Molecular characterization and expression of a cDNA encoding ...

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Molecular characterization and expression of a cDNA encoding fructan:fructan 6G-fructosyltransferase from asparagus (Asparagus officinalis) Blackwell Publishing, Ltd.

Keiji Ueno1, Shuichi Onodera1, Akira Kawakami2, Midori Yoshida2 and Norio Shiomi1 1

Department of Food Production and Utility Development, Graduate School of Dairy Science Research, Rakuno Gakuen University, Bunkyodai-midorimachi,

Ebetsu, 069-8501, Japan; 2Department of Low-temperature Sciences, National Agricultural Research Center for Hokkaido Region, Hitsujigaoka, Sapporo 062-8555, Japan

Summary Author for correspondence: Norio Shiomi Tel: +81 11 3884754 Fax: +88 11 3875848 Email: [email protected] Received: 23 September 2004 Accepted: 4 October 2004

• Fructan:fructan 6G-fructosyltransferase (6G-FFT) catalyses a transfructosylation from fructooligosaccharides to C6 of the glucose residue of sucrose or fructooligosacchrides. In asparagus (Asparagus officinalis), 6G-FFT is important for the synthesis of inulin neoseries fructan. Here, we report the isolation and functional analysis of the gene encoding asparagus 6G-FFT. • A cDNA clone was isolated from asparagus cDNA library. Recombinant protein was produced by expression system of Pichia pastoris. To measure enzymatic activity, recombinant protein was incubated with sucrose, 1-kestose, 1-kestose and sucrose, or neokestose. The reaction products were detected by high performance anion-exchange chromatography. • The deduced amino acid sequence of isolated cDNA was similar to that of fructosyltransferases and vacuolar type invertases from plants. Recombinant protein mainly produced inulin neoseries fructan, such as 1F, 6G-di-β-D-fructofuranosylsucrose and neokestose. • Recombinant protein demonstrates 6G-FFT activity, and slight fructan:fructan 1-fructosyltransferase (1-FFT) activity. The ratio of 6G-FFT activity to 1-FFT activity was calculated to be 13. The characteristics of the recombinant protein closely resemble those of the 6G-FFT from asparagus roots, except for a difference in accompanying 1-FFT activity. Key words: asparagus (Asparagus officinalis), fructan, fructan:fructan 6Gfructosyltransferase (6G-FFT), fructosyltransferase, inulin neoseries, Pichia pastoris. New Phytologist (2005) 165: 813–824 © New Phytologist (2004) doi: 10.1111/j.1469-8137.2004.01294.x

Introduction Fructans (polyfructosylsucrose) are water-soluble and nonstructural polysaccharides consisting of linear or branched fructose chains attached to a sucrose moiety and are found in approx. 15% of flowering plant species (Hendry & Wallace, 1993). They are important storage carbohydrates in plants such as Poaceae (e.g. wheat and barley), Asteraceae (e.g. chicory, Jerusalem artichoke and Cynara cardunculus) and Liliaceae (e.g. onion and asparagus) (Suzuki, 1993; Raccuia & Melilli, 2004), and

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are synthesized in vacuoles (Frehner et al., 1984; Darwen & John, 1989). Fructans are considered to contribute to drought and cold resistance (Hendry, 1993; Pilon-Smits et al., 1995) and snow mold resistance (Yoshida et al., 1998). We previously reported that asparagus (Asparagus officinalis) and onion (Allium cepa) plants contain two types of fructans distinguished from each other by their structures (Shiomi, 1989). One is an inulin-type fructan (1F(1-β--fructofuranosyl)m sucrose) which is a β -2,1-linked fructose-oligomer or -polymer terminated by glucose. The other is a fructan

