AU742048B2 - Plant alkaline and neutral invertases - Google Patents

Description

WO 99/40206 PCT/EP99/00623 Plant Alkaline and Neutral Invertases The present invention relates to DNA encoding proteins which hydrolyze sucrose. In particular, the present invention describes DNA which can be translated into a protein with neutral invertase activity.

In most plant species sucrose (ca-D-glucopyranosyl p-D-fructofuranoside) is the first nonphosphorylated product of photoassimilation and serves as a mobile source of energy and carbon for heterotrophic plant tissues. Metabolism of sucrose is an absolute requirement for the survival of heterotrophic plant organs, where sucrose can only be utilized after cleavage by invertase or sucrose synthase.

Invertases hydrolyze sucrose into glucose and fructose thus feeding the sucrose into various biochemical pathways. There exist several isoforms of invertases with different biochemical properties and subcellular locations. Acid invertases are characterized by acidic pH optima in the range of 4.0 to 5.5. They are found ionically bound to the cell wall (cell wall invertases) or as soluble proteins in the vacuole (vacuolar invertases). The amino acid sequences of said acid invertases share conserved motifs. Analysis of sequence similarities suggests that they are evolutionary related to invertases from yeast and bacteria whereas no counterparts have been found in animal cells. Technically acid invertase is used in the confectionary industry to convert easily crystallized sucrose into the less easily crystallized glucose-fructose mixture. Thereby a hard sucrose core coated for example with chocolate can be turned into the soft center ate. Depending on the specific use invertases with neutral or alkaline pH optima would be preferred.

Neutral and alkaline invertases are characterized by pH-optima in the range of 6.0 to and 7.5 to 8.5, respectively. They are thought to be confined to mature tissues and it is generally assumed that they accumulate in the cytoplasm which is supported by the fact that no N-linked glycans have been detected. Up to now the molecular structure of neutral invertases and their genes had not been elucidated. This, however, is a prerequisite for the biotechnological exploitation of neutral or alkaline invertases. The corresponding enzymes from carrot (Daucus carota cv Queen Anne's Lace) have been purified (Lee and Sturm, Plant Physiol 112: 1513-1522, 1996) and biochemically characterized recently.

Cells of a suspension culture of carrot contain soluble sucrose-cleaving activities with distinct pH optima above and below pH 6 (alkaline and acid invertase, respectively). The two activities were efficiently separated by an ammonium sulphate precipitation at 20-45% saturation. Activity of neutral and alkaline invertase was detected in the protein pellet, whereas that of acid invertase remained in the supematant. The 20-45% ammonium sulphate fraction was chromatographed on Q-Sepharose and two peaks of invertase activity with only poor separation were obtained. Fractions containing activity were combined and further purified by chromatography on HA-Ultrogel followed by affinity chromatography on .,Green 19, leading to the efficient separation of thc two activities. A sucrose-cleaving activity with a neutral pH optimum (neutral invertase, H 1 was identified in the non-bound protein fraction. An activity with a more basic pH optimum (alkaline invertase, H 2 bound to the HA-Ultrogel and Green 19 dye columns and could be eluted with salt-containing buffers. At this stage of the purification, neutral invertase accounted for about one-third of the invertase activity, and alkaline invertase for two-thirds. The pooled fractions containing S neutral and alkaline invertase, respectively, were individually purified further by gel filtration chromatography, a second ion-exchange chromatography, a second gel filtration chromatography and hydrophobic interaction chromatography. Macro-Prep anion-exchange chromatography for neutral invertase was the most effective procedure for the removal of contaminating proteins from the preparations. Although propyl agarose chromatography did not increase the specific activity of alkaline invertase, it was required to obtain electrophoretically pure enzyme. At the end of the purifications less than 10% of the two enzymes were recovered. Said losses are considered to be a consequence of the various purification steps employed and low enzyme stabilities.

Neutral invertase was found to elute from a gel-filtration column as a polypeptide with approximately 456 kD, whereas purified enzmye migrated as a single band of about 57 kD on SDS polyacrylamide gel electrophoresis. Alkaline invertase was found to elute as a polypeptide with approximately 504 kD, whereas purified enzmye migrated as a single band of about 126 kD on SDS polyacrylamide gel electrophoresis. The results suggested that neutral invertase constitutes an octamer and alkaline invertase a tetramer of the corresponding enzymes. Their pH optima were determined to be at pH 6.8 and respectively. In addition neutral invertase was shown to cleave raffinose and stacchyose suggesting a 1-fructofuronidase activity of the enzyme.

pU ^v P:\OPERIKbml27217-99 spe.doc-2 1/9A/() -2A- Advantageously, at least one embodiment of the present invention provides an isolated DNA molecule comprising a nucleotide sequence which can be translated into a protein with neutral invertase activity but lacking 1-fructofuranosidase activity, wherein highest activity is observed in the range 6.0 to 7.5. Although neutral and *o WO 99/40206 PCT/EP99/00623 -3alkaline invertase are believed to be products of different genes, they appear to be immunologically related.

Dynamic programming algorithms yield different kinds of alignments. In general there exist two approaches towards sequence alignment. Algorithms as proposed by Needleman and Wunsch and by Sellers align the entire length of two sequences providing a global alingment of the sequences. The Smith-Waterman algorithm on the other hand yields local alignments. A local alignment aligns the pair of regions within the sequences that are most similiar given the choice of scoring matrix and gap penalties. This allows a database search to focus on the most highly conserved regions of the sequences. It also allows similiar domains within sequences to be identified. To speed up alignments using the Smith- Waterman algorithm both BLAST (Basic Local Alignment Search Tool) and FASTA place additional restrictions on the alignments.

Within the context of the present invention alignments can be conveniently performed using BLAST, a set of similarity search programs designed to explore all of the available sequence databases regardless of whether the query is protein or DNA. Version BLAST (Gapped BLAST) of this search tool has been made publicly available on the internet (currently http://www.ncbi.nlm.nih.gov/BLAST/). It uses a heuristic algorithm which seeks local as opposed to global alignments and is therefore able to detect relationships among sequences which share only isolated regions. The scores assigned in a BLAST search have a well-defined statistical interpretation. Particularly useful within the scope of the present invention are the blastp program allowing for the introduction of gaps in the local sequence alignments and the PSI-BLAST program, both programs comparing an amino acid query sequence against a protein sequence database, as well as a blastp variant program allowing local alignment of two sequences only. Said programs are preferably run with optional parameters set to the default values.

Global or local alignment of the amino acid sequences according to the present invention with known sequences shows less than 40% sequence identity to known sequences of acid invertases or other sucrose-metabolizing enzymes. Examples of DNA comprising a nucleotide sequence which can be translated into a protein with neutral invertase activity are described in SEQ ID NO: 1 and SEQ ID NO: 3. The amino acid sequence of the encoded invertase is given in SEQ ID NO: 2. Related proteins showing more than WO 99/40206 PCT/EP99/00623 -4sequence identity to SEQ ID NO: 2 and their corresponding genes can be isolated from at least any plant from which seeds, fruits or storage organs are harvested. Examples are protein crops, oil crops, and starch storing crops, sugar beet, corn, sweet corn, soybean, sunflower, grasses, oilseed rape, wheat, barley, sorghum, rice, melon, watermelon, squash, chicory, tomato, pepper, broccoli, cauliflower, cabbage, cucumber, daikon, benas, and lettuce.

The protein described in SEQ ID NO: 2 lacks a signal peptide and is very hydrophilic.

