glycopyranoside is cleaved and a reactive oxocarbenium ion of the fructosyl-residue is formed and subsequently attacked by Asp86 of the levansucrase. (c) Covalent fructosylenzyme complex, substituted by C6-OH of the sucrose-analogue (here as the acceptor) to form a trisaccharide. (d) The trisaccharide formed (acceptor position) and the free enzyme active site.

reuteri and Gluconacetobacter diazotrophicus have these three catalytic residues, too (Homann et al., 2007; Ozimek et al., 2004). The levansucrase from B. subtilis has been extensively characterized regarding the reaction mechanism and kinetics by Chambert and Gonzy-Treboul (1976b) and Chambert et al. (1974). The reaction proceeds via a pingpong mechanism. The hydrolysis reaction is slower by two orders of magnitude than that of the fructosyl transfer reaction (for the reversible reaction in 1 M glucose solution), highlighting the differences between glycosyltransferases and hydrolases (Chambert and Gonzy-Treboul, 1976b) We have shown that all reaction steps are reversible; thus the equilibrium of formation of the sucrose analogue galactofructoside (Gal-Fru) can be shifted towards higher yields by eliminating glucose as a reaction byproduct, and when incubating Gal-Fru with glucose sucrose is formed (Baciu et al., 2005; Seibel et al., 2006c). In silico docking experiments have been previously used to explore the mechanism of ordered acceptor and the donor substrate binding to levansucrase (Seibel et al., 2006c). The X-ray structure of levansucrase suggests the equatorial hydroxyl group in position 2 of the glucopyranosyl residue in sucrose directs Glu342 into a productive orientation to protonate the glycosidic bond (Meng and Ftterer, 2003). This explains how the levansucrase recognizes novel substrates (i.e. the sucrose analogues) and interacts with them specically at the glycopyranoside moiety (Scheme 1). Kinetic and docking studies support the hypothesis that the sucrose derivatives bind in a mode similar to sucrose (Scheme 1) (Seibel et al., 2006c). FOSs with prebiotic properties (e.g., kestose and nystose) are fructose oligosaccharides joined by (2 1) or (2 6) link-

ages and terminated with a glucose molecule linked to fructose by an (1 2) bond as seen in sucrose. FOSs and inulin transit through the stomach and small intestine while becoming neither absorbed nor degraded and reach the colon. FOSs and polyfructosides (PSs) are fermented by resident bacterial groups and promote the proliferation of bidobacteria. Beside their positive effects on microorganism growth, nondigestible oligosaccharides like galactooligosaccharides (GOSs) and xylooligosaccharides (XOSs) have additional benets (Boehm et al., 2005; Hsu et al., 2004), such as inhibition of bacterial adherence on hepatocytes, thus reducing the adherence of enteropathogenic Escherichia coli. Our aim was to combine structural features of GOSs and XOS with FOSs to design a new class of FOSs. Very recently we demonstrated the rst examples of the synthesis of 1-kestose analogues by using a combination of sucrose analogues (substrate engineering) as novel substrates and a highly active recombinant -fructofuranosidase from Aspergillus niger (Zuccaro et al., 2008). Here we aimed at expanding our approach with levansucrase enzymes to synthesize 6-kestose analogues. The different glycopyranosyl-residues (i.e. galactose and xylose) that cap kestose analogues may be recognized by carbohydrate binding cell receptors. 2. Materials and methods 2.1. Protein expression and purication E. coli BL21(DE3) cells containing plasmid p24FTF11871 (wildtype) (Seibel et al., 2006c) or p24FTF1187/A5 (N242H) were grown in LuriaBertani medium (Anumula and Taylor, 1992) at 37 C