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(1F(1-β--fructofuranosyl)m-6G(1-β--fructofuranosyl)n sucrose) derived from neokestose (6G-β--fructofuranosylsucrose, 6G-kestotriose) which has β-2,1-linked fructosyl residue(s) on the carbon-6 of the terminal glucosyl residue of inulin-type fructan. The latter, called fructan of the inulin neoseries, is usually found in liliaceous plants. These saccharides are synthesized by activities of sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99), fructan:fructan 1-fructosyltransferase (1-FFT, EC 2.4.1.100) and fructan:fructan 6G-fructosyltransferase (6G-FFT or 6G-fructosyltransferase, 6G-FT) (Shiomi, 1989). 1-SST synthesizes 1-kestose (1-β--fructofuranosylsucrose, 1kestotriose), an inulin-type trisaccharide, from two molecules of sucrose by fructosyltransfer (Edelman & Jefford, 1968; Shiomi & Izawa, 1980; Koops & Jonker, 1996; Lüscher et al., 1996). 1-FFT elongates the fructose chain of inulin-type fructans by fructosyltransfer from 1-kestose to another 1kestose or fructan (Edelman & Jefford, 1968; Shiomi, 1982a; Lüscher et al., 1993; Koops & Jonker, 1994; van den Ende et al., 1996). 6G-FFT catalyses the transfer of a fructosyl residue from 1-kestose to carbon-6 of the terminal glucosyl moiety of sucrose or inulin-type fructan, producing neokestose or inulin neoseries fructan with a higher degree of polymerization (DP), respectively (Shiomi, 1981). It is a key enzyme in the biosynthesis of fructan of the inulin neoseries in asparagus and onion plants (Shiomi, 1989; Wiemken et al., 1995; Vijn & Smeekens, 1999; Ritsema et al., 2003; Fujishima et al., 2005). Recently, many studies with molecular cloning have been performed to investigate the evolution and the function of fructosyltransferases in fructan biosynthesis, and to improve biological potentials of plants by enzymes such as 1-SST (de Halleux & van Cutsem, 1997; Hellwege et al., 1997), 1-FFT (Hellwege et al., 1998; van der Meer et al., 1998), 6G-FFT (Vijn et al., 1997) and sucrose:fructan 6-fructosyltransferase (6-SFT; Sprenger et al., 1995; Kawakami & Yoshida, 2002). Deduced primary sequences of these enzymes and invertases are classified in glycoside hydrolase family 32 (Henrissat, 1991, http://afmb.cnrs-mrs.fr/CAZY/acc.html). A cDNA encoding 6G-FFT from onion has been reported as a 6G-FFT gene (Vijn et al., 1997). The onion recombinant enzyme had 1-FFT activity with 6G-FFT activity, which agreed with our results with native 6G-FFT purified from onion bulbs (Fujishima et al., 2005). However, 6G-FFT purified from asparagus roots (Shiomi, 1981) showed high 6G-FFT activity, while no activity of invertase, 1-SST and 1-FFT were detected under defined reaction conditions (reaction with 0.2  1kestose at pH 5.5 and 30°C for 1 h). Thus, we were interested in cloning a cDNA encoding 6G-FFT from asparagus, which would show different enzymatic characteristics from onion enzymes. The aim of the present study is to obtain information on 6G-FFT from asparagus based on nucleotide sequence and to elucidate enzymatic characteristics of the protein involved in fructan metabolism. Here, we describe isolation of a cDNA clone encoding 6GFFT from asparagus leaves and expression in the methylo-

New Phytologist (2005) 165: 813–824

trophic yeast Pichia pastoris. Characteristics of an enzyme preparation obtained by using a transformant harboring 6G-FFT cDNA from asparagus support the notion that the enzyme system in asparagus differs from that in onion.

Materials and Methods Plant material Asparagus plants (A. officinalis L. cv. Zuiyuu) were sown in pots in March 2001 and grown in a glasshouse for 5 months without supplemental lightning at the National Agricultural Research Center for Hokkaido Region, Sapporo, Japan. Preparation and screening of a cDNA library of asparagus Fresh leaves (1.0 g ) were harvested and ground in liquid nitrogen. Total RNA was isolated from the powder of asparagus leaves using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Poly(A)+RNA was purified with PolyATtract mRNA isolation systems (Promega, Madison, WI, USA). Double-stranded cDNA was synthesized from the poly(A)+RNA, and a cDNA library was made using a Zap cDNA synthesis kit (Stratagene, La Jolla, CA, USA) and Gigapack III Gold cloning kit (Stratagene). To prepare a DNA probe for screening, a DNA fragment was amplified by polymerase chain reaction (PCR) using a pair of degenerate primers and the asparagus cDNA library as template. The primers 5′-GTIGGIATGTGGGA(A/G)TG3′ (forward) and 5′-CCIGTI(C/G)C(A/G)TT(A/G)TT(A/ G)AA-3′ (reverse) were designed on the basis of the amino acid sequences VGMWEC and FNNATG, respectively, which were conserved among several plant invertases and fructosyltransferases (Fig. 1). The PCR conditions were: 1 cycle at 94°C for 2 min, 30 cycles at 94°C for 1 min, 42°C for 2 min and 72°C for 2 min, followed by 1 cycle at 72°C for 7 min using ExTaq polymerase (Takara, Kyoto, Japan). The amplified DNA fragment (about 1.0 kbp) was ligated into pGEM-T vector (Promega) and Escherichia coli DH5α (Toyobo, Osaka, Japan) was transformed by the vector. After preparation of the cDNA fragment inserted, the fragment was labeled with [α-32P]dCTP by using BcaBest labeling kit (Takara) and was used as a probe for screening a cDNA library. The cDNA library was screened by plaque hybridization (Sambrook et al., 1989) according to the Stratagene protocol. The plaques were transferred to a nylon membrane (Hybond N+, Amersham Bioscience, Piscataway, NJ, USA). The membrane was treated with a 32P-labeled probe in a buffer containing 5× Denhardt’s solution, 5× standard saline phosphate EDTA (SSPE), and 0.5% sodium dodecyl sulfate (SDS) at 42°C overnight. The membranes were washed for 15 min at 65°C successively in 2× standard saline citrate (SSC) and 0.1% SDS, 1× SSC and 0.1% SDS and 0.1% SDS. Detection of signals was performed by autoradiography with an X-ray

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Fig. 1 Comparison of deduced amino acid sequence of aoft1 with that of onion fructan:fructan 6G-fructosyltransferase (6GFFT). AoFT1, deduced amino acid sequence of asparagus 6G-FFT; Ac6G-FFT, deduced amino acid sequence of onion 6G-FFT (Accession no. Y07838). Potential Nglycosylation sites in the sequence of AoFT1 are marked by black lines. The underlined sequences with arrows indicate the amino acid sequences used to design the primers for polymerase chain reaction. Two amino acid sequences are aligned by introducing gaps (–) to maximize identity by using DNASIS software. Identical amino acids between both sequences are shaded. Three partial amino acid sequences of invertases from onion, asparagus and carrot are also aligned: AcINV, onion invertase (AJ006067); AoINV, asparagus invertase (AF002656); DcINV, carrot invertase (Y18707).