Furthermore it contains 18 cystein and 15 methionine residues. It shows highest global sequence identity after alingment to the LIM17 protein which is encoded by a partial cDNA clone obtained from Lilium longiflorum. Global alingment to other protein sequences results in less than 40% sequence identity. The DNA sequences encoding LIM proteins were originally identified when screening a library obtained from cDNA derived from microsporocytes of Lilium longiflorum in meiotic prophase using a substraction probe specific to meiotic prophase (Kobayashi et al, DNA Research 1: 15-26, 1994). Using the computer program GAP the amino acid sequence deduced from the partial sequence of the Lilium longiflorum LIM17 protein is 47% identical (58% similiar) to the carrot protein. The related LIM17 protein encoded by the genome of the unicellular cyanobacterium Synechocystis (ORF s110626) is 37% identical (47% similiar) to the sequence of the carrot enzyme after several large gaps had to be introduced for optimal alignment. Thus, it is possible that related proteins, that is proteins showing a sequence identity to the invertases of the present invention of more than 40%, might be found in photosynthetic bacteria. Like the carrot protein, the LIM17 proteins from Lilium and Synechocystis are rich in Cys and Met but their positions within the polypeptide chains do not seem to be conserved. The functions or enzymatic activites of the LIM17 proteins, which are smaller than the carrot sequence homologues, are not known.

Thus, according to the present invention a family of neutral invertases can be defined the members of which after global alingment show a 40% or higher amino acid sequence identity to SEQ ID NO: 2. Preferably the amino acid sequence identity is higher than 50% or even higher than 55%. Sequences more than 55% identical might be considered a subfamily. Sequences according to the present invention can also comprise component sequences of at least 330, 450 or 510 basepairs length which are at least 60%, 70% or even more than 75% identical to locally aligned component sequences of SEQ ID NO: 2.

WO 99/40206 PCT/EP99/00623 When making multiple sequence alignments certain algorithms can take into account sequence similarities such as same net charge or comparable hydrophobicity/hydrophilicity of the individual amino acids in addition to sequence identities. Thus, said algorithms evaluate whether the substitution of one amino acid for another is likely to conserve the physical and chemical properties necessary to maintain the structure and function of the protein or is more likely to disrupt essential structural and functional features of a protein.

Such sequence similarity is quantified in terms of of a percentage of positive amino acids as compared to the percentage of identical amino acids and can help to assign a protein to the correct protein family in border-line cases. Proteins of particular interest within the scope of the present invention are invertases the amino acid sequence of which comprises at least one of the following characteristic amino acid subsequences: VGTVAA (SEQ ID NO: 4) AIGRV (SEQ ID NO: DFGESAIGRVAPVDSGLWWIIL (SEQ ID NO: 6) CMIDRRMGI (SEQ ID NO: 7) PTLLVTDGSCMIDRRMGIHGHPLEIQAL (SEQ ID NO: 8) GGYLIGN (SEQ ID NO: 9) DFRFFTLGN (SEQ ID NO: DNA encoding invertases belonging to said new family of proteins can be produced by the following general method. A single stranded fragment of SEQ ID NO: 1 consisting of at least prefeably 20 to 30 or even more than 100 consecutive nucleotides is used as a probe to screen a DNA library for clones hybridizing to said fragment. The factors determining hybridization are described in Sambrook et al, Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, chapters 9.47-9.57 and 11.45-11.49, 1989. Hybridizing clones are sequenced and DNA of clones comprising an open reading frame encoding a protein with more than 40% sequence identity to SEQ ID NO: 2 are purified. Said DNA can then be further processed by a number of routine methods of recombinant DNA such as restriction enzyme digestion, ligation, or polymerase chain reaction.

DNA comprising a sequence of nucleotides defined by SEQ ID NO: 1 can be cloned in the following way: Comparison of a partial internal tryptic peptide sequence (XNIYPDQIPPWLV, SEQ ID NO: 11) of purified carrot neutral invertase with an est database revealed its presence in -6the amino acid sequence encoded by est t88552 from Arabidopsis (1026 bp 3'-end of an Arabidopsis cDNA). A DNA fragment encoding this peptide sequence (nucleotides 100 to 410) can be isolated by PCR using the primers 5'-TCTAAGGATCTAGAAAGAGCCATTA-3' (SEQ ID NO: 12) and 5'-TTCAATTGAATTCAATATAGCTTC-3' (SEQ ID NO: 13). The PCR product is after cleavage with Xbal and EcoRI ligated into the respective sites of the E. coli plasmid pBluescript II KS (Stratagene). After amplification and purification of the plasmid, tihe 'ragment is excised, purified by agarose gel electrophoresis and electroelution, and randomly labeled with [a- 32 P]ATP. The labeled DNA is used as a probe to screen a library made from rapidly growing suspension cultures of wild carrot (Daucus carota cv Queen Anne's Lace, W001C). Clones obtained are sequenced and for example might reveal a clone comprising a 2447 nucleotide insert containing 29 bp of 5' and 393 bp of 3' noncoding sequences whereas the ORF codes for a protein with 675 amino acids sharing S identity (86% similarity) with the deduced amino acid sequence of the Arabidopsis est o t88552.

A person skilled in the art is able to modifiy said process to make it applicable to any gene encoding a protein belonging to the new family of invertases. Additionally, disclosing SEQ ID NO: 3 enables a person skilled in the art to design oligonucelotides for polymerase chain reactions which attempt to amplify DNA fragments from templates comprising a sequence of nucleotides characterized by any continuous sequence of 15 and preferably 20 to 30 or o more basepairs in SEQ ID NO: 1. Said nucleotides comprise a sequence of nucleotides which represents 20 and preferably 20 to 30 or more basepairs of SEQ ID NO: 1.

Polymerase chain reactions performed using at least one such oligonucleotide and their amplification products constitute another embodiment of the present invention. Further, the disclosed nucleotide sequences enable a person skilled in the art to design transformation vectors which can be used to generate transgenic plants applying art-recognized transformation techniques as described for example in WO 96/27673 (pages 17-20).

OFFICE

P:\OPER\Kbmn27217-99 spc.doc-21/09/01 -6A- Advantageously at least one embodiment of the present invention provides recombinant plant invertase with a neutral pH optimum. This can be achieved by recombinant expression of DNA encoding said invertase, preferably cDNA, in a microbial host such as E.coli or yeast. For example recombinant invertase can be produced the following way: cDNA encoding the enzyme is engineered into an expression vector such as pTrc99 A (Pharmacia Biotech). After o o WO 99/40206 PCT/EP99/00623 -7transformation of bacteria such as E. coli and, if required, induction of protein synthesis with for example IPTG, bacteria are lysed. Neutral invertase activity is determined in the soluble lysate fraction. In particular about 100pl of soluble extract are mixed with 7001 of water, 100 of 0.5 M potassium phosphate, pH 6.8, and 100jl of 0.5 M sucrose, and incubated for min at 37 0 C. Aliquots of this solution are used for the determination of reducing sugars according to Somogyi (Somogyi, J Biol Chem 195: 19-23, 1952). For the determination of the pH dependence of the sucrose-cleaving activity, solutions of 0.5 M potassium phosphate with pH values between 4.5 and 8.5 are used.

Three key biochemical properties of the recombinantly produced invertase are very similiar to those of the enzyme purified from plants, namely a Km value of about 20mM, a pH dependence with a sharp maximum between pH 6.5 and 7.0, and an inhibition by Cu 2 at micromolar concentrations. On the other hand, the recombinant enzyme unexpectedly hydrolyzes only sucrose without cleaving raffinose or stachyose. Thus, the recombinant protein is substantially devoid of 13-fructofuranosidase activity.

