CA2141979A1 - Fructosyltransferase enzyme, method for its production and dna encoding the enzyme - Google Patents

Abstract

Extracellular fructosyltransferase of Acetobacter diazotrophicus was isolated and purified and its enzymatic properties were established. Cloning, sequencing and genetic manipulation of the fructosyltransferase gene so as to produce high levels of the enzyme in recombinant prokaryotic and eukaryotic cells. Both natural and recombinant fructosyltransferase of Acetobacter diazotrophicus produce fructose-containing oligosaccharides and fructans. The enzyme yields in particular high levels of fructo-oligosaccharides from sucrose, such as kestose and kestotetraose which can be used as natural low-calorie sweeteners.

Description

21~197~
Ln/Eur 3579/93081--TITLE: Fructosyltransferase enzyme, method for its production and DNA encoding the enzyme FIELD OF THE INVENTION
The present Lnvention is in the field of biotechnology and is specifically concerned with the isolation, the purification, the characterization and the productlon of a fructosyltransferase.
5 The Lnvention also relates to the cloning, the sequencing and the manipulation of a fructosyitransferase gene so as to allow the production of high levels of recombinant enzyme.
BACKGROUND OF THE INVENTION
10 Production and utilization of microbial fructans by enzymatic transformation of su~:ro3e are~ an important ob ject for the sugar, food, and other industries. Bacterial fructosyltransferases (EC

2.4.1.10) catalyze the synthesis of oligo- and/or polyfructans by transferring fructosyl moieties from sucrose-containing 15 saccharides to acceptor molecules. Different compounds can be used as acceptors which allows the enzymatic production of nondigestible homo- and heterooligo-saccharides with beneficial effects on humans and animals.
20 Most of the ~acterial fructosyltransferases characterized so far are levansucrases (Cote,- G.L. and Ahlgran, J.A., rn Science and Technology of fructans. Metabolism in microorganisms, Part I:
Levan and levansucrase. CRC Press, 1593) . All levansucrases - --catalyze the transfructosylation reaction from sucrose to a ~
25 variety of acceptors such as water (sucrose hydrolysis1, glucose (exchange reactlon), fructose (chaln elongation) and sucrose (synthesis of oligosaccharides~ . However, differences have been noted between these enzymes pertaining to the relative efficiency of each reaction which leads to the accumulation of 30 oligofructans of different polymerization degree.
Several bacterla and fungi have been identified to develop transfructosylation reactions from sucrose (for review see Cot:e, 214~7~

G.L. and Ahlgran, J.A., In Science and Technology of fructans.
Metabolism in miCroorganisms, Part I: Levan and levansucrase.
CP~C Press, 1993~ . Fructosyltransferase genes have been isolated from Bacillus subt~lis (European patent application EP 0117g23 -A1 840905~, Bac~llus amyloliquefaciens (Tang, L.B. et al., Gene 96, 89-93, I990), Streptococ~-us mutans (Shiroza, T. et al., J. Bacteriol. 170, No. 2, 810-816, 1988), Streptococcus salivarius (Rathsam, C. et al., J. Bacteriol. 175, No. 14, 4520 4527, 1993), Zymomonas mobilis (Ki-Bang Song =t al., Blochim.
Biophys. Acta 1173, 320-324, 1993) and E~r:~in~a amylovora (Geier, G. et al., Physiological and Molecular Plant Pathology 42, 387- - -404, 1993) . Low homology is found among the deduced amino acid sequences of fructosyltransferases isolated from different bacterial genera.
The transfructosylation system of the genus Bacillus (Gram-positive bacteria) has been well characterized . The Bacil l us subtil is levansucrase is an inducible exoenzyme which catalyzes the formation of high weight polymer wlthout accumulation of transient oligofructans of low polymerization degree. Recombi-nant levansucrases of Bac~llus subtilis have been obtained in genetically manipulated hosts such as bacteria (Philippe, G. J.
Bacteriol. 153, No. 3, 1424-1431, 1983), yeast (Scotti, P.A. et al., Yeast lQ, No. 1, 29--38, 1994) and plants (Ebskamp, M.J.M.
et al., Biotechnology 12, 272-275, 1994) .
Acetobacter diazotrophicus is the most recently identified species of the genus Acetobacter (Gillis et al., Int. J. Sist.
Bacteriol~ 39, 361-364, 1989) . The cells are Gram-negative, N2-30 fiixing, acid-tolerant, microaerobic, straight rods with rounded ends, about 0.7 to 0.9 by ~ 2 um, motile by lateral or peritrichous flagella. The bacteria are non-pathogenic and further distinguished by their ability to establish beneficial association with sugarcane. E~owever, the molecular biology of 35 the bacterium has ~een poorly investigated.

21~197~1 SUMMARY OF THE INVENTION
~cetobacter diazotrophicus secretes a constitutive fructosyl~
transferase with levansucrase activity. The enzyme has utility 5 for the production of fructose-containing oligosaccharides and levan. Fructose polymers have two characteristic properties, their nondigestibility and selective utilization by beneficial intestinal bacteria, which make them useful as low-calorie uietary fiber for relie~ of constipation, improvement of blood 10 lipid composition, cholesterol reduction, and suppression of intestinal putrefactive substances. During the course of sucrose transformation the Acetobacter diazotrophicus fructosyltrans-ferase accumulates a high yield of fructooligosaccharides, particularly kestose and kestotetraose which can serve as 15 natural low-calorie sweete~ers. l;evan can also be used as a source of fructose, a blood plasma volume extender, an emulsifier, an encapsulatlng agent, etc_ Given the importance of fructooligosaccharides and levan as 20 foodstuffs as well as their industrial applications, it is an object of the invention to produce the fructosyltransferase of Acetobacter diazotroph~cus both by natural and by recombinant procedures .
The invention prov~des a method for isolating, characterizing =
and producing an Acetobacter diazotrophicus fructosyltransferase by natural and recombinarlt procedures.
The fructosyltransferase of Acetobacter diazotrophicus is a constitutive exoenzyme which is accumulated in the culture medium yielding more than 70% of total secrete~ proteins. The culture supernatant can be used as a crude fructosyltransferase solution, or the enzyme be purified by any conventional method applicable here, preferably ion-exchange chromatography.
'rhe fructosyltransferase of Acetobacter d~azotrophicus is a ~ =
levansucrase which yields a high level of oligofructans of low polymerization degree. During the course of sucrose transfor- -21~1~7~