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with 100 g mL1 ampicillin to an OD580 of 0.7; at this OD580 , the expression of sacB gene was induced by the addition of 1 mM isopropyl--d-thiogalactoside (IPTG). Induced cells were cultured for 24 h at 28 C, harvested by centrifugation, and resuspended in 60 mM Na2 HPO4 /NaH2 PO4 (pH 6.2). Cells were disrupted by sonication, and pelleted by centrifugation. The wild-type enzyme and variant A5 (N242H) were puried using a CM-Sepharose column as described previously (Biedendieck et al., 2007). 2.2. Synthesis of sucrose analogues -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-Fru, d-Fuc-Fru, and Xyl-Fru) synthesis, purication and characterisation by NMR and mass spectroscopy (MS) were performed as described previously (Seibel et al., 2005, 2006c). 2.3. General description of the fructosylation reaction using sucrose and sucrose analogues Reactions were performed with puried wild-type levansucrase and A5 variant. Wild-type levansucrase (13 mg L1 ) or variant A5 (N242H; 28 mg L1 ) was added to a reaction mixture containing 90 g L1 sucrose or sucrose analogues (All-Fru, Gal-Fru, d-Fuc-Fru, Xyl-Fru), as substrates in 50 mM phosphate buffer (pH 6) and 50 mg L1 CaCl2 . Man-Fru (90 g L1 ) was incubated with wild-type levansucrase (328 mg L1 ) or variant A5 (696 mg L1 ). Product formation was investigated by discontinuous analysis of aliquots from the reaction mixture at suitable time intervals up to 24 h at 37 C. The enzyme was inactivated by boiling the samples in a water bath for 10 min. After cooling, the inactivated samples were ltered through a 0.22-m nitrocellulose membrane lter (Millipore, Germany). Accurate values for Km and kcat were determined from initial rate data at seven to ten substrate concentrations by a direct t of the data to the MichaelisMenten-equation using the computer program Origin 7.0 (OriginLab Corporation). One unit of total enzyme activity (hydrolysis and transfructosylation) is dened as the release of 1 mol of glycopyranoside per minute at 37 C and substrate saturation. Analysis of the samples was carried out using several different chromatographic systems as described below. The amount of glycopyranoside was determined by thin layer chromatography and HPLC. 2.4. HPLC analysis Enzymatic reactions were analyzed by high-performance liquid chromatography (HPLC). HPLC was performed with a RCM monosaccharide Ca2+ column (300 mm 7.8 mm, Phenomenex , Germany) operated at 80 C and an ion chromatograph (IC) (Metrohm, Germany) with a refractive index detector (ERC-7512, Erma, Germany) using distilled water as eluent at 0.8 mL min1 . Standard sugar solutions were prepared in the range of 0.110 g L1 (fructose, galactose, glucose, xylose, sucrose, melibiose, rafnose, 1-kestose, nystose). The monosaccharides fructose, galactose, glucose, xylose, the disaccharides sucrose and melibiose, the trisaccharides rafnose and 1-kestose, and the tetrasaccharide nystose were used as external standards for peak identication and quantication. The relative standard deviation of this system is approximately 3%.

