CA2319316A1 - Plant alkaline and neutral invertases - Google Patents

Abstract

The present invention provides DNA comprising a sequence of nucleotides which can be translated into a protein with invertase activity, wherein highest activity is observed in the range of pH 6.0 to 7.5, as well as the encoded protein produced recombinantly.

Description

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 (a-D-glucopyranosyl ~-D-fructofuranoside) is the first non-phosphorylated 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 7.5 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 supernatant. 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 the two activities. A sucrose-cleaving activity with a neutral pH optimum (neutral invertase, H,) was identified in the non-bound protein fraction. An activity with a more basic pH optimum (alkaline invertase, H2) 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 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 8.0, respectively. In addition neutral invertase was shown to cleave raffinose and stacchyose suggesting a ~-fructofuronidase activity of the enzyme.
It is the main object of the present invention to provide DNA comprising a nucleotide sequence which can be translated into a protein with invertase activity, wherein highest activity is observed in the range of pH 6.0 to 8.5, preferably 6.0 to 7.5.
Although neutral and alkaline 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 2.0 (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 N0:3. The amino acid sequence of the encoded invertase is given in SEQ ID NO: 2. Related proteins showing more than 40%

sequence 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 (47%) 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 Lllium 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.

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:
(a) VGTVAA (SEQ ID NO: 4) (b) AIGRV
(SEQ ID NO: 5) (c) DFGESAIGRVAPVDSGLWWIIL (SEQ ID NO: 6) (d) CMIDRRMGI (SEQ ID NO: 7) (e) PTLLVTDGSCMIDRRMGIHGHPLEIQAL (SEQ ID NO: 8) (f) GGYLIGN (SEQ ID NO: 9) (g) DFRFFTLGN (SEQ ID NO: 10) 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 15, 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 the amino acid sequence encoded by est t88552 from Arabidopsis (1026 by 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 EcoRl ligated into the respective sites of the E. coli plasmid pBluescript II KS (Stratagene). After amplification and purification of the plasmid, the fragment is excised, purified by agarose gel electrophoresis and electroelution, and randomly labeled with [a-3zP)ATP. The labeled DNA is used as a probe to screen a library made from rapidly growing suspension cultures of wild carrot (Daucus carofa cv Queen Anne's Lace, W001C). Clones obtained are sequenced and for example might reveal a clone comprising a 2447 nucleotide insert containing 29 by of 5' and 393 by of 3' non-coding sequences whereas the ORF codes for a protein with 675 amino acids sharing 80%
identity (86% similarity) with the deduced amino acid sequence of the Arabidopsis est 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 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).
A further object of the present invention is to provide 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 pTrc 99 A (Pharmacia Biotech).
After _7_ transformation 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 100p1 of soluble extract are mixed with 700p.1 of water, 100p.1 of 0.5 M potassium phosphate, pH 6.8, and 100p1 of 0.5 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 (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 Cu2+ 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 ~i-fructofuranosidase activity.
EXAMPLES:
Example 1: Purificatfon of Carrot Neutral and Alkaline Invertase Preparation of Extracfs 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, lOmM
lysine, 0.5mM MgCl2, 0.5% 2-mercaptoethanol and 100mM phenylmethylsulfonyl fluoride).
The homogenate is centrifuged for 20 min at 60008 in a Sorvall GSA-rotor. The supernatant is collected and kept cold. The 60008 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 16,3008 for 30 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 4°C.

_g_ 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 30 min at 16,3008. The precipitate is dissolved in 100m1 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 240m1 of 190-550 mM NaCI in 25mM Hepes-KOH, pH 7.5, containing 0.5% 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 16,3008. The precipitate is dissolved in 5ml of buffer C (5mM K-phosphate 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 200m1 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 16,3008. The two protein pellets are individually dissolved in 5ml of buffer D (25mM K-phosphate buffer, pH 7.5, containing 0.1 % 2-mercaptoethanol) and dialyzed against buffer D overnight.
Affinity Chromatography on Green 19 dye The dialyzed protein solutions (5mi each) are divided into 0.5-ml aliquots, and then applied to 10 prepacked green 19 dye columns (4.5 x 0.7 cm, Sigma, Buchs, Switzerland) _ 9._ equilibrated with buffer D. The columns are washed with l5ml of buffer D, then step eluted with NaCI at 0.35 M and 1.5 M (l5ml and 25m1, 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 16,3008.
Gel-Filtration Chromatography l 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 (V°), thyroglobulin (669 kD), apoferritin (443 kD), f3-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 (25mM
Hepes-KOH, pH 7.2, containing 275mM NaCI and 0.1 % 2-mercaptoethanol).
Anion-Exchange Chromatography ll on Macro-Prep For further purification of alkaline invertase, the dialysate is applied to a Macro-Prep column (1.5cm x 20cm, Bio-Rad Laboratories, Richmond, CA, USA) equilibrated with buffer F. The column is washed with buffer F and eluted with 200m1 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 (l.2cm x 25cm, Bio-Rad Laboratories) equilibrated with buffer G. The column is washed with buffer G and eluted with 200m1 of a linear gradient of 275-360 mM NaCI in 25mM
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 16,3008.