film (Hyperfilm MP, Amersham Bioscience). In vivo excisions of inserted cDNA fragments of positive plaques were performed with Rapid Excision kit (Stratagene). Nucleotide sequences were determined by an automated DNA sequencer (model 377; PE Applied Biosystems, Foster City, CA, USA) with DYEnamic ET terminator cycle sequencing kit (Amersham Biosciences). The nucleotide sequences were analysed by  software (Hitachi Software Engineering, Yokohama, Japan). The nucleotide sequences of full-length cDNA have been submitted to GenBank, EMBL and DDBJ nucleotide sequence databases under accession number AB084283. Expression of recombinant proteins in a methylotrophic yeast The isolated cDNA, named aoft1 was expressed in the methylotrophic yeast Pichia pastoris with the secretory expression vector

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pPICZαB (EasySelect Pichia Expression Kit; Invitrogen). To construct expression plasmids, four cDNA fragments originated from aoft1 were amplified by PCR. The primers for PCR also had adapter sequences of EcoRI and XbaI in forward and reverse primers, respectively. The resulting cDNA fragments named N-M1, N-S50, N-V53 and N-A58 consisted of the nucleotide sequences corresponding to deduced amino acid sequences from Met1 to Asn610, from Ser50 to Asn610, from Val53 to Asn610 and from Ala58 to Asn610 with EcoRI and XbaI sites in each 5′ and 3′ end, respectively. The amplified fragments were digested with EcoRI and XbaI followed by ligation into pPICZαB plasmid vector. The resulting plasmids were sequenced to ensure that no alteration of sequence compared with that of original 6G-FFT cDNA. The plasmids inserted correct sequences were named pNM1, pNS50, pNV53 and pNA58, respectively. Transformation and cultivation of P. pastoris were performed according to the instructions of the manufacturer with minor


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modification. Pichia pastoris X-33 was transformed with 20 µg of the PmeI-linearized vectors by electroporation, and transformants were selected on YPDS (yeast extract peptone dextrose sorbitol)-Zeocin (Invitrogen) agar plates. A freshly prepared single colony was inoculated in 5 ml of YPD (yeast extract peptone dextrose) medium containing Zeocin and was grown at 29°C in a shaking incubator at 200 r.p.m. for 24 h. The culture was then inoculated in 80 ml of preculture medium (buffered glycerol-complex medium, BMGY, pH 6.0), and incubated for 24 h at 29°C with shaking at 200 r.p.m. The cells were collected by centrifugation, transferred to 15 ml of induction medium (buffered methanol-complex medium, BMMY, pH 6.0), and incubated at 29°C for 72 h under aerobic conditions, adding 300 µl of methanol to the culture at intervals of 24 h. The culture was centrifuged and the supernatant was obtained. The supernatant was concentrated to 1.0 ml and desalted with 15 ml of 10 m sodium phosphate buffer (pH 6.5) by ultrafiltration on a VivaSpin concentrator cutting off at 10 kDa (Vivascience, Lincoln, UK). The dialysate was filled up to 1.0 ml with the same buffer and was used as an enzyme solution. All the experiments were done in duplicate. Enzyme assay and carbohydrate analysis 1-Kestose and nystose (1, 1-kestotetraose) were synthesized from sucrose by using asparagus 1-FFT (Shiomi, 1982a) or Scopulariopsis brevicaulis fructosyltransferase (Takeda et al., 1994). Neokestose, 1F,6G-di-β--fructofuranosylsucrose (4c, 1 and 6G-kestotetraose), 6G(1-β--fructofuranosyl)2sucrose (4b, 1,6G-kestotetraose), 1F(1-β--fructofuranosyl)2−6G-β-fructofuranosylsucrose (5c, 1,1 and 6G-kestopentaose) and 1F.-β-D-fructofuranosyl-6G.(1-β--fructofuranosyl)2sucrose (5d, 1 and 1,6G-kestopentaose) were prepared from asparagus roots as described previously (Shiomi et al., 1976, 1979). β2,1-Linked fructooligosaccharides (1F(1-β--fructofuranosyl)3−7 sucrose) (DP5-9) were obtained by carbon Celite column chromatography from an extract of Jerusalem artichoke tuber. An enzyme solution (25 µl) was mixed with 25 µl of 0.1  citric acid-0.2  Na2HPO4 buffer (pH 5.3) and 50 µl of 200 m 1-kestose (final concentration of the substrate, 100 m) and the mixture was incubated at 30°C. The reaction was stopped by boiling for 3 min One unit of 6G-FFT activity was defined as the amount of enzyme that produced 1 µmol of saccharides (a sum of the amount of 4c and neokestose) per min under the reaction conditions mentioned earlier. One unit of 1-FFT activity was defined as the amount of enzyme, which produced 1 µmol of nystose per min under the reaction conditions described. For analysis of fructan oligomers with different glycosidic linkages, high performance anion exchange chromatography (HPAEC) was done on a DX500 chromatograph (Dionex, Sunnyvale, CA, USA) with a CarboPac PA-1 anion exchange column (Dionex) and a pulsed amperometric detector (PAD), as described previously (Shiomi et al., 1991).


