JP2002505084A - Plant alkaline and neutral invertase - Google Patents

Abstract

  (57) [Summary] The present invention provides a DNA comprising a nucleotide sequence that can be translated into a protein having an invertase activity having the highest activity in the pH range of 6.0 to 7.5, and an encoded protein produced by a recombinant method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The present invention relates to a DNA encoding a protein that hydrolyzes sucrose. In particular, the present invention describes DNA that can be translated into a protein having neutral invertase activity.

[0002] In many plant species, sucrose (α-D-glucopyranosyl β-D-fructofuranoside) is the first non-phosphorylated product of photo-anabolism and is the energy source for organotrophic plant tissues. And a mobile source of carbon. Sucrose metabolism is an absolute requirement for the survival of organotrophic plant organs, and sucrose can only be used after being cleaved by invertase or sucrose synthase.

[0003] Invertase hydrolyzes sucrose into glucose and fructose, which is then supplied to various biochemical pathways. There are several isoforms of invertase that exhibit different biochemical properties and subcellular distribution. Acid invertase is characterized by an acidic pH optimum in the range of 4.0 to 5.5. They are either attached to the cell wall (cell wall invertase) or found in vacuoles as soluble proteins (vacuolar invertase). The amino acid sequence of the acid invertase shares a conserved motif. Analysis of sequence similarity suggests that they are evolutionarily related to yeast and bacterial invertases, but no counterpart has been found in animal cells. Technically, acid invertase is used in the confectionery industry to convert easily crystallizing sucrose into glucose-
It has been used to convert to fructose mixtures. Thereby, for example, a hard sucrose core coated with chocolate is converted into a soft central ate. For certain applications, invertases having a neutral or alkaline optimum pH are preferred.

[0004] Neutral and alkaline invertases are 6.0 to 7.5 and 7.5 to 8 respectively.
Characterized by an optimum pH in the range of .5. These are thought to be restricted to mature tissues and are generally assumed to accumulate in the cytoplasm, supported by the fact that N-linked glycans have not been detected. To date, the molecular structure and gene of neutral invertase has not been elucidated. However, this is an essential prerequisite for the biotechnological development of neutral and alkaline invertase. Recently, carrots (Dauc
The corresponding enzyme was purified from us carota cv Queen Anne's Lace (Lee and Stum
, Plant Physiol. 112: 1513-1522 (1996)).

[0005] Carrot suspension culture cells have soluble sucrose cleavage activity with different optimum pH above and below pH 6 (alkaline and acid invertase, respectively). The two activities were effectively separated by ammonium sulfate precipitation at 20-45% saturation. Neutral and alkaline invertase activity was detected in the protein pellet, whereas that of acid invertase remained in the supernatant. The 20-45% ammonium sulfate fraction was chromatographed on Q-Sepharose to obtain two poorly separated peaks of invertase activity. The active fractions were combined, chromatographed on HA-Ultrogel, and then affinity chromatographed on green 19 to effectively separate the two peaks.

[0006] Sucrose cleavage activity with a neutral optimum pH (neutral invertase, H ₁ ) is identified in the non-binding protein fraction. Activities with a more basic optimal pH (alkaline invertase, H ₂ ) can be bound to HA-Ultrogel and Green 19 dye columns and eluted with a salt-containing buffer. At this stage of the purification, neutral invertase represented approximately one third of the invertase activity and alkaline invertase represented two thirds. Fractions containing neutral and alkaline invertase were each pooled and individually further purified by gel filtration chromatography, second ion exchange chromatography, second gel filtration chromatography and hydrophobic interaction chromatography. Macro-Prep anion exchange chromatography on neutral invertase is the most effective procedure for removing contaminating proteins from preparations. Propyl agarose chromatography did not improve the specific activity of alkaline invertase, but was necessary to obtain electrophoretically pure enzyme. At the end of the purification, less than 10% of the two enzymes were recovered. This low yield is believed to be due to the various purification steps employed and the poor stability of the enzyme.