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 (25mM
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 (l0cm x l.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-ml fractions are collected. Fractions containing enzyme activity are pooled, dialyzed against lOmM
Hepes-KOH, pH 8.0, containing 0.1 % 2-mercaptoethanol and stored in 50%
glycerol at -20°C.
Gel-Filtration Chromatography II 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 1 OmM K-phosphate buffer, pH 7.0, containing 0.1 % 2-mercaptoethanol and stored in 50% glycerol at -20°C.
Example 2: Isolation of a cDNA Clone Encoding Carrot Neutral lnvertase 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 by 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: i 3). Amplification is achieved in a DNA Thermal Cycler (Perkin Elmer Cetus) with the following conditions: 10 cycles of denaturation at 95°C for 1 min, annealing at 40°C for 0.5 min, and elongation at 72°C for 1.5 min, followed by 20 cycles of denaturation at 95°C
for 1 min, annealing at 60°
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 EcoRl ligated into the respective sites of the E. coli plasmid pBluescript II KS (Stratagene). After amplification and purification of the plasmid, the fragment is excised, purified by agarose gel electrophoresis and electroelution, and randomly labeled with [a-32P]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, W001 C) 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 chain-termination method. Computer-assisted analysis of DNA and protein sequences as described in Examples 2 and 3 is performed using the Wisconsin Package Version 9.0, 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 (J. 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 by of 5' and 393 by 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 (car) with the sequences of the LIM17 proteins from L. longiflorum (lil) and Synechocystis (bac) (Table 1) identifies three conserved sequence domains (boxes 1-3). When the sequence of box 2 is used for a database search, a protein of known function is identified, namely ceHobiose phosphorylase from Clostridium stercorarium, which cleaves cellobiose [[i-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 cDNA-derived amino acid sequences of neutral invertase from carrot (car) with the amino acid sequences of the LIM17 proteins form Lilium longiflorum (lil) and Synechocystis (bac). The amino acid sequences are in one-letter-code and have been aligned by introducing gaps (..) to maximize identity. The amino acid residues in bold face indicate conserved domains (boxes 1-3). The asterisks below the sequence mark amino acid residues identical in all three sequences.
car ~I~CIAVSN N~tPCGRN~LS CIQ~TSSIFGYS FRKCDHRN~"I' NLSKKQFKVY GLRGYVSCRG
lil PPQISARSAV DQVR~FERP LSFWVMPSW NmQGHI~I~TP DSGQDKPDEF DFSHI.~LHIKP 120 car GKGI~GY'RCGI DI~FCGFFGS GS17VVGQPRVL . . . . . . . . . . TSGC~RVDSG
GRS~7LVNVAS
lil RVLNIDRQTS ..CDERSLLE HSTGIGIIYP PLVFKNPESN SSRLLLFTPEI VSTPQ~tSAV 180 car DYR~STSVE GHVNDKSFER IYVRGGLd~IVIC PLVIERVE.K GEfCVRF~~7GR VGUNGSNVnTI
lil 1~TI'PKAFN. . . ~HPM Nm. EC~nIDAI~C RSLYYFRGQP 'VC~IAALI~1S EEA.INY~V 240 car GDSKGL~GK VLSPKREVSE VEf~ GAVVDY~GiI~ VC~VAASDFA DSTPT,i~YDQV
bac --_~_~~_~_~ ~pQp,QS2I LYE KAMV~C'~Y VQPVAAIPQS ~LrIY'l~V
* * *** ** ***
lil FVRZ~t~P9GC, AI~Ti~E~EI Vt~3~PLR IRSWECQCIDR FKLGEGANiPA SFKV~PV. 300 car FIRD~'VPSAL AFLLNGaGEI ViI~Q LQSWFdCTVDC HSPGQGLMPA SFfCVKIV~IAID
bac FZ~NVP'Vl~ ~T'I V~LEICIIP LQS . . . . . . K GFP'T'YGIFPT SF . . . . . . .
.
* ** * * * ** * *** * ***
lil . . . .RNQETL I~1DFC~P'SAICi RVApVDSGFy~ WIILLRAM3t STGDT~AEri pDC:Q,I 360 car GKIGESEDIL DP'D ~ ~ (3 RVAPVD6GLH1 WIILI~RA'YTtt Il~7tCRaQAR VDV~TGIRI,I
bac . . VETEL~ IWDYC~QRAIC3 RVCSVLi~ISI~1 TAPILAYYYV~ R ~' THVQI~,QI~' * * * *** ** ** ** * ** **
lil IJt~S~7GFD ~ pCi~DI y~GypIEI~i, pZ~, 1~KQ.. ... 420 car Ii~DGP'D 1~~'P~L'I~LV'1'DG sC~R~I I~C~I~I~AL FYSALRCSRE I~d.. IV . . . . .
bac Ii~.~'VFR L1APTLivPDG AF'~tpT!~7V WGAPLEIQ"I't~ LYGAIxSAAG LLLIDLKAKG
* * * * *** * **** * * * *** * **