EXAMPLES:

Example 1: Purification of Carrot Neutral and Alkaline Invertase Preparation of Extracts Carrot cells (400g) collected from suspension cultures in the exponential growth phase are homogenized four times for 20 sec at full speed with a Polytron homogenizer in 2.5 volumes of ice-cold buffer A (50mM Hepes-KOH, pH 7.5, containing 0.5mM EDTA, 10mM lysine, MgCI 2 0.5% 2-mercaptoethanol and 100mM phenylmethylsulfonyl fluoride). The homogenate is centrifuged for 20 min at 6000g in a Sorvall GSA-rotor. The supernatant is collected and kept cold. The 6000g pellet is resuspended in 2.5 volumes of ice-cold buffer A, homogenized with a Polytron homogenizer three times for 20 sec at full speed and centrifuged for a further 20 min. The combined supernatants are centrifuged at 16,300g for min and then poured through four layers of Miracloth (Calbiochem-Behring Corporation, La Jolla, USA). The filtrate is used for further protein purification. If not stated otherwise, all steps are carried out at WO 99/40206 PCT/EP99/00623 -8- Ammonium Sulfate Precipitation Solid ammonium sulfate is slowly added to the crude extract with gentle stirring, and the protein that precipitates between 20% and 45% saturation is collected by centrifugation for min at 16,300g. The precipitate is dissolved in 100ml of buffer B (25mM Hepes-KOH, pH 7.5, containing 190mM NaCI, 0.5% 2-mercaptoethanol and 100mM phenylmethylsulfonyl fluoride), and dialyzed against buffer B overnight.

Anion-Exchange Chromatography on Q-Sepharose The dialysate is loaded onto a Q-Sepharose column (2.5cm x 25cm, Pharmacia LKB Biotechnology, Uppsala, Sweden) equilibrated with buffer B. The column is washed with buffer B until the absorbance at 280nm is less than 0.01. Bound protein is eluted with a linear gradient of 240ml of 190-550 mM NaCI in 25mM Hepes-KOH, pH 7.5, containing 2-mercaptoethanol and 100mM phenylmethylsulfonyl fluoride. Active fractions (fraction size 5 ml) are pooled, precipitated with ammonium sulfate at 60% saturation, and centrifuged for 30 min at 16,300g. The precipitate is dissolved in 5ml of buffer C (5mM Kphosphate buffer, pH 7.5, containing 0.1% 2-mercaptoethanol), and dialyzed against buffer C overnight.

Chromatography on HA-Ultrogel The dialysate is applied to an HA-Ultrogel column (2.5cm x 25cm, Sigma, Buchs, Switzerland) equilibrated with buffer C. The column is washed with buffer C and eluted with 200ml of a linear gradient of 5-500 mM K-phosphate buffer, pH 7.5, containing 0.1% 2-mercaptoethanol. The column is eluted at a flow rate of 40 ml/h and fractions of 5ml are collected. Fractions in the flow-through containing neutral invertase activity and fractions in the eluate containing alkaline invertase activity are combined separately, precipitated with ammonium sulfate at 60% saturation and centrifuged for 30 min at 16,300g. The two protein pellets are individually dissolved in 5ml of buffer D (25mM K-phosphate buffer, pH containing 0.1% 2-mercaptoethanol) and dialyzed against buffer D overnight.

Affinity Chromatography on Green 19 dye The dialyzed protein solutions (5ml each) are divided into 0.5-mi aliquots, and then applied to 10 prepacked green 19 dye columns (4.5 x 0.7 cm, Sigma, Buchs, Switzerland) WO 99/40206 PCT/EP99/00623 -9equilibrated with buffer D. The columns are washed with 15ml of buffer D, then step eluted with NaCI at 0.35 M and 1.5 M (15ml and 25ml, respectively), and the eluate is collected in 2ml fractions. Neutral invertase activity is detected in the flow-through, whereas alkaline invertase activity is eluted by 1.5 M NaCI. Fractions containing enzyme activity are pooled and precipitated with ammonium sulfate at 60% saturation. The precipitated proteins are collected by centrifugation for 30 min at 16,300g.

Gel-Filtration Chromatography I on Sephacryl S-300 Each protein pellet is dissolved in 7ml of buffer E (100 mM Hepes-KOH, pH 7.5, containing 0.1% 2-mercaptoethanol). The protein solutions are individually applied to a Sephacryl S-300 column (2.6cm x 100cm, Pharmacia LKB Biotechnology, Uppsala, Sweden) equilibrated with buffer E and calibrated with blue dextran thyroglobulin (669 kD), apoferritin (443 kD), B-amylase (200 kD), alcohol dehydrogenase (150 kD), BSA (66 kD), and carbonic anhydrase (29 kD). The column is eluted at a flow rate of 110 ml/h and fractions of 5ml are collected. Fractions containing enzyme activity are pooled, dialyzed overnight against buffer F for alkaline invertase (25mM Hepes-KOH, pH 8.0, containing 200mM NaCI and 0.1% 2-mercaptoethanol), and buffer G for neutral invertase Hepes-KOH, pH 7.2, containing 275mM NaCI and 0.1% 2-mercaptoethanol).

Anion-Exchange Chromatography II on Macro-Prep For further purification of alkaline invertase, the dialysate is applied to a Macro-Prep column x 20cm, Bio-Rad Laboratories, Richmond, CA, USA) equilibrated with buffer F. The column is washed with buffer F and eluted with 200ml of a linear gradient of 200-450 mM NaCI in 25mM Hepes-KOH, pH 8, containing 0.1% 2-mercaptoethanol.

For further purification of neutral invertase, the dialysate is applied to a Macro-Prep column (1.2cm x 25cm, Bio-Rad Laboratories) equilibrated with buffer G. The column is washed with buffer G and eluted with 200ml of a linear gradient of 275-360 mM NaCI in Hepes-KOH, pH 7.2, containing 0.1% 2-mercaptoethanol.

Fractions containing the relevant enzyme activity are combined separately and precipitated with ammonium sulfate at 60% saturation and centrifuged for 30 min at 16,300g.

WO 99/40206 PCT/EP99/00623 Hydrophobic Interaction Chromatography on Propyl Agarose The protein pellet with alkaline invertase activity is dissolved in 5ml of buffer H Hepes-KOH, pH 8.0, containing 1.5 M ammonium sulfate and 0.1% 2 -mercaptoethanol).

The solution is applied to a Propyl Agarose column (10cm x 1.5cm, Sigma, Buchs, Switzerland) equilibrated with buffer H. The column is washed with buffer H, then eluted with 25mM Hepes-KOH, pH 8.0, containing 0.1% 2-mercaptoethanol, and 3-mi fractions are collected. Fractions containing enzyme activity are pooled, dialyzed against Hepes-KOH, pH 8.0, containing 0.1% 2-mercaptoethanol and stored in 50% glycerol at -200C.

Gel-Filtration Chromatography I on Sephacryl S-300 The protein pellet with neutral invertase activity is dissolved in 5ml of buffer I (100mM K-phosphate buffer, pH 7.0, containing 0.1% 2-mercaptoethanol) and applied to a Sephacryl S-300 column (2.6cm x 100cm, Pharmacia LKB) equilibrated with buffer I.