In the pH activity and stability profile experiments, 0.1  citric acid−0.2  Na2HPO4 buffer (pH 3.7, 4.2, 4.8, 5.3, 5.8, 6.3, 6.7 or 7.1) was used, and the reaction was stopped by addition of 900 µl of 150 m NaOH. For pH-stability profiles, preincubation was performed for 24 h at 4°C in the same buffer. The reaction mixture was adjusted to pH 5.3 and was incubated with 1-kestose at 30°C for 1 h. In the temperature-stability profile experiment, enzyme solutions were preincubated with 0.1  citric acid−0.2  Na2HPO4 buffer (pH 5.3) for 10 min at 4, 20, 30, 37, 45, 50 or 60°C, and then the preincubated solution was cooled to 0°C. After the preincubation, the mixtures were incubated with 1-kestose at 30°C for 1 h. All the experiments were done in duplicate.

Results Molecular characterization of asparagus fructosyltransferase To isolate a cDNA clone for 6G-FFT, poly(A)+ RNA was obtained from fresh leaves of growing asparagus and reverse transcribed. The double-stranded cDNA mixture was synthesized and then used as a template for PCR with a forward and a reverse primer covering sequences highly conserved in invertases and fructosyltransferases. The sequence covered by the forward primer was considered to be one of the catalytic sites in enzymes belonging to glycoside hydrolase family 32. Multiple rounds of screening of the cDNA library resulted in the isolation of 16 positive clones. From the sequence analysis, all clones encoded the same protein, except for a difference in the nucleotide length. The longest cDNA clone consisted of 2201 bp and contained an open reading frame (ORF) of 1833 bp and poly(A) sequence at the 3′ end. The ORF named aoft1 encoded a polypeptide of 610-amino acids (Fig. 1). The molecular mass and pI of the deduced polypeptide, named AoFT1, were calculated to be 68311 Da and 5.4, respectively. The deduced polypeptide had six potential N-glycosylation sites. The primary sequence of AoFT1 showed the greatest identity (68%) with that of an onion 6G-FFT (Fig. 1). Also, except for the sequence from Tulipa gesneriana invertase, AoFT1 had greater identity with sequences from liliaceous plants (Group A), such as 1-SSTs from onion (58%) and garlic (57%), and invertases from asparagus (59%) and onion (58%) than with sequences for the same enzymes from asteraceous (Group B, 48–51%) or gramineous plants (Group C, 51–53%). Further, the sequence of AoFT1 had lower identity (39–42%) with cell-wall invertases and fructan 1-exohydrolases (1-FEH) (van den Ende et al., 2000, 2001) (Fig. 2). The primary sequence of AoFT1 shows greater homology with that of vacuole-type enzymes, such as invertases and fructosyltransferases, than with cell wall invertases, indicating that AoFT1 is one of the vacuolar-type enzymes. The result agreed well with a previous report (Wei & Chatterton, 2001). AoFT1 also belongs to the


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Fig. 2 Phylogenetic tree of plant fructosyltransferases, invertases, and fructan 1-exohydrolases (1-FEH). Alignment of the sequences was created with the CLUSTAL W program and used to construct a phylogenetic tree. The sequences were obtained from the GenBank/EMBL/DDBJ databases: Ac6G-FFT, Allium cepa fructan:fructan 6G-fructosyltransferase (6G-FFT) (accession no. Y07838); AoINV, Asparagus officinalis invertase (AF002656); AcINV, A. cepa invertase (AJ006067); Ac1-SST, A. cepa sucrose:sucrose 1-fructosyltransferase (1-SST) (AJ006066); As1SST, Allium sativum 1-SST (AY098442); Ht1-FFT, Helianthus tuberosus fructan:fructan 1-fructosyltransferase (1-FFT) (AJ009756); Ci1-FFT, Cichorium intybus 1-FFT (U84398); Cs1-FFT, Cynara scolymus 1-FFT (AJ000481); Cs1-SST, C. scolymus 1-SST (Y09662); Ci1-SST, C. intybus 1-SST (U81520); Ht1-SST, H. tuberosus 1-SST (AJ009757); Ta6-SFT, Triticum aestivum sucrose:fructan 6-fructosyltransferase (6-SFT) (AB029887); Hv6-SFT, Hordeum vulgare 6-SFT (X83233); Fa1-SST, Festuca arundinacea 1-SST (AJ297369); TgINV, Tulipa gesneriana invertase (X95651); DcWINV, Daucus carota cell-wall invertase (X69321); TaWINV, T. aestivum cell-wall invertase (AF030420); LeWINV, Lycopersicon esculentum cell-wall invertase (AJ272304); Ci1-FEH IIb, C. intybus 1-FEH IIb (AJ295034); Ci1-FEH IIa, C. intybus 1-FEH IIa (AJ295033); Ci1FEH I, C. intybus 1-FEH I (AJ242538); Ta1-FEHw1, T. aestivum 1-FEHw1 (AJ516025); Ta1-FEHw2, T. aestivum 1-FEHw2 (AJ508387). Groups A, B and C represent liliaceous, asteraceous, and gramineous plants, respectively, except for T. gesneriana invertase, cell-wall invertases and 1FEHs.