mation 55ga of Eructose transferred by the enzyme is accumulated as kestotriose and kestotretraose. These frllctooligosaccharides are high quality sweeteners with many applicatiDns in the food industry. The enzyme can be efficiently applied, therefore, for =
producing kestose and kestotetraose ~rom sucrose, and it is also useful in the production of hLgh weight levan.
The invention also comprises the nucleotide sequence of the 'ructosyltransferase gene of Acetobacter diazo~roFhicus isolated from a genomic library by complementing EMS-treated mutants of A. diazotrophicus unable to produce levan.
The production of the fructosyltransferase of A diazotrophicus by recombinant procedures allows higher levels of enzyme production as well as the synthesis of the enzymatic reaction products directly in a convenient host. A1SD provided for are strains from ~. coli and the yeast Pic~a pastor~s genetically manipulated so as to produce elevated levels of the recombinant fructosyltransferase.
DETAILED DESCRIPTION OF THE INVENTION
As used herein, the term "Acetobacter" refers to a particular genus of bacteria described in detail in Bergey ' s ManuaL of Determinative Bacteriology, Buchanan and Cibbons eds., Williams and Wilkins Publishers. The specific strain of Acetobacter from which the enzyme fructosyltransferase and the corresponding gene were isolated is Ace~obacter d~azotrophicus SRT4 (CBS deposit number CBS 549.94, deposition date 10 November 1994) .
For the purpose of the present invention the term "fructosyl-transferase" refers to one or more polypeptides having fructo- --syltransferase activity. Fructosyltransferase activity refers to the property of enzymatically transferring fructosyi moieties - ~ ~
from sucrose-c~r~taining saccharides to acceptor molecules to --yield fructooligosaccharides and fructans.

2~41~7~

The term "recombinant protein" as used herein intends to cover a polypeptide encoded by genomic, cDNA, semisynthetic or synthetic nucleic acid sequences which, by virtue of their origin or manipulation. (l) are not associated with all or a portion of 5the polynucleotide with which they are associated in nature or =~=
in the form of a libraryi and/or 12~ are linked to a poLynucleo-tide sequence other than that to which they are linked in nature. The recombinant protein displays substantially the same biological properties as the naturally occurr ng protein.
rn a first aspect, the present invention provides an isolated and purified DNA having a nucleotide sequence which essentially corresponds to or hybridizes with a nucleotide sequence comprlsed in the fructosyltransferase gene, shown in Seq. Id.
15No.l, of the bacterium Ace~tobacter ~azotrop~2icus.
The words "a nucleotide sequence comprised in the fructosyl-transferase gene" are intended to include several possibilities. -One option is a DNA which covers the complete fructosyltrans- ~-ferase gene, i.e. the gene inclusive of regulatory and coding sequences. Another option is a DNA essentiaLly consisting of the coding sequencè, or a part of the coding sequence, and lacking the fructosyltransferase promoter region. One such possibility is a ~ructosyltransferase cDN~ obtained by reverse transcription from fructosyltransferase mRNA. Furthermore, the invention relates to oligonucleotides of sufficient length to be specific - - -for fructosyltransferase DNA or RNA. Such oligonucleotides may be useful as fructosyltransferase-speclfic probes or primers and will usually have a length of from about 7 or 8, preferably from about 9 or l0 nucleotides up to about 40 or 50 nucleotides, ~=
preferably to about 25 or 30 nucleotides, or longer, up to the full length of the gene. -The words "a nucleotide sequence which essentially corresponds to or hybrldizes with" are intended to include single stranded and double stranded DNA. In the case of single stranded DNA, both the correspondlng and the compIementary sequence are intended to be included by this wording.

21~1~7~

The correspondence need not be 100%, i.e. DNA having a certain homology to the sequence shown in Seq.Id.No.1 is intended to be included DNA which has a homology of at least 60%, preferably a homology of at least 70% to said sequence ls intended to be 5 covered. It is especially intended to cover DNA having the same function and/or effect, even though the sequence differs from the one shown in Seq. Id.No. l . Thus, the invention is intended to include any changes in the fructosyltransferase coding region which either lead to the same amino acid sequence or to an amino 10 acid sequence which, notwithstanding one or more deviations from the original amino acid sequence, corresponds to an enzyme having essentially fructosyltransferase activity.
For example, the invention intends to cover not only the genomic 15 DNA and cDNA which codes for the fructosyltransferase enzyme of an Aeeto~acter diazotrophicus, but also DNA eoding for related fructosyltransferase enzymes, e.g. DNA coding for the fructosyl-transferase enzyme of reLated bacteria. Fructosyltransferase enzymes are considered to be related when they have a similar 20 fructosyltransferase activity, and/or are recognized by the same anti-fructosyltransferase antibody, and/or are encoded by DNA
having a homology of at least ~0 or 70%.
According to a preferred embodiment o~ the invention, the DNA
25 comprises ~ nucleotide sequence coding for an enzyme having fructosyltransferase activlty. More preferably, sai~ nucleotide sequence codes for an Aceto~acter ~iazotrophicus fructosyltrans-ferase enzyme having the amino acid sequence sho~n in Seq. Id .
No.2. More specifically, said nucleotide sequence essentially 30 consists of the nucleotide sequence shown in Seq. Id.No .1.
A further aspect of the present invention provides a recombinant polynucleotide eomprising a DNA as defined above (a fruetosyl-transferase-specific DNA) and a nueleotide sequenee of a cloning 35 or expression vector, wherein said polynucleotide is able to direet the expression of a protein in a suitable host, more in partieular a protein with fructosyltransferase aetivity. Said host may be a baeterium, sueh as a strain of ~. eoli, in whieh 21419~g ~ 7 case the plasmid pUCLS28 is a good example of a suitable recombinant polynucleotide. The host may also be a yeast, such as the yeast Plchia pastoris, n which case the plasmid pPSLS20 is a good example of a suitable recombinant polynucleotide. The invention is not limited to particular kinds of host5, however.
In princlple, any prokaryotic or eukaryotic cell may be used.
further aspect of this invention is a host cell transformed with a recombinant polynucleotide as defined above. As indicated above, the host cell may for example be a bacterium, such as a --strain of E. coli, e.g. the strain Levl of ~. coli, or a yeast, such as the yeast Pichia pastoris, e.g. the strain Lev2 of Pichia pas~oris.
An important further aspect of this invention is a proteinaceous substance having fructosyltransferase activity which comprises a polypeptide having an amino acid sequence essentially as shown in Seq.Id.No.Z, or a fragment of the same. In a particularly preferred embodiment of the present invention, t~le proteinaceous --substance having fructosyltransferase activity has a molecular ~ ~
weight of about 60D00 daltons and an isoelectric point of about 5 . 5, is stable in a pH range between 4 and 9 and in a temperature= range between 10 and ~0C, is active in the presence of 2% SDS, has an activity recovered after 5M urea treatments, has a specific activity of 2600 U/mg, has a Km for sucrose hydrolysis at 30C and pH 5 . 8 of about 12 mM, is not able to transfer fructosyl moieties to inulin and has a low levanase activity .
The invention covers the enzyme irrespective of how it has been produced, for example by recombinant DNA/genetic engineering technology, chemical synthesis, enzymatic degrada~ion, or a combination thereof. Further, the invention not only covers the enzyme as such, but also in the form of a fusion protein or as a protein physically or chemically bound to any substance and having fructosyltransferase activity.