2.5. Densitometry analysis Aliquots from levansucrase reactions were analyzed using thin layer chromatography (TLC) at room temperature (rt). The solvent system ethylacetate/isopropanol/water was used in a ratio of 6/3/1 (v/v/v) for Gal-Fru and Man-Fru reactions. For Xyl-Fru and FucFru reactions, acetonitrile/water in a ratio of 4/1 (v/v) was used as mobile phase. Reaction samples (nal concentration between 0.05 and 1.0 g L1 ) were applied using end-to-end pipettes (3 L volume, HIRSCHMANN minicaps, Hirschmann, Germany) on silica thin-layer plates (TLC aluminium sheets 20 20 cm, silica gel 60 F254 with concentrating zone 20 2.5 cm, MERCK, Germany). The carbohydrates were separated using one to ve ascents (25 90 min ethylacetate/isopropanol/water or 1 45 min acetonitrile/water). Spots were detected by dipping the plates into the detecting reagent (0.3% (w/v) of N-(1-naphtyl)-ethylenediamine, Fluka, Germany) and 5% (v/v) concentrated sulfuric acid in methanol using a CAMAG Chromatogram Immersion Device III (speed 2, time 4, Camag, Switzerland), followed by heating in an oven at 110 C for 30 min. The sugars were visualized as dark spots on a pale background. The intensity of spots was quantitated (in a range of 1503000 ng sugar) by densitometric scanning the TLC plate using a Bio-Rad Imaging Densitometer using Quantity One Software (Version 4.2). 2.6. Preparative chromatography Separations of oligosaccharides derived from the sucrose analogue reaction were carried out on a 9 cm 46 cm column of BioGel P2 (ne) that was eluted at rt with distilled water at a ow rate of 18 mL h1 . The carbohydrate content of each fraction was analyzed by HPLC. 2.7. High-performance anion-exchange chromatography (HPAEC) Analytical HPAEC was performed on a Dionex DX 300 chromatography system equipped with either a CarboPac PA1 column or a CarboPac PA-100 column. Samples were eluted from the CarboPac PA1 column (4 mm 250 mm, Dionex, USA) at 1 mL min1 using a linear gradient of aqueous sodium acetate (1 M, pH 6.0) linearly from 1 to 8% over 25 min, and then from 8 to 15% over 40 min. Samples were eluted from the CarboPac PA-100 column (4 mm 50 mm, Dionex) with sodium acetate (1 M) in aqueous sodium hydroxide (0.1 M) linearly from 20 to 40% over 65 min. Elution of the oligosaccharides was monitored with pulsed amperometric detection, including a post-column derivatization at pH 6.0 using 1.5 M sodium hydroxide. 2.8. Kinetic analysis The total activity minus the hydrolytic activity (fructose release) reected the transglycosylation enzyme activity (polymer and fructooligosaccharide formation). The reaction products synthesized by levansucrase from B. subtilis with sucrose analogues as substrates were mixed with the reaction products derived from sucrose to highlight novel and identical products. 2.9. Analysis by electrospray ionization MS Aliquots (13 L) corresponding to 220 pmol of oligosaccharides were applied to a nanospray gold-coated glass capillary placed orthogonally in front of the entrance hole of a QTOF-II instrument (Micromass, UK). Then 1000 V was applied to the capillary and ions were separated by the time-of-ight (TOF) analyzer. For MS/MS analysis, parent ions were selected by the quadrupole mass lter

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Fig. 1. (a) Wild-type levansucrase (right) formed from the substrate sucrose dominantly a polyfructoside while variant A5 (left) synthesized the trisaccharide 6-kestose and hydrolysis; (b) view of the active centre of the wild-type levansucrase from B. subtilis with Asn242 (wt). The substitution with His (A5) abrogated the polysaccharide synthesis.

and subjected to collision-induced dissociation. Resulting daughter ions were further separated by the TOF-analyzer. 2.10. GCMS analysis The intact polysaccharides were permethylated as described by Anumula and Taylor (1992), followed by hydrolysis (4 N TFA, 110 C, 2 h), reduction (NaBD4 ), and acetylation. The resulting partially methylated alditol acetates were analyzed on a ThermoQuest GCQ ion trap GC/MS system (MS: EI mode, GC: 30 m DB 5 column). 2.11. Random mutagenesis of the levansucrase gene sacB The E. coli strain BL21 (DE3) (Novagen, Madison, WI, USA) was used for library construction. Plasmid p24FTF1187 (Seibel et al., 2006c) carrying the wild-type sacB gene from B. subtilis NCIMB 11871 was used as template in mutagenic PCR. DNA-isolation, manipulation and -transformation were performed with standard methods (Sambrook et al. 1989). Error prone PCR was carried out using primers 5 -GATATAAACATATGAACATCAAAAAGTTTGCA-3 for the forward primer and 5 -CCAACTCGAGTTTGTTAACGTTTAATTG 3 for the reverse primer with restriction sites for NdeI and XhoI (underlined). Mutagenic PCR was performed in a 100-l reaction mixture containing 16 mM (NH4 )2 SO4 , 67 mM Tris pH 8.8, 0.01% Tween 20, 2.5 mM MgCl2 , 40 M MnCl2 , 0.2 mM (dATP/dGTP),