Fractions of 5 ml are collected. Fractions containing enzyme activity are pooled, dialyzed against 10mM K-phosphate buffer, pH 7.0, containing 0.1% 2-mercaptoethanol and stored in 50% glycerol at -200C.

Example 2: Isolation of a cDNA Clone Encoding Carrot Neutral Invertase Comparison of a partial internal tryptic peptide sequence (XNIYPDQIPPWLV, SEQ ID NO: 11) of the purified carrot neutral invertase with an est database identified its presence in the amino acid sequence encoded by est t88552 from Arabidopsis (1026 bp 3'-end of an Arabidopsis cDNA). A DNA fragment encoding this peptide sequence (nucleotides 100 to 410) is isolated by PCR using the primers 5'-TCTAAGGATCTAGAAAGAGCCATTA-3'

(SEQ

ID NO: 12) and 5'-TTCAATTGAATTCAATATAGCTTC-3' (SEQ ID NO: 13). Amplification is achieved in a DNA Thermal Cycler (Perkin Elmer Cetus) with the following conditions: cycles of denaturation at 95°C for 1 min, annealing at 400C for 0.5 min, and elongation at 72°C for 1.5 min, followed by 20 cycles of denaturation at 950C for 1 min, annealing at 600 for 0.5 min, and elongation at 72°C for 1.5 min. The PCR product is extracted with phenol/chloroform and after cleavage with Xbal and EcoRI ligated into the respective sites of the E. coil plasmid pBluescript II KS (Stratagene). After amplification and purification of the plasmid, the fragment is excised, purified by agarose gel electrophoresis and WO 99/40206 PCT/EP99/00623 -11 electroelution, and randomly labeled with 32 P]ATP. The labeled DNA is used as a probe to screen a cDNA library in a lambda ZAP II vector (Stratagene) made with polyA' mRNA from cells of a rapidly growing suspension culture of wild carrot (Daucus carota cv Queen Anne's Lace, W001C) and a single hybridizing clone is identified.

Example 3: Sequence Analysis of the Carrot Invertase Clone The insert of the cDNA clone of Example 2 is ligated into the pBluescript II KS vector (Stratagene) and both strands are automatically sequenced by the dideoxynucleotide chaintermination method. Computer-assisted analysis of DNA and protein sequences as described in Examples 2 and 3 is performed using the Wisconsin Package Version Genetics Computer Group (GCG), Madison, Wisconsin.

Sequence comparisons are carried out with the computer program GAP, which uses the alignment algorithm of Needleman and Wunsch Mol. Biol. 48; 443-453, 1970) to find the alignment of two complete sequences maximizing the number of matches and minimizing the number of gaps while allowing the introduction of gaps for optimal alignments. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. One provides a gap creation penalty and a gap extension penalty in units of matched bases. In other words, GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If you choose a gap extension penalty greater than zero, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Typical values to use as a point of departure for the gap creation and gap extension penalties are 3.0 and 0.1 for protein sequence comparisons.

The cDNA clone of carrot neutral invertase is found to be 2447 nucleotides long (SEQ ID NO: 3) and contains 29 bp of 5' and 393 bp of 3' non-coding sequences. The ORF codes for 675 amino acids with a molecular mass of 75957 Dalton and a calculated isoelectric point of pl 8.01. The deduced amino acid sequence shares 80% identity (86% similarity) with the deduced amino acid sequence of the Arabidopsis est t88552.

A comparison of the deduced amino acid of the carrot cDNA clone for neutral invertase WO 99/40206 PCT/EP99/00623 12- (car) with the sequences of the LIM17 proteins from L. Ion giflorum (Iii) and Synechocystis (bac) (Table 1) identifies three conserved sequence domains (boxes When the sequence of box 2 is used for a database search, a protein of known function is identified, namely cellobiose phosphoryiase from Clostridium stercorarium, which cleaves cellobiose [j3-D-Glc(1 4)-D-Glc] in the presence of pyrophosphate into glucose 1-phosphate and glucose. This suggests that box 2 may constitute the binding site for the glucose residues of the disaccharides.

Table 1: Comparison of the clDNA-derived amino acid sequences of neutral invertase from carrot (car) with the amino acid sequences of the LIMi 7 proteins form Lilium longiflorum (hil) and Synechocystis (bac). The amino acid sequences are in one-letter-codle and have been aligned by introducing gaps to maximize identity. The amino acid residues in bold face indicate conserved domains (boxes The asterisks below the sequence mark amino acid residues identical in all three sequences.

iii car bac iii car bac iii car bac NN'ICIAVsq MRPCCRML.LS GKNSSIFrGYS FRKCDHRMGT NLSKKQFKVY GLRGYVSCZRG

PPQISARSAV

GKGLZYCGI

]YQVRNRFERP LSFWVNPSWV MDG W DPNRKGFFGS GSDEWGQPRVL DSGQDKPDEF DFSKLLHIKP TSGCRRVDSG GRSVLVNVAs SSRLLDHPEI VSTPGKRSAV GEKVREEEGR VGMVIGSNVNI RVLNIDRQTS .CDERSLLE HSTGIGIIYP DYRNHSTSVE GHVNDKSFER

IYVRGGTINVK

NrPKAFN...

GDSKGLNGCY,

YFEPHGQHPM ND.EGDALK VLSPKREvsE vEKAwELLR -MKSPQAQQI LDARRLLYE lil FVIWFFPSGL car FMEDFvpsAL bac FIPhrVpVM li. PNQEIL car GKIGESEDIL bac .VE'rENHE.A lil LTLCSEGFD car r2UL4MTDGFD bac LZZ2LHPMF

PLVFKNPEN

PLVTERVE K

RSLVYFRGQP

GAVVDYCGNP

Y-AMvxnma3y

LQSWEKKIDR

LQSWEKTVDC

LQS... K

WIILRAYTK

WIIL~LRAYT

WPILAYYYVQ

ANIMPEPEI

AFLLNGEGEI

N1D'GSAIG

DPEFGESAIG

KADYGQRAIEG

VKRL7ZTLR

VQFLEICJT

RVAPVDSGLW

RVrCSVD~SI-W

VOTIAALMM

VGTVAASPA

HSPGQGLvMPA

GFPTYGIFPI'

STGDSA~t

IJDGDYGLQAR

FFWLRCL

PYSALRCSflE

LYGALKSA

DSTrPwYDQV nBSEEZUTEV

SFKVNHDPV.

SFKVENSVAID

SF PnCQKMI

VDVQGILI

LHVQWILQ'

NT2NKQ

LLLIDLKAKG

IFP! ZCAMA CC2'2IRR1I YGYPIEIQMI MFPTLLVTDG. SO DRRWI HGHPLE:QAL Dn.PTLFVPVG AFMaDRtv vrhpLEIQTL WO 99/40206 PCT/EP99/00623 -13lil D DEGRELAERI car N DSITKNLVAA bac YCSNKDHPFD

SFITMEQSHQF

QRLQAL SFLRSYFWL ITRRLNDYR FKTEQYSDTA NRLSAL SFHIREYYWV IKINEIYR YKTEEYSTDA NLSVDWLKKL RTYLLKHYWI NCNIVQALRR RPTEQYGEEA 480 lil nIKnvMPDS car InMFNIYPDQ bac SNEHNVHTEr lil f1DLLEERWP car IINZIEDKWD bac FFRLVILNQR LPMVFD FM