1i1 .........D DDGRELAERI A...QRLQAL SFHLRSYF~ D~'RRIi~IYR Ft~QYSDTA 480 car . . . . . . . . N DSTKNLVAAV N . . . NRLSAL SF33IREYYWV ~C:~IYR
YK'J.~:GYSTLIi~r bac YCSNKDHPFD SFTMEQSHQF NLSVDWLtiKL RZ~~iYWI NCNI'IQALRR RPTEIQYGF~~:A
* * * * **
lil ILLS LPDWVrD~FMP TR~OGYP'IC~W SPAli~ ~ ~ Ci~IAIIS SEA 540 car II~~IYPDQ IPSWLVDG~ E~GYI~IC~ QPAI~~RFr TLt~L~ISIVS SI~FC~Q~S
bac S~NVkI~,T IP11~I~QDHIi.G D~OGYLIC~1I RTatPD~RFF SI~~AII~' DVTSLAQQRS
* * * * * ** *** *** * ***
lil IL~~.LEG'E~1P ELVC~*_~ ~_~~_~_~~~ ~~~__~~~~_ ~~~_~~~~_~ ~_~~__~~_~ 600 car III~IE~1D D~VAI~LKI CYPALEYE~V RVITGSDPKN TPWSYHIQGGS WPTL~LWQFrL
bac FFRLVIi~~IQR ELCAQ~LRI CHPPLKDDDW RSK'IGFDRKN LPVJCYHt~AGH WPCLFWFLW
* * **
lil ~~~~~~~~_~ ~__~~~__~_ _~~~_~~_~_ ~_~~_~_~__ ~~~~~_~_~~ _~~__~~~__ 660 car ACIK...... ......MKKP FLARICAVALA EIQ~LSEDHNTP EYYDTRRGRF IGKQSRLYQT
bac AVLRHSCHSN YGTVEYA~IG NLIRI~YEVL LRRLPKHKWA EYFDGPTGFW VC-~QQSRSYQT
lil __~~__~~~_ ~~_~_~~_~_ ~~______~_ ~~_~~____~ ___~_____~ _~~~_~ 716 car VJI'IAGFLTSK T,r,T.~~ KLFWEmYEL LESCVCAIGK SGRKKCSRFA AKSQW
bac LvlI'IVGLLLVH HITEVNPDDA LM~--~--~-~- -~~--~-----~_ -----~~--~---~ ------~~
Example 4: Steady State Levels of Neutral lnvertase 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 4-, 10-, and 16-week-old leaves, 4-, 10-, and 16-week-old roots, and flower buds (B), flowers (F), small developing seeds (GS), large developing seeds (G,), and mature seeds (S). The blot is hybridized with the 32P-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.

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 5'-CGATTTAGCAAGGTACC
ATAGATATGAATAC-3' (SEQ ID NO: 14) and 5'-CTTATCCTTAAACTAGATCTCCATT
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 95°C 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 pTrc 99 A (Pharmacia Biotech).
Protein biosynthesis in transformed bacteria carrying the expression vector is induced with 1 mM
IPTG for approximately 16 hours, and bacteria are lysed in a small volume of 50mM
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, 100p,1 of soluble extract are mixed with 700w1 of water, 100p,1 of 0.5 M potassium phosphate, pH 6.8, and 100p1 of 0.5 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 lnvertase Acfivity Invertase activity is determined in reaction mixtures containing 50mM K-phosphate buffer (pH 6.8 or 8.0), 100mM sucrose and an appropriate volume of enzyme in a final volume of i 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 {p.mol) 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.