1 mM (dCTP/dTTP), 2 fmol plasmid DNA as template, 40 pmol of each primer and 5 U Tac polymerase (AppliChem, Darmstadt, Germany). Thermal cycling parameters were 97 C for 7 min (1 cycle), 95 C for 1 min, 54 C for 1 min, 72 C for 2 min (25 cycles), and 72 C for 10 min. Puried restricted PCR products were ligated with NdeIXhoI digested expression vector pET24a + and transformed into competent BL21 (DE3) cells using standard methods (Sambrook et al., 1989). Mutated sacB PCR products were cloned into plasmid pET24a + (Novagen, Madison, WI, USA). Error rate of mutagenic PCR was veried by sequencing 10 DNA samples isolated from recombinant clones (Sequiserve, Vaterstetten, Germany). Sequence analysis revealed an error rate of 12 base substitutions per gene.

2.12. Library screening Transformed cells were plated on LauriaBertani (LB) agar plates supplemented with 50 g mL1 kanamycine and grown overnight at 37 C. Single colonies were picked into 364-well microtiter plates containing 100 L medium and incubated for 24 h at 37 C. The master plates were duplicated by transferring a 1 L aliquot to a 96-well microtiter plate containing 100 l LB-Km medium + 1 mM IPTG and grown for 16 h at 30 C. Sucrose solution (50 L, 100 gL1 ) was added and the reaction analysed by TLC after 24 h.

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Scheme 2. Structures of sucrose analogues with glycosidic bonds -(1,2)--fructoside that were synthesized and used for substrate studies.

2.13. -d-Fructofuranosyl-(2 6)--d-fructofuranosyl-(2 1)-d-xylopyranoside (Xyl-(Fru)2 ) White solid, m.p. 235 C: Rf = 0.168 (6:3:1 EtOAcisopropanol water, 3 ascends in 90 min); []D + 4.83 (c 0.725, H2 O); 1 H NMR (600 MHz, D2 O) = 5.27 (d, J1,2 3.8 Hz, 1H, 1-H), 4.174.16 (d, J3 ,4 8.9 Hz, 1H, 3 -H), 4.064.03 (t, J4 ,3 = J4 ,5 8.9 Hz, 1H, 4 -H), 3.923.88 (dt, J5 ,4 = 8.9, J5 ,6 2.8 Hz, 1H, 5 -H), 3.773.74 (2d, J6a ,5 = J6b ,5 2.8 Hz, 2H, 6a -H, 6b -H), 3.84-3.80 (m, 2H, 3-H, 5-H), 3.63 (s, 2H, 1 -H2 ), 3.70 (s, 2H, 1 -H2 ), 3.613.59 (m, 1H, 4-H), 3.503.48 (dd,

J2,3 9.8, J2,1 3.6 Hz, 1H, 2-H), 4.134.11 (d, J3 ,4 8.9 Hz, 1H, 3 -H) 4.044.02 (t, J4 ,3 = J4 5 8.9 Hz, 1H, 4 -H), 3.813.75 (dt, J5 ,4 = 8.9, J5 6 2.8 Hz, 1H, 5 -H) 13 C NMR (600 MHz, D2 O) = 106.75 (C-2 ), 106.54 (C-2 ), 95.20 (C-1), 83.96 (C-5 ), 83.13 (C-5 ), 79.25 (C-3 ), 78.77 (C-3 ), 77.40 (C-4 ), 77.31 (C-4 ), 75.57 (C-3), 73.91 (C-2), 72.10 (C-4), 65.66 (C-6 ), 65.33 (C-6 ), 64.60 (C-5), 63.40 (C-1 ), 62.50 (C-1 ). ESIMS: m/z 497.0 100% [M + Na+ ]. 3. Results 3.1. Enzyme engineering of levansucrase SacB from B. subtilis NCIMB 11871 for FOS synthesis Oligofructans like kestose and nystose are well known for their health benets in human nutrition (Hiramaya et al., 1993). We envisage that the substitution of glucose-residue to another glycopyranosyl-residue like xylose will effect the structural and biochemical properties of the new molecule. In contrast to short chain FOSs the glycopyranosyl-residue in polysaccharides may not inuence its structure and properties tremendously. Thus, our rst aim was to generate a levansucrase that does not produce polysaccharides but instead short-chain FOSs. To increase the oligofructoside formation and to reduce the levan production of the levansucrase from B. subtilis NCIMB 11871, random mutagenesis was applied to the entire levansucrase gene sacB by error-prone PCR with Taq-polymerase. Adjusting the concentration of Mn2+ and the ratio of deoxyribonucleoside triphosphates during PCR led to an error rate of 12 base substitutions per gene, as was obvious from sequence analysis. Recombinant clones of the mutant library were transferred in 96-well microtiter plates and the reaction plates were tested with sucrose on polymer formation by TLC. After screening of 200 mutants of the library (15,000 clones) one variant (A5) was identied, that catalyzed the increased formation of oligofructosides, hydrolysis and in contrast to the wild-type enzyme no levan production was detected (Fig. 1a). Sequence analysis of the variant A5 revealed the presence of two mutations in the gene, V106A and N242H (Fig. 1b). We used these novel insights for a functional characterization of a novel levansucrase from B. megaterium (74% identity with SacB from B. subtilis) (Homann et al., 2007). There we found that V115A (Val106 B. subtilis numbering) mutation has no effect on levansucrases (Homann et al., 2007). 3.2. Kinetic parameters of levansucraseSacB wild-type and variant A5 with sucrose analogues