2~41~7~

Another aspect of this invention is a method for producing a proteinaceou3 3ubstance having fructosyltransferase activity, comprising- the expression in a suitable host of a DNA as defined - herein which codes for a fructosyltransferase enzyme. As stated - -5above, the expression may be in prokaryotic or eukaryotic cells.
This invention includes a method as defined above wherein the fructosyltransferase enzyme produced is recovered from the cells and/or the culture medium.
10The invention furthermore provides a method for producing a proteinaceous ~ubstance having fructosyltransferase activity, comprising culturing a strain of ~cetobacter diazotrophicvs and recovering the fructosyltransferase enzyme produced. Preferably the Acetobacter diazotrophicus strain SRTq (CBS 549 . 94~ is used. -- -15Again the fructosyltransferase enzyme produced may be recovered, preferably from the culture medium. Recovery may be in the form of the culture medium as such, or by isolation and purification of the enzyme from the culture medium.
20The isolated fructosyltransferase is characterized by the following enzymatic properties. ~
(1) The enzyme acts at least on sucrose and raffinose to - - -transfer the fructosyl group to a broad range of acceptor (or ~-receptor) molecules.
25(2) The enzyme hardly exhibits activity on kestose and nistose.
(3) The en~yme shows low levanase activity and does not transfer fructosyl moieties to inulin.

(4) The enzyme has optimal activity at pH 5 and is stable in the range from 4 to 9.
30(5) The enzyme is active in a temperature range from 10 to 70"C.
(6) The enzyme activity is not affected in the presence of 2%
SDS .
(7) The enzyme consists of a single polypeptide of 60000 daltons as estimated by SDS-polyacrylamide gel electrophoresis.
35(8) The isoelectric point of the enzyme is 5.5.
(9) The enzyme is susceptible to the inhibitive effect of the ions of mercury.

214~7~
.
g (10) The Km value for sucrose hydrolysis at 30C and pH 5 . 8 is 11.8 mM.
(11) The specific activity of the enzyme is 2600 U/mg. One unit of enzyme is defined as the amount of enzyme reIeasing 1 umole 5 of glucose per minute at 42C and pH 5 . 2 .
The fructosyltransferase used in the inventive method can be naturally produced by inoculating a suitable culture medium with the strain SRT9 of Acetobacter diazotrophicus and conducting 10 culturing of the microorganlsm under shaking or aeration at a temperature of 20C to 40C, preferably 30C and at a pH value of 5 to 6.5, preferable 5.5 for a length o~ time of 12 hours to 3 days. In the culture medium preferable carbon sources include glucose, fructose, sucrose, glycerol, sorbitol and mannitol.
15 Generally satisfactory results can be obtained by using a culture medium containing 2% of mannitol, 0 . I% of yeast extract, 0.1% of tryptone, 0.12% of KH2PO4, 0.04% of K2HPO4, 0.02% of MgSO4.7H2O, 0.002% of CaCl2.H2O, 0.001% of FeCl3 and 0.0002% of Na2MoO4 having a pH of 6 . 0 .

The naturally occurring fructosyltransferase of Acetobacter diazotrophicus is ArrllmlllAted in the culture medium during the late phase of bacterial growth representing more than 70% of ~he total extracellular proteins. Since it is not inducible with sucrose, the enzyme can be obtained by ~ulturing the micro - --organism in the presence of a preferable carbon source other than sucrose so that levan is not formed in the culture medium, which gives a great practical advanta~e in the recovery of the secreted enzyme.

After culturing, the cells are removed, preferably by centrifu-gation, and the supernatant can be used as a crude enzyme solution or if necessary the enzyme can be purified according to a conventional enzymological procedure, preferably ion-exchange 35 chromatography.
The invention also provides the polynucleotide sequence coding ~~
for the fructosyltransferase of Acetobacter d~azotrophicus. This 21~97~

DNA sequence reveals low homology with known fructosyltrans- -ferase genes. Recombinant DNA molecules comprisLng the provided codlng sequence can be cQnstructed by genetic engineering techniques to express desired levels of the enzyme in a variety 5 of host cells.
Of particular lnterest is the expression of fructosyltransferase in a recombinant host so as to synthesize the transfructosyl-ation products directly in the said host. Cel's o~ interest for 10 fructosyltransferase expression may be eukaryotic or prokary-otic, being preferred eukaryotic cells such as yeast and plant cells. Of interest is also the construction of recombinant DNA
molecules comprising a modified fructosyltransferase gene so as to modulate the catalytic properties of the recombinant enzyme.
The invention further provides recombinant DNA molecules so as to produce elevated levels of the recombinant fructosyltrans-ferase in genetically manipulated cells of i~. coli and the yeast Pichia pastoris. Increased levels of expression may be achieved 20 by a variety of genetic manipulations, including placing the encoded gene on multi-copy plasmids, and/or operably linking high level promoters and other transcriptional control sequences -(operators, attenuators and the like) . Thus the regulation of the fructosyltransferase gene may be altered so as to provide 25 for inducible or constitutive expression in recombinant host cells .
The invention also provides a method for producing fructooligo- ~
saccharides and/or fructans by means of sucrose transfructosyl- ~=
ation comprising contacting sucrose under suitable transructo- = :
sylation conditions with a fructosyltransferase enzyme or with cells producing a fructosyltransferase enzyme and optionally recovering the fructoQligosaccharides produced, wherein said fructosyltransferase enzyme is a proteinaceous s-ubstance as defined herein or a proteinaceous substance obtained by a method as defined herein. Additionally, the fructosyltransferase enzyme or cells producing fructosyltransferase enzyme may be used in an 2~ 41~7~
immobilized form, physlcally or chemically coupled to a carrier - ~~
material .
-The ~ructosyltransferase of Acetobacter diazotrophicus can be -~
used to transfer fructosyl residues to different acceptor compounds, which allows for example the synthesis of homo- and heterooligosaccharides or fructans, the hydrolysis of sucrose, ~ ~etc. The enzyme can be used also to eliminate fructose from any _ilbstance that can function as donor of such residue. Using this _ enzyme it is posslble to change the properties of an organism by changing its carbohydrate profile through the fructosyltrans- - ~
ferase expression. As applications, for example, the products oE
the transfructosylation reaction can be obtained directly from said organism or alternatively the presence of said products, at low levels, could change the form of the crystals of a substance obtained ~rom said organism, so as to facilitate its purif ication .
STRAIN DEPOSITS
An Acetobac~er diazotrop~2icus strain SRT-4 was deposited on 10 November 1994 with the Centraalbureau voor Schimmelcultures - -(CsS), The Netherlands, under the provisions of the Budapest -:
Treaty and received accession number CBS 549.94~.
An F~ coli straln S17-1 containing p21R1 was deposited on 10 November 1994 with the Centraalbureatl voor Schimmelculture5 (CBS), The Netherlands, under the provision5 of the Budapest Treaty and received accession number CBS 550 . 94 .
EXAMPLES
Examples are offered by way o~ illustration, not by way o~
limitation .