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 95% of the activity are lost.
Activity measurement of the recombinant enzyme expressed in E. coli detects about 0.4 units of neutral invertase activity in 100p.1 of the E. coli extract described in example 5.

_y_ S~QUE~E LISTING
<110> Novartis AG
<120> Plant Al3caline and Neutral Invertases <130> S-30364/A
<140>
<141>
<150> GB 9802249.4 <151> 1998-02-03 <160> 15 <170> Patentln Ver. 2.0 <210> 1 <211> 2025 <212> IJNA
<213> Daucus carota <220>
<221> CDS
<222> (1) . . (2025) <400> 1 atg aat act act tgt att get gta tcg aat atg agg cct tgt tgt aga 48 Met Asn Thr Thr Cars Ile Ala Val Ser Asn Met Arg Pro Cars Cys Arg atg tta ctt agc tgt aag aat tca tcg att ttc gga tac tcg ttt cga 96 Met Leu Leu Ser Cys Lys Asn Ser Ser Ile Phe Gly Tyr Ser Phe Arg aaa tgt gat cat aga atg ggg act aat ttg tcg aaa aag caa ttt aag 144 Lys Cys Asp His Arg Met Gly Thr Asn Leu Ser Lys Lys Gln Phe Lys gtg tac ggt ttg cga ggg tat gtt agt tgt agg ggt ggt aaa ggt tta 192 Val Tyr Gly Leu Arg Gly Tyr Val Ser Cps Arg Gly Gly Lys Gly Leu ggt tat agg tgt ggg att gat ccg aat cgg aag ggt ttt ttt ggt tcc 240 Gly Tyr Arg Cps Gly Ile Asp Pro Asn Arg Lys Gly Phe Phe Gly Ser ggt tct gat tgg gga cag cct agg gtt tta aca agt ggt tgt cgt cgt 288 Gly Ser Asp Trp Gly Gln Pro Arg Val Leu Thr Ser Gly Cps Arg Axg gtt gat agt ggt ggt agg agt gta ctt gtt aat gtg gcg tcg gat tat 336 Val Asp Ser Gly Gly Arg Ser Val Leu Val Asn Val Ala Ser Asp Tyr agg aat cat tca act tcg gtt gaa ggt cat gtt aat gat aag agt ttc 384 Arg Asn His Ser Thr Ser Val Glu Gly His Val Asn Asp Lys Ser Phe gag agg att tat gtt cgt gga ggg ttg aat gtg aag ccg ttg gtg att 432 Glu Arg Ile Tyr Val Arg Gly Gly Leu Asn Val Lys Pro Leu Val Ile gaa agg gtg gag aaa ggg gag aaa gta agg gaa gag gag ggt agg gta 480 Glu Arg Val Glu Lys Gly Glu Lys Val Arg Glu Glu Glu Gly Arg Val gga gtt aat ggt tcg aat gta aat att ggt gat tcg aaa ggt tta aat 528 Gly Val Asn Gly Ser Asn Val Asn Ile Gly Asp Ser Lys Gly Leu Asn ggg ggt aag gtt ttg tct ccg aag aga gag gtg tct gag gtc gaa aaa 576 Gly Gly Lys Val Leu Ser Pro Lys Arg Glu Val Ser Glu Val Glu Lys gag get tgg gag tta ctt cga ggt get gtt gtt gat tat tgt gga aac 624 Glu Ala Trp Glu Leu Leu Arg Gly Ala Val Val Asp Tyr CSrs Gly Asn cct gtt ggg act gtt gca get agt gat cca get gat tct aca cca ctc 672 Pro Val Gly Thr Val Ala Ala Ser Asp Pro Ala Asp Ser Thr Pro Leu aac tat gac cag gtg ttt att cgt gat ttt gtc ccc tct get ctt gca 720 Asn Tyr Asp Gln Val Phe Ile Arg Asp Phe Val Pro Ser Ala Leu Ala ttc ttg ctt aat gga gaa ggg gag att gtt aag aat ttt ctg cta cat 768 Phe Leu Leu Asn Gly Glu Gly Glu Ile Val Lys Asn Phe Leu Leu His aca ctg cag tta cag agt tgg gaa aaa act gta gac tgc cat agc cct 816 Thr Leu Gln Leu Gln Ser Trp Glu Lys Thr Val Asp Cps His Ser Pro ggg caa ggg ttg atg ccc gca agt ttc aaa gtt aaa aac gtg get att 864 Gly Gln Gly Leu Met Pro Ala Ser Phe Lys Val Lys Asn Val Ala Ile gat ggg aaa