IPSWLVT&WP

IPWALQDWLG

ELVNGP*--

DVAHMPLKI

ELCAQPLRI

TRGGYFIGNV SPARMDFRWF ETGGYLIGNL QPAHMDFRFF DRGGYLIGNI RGRPDFRFF CYPALEYEEW RVITGSDPKN CHPPLKDDDW RSKTGFDRKN

CLGNCIAIIS

TLGNLWSIVS

SIONCLGAIF

TPWSYHNGGS

LPWCYHNAGH

N.ATAEQSEA

SLDPKQNES

DVTSLAQQRS

WPTLLWQFL

WPCLFWFLVV

IGKQSRLYQT

VGQQSRSYQT

lil car bac lil car bac ACIK

AVLRHSCHSN

WI'IAGFLTSK

WI'IVGLLLVH

MIvKKP ELARKAVALA EKKLSEDHWP EYYDTIRRGRF YGTVEYAEMG NLIRNNYEVL LRRLPKHKWA EYFDGPTGFW LLLEPEMAS KLFWEEDYEL LESCVCAIGK SGRKKCSRFA

AKSQW

HFTEVNPDDA Example 4: Steady State Levels of Neutral Invertase mRNA Steady-state levels of neutral invertase mRNA in leaves and roots at three different developmental stages and in reproductive organs of carrot plants are determined. Total RNA is prepared by the method described by Prescott and Martin (Plant Molecular Biology Reporter 4: 219-224,1987) modified by adding 20 mg of Polyclar AT (Serva) per gram of tissue before grinding in liquid nitrogen. For RNA gel blot analysis, total RNA (10 mg/lane) is separated on a 1.2 agarose gel, containing 6% formaldehyde. The northern blot is loaded with total RNA (10 mg/lane) from 10-, and 16-week-old leaves, 10-, and 16week-old roots, and flower buds flowers small developing seeds large developing seeds and mature seeds The blot is hybridized with the 32 P-labeled cDNA for neutral invertase.

Steady-state transcript levels for carrot neutral invertase are found in all organs at different stages of development with slightly higher levels in developing organs. This finding suggests a more general and possibly growth-related function of the enzyme in carrot sucrose metabolism.

WO 99/40206 PCT/EP99/00623 -14- Example 5: E. coli Expression of Carrot Neutral Invertase To express the cDNA clone of carrot neutral invertase in E.coli strain JM105 (Pharmacia Biotech) the ORF is amplified by PCR using the primers ATAGATATGAATAC-3' (SEQ ID NO: 1'4) and AGACC-3' (SEQ ID NO: 15). Amplification is achieved in a DNA Thermal Cycler (Perkin Elmer Cetus) under the following conditions: 30 cycles of denaturation at 950C for 1 min, annealing at 55°C for 0.5 min, and elongation at 72°C for 1.5 min. The PCR product is extracted with phenol/chloroform and after cleavage with Kpnl and Xbal ligated into the respective sites of the expression vector pTrc99A (Pharmacia Biotech). Protein biosynthesis in transformed bacteria carrying the expression vector is induced with 1mM IPTG for approximately 16 hours, and bacteria are lysed in a small volume of potassium phosphate, pH 6.8, by 1 cycle of freezing and thawing (Johnson and Hecht, Biotechnology 12: 1357-1360, 1994). Neutral invertase activity is determined in a soluble lysate fraction as described by Lee and Sturm, 1996, supra. Briefly, 100l of soluble extract are mixed with 700,l of water, 100pl of 0.5 M potassium phosphate, pH 6.8, and 100pl of M sucrose, and incubated for 30 min at 37°C. Aliquots of this solution are used for the determination of reducing sugars according to Somogyi. For the determination of the pH dependence of the sucrose-cleaving activity, solutions of 0.5 M potassium phosphate with pH values between 4.5 and 8.5 are used.

Example 6: Measurement of Invertase Activity Invertase activity is determined in reaction mixtures containing 50mM K-phosphate buffer (pH 6.8 or 100mM sucrose and an appropriate volume of enzyme in a final volume of 1 ml. The mixture is incubated at 37°C for 30 min. The amount of reducing sugar liberated is determined according to Somogyi. Enzyme activity (units) is expressed as the amount (gmol) of reducing sugar (glucose and fructose) released per minute. Invertase activity is inhibited by high concentrations of ammonium ions, which necessitates that protein solutions prepared after ammonium sulfate precipitation are dialyzed prior to activity determination.

P:\OPER\Kbm\27217-99 spe.doc-24/09/01 From measurement taken during the purification procedure according to Example 1 it can be deduced that 400g of cells express about 240 units neutral invertase activity which means 0.6 units per gramm cells. During the purification procedure of the enzyme about of the activity is lost.

Activity measurement of the recombinant enzyme expressed in E. coli detects about 0.4 units of neutral invertase activity in 100 1l of E. coli extract described in example Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will 10 be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

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OF hC"., I S

CRC

EDITORIAL NOTE NO.27217/99 Sequence listing pages 1 -11, are to be incorporated after the description and before the claims.

WO 99/40206 WO 9940206PCT/EP99/O00623 1- SEQUENCE LISTING <110> <120> <130> <140> <141> <150> <151> <160> <170> <210> <211> <212> <213> <220> Novartis AG Plant Alkaline and Neutral Invertases S-30364/A GB 9802249.4 1998-02-03 Patentln Ver. 1 2025

JJNA

Daucus carota <221> CDS <222> <400> 1 atg aat act Met Asn. Thr 1 atg tta ott Met Leu Leu aaa tgt gat Lys Cys Asp gtg tac ggt Val Tyr Gly ggt tat agg Gly Tyr Mrg ggt tct gat Gly Ser Asp gtt gat agt Val Asp Ser (2025) act tgt att Thr Ci's Ile 5 agc tgt aag Ser Cys Lys cat aga atg His Mrg Met ttg oga, ggg Leu Mrg Gly tgt ggg att Cys Gly Ile 70 tgg gga cag Trp Gly Gin ggt ggt agg Gly Gly Mrg 100 got Ala aat Asn ggg Gly tat 55 gat Asp cct Pro agt Ser gta. tog Val Ser toa. tog Ser Ser 25 act aat Thr Asn 40 gtt agt Val Ser cog aat Pro Asn agg gtt Mrg Val gta ott Val Leu 105 aat atg Asn Met 10 att tto le Phe ttg tog Leu Ser tgt agg Cys Mrg ogg aag Arg Lys 75 tta aca Leu Thr 90 gtt aat Val Asn agg Mrg gga.

Gly aa~a

LYS

ggt.

Gly ggt Gly agt Ser gtg Val cot Pro tao aag

LYS

ggt.

Gly ttt Phe ggt Gly gog Ala tgt tgt aga.

Cys Cys Mrg tog ttt oga Ser Phe Mrg oaa ttt aag Gin Phe Lys aaa ggt tta.

Lys Gly Leu ttt ggt too Phe Gly Ser tgt ogt ogt Cys Mrg Mrg tog gat tat Ser Asp T1yr 110 48 96 144 192 240 288 336 WO 99/40206 WO 9940206PCT/EP99/00623 agg aat cat tea act tcg gtt Arg Asn His Ser Thr Ser Val gag Glu gaa Giu 145 gga Gly ggg Gly gag Giu ect Pro aac Asn 225 ttc Phe aca Thr ggg Gly gat Asp gaa.

Giu 305 ate Ile 115 att Ile gtg Val aat Asn aag Lys tgg Tip 195 ggg Gly gac Asp ctt Leu cag Gin ggg Gly 275 aaa.

LYS

gee Ala ttg Lieu cgt gga Arg Gly 135 ggg gag Gly Glu 150 aat gta Asn Val tct ceg Ser Pro ctt ega.