- . 2141~7~
, E XAMP LE
Isolation of a levan-producing strain of Acetobacter diazo-trophi cus .
5 Stem segments (lcm size~ of sugarcane cultivar Ja60-5 were collected, superficially sterLlized and inoculated into vials with 3 mL of semisolid LGI medium consisted of: K2HPO4 0.02%, KH2PO4 0.06%, MgSO4.7H2O 0.02%, CaCl2-2H2 0-002%~ Na2M4 2H2 ().0002%, FeC13 0.001%, bromothymol blue 0.0025%, sucrose 10%, agar 0.18%, final pH 6Ø Semisolid LGI medium was reported for selective isolation of nitrogen-fixing bacteria of the species Acetobacter diazotrophicus (Cavalcante V.A. et al., Plant Soil 108: 23-31, 1~88) . Vials were incubated at 30C unti~ growth was observed, such vials were replicated into semisolid LGI medium 15 and those showing the typical orange-yellow surface growth were streaked out on LGI medium plates Several mucous colonies were taxonomically characterized and classified as Acetobacter diazotrophicus, one of the isolates was named strain SRT4.

Analysis of the extracellular polymer produced by Acetobacter diazotrophicus strain SRT4 grown on sucrose-~ontaining media.
Bacteria were grown on solid LGI medium at 30C for 7 days. The 25 extracellular polymer synthesized was collected from the medium surface with distilled water. After bacteria were removed by centrifugation, the polymer was precipitated with 2 volumes of ethanol, redissolved in distilled water, treated with saturated phenol, twice precipitated with ethanol, dialyzed against 30 distilled water and freeze-dried . The molecular weight o E the purified polymer was 7X106 daltons, as estimated by gel filtration on a Sephacryl S-500 column (20 x 0.8 cm~ eluted with 0.2 M NaCl at a flow rate of 12 ml h-l. Total acid hydrolysis o~ -purified polymer was performed with 0.5 M H25O4 at 100C for 35 15 min and neutrali~ed with Ba (OH) 2 After total hydrolysis, the polymer composition was found to be fructose, as analyzed by HPLC using a Nucleosil NH2 column (25 x 0 . 8 cm) eluted with 80%
acetonitrile ln water at a flow rate of 0.4 ml min~l in an 2~ 7~

isocratlc way. Polymer analysis by 13C-NMR spectroscopy revealed the presence of ,B-D- (2, 6) -linked fructofuranosyl residues in a ~ : ~ =
greater extent and a low rate of ~3-D (2-1)-linkages. This polymer structure is consistent with bacterlal levans.

Production of the exoenzyme fructosyltransferase of Acetobacter diazotrophicus .
10 The fructosyltransferase of Acetobacter diazotrophicus is a constitutive exoenzyme which is accumulated in the culture medium during the late phase of growth yielding more than 70% of total extracellular protelns.
15 The Acetobacter diazotrophicu~ strain SRT4 was gro~m in a S L
fermenter (3.5 L working volume~ B.E Marubishi (Tokyo, Japan) to stationary phase in a medium consisting of: mannitol 2%, yeast extract 0.1%, tryptone 0.1%, KH2PO4 0.12%, K2HPO4 0.04%, MgSO4.
7H2O 0.02%, CaC12.H2O 0.002%, FeC13 0.001%, Na2MoO4 0.0002~. The 20 fermentation conditions were: temperature 30~C, pH 5 . 5, stirring speed 400 rpm, aeration 1 vvm (1 volume per minute) . The culture reached a stationary OD62~ of 9 . 5 2fter fermentatlon for 60 hrs .
The cells were removed by centrifugation and the culture super-natant was concentrated 5 fold using a rotatory evaporator and 25 dialyzed against 20 mM Tris-HCl pH 7Ø Subsequently, ammonium sulfate was added to give 70% saturation. After centrifugatiQ~, the precipitate was dissolved in 20 mM Tris-HCl pH 7.0, dialyzed against the same buffer and applied to a column (2 . 5 x 13 cm~ of DEAE-Sepharose~C1-6B (fast flow) . The proteins absorbed on the 30 column were selectlvely eluted wlth a linear concentr~Ltion gradient of NaCl in buffer 20 mM Tris-E~Cl pH 7 . O . The fractions with fructosyltransferase activity eluted with the NaCl concentrations of 0 . 2 to 0 . 25 M. The eluted fractiQns were pooled, dialyzed against a solution of 1% NH4HCO3 pH 8 . 0 and 35 lyophilyzed . The purif ied enzymè was -applied on SDS-PAGE showing a slngle band of approxlmately 60 000 daltons. The N-terminal sequence of the mature frllctosyltransferase is Gly Gly Pro Leu 2~ ~19~

Phe Pro &ly Arg Ser 1eU (Seq~. Id.No . 3) as det~rmi nf~f1 by the Edmandegradation procedure.
The following table shows the fructosyltransferase activity, the 5 protein content, the specific fructosyltransfera3e activity and the yield in the fructosyltransferase-active fractions obtained in the individual steps of the purification procedure described above .
10 E'urification Total Total -- Specific Reco- Purifi-step activity protein activity very cation (U) (mg) (U/mg) (%) (fold) Culture super- 449280 288 1560 100 15 natant (10 liters~ -Ammonium sulfate 323880 169 1507 57 0 . 96 ( 7 0 % saturat ion DEAE-Sepharose 128991 49 2618 28 1. 68 CL-6B (fàst flow) The isolated fructosyltransferase is characterized by the following enzymatic properties. : -(1) The enzyme acts at least on sucrose and raffinose to trans-fer the fructosyl group to a broad range of receptor molecules. -(2) The enzyme hardly exhibits activity on kestose and nistose. -(3) The enzyme shows low levanase activity and does not transfer fructosyl moieties to inulin.
(9) The enzyme has optimal activity at pH 5 and is stable in the range f rom 4 to 9 .
(5) The enzyme is active in a temperature range from la to 70C.