[0007] Neutral invertase was found to elute from the gel filtration column as a polypeptide of approximately 456 kD, while the purified enzyme migrated as a single band of approximately 57 kD in SDS polyacrylamide gel electrophoresis. did. Alkaline invertase was found to elute as a polypeptide of approximately 504 kD, while the purified enzyme migrated as a single band of approximately 126 kD on SDS polyacrylamide gel electrophoresis. This result suggested that neutral invertase was composed of the octamer of the corresponding enzyme and alkaline invertase was composed of the tetramer. The optimum pH was determined to be pH 6.8 and 8.0, respectively. In addition, neutral invertase was shown to cleave raffinose and stachyose, suggesting β-fructoflonidase activity of this enzyme.

[0008] The main object of the present invention is to provide a DNA comprising a nucleotide sequence which can be translated into a protein having invertase activity, the highest activity of which is obtained at pH 6.0 to 8.5, preferably 6.0 to 7.5. Neutral and alkaline invertases are believed to be products of different genes, but they appear to be immunologically related.

[0009] Dynamic programming algorithms provide many types of alignments. Generally, there are two methods for aligning sequences. Algorithms proposed by Needleman and Wunsch or by Sellers relate the full lengths of the two sequences and provide an overall alignment of the sequences. On the other hand, Smith-Waterman's algorithm gives a local alignment. Partial alignment aligns the closest set of regions in the sequence by selecting a scoring matrix and gap penalties. This allows a database search to narrow the most conserved regions of the sequence. This also allows the identification of similar domains in the sequence. To accelerate alignment using the Smith-Waterman algorithm, BLAST (Basic Local A
lignment Search Tool) and FASTA both further limit alignment.

In the present invention, the alignment is performed using BLAST, a series of similarity search programs designed to search all available sequence databases, regardless of whether the problem is protein or DNA. Performed conveniently. This search method, BLAST version 2.0 (Gapped BLAST), is generally available on the Internet (current address: http://www.ncbi.nim.gov/BLAST/). It uses heuristic algorithms, seeks partial algorithms as opposed to global algorithms, and thus can detect associations between sequences that share only distant regions. The scores given in BLAST searches have a well-established statistical significance. Within the scope of the present invention, the blastp program and the PSI-BLAST program, which introduce gaps in the alignment of partial sequences, are particularly useful, both programs comparing amino acid problematic sequences with protein sequence databases, and The blastp modification program performs a partial alignment of only two sequences. The program is advantageously executed with the violation values given arbitrary parameters.

[0011] The total or partial alignment of the amino acid sequence with the known sequence according to the present invention is based on the known sequence of the acid invertase or other sucrose metabolizing enzymes.
Shows less than 40% sequence identity. Examples of DNA containing a nucleotide sequence that can be translated into a protein having neutral invertase activity are set forth in SEQ ID NO: 1 and SEQ ID NO: 3. The amino acid sequence of the encoded invertase is shown in SEQ ID NO: 2. Proteins exhibiting greater than 40% sequence identity to SEQ ID NO: 2 and their corresponding genes can be isolated from at least any plant that harvests seeds, fruits or storage organs. Examples include proteinaceous, oily and starch-storing crops, sugar beet, corn, sweet corn, soy, sunflower, grasses, rape, wheat, barley, sorghum, rice, melon, watermelon, squash , Chicory, tomato, pepper, broccoli, cauliflower, cabbage, cucumber,
There are radish, beans and lettuce.

[0012] The protein set forth in SEQ ID NO: 2 lacks a signal peptide and is very hydrophilic. In addition, it has 18 cysteine residues and 15 methionine residues. It shows the highest total sequence identity (47%) in alignment to the LIM17 protein encoded by the partial cDNA clone obtained from Lilium longiflorum. Overall alignment with other protein sequences gives less than 40% sequence identity. The DNA sequence encoding the LIM protein was originally identified by screening a library obtained from micromeispore cells of Lnium longiflorum in meiotic prophase using a meiosis-specific subtraction probe. (Kobayashi
et al, DNA Research 1: 15-26, 1994). Li using computer program GAP
The amino acid sequence obtained from the partial sequence of lium longiflorum LIM17 protein is 47% identical (58% similar) to the present carrot protein.