Loeu Arg gea get Ala Ala 215 ttt att Phe Ile 230 gaa ggg Giu Gly agt. tgg Ser Trp ccc gca Pro Ala gaa tea Glu Ser 295 cgt gtt Arg Val 310 get tat Ala Tyr gaa ggt cat Glu Gly His 120 ggg ttg aat Gly Leu Asn aaa gta agg Lys Val Arg aat att ggt Asn Ile Gly 170 aag aga gag Lys Arg Giu 185 ggt gct gtt Gly Ala Val 200 agt gat eca Ser Asp Pro cgt gat ttt Arg Asp Phe gag att gtt Giu Ile Val 250 gaa. aaa. act Giu Lys Thr 265 agt ttc aaa Ser Phe Lys 280 gag gat att Giu Asp Ile gca cct gtt Ala Pro Val act aag ctc Thr Lys Lieu gtt Val gtg Val gaa Giu 155 gat Asp gtg Val gtt Val gct Ala gte Val 235 aag

LYS

gta Val gtt Val tta Leu gat Asp 315 aca Thr aat Asn aag

LYS

140 gag Giu teg Ser tct Ser gat Asp gat Asp 220 ccc Pro aat Asn gac Asp aaa

LYS

gat Asp 300 tct Ser gga Gly aag agt Lys Ser ttg gtg Leu Val ggt agg Gly Arg ggt tta Gly Leu 175 gte gaa Val Giu 190 tgt gga Cys Gly aca eca Thr Pro gct ctt Ala Leu ctg cta Loeu Loeu 255 cat age His Ser 270 gtg get Val Ala gat ttc Asp Phe tta tgg Leu Tip tat ggg Tyr Gly 384 432 480 528 576 624 672 720 768 816 864 912 960 1008 WO 99/40206 PCT/EP99/00623 -3- 325 330 335 caa gca oga gtg gat gtg cag aca gga ata agg ctg ata ctt aat ctg 1056 Gin Ala Arg Val Asp Val Gin Thr Gly Ile Arg Leu Ile Leu Asn Leu 340 345 350 tgt tta acg gat gga ttc gac atg ttt cct aca ctg tta gtc act gat 1104 Cys Leu Thr Asp Gly Phe Asp Met Phe Pro Thr Leu Leu Vai Thr Asp 355 360 365 ggt tc tgt atg att gac aga agg atg ggc att cat ggt cac cct ctc 1152 Gly Ser Cys Met Ile Asp Arg Arg Met Gly Ile His Gly His Pro Leu 370 375 380 gaa att caa goa ttg ttt tat tca got ttg cgt tgt tot cga gag atg 1200 Glu Ile Gin Ala Leu Phe Tyr Ser Ala Leu Arg Cys Ser Arg Giu Met 385 390 395 400 otc att gtc aat gat too aca aag aat ttg gtt gct gct gtc aac aac 1248 Leu Ile Vai Asn Asp Ser Thr Lys Asn Leu Vai Ala Ala Val Asn Asn 405 410 415 cgg ctt agt gca ctg tcc ttc cac att agg gag tat tat tgg gtg gac 1296 Arg Leu Ser Ala Leu Ser Phe His Ile Arg Giu Tyr Tyr Trp Val Asp 420 425 430 atg aag aag ato aat gaa ata tac cga tao aaa act gaa gaa tac tca 1344 Met Lys Lys Ile Asn Giu Ile Tyr Arg Tyr Lys Thr Glu Giu Tyr Ser 435 440 445 act gat gco atc aat aag tto aao atc tat cog gat caa ata ccc tot 1392 Thr Asp Ala Ile Asn Lys Phe Asn Ile Tyr Pro Asp Gin Ile Pro Ser 450 455 460 tgg otg gta gao tgg atg cot gag acg gga ggg tat otc att ggc aat 1440 Trp Leu Val Asp Trp Met Pro Glu Thr Gly Gly Tyr Leu Ile Gly Asn 465 470 475 480 otg oag cca got oat atg gao ttt aga tto ttt aoo cta gga aat ott 1488 Leu Gin Pro Ala His Met Asp Phe Arg Phe Phe Thr Leu Giy Asn Leu 485 490 495 tgg tot att gto toa toa otg ggt aoa cct aaa oaa aat gag ago att 1536 Trp Ser Ile Val Ser Ser Leu Gly Thr Pro Lys Gin Asn Giu Ser Ile 500 505 510 tta aat ttg ata gaa gat aaa tgg gao gat ott gtg goa oat atg oct 1584 Leu Asn Leu Ile Glu Asp Lys Tip Asp Asp Leu Val Ala His Met Pro 515 520 525 tta aaa ata tgt tao cct got otg gag tat gag gaa tgg oga gta ata 1632 Leu Lys Ile Cys Tyr Pro Ala Leu Glu Tyr Giu Giu Trp Arg Val Ile 530 535 540 aca ggo agt gac ccc aag aat aog cca tgg toa tat oat aat ggg gga 1680 WO 99/40206 WO 9940206PCT/EP99/00623 Thr Giy Ser Asp Pro 545 Lys Asn Thr Pro Trp Ser Tyr His Asn Giy 550 555 tcc Ser aag

LYS

ott Leu ttt Phe ttC Phe 625 ttg Leu att Ile oaa Gin tgg oca Trp Pro aaa coa Lys Pro tog gag Ser Giu 595 att ggg Ile Gly 6i10 tta aca Leu Thr ttt tgg Phe Trp ggc aaa Giy Lys gtg gtC Val Vai 675 aca Thr gag Giu 580 gat Asp aaa Lys tot Ser gag Giu tot Ser 660 ott Leu 565 ott Loeu oat His oaa Gin aag Lys gaa.

Glu 645 ggt Giy oto Leu goa Ala tgg too Ser ttg Loeu 630 gao Asp aga, Arg tgg Trp aga.

Arg oot Pro aga Arg 615 tta Leu tat

T

aag oag Gin aag

LYS

gaa Giu 600 ott Leu ttg Leu gaa.

Giu aag ttt Phe gog Ala 585 tat Tyr tat Tyr gaa Giu otg Leu aoa Thr 570 gtg Val tat oag Gin aat Asn oto Leu 650 tta Leu gog Ala gat Asp aoa Thr ooa Pro 635 gag Giu got.

Mla ttg Leu aoa Thr tgg Trp 620 gag Giu ago Ser tgo Cys goo Ala ogg Arg 605 aoa Thr atg Met tgt Cys att Ile gag Giu 590 ogt Arg att le goa Ala gto Val aag Lys 575 aaa

LYS

gga Giy got Ala toa Ser tgt Cys 655 atg Met aag

LYS

aga.