att gga gaa tca gag gat att tta gat cca gat ttc ggt 912 Asp Gly Lys Ile Gly Glu Ser Glu Asp Ile Leu Asp Pro Asp Phe Gly gaa tca gcc ata ggt cgt gtt gca cct gtt gat tct ggg tta tgg tgg 960 Glu Ser Ala Ile Gly Arg Val Ala Pro Val Asp Ser Gly Leu Trp Trp atc att ttg tta aga get tat act aag ctc aca gga gat tat ggg ctg 1008 Ile Ile Leu Leu Arg Ala Tyr Thr Lys Leu Thr Gly Asp Tyr Gly Leu caagca cga gtg gat gtg cag ata agg ctg ata aatctg 1056 aca gga ctt GlnAla Arg Val Asp Val Gln Ile Arg Leu Ile AsnLeu Thr Gly Leu tgttta acg gat gga ttc gac cct aca ctg tta actgat 1104 atg ttt gtc CpsLeu Thr Asp Gly Phe Asp Pro Thr Leu Leu ThrAsp Met Phe Val ggttcc tgt atg att gac aga ggc att cat ggt cctctc 1152 agg atg cac GlySer Cys Met Ile Asp Arg Gly Ile His Gly ProLeu Arg Met His gaaatt caa gca ttg ttt tat ttg cgt tgt tct gagatg 1200 tca get cga GluIle Gin Ala Leu Phe Tyr Leu Arg C"ys GluMet Ser Ala Ser Arg ctcatt gtc aat gat tcc aca ttg gtt get get aacaac 1248 aag aat gtc LeuIle Val Asn Asp Ser Thr Leu Val Ala Ala AsnAsn Lys Asn Val 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 Glu Tyr Tyr Trp Val Asp atg aag aag atc aat gaa ata tac cga tac aaa act gaa gaa tac tca 1344 Met Lys Lys Ile Asn Glu Ile Tyr Arg Tyr Lys Thr Glu Glu Tyr Ser act gat gcc atc aat aag ttc aac atc tat ccg gat caa ata ccc tct 1392 Thr Asp Ala Ile Asn Lys Phe Asn Ile Tyr Pro Asp Gln Ile Pro Ser tgg ctg gta gac tgg atg cct gag acg gga ggg tat ctc att ggc aat 1440 Trp Leu Val Asp Trp Met Pro Glu Thr Gly Gly Tyr Leu Ile Gly Asn ctg cag cca get cat atg gac ttt aga ttc ttt acc cta gga aat ctt 1488 Leu Gln Pro Ala His Met Asp Phe Arg Phe Phe Thr Leu Gly Asn Leu tgg tct att gtc tca tca ctg ggt aca cct aaa caa aat gag agc att 1536 Trp Ser Ile Val Ser Ser Leu Gly Thr Pro Lys Gln Asn Glu Ser Ile tta aat ttg ata gaa gat aaa tgg gac gat ctt gtg gca cat atg cct 1584 Leu Asn Leu Ile Glu Asp Lys Trp Asp Asp Leu Val Ala His Met Pro tta aaa ata tgt tac cct get ctg gag tat gag gaa tgg cga gta ata 1632 Leu Lys Ile Cars 'I~r Pro Ala Leu Glu Tyr Giu Glu Trp Arg Val Ile aca ggc agt gac ccc aag aat acg cca tgg tca tat cat aat ggg gga 1680 Thr Gly Ser Asp Pro Lys Asn Thr Pro Trp Ser Tyr His Asn Gly Gly tcc tgg cca aca ctt ctc tgg cag ttt aca tta get tgc 1728 att aag atg Ser Trp Pro Thr Leu Leu Trp Gln Phe Thr Leu Ala Cps Ile Lys Met aag aaa cca gag ctt gca aga aag gcg gtg gcg ttg gcc 1776 gag aaa aag Lys Lys Pro Glu Leu Ala Arg Lys Ala Val Ala Leu Ala Glu Lys Lys ctt tcg gag gat cat tgg cct gaa tat tat gat aca cgg 1824 cgt gga aga Leu Ser Glu Asp His Trp Pro Glu Tyr Tyr Asp Thr Arg Arg Gly Arg ttt att ggg aaa caa tcc aga ctt tat cag aca tgg aca 1872 att get ggc Phe Ile Gly Lys Gln Ser Arg Leu Tyr Gln Thr Trp Thr Ile Ala Gly ttc tta aca tct aag ttg tta ttg gaa aat cca gag atg 1920 gca tca aag Phe Leu Thr Ser Lys Leu Leu Leu Glu Asn Pro Glu Met Ala Ser Lys ttg ttt tgg gag gaa gac tat gaa ctg ctc gag agc tgt 1968 gtc tgt gca Leu Phe Trp Glu Glu Asp Tyr Glu Leu Leu Glu Ser Cys Val Cys Ala att ggc aaa tct ggt aga aag aag tgc tct cgg ttt get 2016 gcc aaa tca Ile Gly Lys Ser Gly Arg Lys Lys Cars Ser Arg Phe Ala Ala Lys Ser caa gtg gtc Gln Val Val <210> 2 <211> 675 <212> PRT