[0013] The related LIM17 protein, encoded in the genome of the unicellular cyanobacterium Synechocystis (ORF sll0626), contains the sequence of the carrot enzyme after several large gaps have been introduced for optimal alignment. And 37% identical (47% similar). Therefore, it is possible that related proteins that exhibit greater than 40% sequence identity with the invertases of the present invention may be found in photosynthetic bacteria. Like the carrot protein, the LIM17 protein from Lilium and Synechocystis is rich in Cys and Met, but their position in the polypeptide chain does not appear to be conserved. The function and enzymatic activity of the LIM17 protein smaller than the carrot sequence homolog is unknown.

Thus, according to the present invention, the family of neutral invertases is defined as members exhibiting 40% or more amino acid sequence identity to SEQ ID NO: 2 after global alignment. Preferably, the amino acid sequence identity is at least 50%, more specifically at least 55%. Sequences that are 55% or more identical are considered subfamilies. The sequences of the present invention may also have a length of at least 330, 450 or 510 base pairs and have at least 60%, 70% or 75% of the partially aligned component sequence of SEQ ID NO: 2.
The above includes a component sequence having the same identity.

To perform diversity sequence alignments, some algorithms take into account, in addition to sequence identity, sequence similarities, such as the net charge of individual amino acids and the identity of the hydrophobic / hydrophilic ratio. can do. Therefore, the algorithm considers that substitution of one amino acid with another one is likely to preserve the physical and chemical properties necessary to maintain the structure and function of the protein, Also assesses whether the essential structural and functional characteristics of are likely to be lost. Such sequence similarity is quantified by comparing the percentage of positive amino acids to the percentage of identical amino acids, and helps to classify proteins into the correct protein family in the case of borderlines. A protein of particular interest within the scope of the present invention is an invertase having an amino acid sequence comprising at least one of the following characteristic amino acid sequences: (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)

A DNA encoding invertase belonging to the new family of proteins can be produced by the following general method. Using a single-stranded fragment of SEQ ID NO: 1 consisting of at least 15, preferably 20 to 30, and more preferably 100 contiguous nucleotides as a probe, a clone hybridizing to the fragment is screened from a DNA library. . The factors that determine hybridization are Sambrook, et.
al, Molecular cloning: A laboratory manual, Cold Spring Harbor Laborato
ry Press, chapters 9.47-9.57 and 11.45-11.49, 1989. The hybridizing clone is sequenced and a DNA clone containing an open reading frame encoding a protein having at least 40% sequence identity to SEQ ID NO: 2 is purified. The DNA is then further processed by a series of routine genetic recombination methods, such as restriction digestion, ligation or polymerase chain reaction.

DNA containing the nucleotide sequence defined by SEQ ID NO: 1 can be cloned as follows: Purified carrot neutral invertase partial internal tryptic peptide sequence (XNIYPDQIPPWLV, SEQ ID NO: 11) and est In the database comparison, it is Arabidop
It was found to be present in the amino acid sequence encoded by est t88552 (Arabidopsis cDNA 1026 bp 3 'end) obtained from sis. A DNA fragment (nucleotides 100-410) encoding this peptide sequence was synthesized by PCR using primers 5'-TCTAAGGA TCTAGA AAGAGCCATTA-3 '(SEQ ID NO: 12) and 5'-TTCAATT GAATTC AATATAGCTTC-3' (SEQ ID NO: 13). Could be isolated by This PCR product was cut with XbaI and EcoRI, and then ligated to each position of the E. coli plasmid pBluescript II KS (Stratagene). After amplification and purification of this plasmid, the fragment was excised, purified by agarose electrophoresis and electroelution, and randomly labeled with [α- ³² P] ATP. Using this labeled DNA as a probe, wild carrot (Daucus carota cv Queen)
The library created by high-speed growth suspension culture of Anne's Lace, W001C) was screened. The resulting clones were sequenced, e.g., 5'29 bp and 3'393
A clone was identified that contained a 2447 nucleotide insert containing the non-coding sequence of bp, the ORF of which was identical to the amino acid sequence from Arabidopsis est t88552 and 8
Encodes a protein of 675 amino acids sharing 0% identity (86% similarity).

