WO2005005470A2

WO2005005470A2 - Pectin methylesterase inhibitors for the preparation of fruit juices and derivatives

Info

Publication number: WO2005005470A2
Application number: PCT/IT2004/000390
Authority: WO
Inventors: Daniela Bellincampi; Felice Cervone; Alessandro Raiola; Laura Camardella; Ciro Balestrieri; Giulia De Lorenzo; Luigi Servillo; Lucio Quagliolo; Benedetta Mattei; Alfonso Giovane; Domenico Castaldi.
Original assignee: UNIVERSITA' DEGLI STUDI DI ROMA 'La Sapienza' et al.; Stazione Sperimentale Per Le Industrie Delle Essenze E Dei Derivati Dagli Agrumi
Priority date: 2003-07-15
Filing date: 2004-07-15
Publication date: 2005-01-20
Also published as: ITRM20030346A0; ITRM20030346A1; WO2005005470A3

Abstract

A nucleic acid that encodes a protein or a portion thereof that inhibits pectin methylesterase to be employed in the stabilization of foodstuff products and in combination with other pectolytic enzymes in the preparation of pectins for foodstuffs.

Description

PECTIN METHYLESTERASE TNHTBITORS FOR THE PREPARATION OF FRUIT JUICES AND DERIVATIVES

The present invention concerns pectin methylesterase inhibitors (PMEI) obtained by recombinant methods and the uses thereof. In particular, the inhibitors of the present invention are advantageously utilized in the production of foodstuffs, for example in the preparation of fruit juices, derivatives, concentrates, etc.

Pectins are involved in many industrial processes, such as the preparation of fruit and vegetable juices and concentrates. Pectins are secreted in the cell wall in a highly methyl-esterified form and are de-esterified by pectin methylesterases (PMEs). PMEs remove methyl esters by forming polygalacturonic acid domains able to bind to calcium, thus forming gelatinous macromolecules that can influence the porosity and rigidity of the cell wall. PME activity is also affected by physiologic parameters such as the extracellular pH and represents the basis for the activity of other pectic enzymes such as endo-polygalacturonase (PGs) that degrades the polygalacturonic acid into pectin fragments.

During the production of fruit juices, the intent is to maintain a suspension made of cell fragments together with water stabilized by soluble pectins that confer the juice the desired turbidity and degree of viscosity ("body"), one of consumers' most appreciated feature. If the body is absent, the juice is defined as "thin" and its organoleptic properties are considerably altered. The entire technology of fruit juices and concentrates is aimed at stabilizing the turbidity ("cloud") of the product and preserving it from clarification, sedimentation or flocculation. Clarification of a vegetable juice (cloud destabilization) is due to endogenous PME activity which, by lowering the degree of esterification of the soluble pectins, allows the formation of calcium pectates that precipitate as insoluble pectates. The pectates produce a gel. Gelification of concentrated juices is a problem that strongly affects the quality of the product in that it hampers reconstitution of the juice. In tomato juice and concentrates, for example, to obtain a stable product with elevated consistency, the endogenous PME need to be inactivated at temperatures higher than those in pasteurization using complex and energetically costly techniques. Moreover, the preparation of pectins with a high degree of methylation and polymerization require the inactivation of pectic enzymes including PME and PG. Since it is known that PG act on demethylated pectins, the inhibition of PME also affects the activity of PG. Summarizing, it may be said that the manufacturing technique of fruit and vegetable juices and derivatives is closely connected with the technology and chemistry of pectic substances and enzymes that these substances degrade, among which one of the most important is PME.

The scope of the present invention is to prevent the undesired effects produced by PME activity in juices, concentrates and vegetable derivatives. Experiments conducted by the authors on orange juices not heat treated showed that cloud loss may be efficaciously inhibited by adding pectin methylesterase inhibitor (PMEI) (Castaldo et al., Food Sci 56 1991). Similar results were also observed on other products of vegetable origin. US patent no. 5,053,232 and a published study (Balestrieri et al., 1990) concern natural PMEI in the kiwi (Actinidia chinensis) and its uses in the preparation of fruit and vegetable juices. However the natural protein does not solve the technical problem underlying the invention, in that it cannot be used on a large scale, also because of the impossibility to get a standard result and of high production costs.