<213> Daucus carota <400> 2 Met Asn Thr Thr Cars Ile Ala Val Ser Asn Met Arg Pro Cars Cys Arg Met Leu Leu Ser Cars Lys Asn Ser Ser Ile Phe Gly Tyr Ser Phe Arg Lys Cys Asp His Arg Met Gly Thr Asn Leu Ser Lys Lys Gln Phe Lys Val Tyr Gly Leu Arg Gly Tyr Val Ser Cars Arg Gly Gly Lys Gly Leu Gly Tyr Arg Cars Gly Ile Asp Pro Asn Arg Lys Gly Phe Phe Gly Ser Gly Ser Asp Trp Gly Gln Pro Arg Val Lzu Thr Ser Gly Cars Arg Arg Val Asp Ser Gly Gly Arg Ser Val Leu Val Asn Val Ala Ser Asp Tyr Arg Asn His Ser Thr Ser Val Glu Gly His Val Asn Asp Lys Ser Phe Glu Arg Ile Tyr Val Arg Gly Gly Leu Asn Val Lys Pro Leu Val Ile Glu Arg Val Glu Lys Gly Glu Lys Val Arg Glu Glu Glu Gly Arg Val Gly Val Asn Gly Ser Asn Val Asn Ile Gly Asp Ser Lys Gly Leu Asn Gly Gly Lys Val Leu Ser Pro Lys Arg Glu Val Ser Glu Val Glu Lys Glu Ala Trp Glu Leu Leu Arg Gly Ala Val Val Asp Tyr C'ys Gly Asn Pro Val Gly Thr Val Ala Ala Ser Asp Pro Ala Asp Ser Thr Pro Leu Asn Tyr Asp Gln Val Phe Ile Arg Asp Phe Val Pro Ser Ala Leu Ala Phe Leu Leu Asn Gly Glu Gly Glu Ile Val Lys Asn Phe Leu Leu His Thr Leu Gln Leu Gln Ser 2'zp Glu Lys Thr Val Asp Cars His Ser Pro Gly Gln Gly Leu Met Pro Ala Ser Phe Lys Val Lys Asn Val Ala Ile Asp Gly Lys Ile Gly Glu Ser Glu Asp Ile Leu Asp Pro Asp Phe Gly Glu Ser Ala Ile Gly Arg Val Ala Pro Val Asp Ser Gly Leu Trp Trp Ile Ile Leu Leu Arg Ala Tyr Thr Lys Leu Z'hr Gly Asp Tyr Gly Leu Gln Ala Arg Val Asp Val Gln Thr Gly Ile Arg Leu Ile Leu Asn Leu Cys Leu Thr Asp Gly Phe Asp Met Phe Pro Thr Leu Leu Val Thr Asp _g_ Gly Ser Cys Met Ile Asp Arg Arg Met Gly Ile His Gly His Pro Leu Glu Ile Gln Ala Leu Phe Tyr Ser Ala Leu Arg Cps Ser Arg Glu Met Leu Ile Val Asn Asp Ser Thr Lys Asn Leu Val Ala Ala Val Asn Asn Arg Leu Ser Ala Leu Ser Phe His Ile Arg Glu Tyr Tyr Trp Val Asp Met Lys Lys Ile Asn Glu Ile Tyr Arg 'I'yr Lys Thr Glu Glu Tyr Ser Thr Asp Ala Ile Asn Lys Phe Asn Ile Tyr Pro Asp Gln Ile Pro Ser Trp Leu Val Asp Trp Met Pro Glu Thr Gly Gly Tyr Leu Ile Gly Asn Leu Gln Pro Ala His Met Asp Phe Arg Phe Phe Thr Leu Gly Asn Leu Trp Ser Ile Val Ser Ser Leu Gly Thr Pro Lys Gln Asn Glu Ser Ile Leu Asn Leu Ile Glu Asp Lys Txp Asp Asp Leu Val Ala His Met Pro Leu Lys Ile Cars Tyr Pro Ala Leu Glu Tyr Glu Glu Trp Arg Val Ile Thr Gly Ser Asp Pro Lys Asn Thr Pro Trp Ser Tyr His Asn Gly Gly Ser Trp Pro Thr Leu Leu 2'rp Gln Phe Thr Leu Ala Cars Ile Lys Met Lys Lys Pro Glu Leu Ala Arg Lys Ala Val Ala Leu Ala Glu Lys Lys Leu Ser Glu Asp His Trp Pro Glu Tyr Tyr Asp Thr Arg Arg Gly Arg Phe Ile Gly Lys Gln Ser Arg Leu Tyr Gln Thr Trp Thr Ile Ala Gly Phe Leu Thr Ser Lys Leu Leu Leu Glu Asn Pro Glu Met Ala Ser Lys Leu Phe Trp Glu Glu Asp Tyr Glu Leu Leu Glu Ser Cars Val Cys Ala Ile Gly Lys Ser Gly Arg Lys