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

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

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Table 1 Kinetic parameters determined for wild-type levansucrase from B. subtilis and A5 with sucrose and sucrose analogues Substrate Sucrose (wild-type) Man-Fru (wild-type) All-Fru (wild-type) Gal-Fru (wild-type) D-Fuc-Fru (wild-type) Xyl-Fru (wild-type) Sucrose (variant A5) Overall activation energy ( RT ln
kcat(suc) /Km(suc) kcat(analogue) /Km(analogue)

Km (mM)a , b c14c 30.8 (7.9) 20.3 (4.1) 40.5 (5.7) 28.5 (4.3) 222 (44.5)

kcat (s1 )a , b 85.5 (3.2) 0.4 (0.1) 36.7 (2.8) 70.6 (4.9) 72.6 (3.1) 40.4 (4.2)

kcat /Km (mM1 s1 ) 6.1 0.01 1.8 1.7 2.6 0.2 0 3.8 0.7 0.8 0.5

G (kcal mol1 )

G) was calculated from relative values of kcat /Km using the kinetic constants of sucrose and an analogue in the equation .

G=

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 0.25 U mL1 , Man-Fru 7.4148 U mL1 , Xyl-Fru 0.1 U to 1.0 mL1 , d-Fuc-Fru 0.30.6 U mL1 . Enzyme concentrations used for each substrate were added as estimated to convert about 10% of 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 (Mntsl and Puntala, 1982).

logues containing the same high energy -glycosidic bond like 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, AllFru, 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 fructosyltransferases (Biedendieck et al., 2007; Seibel et al., 2006a). We have determined the kinetic parameters for wild-type levansucrase from B. subtilis and variant A5 with sucrose and sucrose analogues (Table 1). The kcat /Km value reects the rst step, most likely the formation of the fructosyl-enzyme intermediate. Essentially a weak reduction in kcat /Km was seen for most sucrose-analogues as compared to sucrose. The kcat /Km of Xyl-Fru was decreased threefold, that of d-Fuc-Fru and Gal-Fru were decreased fourfold, and that of Man-Fru was reduced by 610-fold relative to sucrose. There was no measurable reaction with All-Fru; less than 1% was transformed, most due to hydrolysis. The kcat value for Man-Fru was lowered 230-fold. The Km value includes the afnity of substrate for enzyme. The Km value for Man-Fru remaining very similar to that of sucrose would suggest that the equatorial position of the hydroxyl group in position 2 is crucial for catalysis. The kcat value for sucrose with variant A5 decreased twofold and that of Km increased 16-fold compared with the wild-type enzyme, leading to a 32-fold reduction of kcat /Km . 3.3. FOS and PS synthesis using substrate analogues and the levansucrase variant Sucrose analogues (90 gL1 ) were incubated with puried wildtype levansucrase and levansucrase variant A5 (1.48 U mL1 for most analogues, 37.1 U mL1 for Man-Fru) at 37 C and the reaction progress was followed by TLC and HPAEC (Fig. 2). All substrates except All-Fru were consumed within 24 h. The wild-type enzyme converted 54% of the sucrose into transglycosylation products, 36% to levan and 18% to FOSs. Variant A5 converted only 38% to transfructosylation products, nearly exclusively to 6-kestose and 6-nystose. The wild-type enzyme converted 52% of the GalFru into transglycosylation products, almost exclusively levan-type polysaccharide, and 48% of Gal-Fru was hydrolysed. Reaction with Gal-Fru and variant A5, which is unable to produce levan, yielded only 3% FOS, the dominant reaction was hydrolysis. Xyl-Fru yielded 53% and d-Fuc-Fru 49% transglycosylation products with wild-type levansucrase. Variant A5 yielded 17% transglycosylation products from Xyl-Fru, the rest was hydrolyzed as well. Man-Fru