(6) The enzyme activity is not affected by 2% SDS.

(7) The isoelectric point of the enzyme is 5.5.

(8) The enzyme i9 susceptible to the inhibitive effect of the ions of mercury.

(9) The ECm value ~or sucrose hydrolysis at 30C and pH 5 . 8 is 11.8 mM.

(10) The specific activity of the enzyme is 2600 U/mg.

21~97~
- ~ 15 Production of fructooligosaccharides by the action of the A.
diazotrophicus fructosyltransferase on sucrose. --~~
5 Levansucrase was added to a reaction mixture containing sucrose -- ~
1 M in acetate buffer 0.1 M, p.H 5.8, in a ratio of 30 units of enzyme per gram of sucrose. The reaction was conducted at 30C
for 3 hours~.: Analysis of sugar products by paper chromatography on Whatman 3 MM paper in 4: 6: 3 pyridine: butan-1-ol: water 10 revealed that 55% of total released fructose was accunulated in kestose in the reaction mixture. A further analysis of the reaction products was carried out by high performance li~uid chromatography ~HPLC) on Dionex carbopac TM PAL ~column 4 x 250 :~
mm) showed~ the following sugar composition: 31% glucose ~G); 17 15fructose ~F) i 23% sucrose; 18% kestose ~GF2); 6% nistose (GF3);
3% fructosylnistose ~GF4); 296 fructan ~GF>4) .
EXAMPLE 5:
Isolation of Acetobacter diazotrophicus mutants def icient in levan synthesis ~Lev- phenotype) by EMS mutagenesis.
Lev~ mutants were obtained by mutagenesis with ethylmethane-sulfonate ~EMS) according to the method described by Miller ~Miller, J.H. 1972, Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor. N. Y. ) with some modifications. The A. dlazotrophicus strain SRT4 was grown aerobically in SB medium for 48 hours at 30C. Bacteria ~1. 5 ml culture in an eppendorf tube~ were harvested by centrifugation, washed ln 1.5 ml o~ LGI medium salts, resuspended in 0 5 ml of 2% EMS in 0.2M Tris-HC1 pH 7.4 and incubated for 90 minutes at 30C. The treated cells were collected by centrifugation, washed with 1 ml of 5% NaS203 in 0.2M Tris--HCl pH7.4, resaspended in 3 mL of SB medium supplemented with glycerol 196 and grown with aeration for 16 hours at 30C. The bacterial culture was plated onto LGIE medium ~LGI medium salts, tryptone 0.1%, yeast extract 0.02%~ with sucrose 5% and glycerol 1% and incubated at 30C.
Colonies showlng a non-mucous phenotype were picked, grown in liquid LGIE medium and assayed for fructosyltransferase 2~19~

activity. Nine Lev- mutants were obtained at a frequency of 2 . 5 x 10-~ . Two classes of mutants were LsQlated according to the phenotype: class I mutants dld not show neither extracellular nor intracellular fructosyltransferase activity, probably due tQ - -5 mutations in the enzyme gene; class II mutants did not show fructosyltransferase activity in the culture supernatant but --still maintain the intrace~lular activity.
EXAMPLE 6:
Construction of a genomic clone coding for the A. diazotrophicus fructosyltransferase .
Routine recombinant DNA procedures were performed by standard methods essen~ially as described in Maniati~3 1989, Molecular Cloning, CSE~, N . Y ., USA.
A genomic library of A. diazotrophicus strain SRT4 was constructed in the broad-host-range cosmid pPW12, a derivative of pLAFR1 (Friedman et al., Gene 18, 289-296, 1982) with a synthetic sequence cohtalning the EcQRI-BamHI-EcoRI sites inserted at the EcoRI site (AFRC-IPSR Nitrogen Fixation Laboratory, University of Sussex, Brighton, East Sussex, BN19XQ, United Kingdom) .
Total DNA frQm A. diazotropllicus strain S~T4 was partially digested with the restriction endonuclease Sau3A I. Fragments of 15-30 kb were isolated from a low melting temperature agarose gel and cloned into BamHI-cleaved and dephosphorylated vec~or - -pPW12. The library was packaged into lambda phage particles, transferred by infection to ~ coli strain S17-1 (Simon, R. et -al., pp g8--106, en A. Puhler ed. Molecular C;enetics of the bacteria-plant interaction, Springer-Verlag, Berlin) and plated onto LB medium supplemented with tetracycline 12 ocg/ml. Then the gene bank was collected and transferred by con~ugation to a Lev~ ~=
class I mutant strain isolated from the Acetooacter - ---d~azotrophicus strain SRT4.