One of skill in the art can modify the above methods to apply to any gene encoding a protein belonging to the new family of invertases. In addition, SEQ ID NO:
No. 3 discloses a polymerase chain reaction that attempts to amplify a DNA fragment from a template comprising a sequence of nucleotides characterized by 15, preferably 20-30 or more contiguous sequences of SEQ ID NO: 1. The skilled artisan in the design of oligonucleotides for The nucleotide is 2 of SEQ ID NO: 1.
It encompasses nucleotide sequences consisting of 0, preferably 20-30 or more base pairs. The polymerase chain reaction performed using at least one such oligonucleotide and its amplification product constitutes another aspect of the present invention. In addition, the disclosed nucleotide sequences allow one of ordinary skill in the art to design transformation vectors, which apply the established transformation techniques described, for example, in WO 96/27673 (pages 17-20). Can be used to generate transformed plants.

A further object of the present invention is to provide a recombinant plant invertase having a neutral optimum pH. This is achieved by recombinant expression of a DNA encoding the invertase, preferably a cDNA, in a microbial host such as E. coli or yeast. For example, a recombinant invertase is produced as follows: cDNA encoding the enzyme is engineered into an expression vector such as pTrc99A (Pharmacia Biotech). E.
A bacterium such as E. coli is transformed and, if necessary, the bacterium is lysed after inducing protein synthesis with, for example, IPTG. Neutral invertase activity is found in the soluble lysate fraction. In particular, about 100 μl of the soluble extract is mixed with 700 μl of water, 0.5 M potassium phosphate (pH 6.8) 100
Mix with μl and 100 μl 0.5M sucrose and incubate at 37 ° C. for 30 minutes. An aliquot of this solution is used for a reducing sugar assay by Somogyi (Somogyi, J. Biol. Chem., 195; 19-23, 1952). To determine the pH dependence of this sucrose cleavage activity
0.5M potassium phosphate with a pH value ranging from 4.5-8.5 is used.

Important biochemical properties of recombinantly produced invertase are similar to those of enzymes purified from plants. That is, the _Km value is about 20 mM, and the pH dependency is pH 6.0.
It shows a sharp maximum between 7.0 and is inhibited by Cu ²⁺ at micromolar concentrations. On the other hand, this recombinant enzyme surprisingly hydrolyzes only sucrose without cleaving raffinose or stachyose. Therefore, this recombinant protein substantially lacks β-fructofuranosidase activity.

EXAMPLES Example 1 Purification of Carrot Neutral and Alkaline Invertase Preparation of Extracts Carrot cells (400 g) taken from an exponentially growing suspension culture were combined with 2.5 volumes of ice-cold buffer A (50 mM) Hepes-KOH, pH7.5, 0.5mM EDTA, 10mM lysine, 0.5mM MgCl ₂ ,
Homogenize in 0.5% 2-mercaptoethanol and 100 mM phenylmethylsulfonyl fluoride using a Polytron homogenizer at full speed for 20 seconds every 4 seconds. The homogenate is centrifuged at 6000 g for 20 minutes in a Sorvall GSA-rotor, and the supernatant is collected and kept cool. The 6000 g pellet was resuspended in 2.5 volumes of ice-cold buffer A and
On a homogenizer, homogenize at full speed for 20 seconds, 3 times, and centrifuge for another 20 minutes.
The combined supernatant was centrifuged at 16,000 g for 30 minutes, and then Miracloth (Calbiochem-Behring
Corporation) four layers. The filtrate is used for further protein purification. Unless otherwise stated, all steps are performed at 4 ° C.

Ammonium Sulphate Precipitation Solid ammonium sulphate is added slowly with gentle agitation of the crude extract, and proteins that precipitate between 20% and 40% saturation are collected by centrifugation at 16,300 g for 30 minutes. The precipitate was added to 100 ml of buffer B (25 mM Hepes-KOH, pH 7.5, 190 mM NaCl, 0.5% 2-mertaptoethanol and 100 mM phenylmethylsulfonyl fluoride).
And dialyzed against buffer B overnight.