was predominantly hydrolyzed (95%) by the wild-type enzyme. Only 5% transglycosylation products were synthesized during the reaction with Man-Fru. These were also hydrolyzed when the reaction time was extended. The levan formed in the reaction may be a better substrate for the levansucrase than Man-Fru itself (Table 2). A more detailed analysis of the transfructosylation products highlighted large differences between the products formed from sucrose analogues. Although very small amounts of FOSs were produced from the substrates Gal-Fru and d-Fuc-Fru by wild-type levansucrase, new xylo-fructooligosaccharides (XFOSs), including Xyl-(Fru)n -Fru (n = 126), were formed from Xyl-Fru with the wildtype enzyme. The decrease of the lower molecular weight XFOSs during extended reaction times correlated with the appearance of higher molecular weight XFOSs (Fig. 3). Wild-type levansucrase produces a wide range of XFOS with Xyl-Fru, but variant A5 does not. The main transfructosylation product with Xyl-Fru is Xyl-Fru2 (Fig. 4) and a high rate of hydrolysis is detected. The 17% of transglycosylation products consist of 15% Xyl-Fru2 and only 2% other products, mainly Xyl-Fru3 . 3.4. Oligosaccharide analysis by ESI-MS The reaction products from Xyl-Fru and wild-type enzyme were fractionated into ve groups using a BioGel P2 gel permeation column chromatography. The unit length was determined by MS analysis. The ESI-MS analysis results agree with the structural assignments proposed from HPAEC and TLC analysis. As exem-

Table 2 Endpoint reactions of different substrates with wild-type levansucrase and variant A5 Substrate Sucrose (wild-type) Sucrose (A5) Man-Fru (wild-type) All-Fru (wild-type)a Gal-Fru (wild-type) Gal-Fru (A5) D-Fuc-Fru (wild-type) Xyl-Fru (wild-type) Xyl-Fru (A5) Hydrolysis (%) 46 62 95 <1 52 97 51 47 83 Transglycosylation products (%) 54 38 5 <1 48 3 49 53 17

Reaction conditions as described in Section 2. Reaction times and enzyme concentrations used for each substrate were as follows: 1345 min Gal-Fru and Man-Fru, 510 min others; 90 g L1 initial substrate concentration, pH 6, 37 C, 0.05 g L1 CaCl2 , 37.1 U mL1 for Man-Fru, 1.48 U mL1 for others. a Reaction with All-Fru is very slow compared with sucrose.