The authors of the present invention have produced by recombinant route the PME inhibitor from various plant species and selected the recombinant product that may be advantageously used on a large scale, in terms of lower production costs, simpler purification of the protein from cultural mediums, and the higher production levels. The object of the present invention is a nucleic acid that encodes a protein or a portion thereof that inhibits pectin methylesterase. Preferably, the protein or portion thereof that inhibits the pectin methylesterase derives from Arabidopsis ox Actinidia. In a preferred embodiment, the protein or portion thereof that inhibits pectin methylesterase has a sequence comprised in one of the following amino acid sequences: AtPMEI-1 (Seq. Id. No. 2)

QVADIK CGKAKNQSFCTSYMKSNPKTSGADLQTLANITFGSAQTSASEGFRK IQSLVKTATNPTM -KAYTSCVQ1TΪXSAISSLNDAKQSLASGDGKGLNIKNSAA MEGPSTCEQDMADFKNDPSAVKΝSGDFQΝICGIVLVISΝMM; AtPMEI-2 (Seq. Id. No. 4) ITSSEMSTICDKTLNPSFCLKFLNTKFASPNLQALAKTTLDSTQARATQTLKKLQ

SITDGGNDPRSKLAYRSCNDEYESAIGNLEEAFEHLASGDGMGMNMKNSAALD GADTCLDDVKRLRSVDSSNVΕIΝSKTIKm-CGLΛ-LNISΝMLPRΝ: or synAcPMEI (Seq. Id. No. 6)

ENHLISEICPKTRNPSLCLQAJ.ESDPRSAS1^LKGLGQFSIDIAQASAKQTSKIIAS LTNQATDPKLKGRYETCSENYADAΓDSLGQAKQFLTSGDYNSLNΓYASAAFDG AGTCEDSFEGPPNIPTQLHQADLKLEDLCDINLNISNLLPGS.

In another embodiment of the invention, the nucleic acid of the invention has a sequence comprised in one of the following amino acid sequences: AtPMEI-1 (Seq Id. No. 1)

CAAGTGGCAGACATAAAAGCGATATGTGGAAAAGCGAAAAACCAATCCTTC TGTACGAGCTACATGAAATCCAACCCAAAGACCTCAGGTGCTGATCTTCAA ACGCTTGCAAATATCACATTTGGTTCTGCACAAACAAGTGCATCAGAAGGTT TCAGGAAAATTCAATCTCTAGTCAAGACAGCAACCAACCCCACTATGAAGA AAGCATACACCTCATGTGTACAACATTATAAGAGTGCAATAAGCAGTCTCA ATGATGCTAAGCAGAGCCTGGCGTCAGGCGATGGCAAAGGGTTGAACATTA AGGTTTCAGC AGCTATGGAAGGACCTTCAACATGTGAACAAGAC ATGGCGG

ATTTCAAAGTTGATCCTTCAGCTGTGAAGAACAGTGGTGATTTTCAGAATAT TTGTGGCATTGTACTTGTCATCTCAAACATGATGTGA; AtPMEI-2 (Seq Id. No. 3) ATCACAAGTTCAGAAATGAGCACAATCTGTGACAAAACCTTAAATCCATCT TTCTGTCTTAAGTTCCTCAATACGAAATTCGCATCGCCTAATCTTCAAGCCTT GGCAAAAACCACACTTGATTCTACACAAGCGAGAGCTACACAAACGTTAAA GAAACTCCAATCTATTATCGATGGAGGAGTCGACCCTCGATCTAAGTTAGCT TACAGGTCATGCGTAGATGAATACGAGAGCGCGATTGGAAACCTCGAGGAA GCTTTTGAGCATTTAGCTTCAGGAGATGGTATGGGGATGAACATGAAAGTTT CTGCTGCATTGGATGGAGCTGATACATGTTTAGATGATGTGAAGAGATTGA

GATCAGTAGATTCTTCGGTTGTGAATAACAGTAAAACAATTAAGAATCTTTG TGGTATTGCTCTTGTTATCTCTAACATGTTACCACGTAATTAA; or SynAcPMEI (Seq Id. No. 5) GAAAATCATCTTATTTCTGAAATTTGTCCAAAGACTAGGAATCCATCTCTTT