Arg ggo Giy aag

LYS

640 goa Ala 1728 1776 i1824 1872 192 0 1968 2016 tgo tot ogg ttt got goo aaa. toa LYS LYS Cys 665 Ser Arg Phe Ala Ala Lys Ser 670 2025 <210> 2 <211> 675 <2i2> PRT <2i3> Dauous oarota <400> 2 Met Asn Thr Thr Cys i 5 Met Leu Leu Ser Cys Lys Cys Asp His Arg Vai Ty~r Gly Leu Arg Gly Tyr Arg Cys Gly le Ala Lys Asn Met Giy Gly Tyr 55 Ile Asp Asn Ile Leu Cys Arg Pro Tys Gly Phe Cys Ser Gin

LYS

Phe WO 99/40206 PCT/EP99/00623 70 75 Gly Ser Asp Trp Gly Gin Pro Arg Val Leu Thr Ser Gly Cys Arg Arg 90 Val Asp Ser Gly Gly Arg Ser Val Leu Val Asn Val Ala Ser Asp Tyr 100 105 110 Arg Asn His Ser Thr Ser Val Glu Gly His Val Asn Asp Lys Ser Phe 115 120 125 Glu Arg Ile Tyr Val Arg Gly Gly Leu Asn Val Lys Pro Leu Val Ile 130 135 140 Glu Arg Val Glu Lys Gly Glu Lys Val Arg Glu Glu Glu Gly Arg Val 145 150 155 160 Gly Val Asn Gly Ser Asn Val Asn Ile Gly Asp Ser Lys Gly Leu Asn 165 170 175 Gly Gly Lys Val Leu Ser Pro Lys Arg Glu Val Ser Glu Val Glu Lys 180 185 190 Glu Ala Trp Glu Leu Leu Arg Gly Ala Val Val Asp Tyr Cys Gly Asn 195 200 205 Pro Val Gly Thr Val Ala Ala Ser Asp Pro Ala Asp Ser Thr Pro Leu 210 215 220 Asn Tyr Asp Gin Val Phe Ile Arg Asp Phe Val Pro Ser Ala Leu Ala 225 230 235 240 Phe Leu Leu Asn Gly Glu Gly Glu Ile Val Lys Asn Phe Leu Leu His 245 250 255 Thr Leu Gin Leu Gin Ser Trp Glu Lys Thr Val Asp Cys His Ser Pro 260 265 270 Gly Gin Gly Leu Met Pro Ala Ser Phe Lys Val Lys Asn Val Ala Ile 275 280 285 Asp Gly Lys Ile Gly Glu Ser Glu Asp Ile Leu Asp Pro Asp Phe Gly 290 295 300 Glu Ser Ala Ile Gly Arg Val Ala Pro Val Asp Ser Gly Leu Trp Trp 305 310 315 320 Ile Ile Leu Leu Arg Ala Tyr Thr Lys Leu Thr Gly Asp Tyr Gly Leu 325 330 335 Gin Ala Arg Val Asp Val Gin Thr Gly Ile Arg Leu Ile Leu Asn Leu 340 345 350 Cys Leu Thr Asp Gly Phe Asp Met Phe Pro Thr Leu Leu Val Thr Asp 355 rn" WO 99/40206 -6- Gly Ser Cys Met Ile Asp Arg Arg Met Gly Ile His Gly His Pro Leu 370 375 380 Glu Ile Gin Ala Leu Phe Tyr Ser Ala Leu Arg Cys Ser Arg Glu Met 385 390 395 400 Leu Ile Val Asn Asp Ser Thr Lys Asn Leu Val Ala Ala Val Asn Asn 405 410 415 Arg Leu Ser Ala Leu Ser Phe His Ile Arg Glu Tyr Tyr Trp Val Asp 420 425 430 Met Lys Lys Ile Asn Glu Ile Tyr Arg Tyr Lys Thr Glu Glu Tyr Ser 435 440 445 Thr Asp Ala Ile Asn Lys Phe Asn Ile Tyr Pro Asp Gin Ile Pro Ser 450 455 460 Tr Leu Val Asp Trp Met Pro Glu Thr Gly Gly Tyr Leu Ile Gly Asn 465 470 475 480 Leu Gin Pro Ala His Met Asp Phe Arg Phe Phe Thr Leu Gly Asn Leu 485 490 495 Trp Ser Ile Val Ser Ser Leu Gly Thr Pro Lys Gin Asn Glu Ser Ile 500 505 510 Leu Asn Leu Ile Glu Asp Lys Trp Asp Asp Leu Val Ala His Met Pro 515 520 525 Leu Lys Ile Cys Tyr Pro Ala Leu Glu Tyr Glu Glu Trp Arg Val Ile 530 535 540 Thr Gly Ser Asp Pro Lys Asn Thr Pro Trp Ser Tyr His Asn Gly Gly 545 550 555 560 Ser Trp Pro Thr Leu Leu Trp Gin Phe Thr Leu Ala Cys Ile Lys Met 565 570 575 Lys Lys Pro Glu Leu Ala Arg Lys Ala Val Ala Leu Ala Glu Lys Lys 580 585 590 Leu Ser Glu Asp His Trp Pro Glu Tyr Tyr Asp Thr Arg Arg Gly Arg 595 600 605 Phe Ile Gly Lys Gin Ser Arg Leu Tyr Gin Thr Trp Thr Ile Ala Gly 610 615 620 Phe Leu Thr Ser Lys Leu Leu Leu Glu Asn Pro Glu Met Ala Ser Lys 625 630 635 640 Leu Phe Trp Glu Glu Asp Tyr Glu Leu Leu Glu Ser Cys Val Cys Ala 645 650 655 PCT/EP99/00623 WO 99/40206 PCT/EP99/00623 -7- Ile Gly Lys Ser Gly Arg Lys Lys Cys Ser Arg Phe Ala Ala Lys Ser 660 665 670 Gin Val Val 675 <210> 3 <211> 2447 <212> MJNA <213> Daucus carota <400> 3 gaattccgat ttagcaaatt gttatagata tgaatactac ttgtattgct gtatcgaata tgaggccttg ttgtagaatg ttacttagct ttcgaaaatg gtttgcgagg atccgaatcg caagtggttg attataggaa tttatgttcg agaaagtaag attcgaaagg aaaaagaggc ggactgttgc ttcgtgattt agaattttct gCcctgggca aaattggaga ttgcacctgt caggagatta atctgtgttt gtatgattga attcagcttt tgatcatage gtatgttagt gaagggtttt tcgtcgtgtt tcattcaact tggagggttg ggaagaggag tttaaatggg ttgggagtta agctagtgat tgtcccctct gctacataca agggttgatg atcagaggat tgattctggg tgggctgcaa aacggatgga cagaaggatg gcgttgttct Latggggacta tgtaggggtg tttggttccg gatagtggtg tcggttgaag aatgtgaagc ggtagggtag ggtaaggttt cttcgaggtg ccagctgatt gctcttgcat ctgcagttac cccgcaagtt attttagatc ttatggtgga gcacgagtgg ttcgacatgt ggcattcatg cgagagatgc gtaagaattc atttgtcgaa gtaaaggttt gttctgattg gtaggagtgt gtcatgttaa cgttggtgat gagttaatgg tgtctccgaa ctgttgttga ctacaccact tcttgcttaa agagttggga tcaaagttaa cagatttcgg tcattttgtt atgtgcagac ttcctacact gtcaccctct tcattgtcaa atcgattttc aaagcaattt aggttatagg gggacagcct acttgttaat tgataagagt tgaaagggtg ttcgaatgta gagagaggtg ttattgtgga caactatgac tggagaaggg aaaaactgta aaacgtggct tgaatcagcc aagagcttat aggaataagg gttagtcact cgaaattcaa tgattccaca ggatactcgt 120 aaggtgtacg 180 tgtgggattg 240 agggttttaa 300 gtggcgtcgg 360 ttcgagagga 420 gagaaagggg 480 aatattggtg 540 tctgaggtcg 600 aaccctgttg 660 caggtgttta 720 gagattgtta 780 gactgccata 840 attgatggga 900 ataggtcgtg 960 actaagctca 1020 ctgatactta 1080 gatggttcct 1140 gcattgtttt aagaatttgg 1200 1260 WO 99/40206 WO 9940206PCT/EP99/00623 ttgctgctgt tggacatgaa.