glycoside hydrolase family 32, which includes invertase and fructosyltransferase from plants or invertase, inulinase and levanase from bacteria and fungi. The AoFT1 contained some conserved amino acid sequences in various fructosyltransferases and invertases (Fig. 3). Two catalytic motifs were confirmed to be NDPN and EC in the primary sequence of yeast invertase by affinity labeling and site-directed mutagenesis (Reddy & Maley, 1990, 1996). The Asp (D) and the Glu (E) residues in each motif in yeast invertase were identified as a nucleophile and a proton donor, respectively. The AoFT1 conserved the putative catalytic residues, as do other plant enzymes belonging to the family, and the sequences around the residues were also highly conserved. Expression of recombinant AoFT1 protein in P. pastoris A recombinant protein was obtained by expression of aoft1 in P. pastoris to investigate the enzymatic properties of AoFT1 protein. Because the N-terminal amino acid of the mature protein was not clear, four cDNA fragments, N-M1, N-S50,


N-V53 and N-A58 were designed for expression of aoft1. These fragments encoded the same protein with different 5′-deletions (Fig. 4). Each cDNA fragment was synthesized by PCR and ligated into pPICZαB vector, named pNM1, pNS50, pNV53 and pNA58, respectively. Pichia pastoris was transformed with those vectors by electroporation. After cultivation of transformed P. pastoris for 72 h at 29°C, each enzyme solution was prepared from the culture broth. The 6G-FFT activity of the four enzyme solutions was assessed, regardless of the secretion levels of the recombinant protein. From the enzyme assay with 100 m 1-kestose as a substrate, the highest 6G-FFT activity was detected in the enzyme solution prepared from the culture broth of P. pastoris harboring pNS50 plasmid (Fig. 4). Although a high 6G-FFT activity was also detected in enzyme solution prepared by using pNV53 plasmid, enzyme solutions prepared by using pNM1 and pNA58 plasmids showed the lower activity. Further investigations were done with the enzyme solution prepared from the culture broth of P. pastoris harboring pNS50 plasmid, which contained the recombinant protein, named AoFT1-NS50.


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Fig. 3 Comparison of amino acid sequences of highly conserved regions of AoFT1 with those of plant fructosyltransferases and invertases. The origins of sequences and abbreviations for deduced amino acid sequences are: Ac6G-FFT, Allium cepa fructan:fructan 6G-fructosyltransferase (6G-FFT) (accession no. Y07838); Ta6-SFT, Triticum aestivum sucrose:fructan 6-fructosyltransferase (6-SFT) (AB029887); Hv6-SFT, Hordeum vulgare 6-SFT (X83233); Ac1-SST, A. cepa sucrose:sucrose 1-fructosyltransferase (1-SST) (AJ006066); As1-SST, Allium sativum 1-SST (AY098442); AcINV, A. cepa invertase (AJ006067); AoINV, Asparagus officinalis invertase (AF002656); DcINV, carrot invertase (Y18707); DcWINV, D. carota cell-wall invertase (X69321); TaWINV, T. aestivum cell-wall invertase (AF030420); LeWINV, Lycopersicon esculentum cell-wall invertase (AJ272304); DcWINV, D. carota cell-wall invertase (X69321). Amino acid sequences are aligned by introducing gaps (–) to maximize identity by using DNASIS software. The asterisked amino acid residues are putative catalytic residues (Reddy & Maley, 1990, 1996). Identical amino acid sequences are shaded.

Fig. 4 Constructs for transformation of Pichia pastoris to express asparagus fructan:fructan 6G-fructosyltransferase (6G-FFT) and relative activities of transformants producing 1F, 6G-di-β-D-fructofuranosylsucrose (4c, 1 and 6G-kestotetraose) and neokestose (6G-kestotriose) from 1-kestose (1-kestotriose). Each cDNA fragment consist of the nucleotide sequences of aoft1 corresponding to deduced primary sequences from Met1 to Asn610 (N-M1), from Ser50 to Asn610 (N-S50), from Val53 to Asn610 (N-V53), and from Ala58 to Asn610 (N-A58), respectively. Each cDNA fragment has adaptor sequences added to create EcoRI and XbaI sites at the 5′ and 3′ end, respectively. The enzyme activity of each protein extract obtained from the culture broth of each transformed P. pastoris was measured by high performance anion-exchange chromatography. Each enzyme activity is the relative value to that of the broth obtained by using pNS50 plasmid.