7 ~

E or con~ugal matlng the Lev- mutant straln of A . diazotrophicus was grown aerobically for 36 hours at 3aoC in SB medlum with 1%
glycerol as carbon source, l . 5 ml of bacterial culture were sedimented by centrifugation for 5 minutes at 12000 rpm and the 5 cells resuspended in 0 . 3 mL of LGI medium salts . Simultaneously l ml of the gene library kept in glycerol was inoculated to 2 ml of LB medium and incubated with aeration at 37C for 3 hours.
The cells were harvested by centrifugation and resuspended in 0.3 ml of M9 medium salts. The A. diazotrophicus mutant strain lO and the E. coli cells carrying the gene library were mixed on GYC agar plates ~5% glucose, 1% yeast extract, 3% CaCO3) (De Ley, J et al., pp 268-274, en N.R. Krieg & J.G. Holt ed Bergey's manual of Systematic Bacteriology, 8th ed. The Williams &
Wilkins Co., Baltimore, lg84~ and grown for 48 hours at 30C.
15 The mating mixture was collected and plated onto LGIE agar medium with 5% sucrose supplemented with ampicillin 25 ~Lg/m~
~included to select against E. coli) and tetracycline 20 llg/ml for transcon ~ugant selection. Individual colonies which recovered the mucous phenotype were picked. Plasmidic DNA from 20 these revertants was purified, used to transform the E. coli strain Sl7-l and transferred back by con~ugation to the A.
diazotrophicus mutant strain ascertaining that the phenotypic complementation of the Lev- mutant was due to the information carried on the plasmids.
As a result of restriction endonuclease mapping, two recombinant cosmids identified as p21R1 and p21R2 were found to share a common 7 . 8-kb region. By several subcloning and com-plementation experiments, the fructosyltransferas~e gene was located in a 30 2.3 kb BglII fragment. This fragment was cloned into pUC18 (C. Yanlsch-Perron et al., 1985, Gene 33: 103-ll~ and named pUCLS23. Nucleotide sequencing of this region revealed the presence of an op-en reading frame which codes :for a protein with certain homology to the fructosyltransferases published so far.
35 A smaller 2.0 kb SmaI fragment from pUCLS23 containing only this open reading frame was ligated in-frame into the 5 '-region of lacZ ' of pUC18 vector (C. Yanisch-Perron et al., 1985, Gene 33 . ~ --103-ll9~ under the control of the PlaC promoter. The constructed 21~1~7g plasmid named pUCl,S20 was used to transform the EM co7i strain 71-18 (C. Yanisch-Perron et al., l9B5, Gene~33: 103-119~ . The resulting recombinant strain identified as Levl was grown in LB
medium at 37C and the expression of the cloned gene was induced 5 with isopropylthiogalactoside (IPTG) . The polypeptide e~pressed in E. coli was detected in westerr; blot by rabbit antibodies raised against the A. diazotrophicus fructosyltranferase The recombinant protein exhlbited the expected molecular mass ~ =
(60 00~ daltons) and showed fructosyltransferase activity as the natural enzyme.
These results demonstrate that the identified open reading frame in the recombinant plasmid pUCLS23 codes for the fructosyltrans-ferase of Acetobacter diazotrophicus. The nucleotide sequence of the fructosyltransferase gene corresponds to the Seq. Id.No .1.
The amino acid sequence deduced from the nucleotide sequence of said gene corresponds to the Seq . Id . No . 2 .
EXAMPLE 7:
Production of an enzymatic preparation with fructosyltransferase activity in recorbinant E. coll.
The recombinant E. coli strain Levl was inoculated to an erlenmeyer containing 300 ml of LB medium supplemented with 0 . 5%
glucose and grown in a rotatory shaker at 37C for 16 ~.ours.
This culture was used to inoculate a 5 L fermenter (working volume 3 . 5 L) B . E Marubishi (Tokyo, Japan j containlng LB medium supplemented with 0.5% glucose at an ~nitial OD530 of 0.05. The fermentation conditions were as ~ollows: 37C, pE~ 6.8-7.2, stirring speed 350 rpm and aeration 1 WM (1 volume per minute) .
When the OD530 reached a .value of 2 the culture was induced wi~h 1 mM of isopropylthiogalactoside (IPTG) and kept in the same conditions for further 6 hours. The cells collected by centri-fugation were resuspended in an equal volume of buffer 0.1 M
sodium acetate, pE~ 5 . 8 and sonicated in a B . BP~AUN, model :
LABSONIC 2000 apparatus for five cycles of 30 seconds each with 1 min intervals on ice. The production of the recombinant enzyme was determined lmmunologically in the cellular crude, 21~7~

representing approxlmately the 5% of tQtal soluble proteins. The recombinant fructosyltransferase expressed in ~. coli displayed the same cataLytic properties as the natural enzyme.
EXAMPLE 8:
Production of an enzymatic preparation with fructosyltransferase activity in recombinant yeast Pichia pastoris.
rn order to obtaln extracellular expression of the recombinant fructosyltransferase in the methylotrophic yeast Plchla pastoris, the following experiments were performed.
Two oligonucleotides were synthesized: 5 ' -CATGGCGGCCCGCTCTTCCCC-3 ' ( Seq . Id . No . 4 ) and 5 ' -GGGGAAGAGCGGGCCGC-3 ' ( Seq . Ia . No . 5 ) and hybridized to each other, resulting in a double stranded DNA
fragment with one extremity compatible with the NcoI recognition site and one blunt-ended ~xtremity. The sequence of the oligo-nucleotides was chosen such that the fragment coded for the first six amino acids of the N-terminal region of the mature fructosyltransferase. This synthetic fragment was blunt ligated to the 2 . 0-kb SmaI fragment of the plasmid pUCLS23 obtaining a NcoI fragment with the entire codlng sequence o~ the mature enzyme, which was then inserted into the cleaved NcoI site of the integrative vector pPS7 (l~uropean patent application EP 438 200 A1) resulting in an "in frame" fusion of the Saccharomyces cerevls~ae Suc2 signal peptide cQding sequence and the fructosyltransferase gene under the control of the alcohol oxidase 1 IAOX1) methanol inducible promoter. This construction named pPSLS20 was PvuII digested and used to transform the Pichia pastorls mutant strain MP36 (his3-) (European patent application EP 438 Z00 A1) according to the procedure described ~:
by Meilhoc et al. (Meilhoc et al. (1990), ~io/Technology 8: 223-227) . The transformed cells were selected on minimal medium G
(P. Galzy, 1957, Paris. C.R. Acad. sci. 245: 2423-2427) with 2%
glucose as c-arbDn source and immunologically screened for fructosyltransferase production. A recombinant yeast expressing high levels of fructosyltransferase after induction with methanol was named strain Lev2.

2141~g The production of re~:ombinant fructosyltransferase by Pichia pastor~ strain Lev2 was studied in a 5 L fermenter (3.5 1 working volume) B.E Marubishi (Tokyo, Japan) in YPG medium (1 yeast extract 1%, peptone 2%, glucose 2%), inoculated at an initial OD530 of 0.2, pH 5.2, temperature 30C, aeration 1 VVM
(1 volume per minute), stirring speed 350 rpm. At an optical density of ~0 a gradually increasing flow of methanol was fed reaching 3 5 g/h/L, stirring speed was lncreased from 350 to ~50 rpm. The culture was induc~d during 120 ho~rs.
The enzymatic activity in the culture supernatant increased during induction reaching 5000 U/mL (2 g/L) . FolIowing cell disruption with glass beads (Maniatis et al. (1989) Molecular Cloning, CSH, NY, USA) it was determined that approximately 35%
of the total enzyme produ-ced was secreted to the culture medlum. =