Anion exchange chromatography on Q-Sepharose The dialysate was applied to a Q-Sepharose column (2.5 cm × 25 cm, Pharmacia LKB).
Biotechnology, Uppsala, Sweden) and equilibrate with buffer B. The column is washed with buffer B until the absorbance at 280 nm is less than 0.01. The binding protein is
Elution is performed with a linear gradient of 240 ml of 25 mM Hepes-KOH, pH 7.5, containing NaCl 190-550 mM containing 0.5% 2-mertaptoethanol and 100 mM phenylmethylsulfonyl fluoride. The active fractions (fraction size 5 ml) are pooled, and ammonium sulfate 60%
Precipitate at saturation and centrifuge at 16,300 g for 30 minutes. The precipitate is dissolved in 5 ml of buffer C (5 mM K-phosphate buffer, pH 7.5, containing 0.1% 2-mercaptoethanol) and dialyzed against buffer C overnight.

Chromatography on HA-Ultrogel The dialysate was applied to an HA-Ultrogel column (2.5 cm x 25 cm, Sigma, Buchs, S
witzerland) and equilibrate with buffer C. Wash column with buffer C, 0.1%
Elution is performed using a 200 ml linear gradient of 5-500 mM K-phosphate buffer containing 2-mercaptoethanol, pH 7.5. The column elutes at a flow rate of 40 ml / h and collects 5 ml fractions. The flow-through fraction with neutral invertase activity and the eluted fraction with alkaline invertase activity are separately combined, precipitated with 60% saturation of ammonium sulfate, and centrifuged at 16,300 g for 30 minutes. The two protein pellets were individually buffered (D
Dissolve in 5 ml of 5 mM K-phosphate buffer (pH 7.5, containing 0.1% 2-mercaptoethanol) and dialyze against Buffer D overnight.

Affinity Chromatography on Green 19 Dye The dialyzed protein solution (5 ml each) was divided into 0.5 ml aliquots and equilibrated with buffer D in a Green 19 dye column (4.5 × 0). .7cm, Sigma, Buch
s, Switzerland) Applies to 10. The column is washed with 15 ml of buffer D, then 0.35M
Stepwise elute with 1.5M and 1.5M NaCl (15ml and 25ml respectively) and collect 2ml fractions. Neutral invertase activity is detected in the flow-through, and alkaline invertase activity is eluted at 1.5 M NaCl. Fractions containing enzymatic activity are pooled and precipitated at 60% saturation with ammonium sulfate. The precipitated protein is collected by centrifugation at 16,300 g for 30 minutes.

Gel Filtration Chromatography I on Sephacryl S-300 Each protein pellet is dissolved in 7 ml of buffer E (100 mM Hepes-KOH, pH 7.5, containing 0.1% 2-mercaptoethanol). Protein solutions were individually separated by Sephacryl S-300 (S
ephacryl S-300) column (2.6cmx100cm, Pharmacia LKB Biotechnology, Upps)
ala, Sweden). The column was equilibrated with buffer E and blue dextran (V ₀ ),
thyroglobulin (669 kD), apoferritin (443 kD), β-amilase (200 kD), alcoh
ol dehydrogenase (150 kD), BSA (66 kD) and carbonic anhydrase (29 kD)
Is calibrated by Elute the column at a flow rate of 110 ml / hr and collect 5 ml fractions. The fractions containing enzyme activity were collected, and alkaline invertase was added to buffer F (25 mM Hepes-KOH, pH 8.0, containing 200 mM NaCl and 0.1% 2-mercaptoethanol), and neutral invertase was added to buffer G (25 mM Hepes-KOH). -KOH, pH7.2, 275mM
Dialyze overnight against NaCl and 0.1% 2-mercaptoethanol).