In the reaction of AoFT1-NS50 (6G-FFT activity, 0.001 U) with 100 m 1-kestose for 1 h, the initial products were 4c and sucrose (Fig. 5a). After reaction for 2 h, neokestose was produced. In addition to oligosaccharides of the inulin


neoseries resulting from 6G-FFT activity, prolonged reaction of AoFT1-NS50 with 1-kestose also produced a small amount of nystose, indicating that the protein also had weak 1-FFT activity. The reaction mixture at 24 h also contained 4b,


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Fig. 5 High performance anion-exchange chromatograms of the reaction products formed from 1-kestose (1-kestotriose), neokestose (6G-kestotriose), or sucrose by AoFT1-NS50. Reaction mixtures containing AoFT1-NS50 were incubated with 100 mM 1-kestose (a), 100 mM neokestose (b), or 100 mM sucrose (c) for up to 24 h at 30°C. As a control, reaction mixtures containing protein extract obtained by using the empty vector was incubated with each substrate for 24 h. S, sucrose; 1-K, 1-kestose; N-k, neokestose; Nys, nystose (1,1-kestotetraose); 4c, 1F,6G-di-β--fructofuranosylsucrose (1 and 6G-kestotetraose); 4b, 6G(1-β-Dfructofuranosyl)2sucrose (1,6G-kestotetraose); 5c, 1F(1-β-D-fructofuranosyl)2-6G-β-Dfructofuranosylsucrose (1, 1 and 6Gkestopentaose); 5d, 1F-β-D-fructofuranosyl6G(1-β-D-fructofuranosyl)2sucrose (1 and 1,6G-kestopentaose). Elution patterns of standard saccharides are represented in (d).

5c and 5d. Furthermore, in the reaction of AoFT1-NS50 with 100 m neokestose, 4c and small amount of 4b and sucrose were produced at 2 h (Fig. 5b). Also, 5c, 5d and 1-kestose were formed after 24 h reaction. These oligosaccharides were previously reported to be synthesized from 1-kestose or neokestose in some reaction steps by 6G-FFT and 1-FFT activities in asparagus (Shiomi, 1981, 1982b, 1989) or in onion (Shiomi et al., 1997). The enzyme solution did not show any 1-SST and 6-SFT activities in the reaction with 100 m sucrose as a sole substrate (Fig. 5c). Consequently, AoFT1-NS50 expressed in P. pastoris was confirmed to be 6G-FFT and was secreted into the culture broth.


Functional characterization of recombinant 6G-FFT The enzymatic properties of recombinant 6G-FFT were investigated. The recombinant 6G-FFT showed the highest activity at pH 5.3 (Table 1). The optimum pH almost agreed with that of the native enzyme (Shiomi, 1981). After the recombinant 6G-FFT was preincubated from pH 3.7 to pH 7.1, the residual activity was measured at pH 5.3. Enzymatic activities remaining at pH 3.7, 4.2, 4.8, 5.3, 5.8, 6.3, 6.7 and 7.14 were 32, 85, 99, 98, 98, 100 and 86%, respectively. Therefore, the enzyme was stable from pH 5.3 to 6.7, although the native enzyme was stable from pH 5.0 to


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Optimum pH pH Stability Temperature stability

AoFT1-NS50

Native 6G-FFTa

5.3 5.3– 6.7b Up to 37°C

5.5 5.0– 6.0c Up to 37°C

a

Reported previously (Shiomi, 1981). The value was obtained by preincubation at 4°C for 24 h. c The value was obtained by preincubation at 45°C for 20 min. b

6.0 under different condition (preincubation for 20 min at 45°C). The enzyme solution was incubated in 0.1  citric acid−0.2  Na2HPO4 buffer (pH 5.3) for 10 min at various temperatures. The residual activities of the enzyme were 99–100% at 4–30°C, 97% at 37°C, 53% at 45°C and 6% at 50°C. The enzyme was inactivated at 60°C. The enzyme was stable at up to 37°C. The temperature stability also agreed with that of the native enzyme. The effects of pH and temperature on 1-FFT activity of the AoFT1-NS50 were the same as 6G-FFT activity. Recombinant 6G-FFT (0.004 U) was incubated with 100 m 1-kestose, 100 m 1-kestose and 100 m sucrose, or 100 m neokestose at 30°C and the products were measured for up to 48 h (Fig. 6). When 100 m 1-kestose was given as a sole substrate, 4c and sucrose were the initial products, and were produced proportionally up to 8 h. Neokestose was also produced with an initial lag phase. The result indicated that 6G-FFT produced 4c from two molecules of 1-kestose with liberation of sucrose, and then produced neokestose by fructosyltransfer from 1-kestose to sucrose. The amount of neokestose gradually increased up to 48 h, although the amount of 4c was decreased compared with amounts of 4b, 5c and 5d (Fig. 6a). The three saccharides, 4b, 5c and 5d were synthesized by the reverse reaction using neokestose as a substrate. In addition to the major products of 4c, sucrose and neokestose, a small amount of nystose was detected in the prolonged reaction, indicating that recombinant protein had weak 1-FFT activity, as indicated in Fig. 5. The enzymatic activities of 6G-FFT and 1-FFT in the 1-h reaction were 0.143 U ml−1 and 0.011 U ml−1. The ratio of the activity of

Fig. 6 Courses of the reaction products formed from 100 mM 1kestose (1-kestotriose) (a), 100 mM 1-kestose and 100 mM sucrose (b), or 100 mM neokestose (6G-kestotriose) (c) by AoFT1-NS50. Open circles, triangles and squares indicate sucrose, 1-kestose, and nystose (1,1-kestotetraose), respectively. Closed diamonds, circles, squares and triangles indicate neokestose (6G-kestotriose), 4c (1F,6G-i-β-D-fructofuranosylsucrose, 1 and 6G-kestotetraose), 4b (6G(1-β-D-fructofuranosyl)2sucrose, 1,6G-kestotetraose) and 5c (1F(1-β-D-fructofuranosyl)2-6G-β-D-fructofuranosylsucrose, 1,1


and 6G-kestopentaose) +5d (1F-β-D-fructofuranosyl-6G(1-β-Dfructofuranosyl)2sucrose, 1 and 1,6G-kestopentaose), respectively. The saccharides with closed symbols indicate fructans of the inulin neoseries.