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Fig. 3. HPAEC of the XFOS products of Xyl-Fru (90 g L1 ) at 37 C after (a) 10 min, (b) 45 min, (c) 510 min and (d) 1345 min incubation using wild-type levansucrase at 1.48 U mL1 . The chromatographic analysis shows a signicant shift of the formation of low molecular weight (a and b) towards higher molecular weight oligosaccharides (c and d) with increasing reaction time.

plied in the ESI spectrum, oligosaccharides produced by this levansucrase from Xyl-Fru yielded a major molecular ion series corresponding to FOSs [Fru110 + Na]+ at m/z 203, 365, 527, 689, etc. The corresponding derivatives bearing one additional xylose residue ([Xyl-Fru19 + Na]+ appeared at m/z 335, 497, 659, etc. The xylose containing FOS ([Xyl-Fru3 + Na]+ at m/z 659 was further subjected to tandem mass analysis (MS/MS) (data not shown). The spectra showed a characteristic fragment ion at m/z 527 for [Fru3 + Na]+ , which provided evidence for the sequence Xyl-Fru3 . These results were conrmed by an analogous analysis of the permethylated derivatives (data not shown). The trisaccharide obtained from the reaction of variant A5 and Xyl-Fru was isolated by BioGel P2 gel permeation column chromatography. The ESI-MS analysis showed the adduct ion at m/z 497 [M + Na]+ which characterize a xylofructooligosaccharide with the molecular formula C17 H30 O15 (Xyl-Fru2 ). Further structural evidence of the -d-fructofuranosyl-(2 6)--d-fructofuranosyl(2 1)--d-xylopyranoside (Xyl-Fru2 , a 6-kestose analogue) was veried by 1 H NMR and 13 C NMR spectroscopy. 3.5. Methylation analysis It was expected that the xylofructooligosaccharides composed of (2 6) linked fructose units (levan). To unequivocally identify the linkage pattern of the monosaccharide units of the XFOSs the polysaccharides were permethylated (Anumula and Taylor, 1992), followed by hydrolysis with triuoro acetic acid (TFA). The monosaccharide mixture of fructose and xylose units were reduced

by NaBD4 to glucitol and mannitol derivatives (originating from the fructose residues) and xylitol (from xylose residue) and peracetylated. The resulting partially methylated alditol acetates were analyzed by GC/MS (Table 3). The derivatives characteristic for 2,6substituted fructose (1,3,4-Tri-O-methyl glucitol, mannitol) were detected, corresponding to the inner fructose residues of the fructooligosaccharide chains. In contrast, trace amounts of Fru(2 1) linkages were observed. The terminal position of the xylosyl residue was veried as 2,3,4-Tri-O-methyl-xylitol derivative. 4. Discussion Levansucrase essentially converts sucrose into a large levan polymer, only few intermediate oligosaccharides can be detected and polysaccharides of high molecular weight are obtained from the start of the reaction (Ozimek et al., 2006b). Here we tested sucrose analogues as alternative substrates for B. subtilis levansucrase. Our sucrose analogues Man-Fru, Gal-Fru, Xyl-Fru, and Fuc-Fru were accepted as substrates by B. subtilis levansucrase. With these new substrates we have obtained novel oligosaccharide products that may be of scientic and/or practical interest. The fructan synthesis with sucrose-analogue Gal-Fru by levansucrase proceeded in the same manner like sucrose. Gal-(Fru)n -Fru (n > 12) was the dominate polymer from Gal-Fru with similar transfructosylation efciency. In contrast the use of Xyl-Fru effected the formation of the oligosaccharide XFOS series Xyl(Fru)n -Fru (n = 18). This was not observed with sucrose or Gal-Fru. Thus, the substrate analogues inuence the lengths

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R. Beine et al. / Journal of Biotechnology 138 (2008) 3341

Fig. 4. HPAEC of the XFOS products using (a) 90 g L1 Xyl-Fru with 2.00 U mL1 wild-type levansucrase at 37 C after 115 min incubation, and using (b) 90 g L1 XylFru with 0.78 U mL1 variant A5 at 40 C after 120 min incubation. The selective formation of the trisaccharide by the variant A5 is obvious.