GTCTTCAAGCTCTAGAATCTGATCCAAGGTCTGCTTCTAAGGATCTTAAGGG TCTTGGTCAATTTTCTATTGATATTGCTCAAGCATCTGCTAAGCAAACTTCA AAGATTATTGCTTCTCTTACTAATCAAGCTACTGATCCAAAACTTAAGGGTA GGTATGAAACTTGTTCTGAAAATTATGCTGATGCTATTGATTCTCTTGGTCA AGCTAAGCAATTTCTTACTTCTGGTGATTATAATTCTCTTAATATTTATGCTT CTGCTGCTTTTGATGGTGCTGGTACCTGTGAAGATTCTTTTGAAGGTCCACC AAATATTCCAACTCAACTTCATCAAGCTGATCTTAAACTTGAAGATCTTTGT

GATATTGTTCTTGTTATTTCTAATCTTCTTCCAGGTTCTTAA. An object of the invention is a recombinant vector for the correct and efficient expression in recombinant organisms of the nucleic acid of the invention. Preferably, the recombinant organisms are eukaryotes, more preferably yeasts, and more preferably Pichiapastoris.

Another object of the invention is a process for the recombinant production of the protein or a portion thereof that inhibits pectin methylesterase.

Another object of the invention is the use of a protein or a portion thereof that inhibits pectin methylesterase in the stabilization of foodstuff products. Another object of the invention is the use of a protein or portion thereof that inhibits pectin methylesterase, also in combination with other pectolytic enzymes in the preparation of pectins for food industry products. Preferably, food industry products are comprised in the group of juices, derivatives, concentrations and purees of fruit and/or vegetables as preserves or frozen foods and others. It is another object of the invention a stabilized fruit or vegetable derivative containing the protein or a portion thereof that inhibits pectin methylesterase produced by the method of the invention.

The authors have isolated the DNA encoding the two PMEI of Arabidopsis (AtPMEI-1 and AtPMEI-2) on the basis of two genes (At3gl7220 and Atlg-48020, respectively, of unknown function: the Arabidopsis Genome Initiative, 2000). The deduced amino acid sequences show only 38% homology with the amino acid sequence of the protein inhibitor isolated from kiwi and previously determined (Camardella et al., 2000), maintain the conservation of five cysteines, four of which are involved in the formation of disulfide bridges and may be important in the definition of the protein structure. The coding gene fragments, with no intron sequences, were independently isolated by PCR using specific primers and cloned in appropriate plasmid vectors. The authors have also in vitro synthesized a coding sequence gene based on the amino acid sequence of the kiwi inhibitor (Camardella et al., 2000).

On the basis of the nucleotide sequence related to the mature protein, the gene fragments corresponding to various genes were independently cloned in an appropriate vector (pPICZoA, J-nNitrogen, CA, USA) for expression in Pichia pastoris and secretion of the proteins in the culture medium. Based on the sequencing of the Ν- terminal portions of purified synAcPMEI, AtPMEI-1 and AtPMEI-2 inhibitors, there were four additional predominantly present amino acids derived from the proteolytic action of the specific enzymes that eliminated the yeast secretion factor. SDS-PAGE analysis showed that the biochemically purified proteins had the following molecular masses: SynAcPMEI: about 16,000 Da, AtPMEI-1: about 25,000 Da and AtPMEI-2: about 18,000 Da. In vitro assay showed that recombinant proteins were able to inhibit PMEs from tomato, kiwi, carrot, Arabidopsis, tobacco and Nicotiana benthamiana. Since the Arabidopsis protein seems to show the same function as the kiwi PMEI, it is advantageously employed in the preparation of fruit and vegetable juices and pectin products. The purified recombinant PMEI can therefore be added to any plant derived product to inhibit or to reduce PME activity, thus providing the "cloud" stability in vegetable juices and concentrates and aiding in obtaining high molecular mass pectins. The invention is described below by way of example and in reference to the following figures and tables:

Figure 1: schematic diagram of the AcPMEI gene; A, B, and C denote the gene fragments subsequently assembled A (start - Xbal); C (Kpnl - Stop): the synthesized oligonucleotides used to generate fragments A and C are highlighted; the arrows indicate the amplification primers. B (Xba I - 265 bp — Kpn I): the synthesized oligonucleotides used to generate fragment B are highlighted; the arrows indicate the primers designed to fuse the two subfragments and their amplification. Figure 2: SDS/PAGE of the recombinant PMEI after purification. 1- molecular mass markers; 2- AtPMEI-1; 3-AtPMEI-2; 4-SynAcPMEI. Figure 3: reverse phase column HPLC (Vydac C4) of AtPMEI-1 purified by Arabidopsis. Chromatography was developed on a linear gradient of solvent B (0.08% trifluoracetic acid in acetonitrile in solvent A (0.1% trifluoracetic acid in water) at a flow rate of 1 ml/min following absorbance at 220 nm. Under these conditions, the retention time of AtPMEI-1 is 35.6, that of AtPMEIJ is 38.8, and that of PMEI of Actinidia is 40.5.

Synthesis of the kiwi PME inhibitor gene (synAcPMEI^')

The synthetic AcPMEI gene encoding the kiwi inhibitor was obtained on the basis of the amino acid sequence of the natural protein isolated from mature kiwis (PMEI_ACTCH accession number P83326 NCBI database; http://www.ncbi.nlm.nih.gov/). Gene synthesis was obtained by PCR using the PWO DNA Polymerase enzyme (Roche, Germany). The gene was synthesized in 3 different fragments, called A, B and C, which had 68 bp, 271 bp and 126 bp, respectively (Figure 1). The entire gene was assembled by fusing all the fragments after introducing Xbal and Kpnl, respectively, into the two internal restriction sites of the pUC19 vector.

The complete gene was then cloned in frame in the pPICZoA and downstream from the factor secretion signal, and the construct was employed to transform E. coli. The constructs isolated from the positive colonies were then aligned and used to transform Pichia pastoris.

Cloning of AtPMEI-1 and AtPMEI-2

The nucleotide sequences of the genes AtPMEI-1 (accession number in TATR database: At3gl 17220; http://www.arabidopsis.org/) and AtPMEI-2 (accession number in TAIR database: Atlg48020; http://www.arabidopsis.org/) were amplified by genomic DNA isolated from Columbia ecotype Arabidopsis plants using the following primer pairs:

- for AtPMEI-1

Atpmei-1/F (5'-ATCGAGAATTCCAAGTGGCAGACATAAAAG-3') (Seq Id No. 7) Atpmei- 1/R (5⁵-ATCGATCTAGATCACATCATGTTTGAGATG-31 (Seq Id No. 8)

- for AtPMEI-2 Atpmei-2/F (5'-ATCGAGAATTCATCACAAGTTCAGAAATGAG-3') (Seq Id No. 9)

Atpmei-2/R (5'-ATCGATCTAGATTAATTACGTGGTAACATGT-3⁵)(Seq Id No. 10) The amplified products were isolated and cloned in frame between the restriction sites EcoRI and Xbal in the pPICZoA vector and downstream from the factor secretion signal, and the construct was employed to transform E. coli. The constructs isolated from the positive colonies were then linearized and used to transform Pichia pastoris.

Purification of the AtPMEI-1- AtPMEI-2 and synAcPMEI inhibitors expressed in Pichia pastoris AtPMEI-1

Culture broth of Pichia pastoris was filtered and precipitated with 80% saturated ammonium sulphate. The precipitate was collected by centrifugation at 20,000 x g for 20 min, resuspended in a 10 nM Na acetate buffer, pH 5.0 and dialyzed exhaustively against the tampon. After dialysis, the solution was centrifuged at 20,000 x g for 20 min and loaded on a MonoS ® column (HR 10/10), Pharmacia) and equilibrated with the same tampon. The PMEI was then eluted with a linear gradient from 0 to 0.5 M NaCL in 30 min at a flow rate of 1.5 ml/min. The fractions with inhibitory activity were pooled, concentrated by ultrafiltration on Centricon 3 filters (Amicon) and loaded on a Sephadex ® G75 column (1.5 x 90 cm) equilibrated in 10 mM TrisCl, pH 7.5, 250 mM