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Fig. 7 Possible pathway for production of fructooligosaccharides detected in the reaction of recombinant fructan:fructan 6Gfructosyltransferase (6G-FFT) with 1-kestose (1-kestotriose), 1-kestose and sucrose, or neokestose (6G-kestotriose). S, sucrose; 1-K (GFF), 1-kestose; N-k (FGF), neokestose; Nys (GFFF), nystose (1,1-kestotetraose); 4c (FGFF), 1F,6G-di-β-D-fructofuranosylsucrose (1 and 6G-kestotetraose); 4b (FFGF), 6G(1-β-D-fructofuranosyl)2sucrose (1,6G-kestotetraose); 5c (FGFFF), 1F(1-β-D-fructofuranosyl)2-6G-β-Dfructofuranosylsucrose (1,1 and 6G-kestopentaose); 5d (FFGFF), 1F-β-D-fructofuranosyl-6G(1-β-Dfructofuranosyl)2sucrose (1 and 1,6Gkestopentaose). Arrows with solid line and with dashed line indicate forward or reverse reaction of 6G-FFT activity, respectively; arrow with dotted line indicates 1-FFT activity. Asterisk indicates the reaction reported previously (Shiomi, 1981, 1982b). Bold line indicates a major pathway.

6G-FFT to 1-FFT of the recombinant 6G-FFT was calculated to be 13. The 6G-FFT activity of the enzyme was over 10 times higher than 1-FFT activity. Although the native 6GFFT from asparagus roots showed a negligible 1-FFT activity (Shiomi, 1981), the recombinant enzyme had weak 1-FFT activity. The reason for the difference of 1-FFT activity between both enzymes is discussed below. In the reaction with 100 m 1-kestose and 100 m sucrose as mixed substrates, neokestose was produced proportionally up to 24 h (Fig. 6b). The amount of 4c was lower than that in the reaction with the 1-kestose only because of the competition of acceptors between sucrose and 1-kestose. Nystose was also produced in the reaction mixture as in the reaction with the 1-kestose as a sole substrate. These results indicate that the recombinant enzyme catalyses a fructosyltransfer from terminal fructosyl residue of β-2,1-linked fructooligosaccharides to HO-6 of the glucosyl residue of similar saccharides. In the reaction with 100 m neokestose as a sole substrate, 4c, 4b and sucrose were the initial products (Fig. 6c). Production of these saccharides showed that recombinant enzyme catalysed the transfer of a fructosyl residue linked to HO-6 of the glucosyl residue of neokestose (donor substrate) to HO-1 of the fructosyl residues of another neokestose (acceptor substrate). This


is a reverse reaction of 6G-FFT (Shiomi, 1982b). 1-Kestose, 5c and 5d were also produced with an initial lag phase. These saccharides were estimated to be produced by fructosyltransfer from neokestose to sucrose or 4c. Both saccharides of 5c and 5d are also considered to be formed by self-transfer between two molecules of 4c liberating 1-kestose. From the results in these experiments, a possible pathway for oligosaccharides of inulin neoseries formed by the recombinant 6G-FFT is summarized in Fig. 7. Moreover, the recombinant 6G-FFT was incubated with the β-2,1-linked fructooligosaccharides (from DP 5-9) from Jerusalem artichoke. The formation oligosaccharides with DP higher than the substrate was detected by HPAEC analysis. These saccharides were estimated to be inulin neoseries fructooligosaccharides (Fig. 8).

Discussion The present study shows that a cDNA clone named aoft1 encoding 6G-FFT has been isolated from a cDNA library of asparagus leaves and that a recombinant protein has been successively expressed in P. pastoris. The aoft1 encoded a protein named AoFT1 consisted of 610-amino acids, and the


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Fig. 8 High performance anion-exchange chromatograms of the reaction products formed from β-2,1-linked fructooligosaccharides (DP5-9) by AoFT1-NS50. Reaction mixtures containing AoFT1-NS50 (b) or protein extract obtained by using the empty vector (a) were incubated with 2.5% β-2,1-linked fructooligosaccharides (1F(1-β-Dfructofuranosyl)3−7 sucrose) (DP5-9) from Jerusalem artichoke for 48 h. Asterisked peaks are inulin neoseries fructooligosaccharides compared with the saccharides from asparagus roots (c) as standards.