of the products, the contribution and the efciency of the enzymatic reaction. What could be a reasonable explanation? Meng and Ftterer reported recently about the rafnose-bound levansucrase, where they observed that the galactosyl-residue of rafnose make water mediated H-bonds of the 6 -hydroxyl with Asn242 and Tyr237, whereas the 2 -, 3 - and 4 -hydroxyl groups point into solvent (Meng and Ftterer, 2008). Thus, Asn242 located at subsite +2 may contribute to acceptor binding and the coordination of the growing fructan chain. In the substrate Gal-Fru, the galactosyl residue may bind as acceptor over 6-hydroxyl of the galactosyl residue in the same way observed for rafnose. As a consequence the polymer can be formed. The xylosyl-residue

of Xyl-Fru has no 6-hydroxyl group and cannot be coordinated as an acceptor in the same manner like Gal-Fru or sucrose. This may explain the appearance of xylofructooligosaccharides. With dFuc-Fru as substrate higher amounts of oligofructosides compared to the substrate sucrose was observed, but also polymer formation took place. This indicates that the 6-hydroxyl group of the glycopyranosyl-residue determines not exclusively between polymer or oligosaccharide formation, but at least it contributes to the acceptor binding. Further insights about the +2 subsite result from the variant A5. Mutant enzyme A5 (N242H) is unable to produce polymers and shifts its product pattern to short chain oligosaccharides and hydrolysis. So far most site directed mutagenesis has been done on residues directly involved in binding sucrose in the active site. Mutations at subsite +1 led to 1-kestose formation (Chambert and Petit-Glatron, 1991), while mutations at subsite-1 in FOS producing enzymes led to a shift to polymer formation (Ozimek et al., 2006a). But Asn242His mutation in variant A5 located at the +2 sugar binding subsite plays an import role in polymer versus oligosaccharide synthesis as recently demonstrated (based on this study) with the homologue B. megaterium levansucrase (Homann et al., 2007) and supported by a recent study of Meng and Ftterer (2008). Due to its position at the +2 subsite, it might stabilise the third fructosyl unit of the growing oligosaccharide chain and direct it as an acceptor substrate to an optimal position for further transfructosylation (Scheme 1) (Homann et al., 2007). The substitution of asparagine against histidine may effect that the coordination of this position with the third sugar residue is abolished. This effect was supported by a mutation of Asn252 to aspartate (corresponding to Asn 242 in B. subtilis) where the polymerase activity was preserved (Homann et al., 2007). The exact mode of the acceptor binding is still unclear. These previous discussed effects combined with Xyl-Fru as substrate and levansucrase variant A5 resulted in the production of one main product, a 6-kestose analogue (Xyl-(1,2)--Fru-(2,6)-Fru). As 6-kestose and XOFs have strong prebiotic activity, the impact of this novel product on the probiotic properties should be tested in mammalian gut system. However, the efciency and the transfer-rate of the enzyme need to be improved in future as the high hydrolysis rate may limit the scope of oligosaccharide synthesis. The data obtained from these studies could be subsequently used to create tailor-made enzymes, producing novel fructans with specic sizes and containing specic glycopyranosyl-residues for the preparative synthesis of interesting and promising hetero-FOSs, polymers and glycoconjugates as biomaterials.

Table 3 The fructooligosaccharide mixtures were permethylated, and then hydrolyzed (TFA), followed by reduction (NaBD4 ), and acetylation Peracetylated derivative of Xylitol 2,3,4-Tri-O-methylGlucitol 1,2,3,4,5-Penta-O-methyl-(2-d)1,3,4,6-Tetra-O-methyl2,3,4,6-Tetra-O-methyl 1,3,4-Tri-O-methyl 3,4,6-Tri-O-methyl Mannitol 1,2,3,4,5-Penta-O-methyl-(2-d)1,3,4,6-Tetra-O-methyl1,3,4-Tri-O-methyl 3,4,6-Tri-O-methyl Linkage type ta -d-Xylp 6-Linked d-Fruf ta -d-Fruf ta -d-Glcp (26) d-Fruf (21) d-Fruf 6-Linked d-Fruf ta -d-Fruf (26) d-Fruf (21) d-Fruf FOS-mixture 0.3 0.7 1.0 0.9 0.8 0.5 0.6 1-Kestose 0.3 1.0 0.9 0.2 0.9 Nystose 0.3 0.95 1.0 0.2 1.0

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

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Acknowledgement Financial support by the German Research Foundation via Sonderforschungsbereich 578 From Gene to Product is gratefully acknowledged. References
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