214197~

SEQUENCE LISTING:
SEQ ID NO.1:
5 TYPE: Nucleic acid LENGTH: 1632 base pairs STRANDEDNESS: Double TOPO10GY: Linear MOLECIJLE TYPE: DNA (genomic) 10 ORIGINAL SOURCE: Acetobacter diazotroph~cus strain SRT4 DI~ECT SOURCE: plasmid pUCLS23 PROPERTIES: Extracellular ~ructosyltransi~erase gene oi: Aceto-bacter diazotrop~icus 15ATGGCGCACC GTCCGGGTGT GATGCCTCGT GGCGGCCCGC ~ C~ GG 50 GCGGTCGCTG bCCbt,bbl'bC CGGGCTTCCC GCTGCCCAGC ATTCATACGC 100 AGCAGGCGTA TGACCCGCAG TCGGACTTTA CCGCCCGCTG GACACGTGCC :~50 GACGCATTGC A-GATCAAGGC GCATTCGGAT GCGACGGTCG CGGCCGGGCA '200 GAATTCCCTG CCGGCGCAAC TGAeCATGCC GAACATCCCG GCGGACTTCC 250 AAGCACGCCG ATCAGTTCAG CTATAACGGC TGGGAAGTCA ~ b~ 350 GACGGCCGAC CCCAATGCCG GATACGGTTT CGACGACCGC CACGTGCATG ~00 CCCGCATCGG ~:'l"L~ .lAT Cblcb~bCGG GTATTCCCGC CAGCCGGCGG 450 CCGGTGAATG GCGGCTGGAC CTATGGCGGC CATeTCTTCC CCGACGGAGC, .` ~5'00 25CAGCGCGCAG GTCTACGCCG GCC'AGACCTA r;~rr.~rr~r. GCGGAATGGT ' 550 CCGGTTCGTC GCGTCTGATG r~r~T~r~TG GCAATACCGT ATCGGTCTTC 600 TATACCGACG TGGCGTTCAA CCGTGACGCC AACGCCAACA AcATcAcccc 650 TGTTCGAGGG CAATACCGCG GG-CCAGCGTG GCGTCGC-CA~ CTGCACCGAG 900 GCCGATCTGG GCTTCCGCCC GAACGATCCC AATGCGGA~A CCCTGCAGGA 9 5 0 AGTCCTGGAT AGCGG~GCCT ATTACCAGAA GGCCAATATC GGCCTGGCCA 1000 35TCGCCACGGA TTCGACCCTG TCGAaATGGA AbLlC~:lblC GCCGCTGATT 1050 TCGGCCAACT GCG~CAATGA rr.~r.~rrr~ CGGCCGCAGG TGTACCTCCA lloo TAACGG~AAA TACTATATCT TCACCATCAG CCACCGCACG ACCTTCGCGG 1150 CCbbll,L~:GA TGG-ACCGGAC bG~:b-'L~:'LACG GCTTCGTGGG TGACGGCATC 1200 21~97~

TCCGACCGAC CTCAACACGG CGGCAGGCAC GGATTTCGAT CCCAGCCCGG 13~1~
~f~r~G~r.rr ~ ,cll~ CAGTCCTATT CGCACTACGT CATGCCGGGG 1350 GGACTGGTTG AATCGTTCAT CGACACGGTG GAAAACCGTC GCGGGGGTAC 1400 ~:
CCTGGCGCCC ACGGTCCGGG TGCGCATCGC CCAGAACGCG TCCG'CGGTCG L450 :~

GCCCACGTCG ~GGTCT-GGCGG CGCAGGCGTC CACCAACAAT'''GCCCAGGTGC L600 SEQ ID NO . 2:
TYPE: Amino acid LENGTH: 543 amino aclds TOPOLOGY: Linear MOLECULE TYPE: protein ORIGINAL SOURCE: Aceto~acter diazotrophicus strain SRT4 DIRECT SOURCE: Deduced from the nucleotide sequence coding for the Acetobacter diazotrophicus fructosyltransferase 20PROPERTIES: Protein with fructosyltransferase activity from Acetobacter diazotroph~cus Met Ala His Arg Pro Gly Val Met Pro Arg Gly Gly Pro Leu Phe 5 10 15 =~
25Pro Gly Arg Ser Leu Ala Gly Val Pro Gly Phe Pro 1eu Pro Ser 20 25 30 ~~
Ile His Thr Gln Gln Ala Tyr Asp Pro Gln Ser Asp Phe Thr Ala Arg Trp Thr Arg Ala Asp Ala Leu Gln Ile Lys Ala` His Ser ASp Ala Thr Val Ala Ala Gly Gln Asn Ser Leu Pro Ala Gln Leu Thr Met Pro Asn Ile Pro Ala Asp Phe Pro Val Ile Asn Pro Asp Val 35Trp Val Trp Asp Thr Trp Thr Leu IIe Asp Lys His Ala Asp Gln Phe Ser Tyr Asn Gly Trp Glu Val Ile Phe Cys Leu Thr Ala Asp 21~Q~9 Pro Asn Ala &ly Tyr Gly Phe Asp Asp Arg His Val His Ala Arg Ile Gly Phe Phe Tyr Arg Arg Ala Gly Ile Pro Ala Ser Arg Arg 5 Pro Val Asn Gly Gly Trp Thr Tyr Gly Gly His Leu Phe Pro Asp Gly Ala Ser Ala Gln Val Tyr Ala Gly Gln Thr Tyr Thr Asn Gln 170 ~75 180 Ala Glu Trp Ser Gly Ser Ser Arg Leu Met Gln Ile His GLy Asn 185 190 ~ 195 Thr Val Ser Val Phe Tyr Thr Asp Val Ala Phe Asn Arg Asp Ala Asn Ala Asn Asn Ile Thr Pro Pro Gln Ala Ile IIe Thr Gln Thr Leu Gly Arg Ile His Ala Asp Phe Asn His Val Trp Phe Thr Gly Phe Thr Ala His Thr Pro Leu Leu Gln Pro Asp Gly Val Leu Tyr Gln Asn GIy Ala Gln Asn GIu Phe Phe Asn Phe Arg Asp Pro Phe Thr Phe Glu Asp Pro Lys His Pro Gly Val Asn Tyr Met Val Phe Glu Gly Asn Thr Ala Gly Gln Arg Gly Val Ala Asn Cys Thr Glu Ala Asp Leu Gly Phe Arg Pro Asn Asp Pro Asn Ala Glu Thr Leu Gln Glu Val Leu Asp Ser Gly Ala Tyr Tyr Gln Lys Ala Asn Ile Gly Leu Ala Ile Ala Thr Asp Ser Thr Leu Ser Lys Trp Lys Phe 335 ~ 340 345 Leu Ser Pro Leu Ile Ser Ala Asn Cys Val Asn Asp Gln Thr Glu Arg Pro Gln Val Tyr Leu His Asn Gly Lys Tyr Tyr Ile Phe Thr Ile Ser His Arg Thr Thr Phe Ala Ala Gly Val Asp Gly Pro Asp 3ao 385 390 Gly Val Tyr Gly Phe Val Gly Asp Gly Ile Arg Ser Asp Phe Gln 395 . 400 405 21~197~
_ 24 Pro Met Asn Tyr Gly Ser Gly Leu Thr Met Gly Asn Pro Thr Asp Leu Asn Thr Ala Ala Gly Thr Asp Phe Asp Pro Ser Pro Asp Gln 425 430 435 Asn Pro Arg Ala Phe Gln Ser Tyr Ser His Tyr Val Met Pro Gly Gly Leu Val Glu Ser Phe Ile Asp Thr Val Glu Asn Arg Arg Gly Gly Thr Leu Ala Pro Thr Val Arg Val Arg Ile Ala Gln Asn Ala Ser Ala Val Asp Leu Arg Tyr Gly Asn Gly Gly Leu Gly Gly Tyr Gly Asp Ile Pro Ala Asn Arg Ala Asp Val Asn Ile Ala Gly Phe 500 505 510 5 Ile Gln Asp Leu Phe Gly Gln Pro Thr Ser Gly Leu Ala Ala Gln Ala Ser Thr Asn Asn Ala Gln Val Leu Ala Gln Val Arg Gln Phe Leu Asn Gln SEQ. ID.NO: 3 SEQUENCE TYPE: Amino acid 25 ~ ;QU~;NI :~; LENGTH: 10 amino aclds TOPOLOGY: linear Gly Gly Pro Leu Phe Pro Gly Arg Ser Leu SEQ . ID . NO: 4 SEQUENCE TYPE: Nucleic acid SEQUENCE LENGTH: 2 1 bp 35 STRANDEDNESS: single TOPOLOGY: linear CATGGCGGCC ~:~I,'L~.'L'l'CCC C 21 214197g SEQ.ID.NO: 5 SEQUENCE TYPE: N~cleic aci~
SEQUENCE LENGTE~: 17 bp STRANDEDNESS: linear 5 TOPOLOGY: Linear