molecular mass and pI of the protein were calculated to be 68311 Da and 5.4, respectively. The deduced amino acid sequences of aoft1 showed the greatest identity (68%) and similarity (79%) to those of onion 6G-FFT. The N-terminal amino acids of mature 6G-FFTs from both asparagus and onion are not clear. Vijn et al. (1997) compared their results with barley 6-SFT (Sprenger et al., 1995) and carrot vacuolar invertase (Unger et al., 1994). Homology of the primary sequence of onion 6G-FFT with those sequences started at


the 58th codon, and onion 6G-FFT was suggested to be made from a larger precursor protein containing a signal (for vacuolar-targeting) peptide located at N-terminus. The putative signal peptides in the three sequences were also revealed to be less conserved with one another. In the first 52-amino acid sequences of N-terminus of AoFT1, 18 amino acid residues were identical with those of onion 6G-FFT and other 12 amino acid residues were conservatively substituted, indicating that AoFT1 also has a signal peptide consisting of around 52 amino acids. This is expected for AoFT1, because the synthesis of fructan in plants is known to be located in vacuole (Frehner et al., 1984; Darwen & John, 1989). The glycoside hydrolase family 32 (Henrissat, 1991, http:// afmb.cnrs-mrs.fr/CAZY/acc.html) contains enzymes such as invertase, FEH, 1-SST, 1-FFT, 6G-FFT and 6-SFT. These enzymes are considered to have evolved from invertases. Moreover, primary structures of enzymes catalysing the different transfructosylation in liliaceous plant are more closely related to one another than those of enzymes catalysing the same reaction in asteraceous and gramineous plants (Fig. 2). Recombinant AoFT1 protein obtained from P. pastoris showed 6G-FFT activity. Although four recombinant proteins, named AoFT1-NM1, AoFT1-NS50, AoFT1-NV53, and AoFT1-NA58, respectively, showed nearly the same patterns of products (data not shown), deletion of N-terminal sequences of the protein had an effect on the amount of enzymatic activity contained in the culture broth from Pichia transformants (Fig. 4). The effect may suggest that a deletion of N-terminal amino acid sequences at an appropriate position in AoFT1 is important for secretion of a protein into the culture broth. A similar result, that addition of an original signal peptide decreased enzyme activity in the culture broth, was reported in expression of barley 6-SFT in Pichia (Hochstrasser et al. 1998). However, the possibility that the signal or prosequence interfered with the enzyme activity still remained, because the enzyme has not yet been purified. To clarify this, purification of enzymes with different N-terminal sequence is needed. The recombinant enzyme had not only 6G-FFT activity but also 1-FFT activity. The ratio of the activity of 6G-FFT to 1-FFT in the recombinant 6G-FFT from asparagus was calculated to be 13. The recombinant onion 6G-FFT from stable transformed tobacco plants also catalysed the production of the same saccharides including nystose from 1-kestose (Vijn et al., 1997) and Ritsema et al. (2003) demonstrated that the recombinant 6G-FFT from onion was a bifunctional enzyme that showed both 6G-FFT and 1-FFT activity. In purified native 6G-FFT from onion, the ratio of the 6G-FFT activity to 1-FFT in the native enzyme was calculated to be 2.3 (Fujishima et al., 2005). The 6G-FFT from onion was distinguished from the asparagus enzyme by relatively high 1-FFT activity. With regard to 1-FFT activity of 6G-FFT from asparagus, the native 6G-FFT from asparagus roots (Shiomi, 1981) produced only a trace of nystose from 1-kestose in prolonged reaction. Although the reason for the difference in


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1-FFT activity of 6G-FFT between the native and the recombinant 6G-FFT from asparagus is not clear, most of 1-FFT activity of the recombinant 6G-FFT from asparagus may result from the expression system in P. pastoris. Indeed, a barley 6-SFT expressed in P. pastoris was reported to show unexpected 1-SST activity, and it was described that the differences in enzymatic characteristics between the native and recombinant 6-SFT might result from to difference in folding or glycosylation between the Pichia and the plant system (Hochstrasser et al., 1998). In asparagus, three enzymes, 1-SST, 1-FFT and 6G-FFT participate in syntheses of fructans (Shiomi, 1989). The recombinant 6G-FFT from asparagus successively produced 4c and neokestose from 1-kestose, and showed the reverse reaction from neokestose (Figs 5 and 6). The properties of the recombinant 6G-FFT were similar to the native enzyme (Shiomi, 1981, 1982b). The enzyme also synthesized corresponding fructans of inulin neoseries from β-2,1-linked fructooligosaccharides (DP 5-9) from Jerusalem artichoke (Fig. 8). Effects of pH and temperature on the activities of both the native and the recombinant 6G-FFTs from asparagus agreed well with each other. The AoFT1 might actually play an important role in fructan metabolism of asparagus, acting as 6G-FFT in the three-enzyme system. In onion plant, however, no 1-FFTs have been purified or 1-FFT cDNAs cloned, although recombinant 6G-FFT has 1-FFT activity (Vijn et al., 1997; Ritsema et al., 2003). These results suggest that the three-enzyme system of fructan synthesis in asparagus may differ from the two-enzyme system in onion. It remains unknown which amino acid residues are responsible for the differences of 1-FFT activity between asparagus 6G-FFT and the onion enzyme, while the deduced primary sequence of AoFT1 shows 68% identity to that of onion 6GFFT. The same problems arise in fructosyltranferases involved in fructan metabolism, with high identities of primary sequences, but with a range of different enzymatic characteristics. To resolve these questions, further investigations are needed on the structures of domains in the enzymes corresponding to the binding of donor and acceptor saccharides.

Acknowledgements This work was supported in part by Grant-in Aid for the promotion of high-technology centered projects from the Ministry of Education Culture, Sports, Science and Technology of Japan (2003) and for Cooperative Research from Rakuno Gakuen University (2004), for which the authors express their appreciation.

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