Anion exchange chromatography on macro-prep II For further purification of alkaline invertase, the dialysate was applied to a Macro-Prep column (1.5 cm x 20 cm, Bio-Prep) equilibrated with buffer F. Rad Laborator
iers, Richmond, CA, USA). Column is 25 mM Hepes-KOH, pH8, 0.1%
Elute with a 200 ml linear gradient of 200-450 mM NaCl dissolved in 2-mercaptoethanol. For further purification of neutral invertase, the dialysate is applied to a macro-prep column (1.5 cm x 25 cm, Bio-Rad Laboratoriers) equilibrated with buffer G. The column was dissolved in 25 mM Hepes-KOH, pH 7.2, containing 0.1% 2-mercaptoethanol.
Elute with a 200 ml linear gradient of 275-360 mM NaCl. The fractions containing the corresponding enzyme activity are combined individually, precipitated at 60% saturation with ammonium sulfate and centrifuged at 16,300 g for 30 minutes.

Hydrophobic Interaction Chromatography on Propyl Agarose A protein pellet of alkaline invertase activity was added to 5 ml of buffer H (25 mM Hepes
-KOH, pH8.0, containing 1.5M ammonium sulfate and 0.1% 2-mercaptoethanol). This solution is applied to a propyl agarose column (10 cm × 1.5 cm, Sigma, Bichs, Switzerland) equilibrated with buffer H. The column was washed with buffer H and eluted with 25 mM Hepes-KOH, pH 8.0, containing 0.1% 2-mercaptoethanol.
Collect 3 ml fractions. Fractions containing enzyme activity are pooled and 10 mM Hepes-KOH, pH8
Dialysis against 0.0, 0.1% 2-mercaptoethanol, 50% glycerol, -2
Store at 0 ° C.

Gel Filtration Chromatography II on Sephacryl S-300 II A protein pellet with neutral invertase activity is dissolved in 5 ml of buffer I (100 mM K-phosphate buffer, pH 7.0, containing 0.1% 2-mercaptoethanol). , Buffer
Apply to a propyl agarose column (2.6 cm x 100 cm, Pharmacia LKB) equilibrated with I. Fractions of 5 ml each were collected, the fractions having enzyme activity were pooled, dialyzed against 10 mM K-phosphate buffer, pH 7.0, containing 0.1% 2-mercaptoethanol, and -20% in 50% glycerol. Store at ° C.

Example 2: Isolation of a cDNA clone encoding a carrot neutral invertase A partial internal tryptic peptide sequence of purified carrot neutral invertase (XNIYPDQIPPWLV, SEQ ID NO: 11) compared to the est database, from Arabisopsis It was identified as being present in the amino acid sequence encoded by est t88552 (1026 bp at the 3 'end of the Arabidopsis cDNA). DN encoding this peptide sequence
A fragment (nucleotides 100-410) was synthesized with the primer 5'-TCTAAGGA TCTAGA AAGAGCCATTA-
Isolated by PCR using 3 '(SEQ ID NO: 12) and 5'-TTCAATT GAATTC AATATAGCTTC-3' (SEQ ID NO: 13). Amplification is performed using a DNA thermal cycler (Perkin Elmer).
(Cetus) under the following conditions: 10 cycles of denaturation at 95 ° C for 1 minute, annealing at 40 ° C for 0.5 minutes and extension at 72 ° C for 1.5 minutes, then denaturation at 95 ° C for 1 minute, annealing at 60 ° C for 0.5 minutes and extension 20 cycles of 1.5 minutes at 72 ° C. The PCR product was extracted with phenol / chloroform, cleaved with Xbal and EcoRI and the E. coli plasmid pBluescript II KS (Stratagene
) To ligate each position. After amplification and purification of this plasmid, the fragment was excised, purified by agarose gel electrophoresis and electroelution, and [α- ³² P]
Label randomly with ATP. Using this labeled DNA as a probe, wild carrot (
A single hybridizing clone is obtained by screening a cDNA library of a lambda ZAP II vector made with poly A ⁺ mRNA obtained from high-speed suspension culture cells of Daucus carota cv Queen Anne's Lace (W001C).