Lys Cps Ser Arg Phe Ala Ala Lys Ser Gln Val Val <210> 3 <211> 2447 <212> L~TP, <213> Daucus carota <400> 3 gaattccgat ttagcaaatt gttatagata tgaatactac ttgtattgct gtatcgaata 60 tgaggccttg ttgtagaatg ttacttagct gtaagaattc atcgattttc ggatactcgt 120 ttcgaaaatg tgatcataga atggggacta atttgtcgaa aaagcaattt aaggtgtacg 180 gtttgcgagg gtatgttagt tgtaggggtg gtaaaggttt aggttatagg tgtgggattg 240 atccgaatcg gaagggtttt tttggttccg gttctgattg gggacagcct agggttttaa 300 caagtggttg tcgtcgtgtt gatagtggtg gtaggagtgt acttgttaat gtggcgtcgg 360 attataggaa tcattcaact tcggttgaag gtcatgttaa tgataagagt ttcgagagga 420 tttatgttcg tggagggttg aatgtgaagc cgttggtgat tgaaagggtg gagaaagggg 480 agaaagtaag ggaagaggag ggtagggtag gagttaatgg ttcgaatgta aatattggtg 540 attcgaaagg tttaaatggg ggtaaggttt tgtctccgaa gagagaggtg tctgaggtcg 600 aaaaagaggc ttgggagtta cttcgaggtg ctgttgttga ttattgtgga aaccctgttg 660 ggactgttgc agctagtgat ccagctgatt ctacaccact caactatgac caggtgttta 720 ttcgtgattt tgtcccctct gctcttgcat tcttgcttaa tggagaaggg gagattgtta 780 agaattttct gctacataca ctgcagttac agagttggga aaaaactgta gactgccata 840 gccctgggca agggttgatg cccgcaagtt tcaaagttaa aaacgtggct attgatggga 900 aaattggaga atcagaggat attttagatc cagatttcgg tgaatcagcc ataggtcgtg 960 ttgcacctgt tgattctggg ttatggtgga tcattttgtt aagagcttat actaagctca 1020 caggagatta tgggctgcaa gcacgagtgg atgtgcagac aggaataagg ctgatactta 1080 atctgtgttt aacggatgga ttcgacatgt ttcctacact gttagtcact gatggttcct 1140 gtatgattga cagaaggatg ggcattcatg gtcaccctct cgaaattcaa gcattgtttt 1200 attcagcttt gcgttgttct cgagagatgc tcattgtcaa tgattccaca aagaatttgg 1260 _g_ ttgctgctgt caacaaccgg cttagtgcac tgtccttcca cattagggag tattattggg 1320 tggacatgaa gaagatcaat gaaatatacc gatacaaaac tgaagaatac tcaactgatg 1380 ccatcaataa gttcaacatc tatccggatc aaataccctc ttggctggta gactggatgc 1440 ctgagacggg agggtatctc attggcaatc tgcagccagc tcatatggac tttagattct 1500 ttaccctagg aaatctttgg tctattgtct catcactggg tacacctaaa caaaatgaga 1560 gcattttaaa tttgatagaa gataaatggg acgatcttgt ggcacatatg cctttaaaaa 1620 tatgttaccc tgctctggag tatgaggaat ggcgagtaat aacaggcagt gaccccaaga 1680 atacgccatg gtcatatcat aatgggggat cctggccaac acttctctgg cagtttacat 1740 tagcttgcat taagatgaag aaaccagagc ttgcaagaaa ggcggtggcg ttggccgaga 1$00 aaaagctttc ggaggatcat tggcctgaat attatgatac acggcgtgga agatttattg 1860 ggaaacaatc cagactttat cagacatgga caattgctgg cttcttaaca tctaagttgt 1920 tattggaaaa tccagagatg gcatcaaagt tgttttggga ggaagactat gaactgctcg 1980 agagctgtgt ctgtgcaatt ggcaaatctg gtagaaagaa gtgctctcgg tttgctgcca 2040 aatcacaagt ggtctaatgg aggcccagtt taaggataag tatcaataac agatgaggcg 2100 ttctttctaa tccactactc tttagataga gcttcacagt tttagactga cagtgattga 2160 ttaagaggct gttgtgaata gccacatctg gatttaaaac ttctaagaat aagtatctag 2220 ctgacttgat tagatttatg agtctgaaga caatagcaga gcagtgttga acttattata 2280 gttttcttct tgctctgttt atggttagaa atccgcattt tctttctaac cataacagca 2340 tgattctttg tttctttggt gagcaaacac tttagaacct ggtttgagga atgaagcagg 2400 gtgacatttt cttaaaaaaa aaaaaaaaaa aaaaggaatt ggaattc 2447 <210> 4 <211> 6 <212> PRT