ccatcaataa ctgagacggg ttaccctagg gcattttaaa.

tatgttaccc atacgccatg tagcttgcat aaaagctttc ggaaacaatc tattggaaaa.

agagctgtgt aatcacaagt ttctttctaa.

ttaagaggct Ctgacttgat gttttcttct tgattctttg gtgacatttt caacaaccgg gaagatcaat gttcaacatc agggtatctc aaatctttgg tttgatagaa tgctctggag gtcatatcat taagatgaag ggaggatcat cagactttat tccagagatg ctgtgcaatt ggtctaatgg tccactactc gttgtgaata tagatttatg tgctctgttt tttctttggt cttaaaaaaa cttagtgcac gaaatatacc tatccggatc attggcaatc tctattgtct gataaatggg tatgaggaat aatgggggat aaaccagagc tggcctgaat cagacatgga gcatcaaagt ggcaaatctg aggcccagtt tttagataga, gccacatctg agtctgaaga atggttagaa gagcaaacac aaaaaaaaaa.

tgtccttcca gatacaaaac aaataccctc tgcagccagc catcactggg acgatcttgt ggcgagtaat cctggccaac ttgcaagaaa attatgatac caattgctgg tgttttggga gtagaaagaa taaggataag gcttcacagt gatttaaaac caatagcaga atccgcattt tttagaacct aaaaggaatt cat tagggag tgaagaatac ttggctggta tcatatggac tacacctaaa ggcacatatg aacaggcagt acttctctgg ggcggtggcg acggcgtgga cttcttaaca ggaagactat gtgctctcgg tatcaataac tttagactga ttctaagaat gcagtgttga tctttctaac ggtttgagga ggaattc tattattggg 1320 tcaactgatg 1380 gactggatgc 1440 tttagattct 1500 caaaatgaga 1560 cctttaaaaa 1620 gaccccaaga. 1680 cagtttacat 1740 ttggccgaga. 1800 agatttattg 1860 tctaagttgt 1920 gaactgctcg 1980 tttgctgcca 2040 agatgaggcg 2100 cagtgattga 2160 aagtatctag 2220 acttattata. 2280 cataacagca 2340 atgaagcagg 2400 2447 <210> 4 <211> 6 <212> PRT <213> Unknown <220> <223> Description of Unkniown organism: protein fragment <400> 4 Val Gly Thr Val Ala Ala 1 <210> WO 99/40206 -9- <211> <212> PRT <213> Unknown <220> <223> Description of Unknown Organism: protein fragment <400> Ala Ile Gly Arg Val 1 <210> 6 <211> 22 <212> PRT <213> Unknown <220> <223> Description of Unknown Organism: protein fragment <400> 6 Asp Phe Gly Glu Ser Ala Ile Gly Arg Val Ala Pro Val Asp Ser Gly 1 5 10 Leu Trp Trp Ile Ile Leu <210> 7 <211> 9 <212> PRT <213> Unknown <220> <223> Description of Unknown Organism: protein fragment <400> 7 Cys Met Ile Asp Arg Arg Met Gly Ile 1 <210> 8 <211> 28 <212> PRT <213> Unknown <220> <223> Description of Unknown Organism: protein fragment <400> 8 Pro Thr Leu Leu Val Thr Asp Gly Ser Cys Met Ile Asp Arg Arg Met 1 5 10 Gly Ile His Gly His Pro Leu Glu Ile Gin Ala Leu PCT/EP99/00623 WO 99/40206 PCT/EP99/00623 <210> 9 <211> 7 <212> PRT <213> Unknown <220> <223> Description of Unknown Organism: protein fragment <400> 9 Gly Gly Tyr Leu Ile Gly Asn 1 <210> <211> 9 <212> PRT <213> Unknown <220> <223> Description of Unknown Organism: protein fragment <400> Asp Phe Arg Phe Phe Thr Leu Gly Asn 1 <210> 11 <211> 13 <212> PRT <213> Unknown <220> <223> Description of Unknown Organism: protein fragment <400> 11 Xaa Asn Ile Tyr Pro Asp Gin Ile Pro Pro Trp Leu Val 1 5 <210> 12 <211> <212> DNA <213> Unknown <220> <223> Description of Unknown Organism: oligonucleotide <400> 12 tctaaggatc tagaaagagc catta <210> 13 <211> 24 WO 99/40206 PCT[EP99/00623 <212> EM@ <213> Unknown <220> <223> Description of Unknown Organism: oligonucleotide <400> 13 ttcaattgaa ttcaatatag cttc 24 <210> 14 <211> 31 <212> DNsA <213> Unknown <220> <223> Description of Unknown Organism: oligonucleotide <400> 14 cgatttagca aggtaccata gatatgaata. c 31 <210> <211> <212> M1A <213> Unknown <220> <223> Description of Unknown Organism: oligonucleotide <400> cttatcctta. aactagatct ccattagacc

Claims (11)

1. An isolated DNA molecule comprising a sequence of nucleotides which can be translated into a protein with neutral invertase activity but lacking P- fructofuranosidase activity, wherein highest activity is observed in the range of pH to

2. The DNA according to claim 1 coding for a plant protein.

3. The DNA according to claim 1 comprising a sequence of nucleotides encoding an amino acid sequence selected from the group of amino acid sequences described 10 in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO:

4. The DNA according to claim 1 comprising a sequence of nucleotides coding for a protein as described in SEQ ID NO: 2. The DNA according to claim 1 comprising a sequence of nucleotides as described 15 in SEQ ID NO: 1.

6. An isolated protein having neutral invertase activity but lacking P- fructofuranosidase activity, wherein highest invertase activity is observed between S: pH 6.0 and 7.5 and wherein said protein has more than 50% sequence identity with SEQ ID NO: 2. 20 7. An isolated protein having neutral invertase activity but lacking P- fructofuranosidase activity wherein highest invertase activity is observed between pH 6.0 and 7.5 and wherein said protein comprises an amino acid sequence selected from the group of amino acid sequences described in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO:

8. A plant protein according to claim 6 having the amino acid sequence described in SEQ ID NO: 2.

9. A method of producing DNA according to claim 1, comprising screening a DNA library for clones which are capable of hybridizing to a fragment of the DNA defined by SEQ ID NO: 3, wherein said fragment has a length of at least 15 nucleotides; A sequencing hybridizing clones; purifying vector DNA of clones comprising an open reading frame encoding a protein with more than 40% sequence identity to SEQ ID NO: 2 P:\OPER\Kbml27217-99 spc.doc-24/09/01 -17- optionally further processing the purified DNA. A polymerase chain reaction wherein at least one oligonucleotide used comprises a sequence of nucleotides which represents 15 or more basepairs of SEQ ID NO: 1 or SEQ ID NO: 3.

11. An isolated DNA according to claim 1, substantially as hereinbefore described with reference to the Examples.

12. An isolated protein having neutral invertase activity but lacking P- fructofuranosidase activity according to claim 6 or claim 7, substantially as hereinbefore described with reference to the Examples. 10 13. A method of producing DNA according to claim 9, substantially as hereinbefore described with reference to the Examples.

14. An isolated DNA produced by the method of claim 9 or claim 13.

15. A polymerise chain reaction according to claim 10, substantially as hereinbefore described with reference to the Examples. DATED this 2 1 st day of September, 2001 NOVARTIS AG its Patent Attorneys DAVIES COLLISON CAVE O C< i pRC-