Example 3: Sequence analysis of carrot invertase clone The insert of the cDNA clone of Example 2 was inserted into the pBluescript II KS (+/-) vector (S
ligated to tratagene), and both strands are automatically sequenced by the dideoxynucleotide chain termination method. Computer-assisted analysis of DNA and protein sequences described in Examples 2 and 3 was performed by Genetic Computer Group (GCG).
, Madison, Wisconsin, Wisconsin Package version 9.0. Sequence comparisons were performed using the alignment algorithm of Needleman and Wunsch (J. Mol.
Biol. 48: 443-453, 1970), two complete sequences that maximize the number of matches and minimize the number of gaps, while allowing the introduction of gaps for optimal alignment. Find the alignment of GAP considers all possible alignments and gap positions and creates an alignment with the largest matching bases and the smallest gap. Then, a gap creation penalty and a gap extension penalty are provided for each matched base unit. In other words, the GAP must benefit from a gap creation penalty in the number of matches for each gap inserted. If you choose a gap extension penalty greater than 0, GAP additionally adds a gap extension penalty to its length for each gap inserted.
You have to get the benefit of penalty. Gap Creation
Typical values used as break-off points for penalties and gap extension penalties are 3.0 and 0.1 for protein sequence comparisons.

The carrot neutral invertase cDNA clone was found to be 2447 nucleotides in length (SEQ ID NO: 13) and contained 29 bp of 5 ′ and 393 bp of 3 ′ non-coding sequence.
The ORF encodes 675 amino acids with a molecular weight mass of 75957 daltons and a calculated isoelectric point pI of 8.01. The deduced amino acid sequence shares 80% identity (86% similarity) with the deduced amino acid sequence of Arabidopsis est t88552.

The putative amino acids of the carrot cDNA of neutral invertase (car) and L. longifloru
Comparison with the sequence of LIM17 from m (lil) and Synechocystis (bac) (Table 1) identifies three conserved sequence domains (boxes 1-3). A search of the database using the sequence in Box 2 reveals a protein of known function, namely Clostridium stercorari.
um cellobiose phosphorylase can be identified. It cleaves cellobiose [β-D-Glc (1 → 4) -D-Glc] into glucose 1-phosphate and glucose in the presence of pyrophosphate. This suggests that box 2 constitutes a binding site for disaccharide glucose residues.

Table 1. Amino acid sequence of neutral invertase cDNA from carrot (car) and Li
Comparison of the LIM17 protein from lium longiflorum (lil) and Synechocystis (bac) with the amino acid sequence. Amino acid sequences are shown in single letter code and are aligned by inserting gaps (...) to maximize identity. Bold amino acid residues indicate conserved domains (boxes 1-3). The asterisk indicates the same amino acid residue in all three sequences.

[0035]

[Table 1]

Example 4: Steady-state levels of neutral invertase mRNA The steady-state levels of neutral invertase mRNA in the leaves and roots, as well as in the reproductive organs of carrot plants at three different developmental stages are examined. Total
RNA was purified from 20 mg of polyclar AT (Se) per gram of tissue prior to trituration in liquid nitrogen.
rva) by Prescott and Martin (Plant Molecul)
ar Biology Reporter 4: 219-224, 1987). For RNA gel blot analysis, total RNA (10 mg / lane) is separated on a 1.2% agarose gel containing 6% formaldehyde. Northern blots showed 4-, 10- and 16-week-old leaves, 4-, 10- and 16-week-old roots, flower buds (B), flowers (F) and small developing seeds (G _s ), Load total RNA (10 mg / lane) from large developing seeds ( _Gl ) and mature seeds (S). Blots are hybridized with ³² P-labeled neutral invertase cDNA.

Steady-state transcript levels of carrot neutral invertase are found in all organs at different developmental stages and are slightly higher in developing organs. This finding suggests a more comprehensive and possible growth-related function of this enzyme in carrot sucrose metabolism.