Claims (28)

1. An isolated and purified DNA having a nucleotide sequence which essentially corresponds to or hybridizes with a nucleotide sequence comprised in the fructosyltransferase gene, shown in Seq.Id.No.1, of the bacterium Acetobacter diazotrophicus.

2. An isolated and purified DNA according to claim 1 which comprises a nucleotide sequence coding for an enzyme having fructosyltransferase activity.

3. An isolated and purified DNA according to claim 1 which comprises a nucleotide sequence coding for an Acetobacter diazo-trophicus fructosyltransferase enzyme having the amino acid sequence shown in Seq.Id.No.2.

4. An isolated and purified DNA according to claim 1 wherein said nucleotide sequence essentially consists of the nucleotide sequence shown in Seq.Id.No.1.

5. A recombinant polynucleotide comprising the DNA of any one of the claims 1 to 4 and a nucleotide sequence of a cloning or expression vector, wherein said polynucleotide is able to direct the expression of a protein in a suitable host.

6. A recombinant polynucleotide according to claim 5 which is able to direct expression of a protein with fructosyltransferase activity in a suitable host.

7. A recombinant polynucleotide according to claim 5 which is able to direct expression of a protein with fructosyltransferase activity in a bacterium.

8. A recombinant polynucleotide according to claim 5 which is able to direct expression of a protein with fructosyltransferase activity in E. coli.

9. A recombinant polynucleotide according to claim 8 which is the plasmid pUCLS28.

10. A recombinant polynucleotide according to claim 5 which is able to direct expression of a protein with fructosyltransferase activity in a yeast.

11. A recombinant polynucleotide according to claim 5 which is able to direct expression of a protein with fructosyltransferase activity in the yeast Pichia pastoris.

12. A recombinant polynucleotide according to claim 11 which is the plasmid pPSLS20.

13. A host cell transformed with the recombinant polynucleotide of any one of the claims 5 to 12.

14. A host cell according to claim 13 which is a bacterium.

15. A host cell according to claim 13 which is E. coli.

16. A host cell according to claim 13 which is the strain Lev1 of E. coli.

17. A host cell according to claim 13 which is a yeast.

18. A host cell according to claim 13 which is the yeast Pichia pastoris.

19. A host cell according to claim 13 which is the strain Lev2 of Pichia pastoris.

20 . A proteinaceous substance having fructosyltransferase activity which comprises a polypeptide having an amino acid sequence essentially as shown in Seq.Id.No.2, or a fragment of the same.

21. A proteinaceous substance having fructosyltransferase activity according to claim 20 which has a molecular weight of about 60000 daltons and an isoelectric point of about 5.5, is stable in a pH range between 4 and 9 and in a temperature range between 10 and 70°C, is active in the presence of 2% SDS, has an activity recovered after 5M urea treatments, has a specific activity of 2600 U/mg, has a Km for sucrose hydrolysis at 30°C
and pH 5.8 of about 12 mM, is not able to transfer fructosyl moieties to inulin and has a low levanase activity.

22. A method for producing a proteinaceous substance having fructosyltransferase activity, comprising the expression in a suitable host of a DNA according to any one of claims 1 to 4 coding for a fructosyltransferase enzyme.

23. A method according to claim 22 wherein said expression is in prokaryotic or eukaryotic cells.

24. A method according to claim 22 wherein the fructosyltrans-ferase enzyme produced is recovered from the cells and/or the culture medium.

25. A method for producing a proteinaceous substance having fructosyltransferase activity, comprising culturing a strain of Acetobacter diazotrophicus and recovering the fructosyltrans-ferase enzyme produced.

26. A method according to claim 25 wherein Acetobacter diazo-trophicus strain SRT4 (CBS 549.94) is used.

27. A method according to claim 25 wherein the fructosyltrans-ferase enzyme produced is recovered from the culture medium.

28. A method for producing fructooligosaccharides and/or fructans by means of sucrose transfructosylation comprising contacting sucrose under suitable transfructosylation conditions with a fructosyltransferase enzyme or with cells producing a fructosyltransferase enzyme and optionally recovering the fructooligosaccharides produced, wherein said fructosyltrans-ferase enzyme is a proteinaceous substance according to claim 20 or 21 or a proteinaceous substance obtained by a method according to any one of claims 22-27.