<213> Un)mown <220>
<223> Description of Ur~mown Organism: protein fragment <400> 4 Val Gly Thr Val Ala Ala <210> 5 <211> 5 <212> PRT
<213> Unlazown <220>
<223> Description of Un~own Organism: protein fragment <400> 5 Ala Ile Gly Arg Val <210> 6 <211> 22 <212> PRT
<213> Unlmown <220>
<223> Description of Unlmown Organism: protein fragment <400> 6 Asp Phe Gly Glu Ser Ala Ile Gly Arg Val Ala Pro Val Asp Ser Gly Leu Tzp Tzp Ile Ile Leu <210> 7 <211> 9 <212> PRT
<213> Unlmown <220>
<223> Description of Un~own Organism: protein fragment <400> 7 Cys Met Ile Asp Arg Arg Met Gly Ile <210> 8 <211> 28 <212> PRT
<213> Unlmown <220>
<223> Description of Unlmown Organism: protein fragment <400> 8 Pro Thr Leu Leu Val Thr Asp Gly Ser Cars Met Ile Asp Arg Arg Met Gly Ile His Gly His Pro Leu Glu Ile Gln Ala Leu <210> 9 <211> 7 <212> PRT
<213 > Unlmown <220>
<223> Description of Unlazown Organism: protein fragment <400> 9 Gly Gly Tyr Leu Ile Gly Asn <210> 10 <211> 9 <212> PRT
<213> Ux~lmown <220>
<223> Description of Unlmown Organism: protein fragment <400> 10 Asp Phe Arg Phe Phe Thr Leu Gly Asn <210> 11 <211> 13 <212> PRT
<213> Lh~mown <220>
<223> Description of Unlmown Organism: protein fragment <400> 11 Xaa Asn Ile Tyr Pro Asp Gln Ile Pro Pro Trp Leu Val <210> 12 <211> 25 <212> I~IA
<213> Unlazown <220>
<223> Description of Unamown Organism: oligonucleotide <400> 12 tctaaggatc tagaaagagc catta 25 <210> 13 <211> 24 _ii_ <212 > . INA
<213> Ur~3aZO~nm <220>
<223> Description of Unknown Organism: oligonucleotide <400> 13 ttcaattgaa ttcaatatag cttc 24 <210> 14 <211> 31 <212> L8~1P.
<213> Unknown <220>
<223> Description of Unknown Organism: oligonucleotide <400> 14 cgatttagca aggtaccata gatatgaata c 31 <210> 15 <211> 30 <212> I~
<213> Unknown <220>
<223> Description of Unknown Organism: oligonucleotide <400> 15 cttatcctta aactagatct ccattagacc 30

Claims (10)

What is claimed is:

1. DNA comprising a sequence of nucleotides which can be translated into a protein with invertase activity, wherein highest activity is observed in the range of pH
6.0 to 7.5.

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 form 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:10.

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

5. The DNA according to claim 1 comprising a sequence of nucleotides as described in SEQ ID NO:1.

6. A protein having invertase activity but lacking .beta.-fructofuranosidase activity, wherein highest invertase activity is observed between pH 6.0 and 7.5.

7. A plant protein according to claim 6 comprising 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:10.

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 nucleotides;

- 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 - optionally further processing the purified DNA.

10. 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.