Example 5: E. coli expression of carrot neutral invertase To express a carrot neutral invertase cDNA clone in E. coli strain JM105 (Pharmacia Biotech), primer 5'-CGATTTAGCAA GGTACC ATAGATATGAATA
C-3 '(SEQ ID NO: 14) and 5'-CTTATCCTTAAACT AGATCT CCATTAGACC-3' (SEQ ID NO: 1)
Amplify by PCR using 5). Amplification is performed using a DNA thermal cycler (Perkin
Elmer Cetus) under the following conditions: 30 cycles of denaturation at 95 ° C for 1 minute, annealing at 55 ° C for 0.5 minute and extension at 72 ° C for 1.5 minutes. The PCR product is extracted with phenol / chloroform and after cleavage with KpnI and Xbal, the expression vector pTrc99A (Pharmacia Biotech)
To the corresponding site. Protein biosynthesis in transformed bacteria harboring the expression vector is induced with 1 mM IPTG for about 16 hours, and the bacteria are lysed in a single cycle of freeze-thaw in a small volume of 50 mM potassium phosphate (Johnson and Hecht).
, Biotechnoliogy 112: 1357-1360, 1994). Neutral invertase activity is
Confirmed in soluble lysate fraction as described by nd Sturm (above). Briefly, 100 μl of the soluble extract was mixed with 700 μl of water, 100 μl of 0.5 M potassium phosphate, pH 6
Mix with 100 μl of .8 and 0.5 M sucrose and incubate at 37 ° C for 30 minutes. An aliquot of this solution is used to measure reducing sugars by Somogyi. To test the pH dependence of sucrose cleavage activity, use 0.5M potassium phosphate with a pH value of 4.5-8.5.

Example 6: Measurement of invertase activity Invertase activity was measured using 50 mM K-phosphate buffer (pH 6.8 or 8.0), 1
Measure in a reaction mixture containing 00 mM sucrose and an appropriate amount of enzyme to a final volume of 1 ml. The mixture is incubated at 37 ° C. for 30 minutes. The amount of reducing sugar released is measured by Somogyi. Enzyme activity (units) is expressed as the amount (μmol) of reducing sugars (glucose and fructose) released per minute. Since invertase activity is inhibited by high concentrations of ammonium ions, protein solutions obtained by ammonium sulfate precipitation need to be dialyzed before measuring the activity. From the measurements performed in the purification step of Example 1, 400 g of cells express about 240 units of neutral invertase activity, which means 0.6 units per gram of cells. 95% of the activity is lost during the enzyme purification step. In the activity measurement of the recombinant enzyme expressed in E. coli, about 0.4 unit of neutral invertase activity was detected in 100 μg of the E. coli extract described in Example 5.

[0040]

[Sequence list]

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE , KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZWF terms (reference) 4B024 AA03 AA05 BA12 CA04 CA09 CA20 DA06 EA04 HA13 HA14 4B050 CC01 CC03 DD13 EE01 FF03E FF04E FF05E FF09E FF10E FF12E FF14E LL02 LL03 LL05 4B063 QA01 QQ44 QQ53 QR56 QS16 QX01 QX07

Claims (10)

[Claims] 1. A DNA comprising a nucleotide sequence which can be translated into a protein having invertase activity, which exhibits the highest activity in the pH range of 6.0 to 7.5. 2. The DNA according to claim 1, which encodes a plant protein. 3. Encoding 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. 2. The DNA according to claim 1, comprising a nucleotide sequence that: 4. The DNA according to claim 1, which comprises a nucleotide sequence encoding the protein set forth in SEQ ID NO: 2. 5. The DNA according to claim 1, comprising the nucleotide sequence set forth in SEQ ID NO: 1. 6. A protein having invertase activity, but lacking β-fructofuranosidase activity, and having the highest invertase activity between pH 6.0 and 7.5. 7. It 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: 10. 7. The plant protein according to claim 6. 8. The plant protein according to claim 6, which has the amino acid sequence set forth in SEQ ID NO: 2. 9. The method for producing DNA according to claim 1, comprising:-a DNA fragment defined by SEQ ID NO: 3, which is capable of hybridizing with a fragment having a length of at least 15 nucleotides. Screening from the DNA library,-sequencing of hybridizing clones,-purification of vector DNA of clones containing an open reading frame encoding a protein having more than 40% sequence identity with SEQ ID NO: 2. -Optionally, further processing the purified DNA. 10. A polymerase chain reaction wherein at least one oligonucleotide used comprises a nucleotide sequence representing 15 or more base pairs of SEQ ID NO: 1 or SEQ ID NO: 3.