NaCl at a flow rate of 5 ml h. The fractions with inhibitory activity tested pure when analyzed by 12.5% SDS-gel electrophoresis and stained with a solution of Coomassie ® Brilliant blue G250 (Figure 2). The calculated molecular mass is about 25 kDa, whereas that deduced from the sequence is 16,092 Da, suggesting an elevated level of glycosylation. The protein presents a single peak when analyzed by reverse phase column HPLC (Figure 3). The N-terminal sequence (EAEFQVADIKAI-GKAK-Q) corresponds to the cloned and expressed gene sequence, and shows the presence of four additional amino acids (bold) corresponding to the signal peptide sequence of Pichia. AtPMEI-2 Culture broth of Pichia pastoris was filtered and precipitated with 80% saturated ammonium sulphate. The precipitate was collected by centrifugation at 20,000 x g for 20 min, resuspended in a 10 mM TrisCl buffer, pH 8.5 and dialyzed exhaustively against the tampon. After dialysis, the solution was centrifuged at 20,000 x g for 20 min and loaded on a MonoS ® column (HR 10/10), Pharmacia) and equilibrated with the same tampon. The PMEI was then eluted with a linear gradient for 0 to 0.5 M NaCL in

30 min at a flow rate of 1.5 ml/min. The fractions with inhibitory activity were pooled, concentrated by ultrafiltration on Centricon 3 filters (Amicon) and loaded on a Sephadex ® G75 column (1.5 x 90 cm) equilibrated in 10 mM TrisCl, pH 7.5, 250 mM NaCl at a flow rate of 5 ml/h. The fractions with inhibitory activity tested pure when analyzed by 12.5% SDS-gel electrophoresis and stained with a solution of Coomassie ®

Brilliant blue G250 (Figure 2). The calculated molecular mass is about 18 kDa, whereas that deduced from the sequence is 16,743 Da. The protein has one putative glycosylation site. The protein has a single peak when analyzed by reverse phase column HPLC, with a longer retention time than that of the AtPMEI-1 protein (Figure 3). The N-terminal sequence (EAEFITSSEMSTI) corresponds to the cloned and expressed gene sequence, and shows the presence of four additional amino acids (in bold) corresponding to the peptide signal residue of Pichia.

PMEI of Actinidia (SynAcPMEI)

Culture broth of Pichia pastoris was filtered and precipitated with 80% saturated ammonium sulphate. The precipitate was collected by centrifugation at 20,000 x g for 20 min, resuspended in a 10 nM TrisCl buffer, pH 8.5 and dialyzed exhaustively against the same buffer. After dialysis, the solution was centrifuged at 20,000 x g for 20 min and loaded on a MonoS ® column (HR 10/10), Pharmacia) and equilibrated with the same buffer. The PMEI was then eluted with a linear gradient from 0 to 0.5 M NaCL in 30 min at a flow rate of 1.5 ml/min. The fractions with inhibitory activity were pooled, concentrated by ultrafiltration on Centricon 3 filters (Amicon) and loaded on a Sephadex ® G75 column (1.5 x 90 cm) equilibrated in 10 mM TrisCl, pH 7.5, 250 mM

NaCl at a flow rate of 5 ml/h. The fractions with inhibitory activity tested pure when analyzed by 12.5% SDS-gel electrophoresis and stained with a solution of Coomassie ® Brilliant blue G250 (Figure 2). The calculated molecular mass of about 16 kDa corresponds to that deduced from the amino acid sequence (16,753 Da; in fact, the protein has no glycosylation sites. The protein presents a single peak when analyzed by reverse phase column HPLC, with a longer retention time than that of the Arabidopsis protein (Figure 3). The N-terminal sequence (EAEFENHLISEI-PK) corresponds to the cloned and expressed gene sequence, and shows the presence of four additional amino acids (in bold) corresponding to the peptide signal residue of Pichia. Measurement of the activity of PMEI in relation to pH and temperature

Inhibitory activity was measured at room temperature using an automatic titrator (Mettler model DL50) connected to a computer and onboard software package. The incubated mix (20 ml) contained 0.1% pectin with a minimum percentage of methylation of 70%, 0J0 M NaCl and 1 unit of PME. The titration solution contained 0.01 m NaOH. Pectin methylesterase activity, expressed in PME units (micromoles of acid produced per minute) was determined as the quantity of NaOH, in ml, needed to maintain the pH of the solution at the preset value (pH=7.50) according to the formula:

whejέ:

Vs mL NaOH to titrate the sample Jo = mL NaOH to titrate the dry run M_Na_OH ⁼ Concentration of NaOH (expressed in molarity) t = time of analysis in minutes

PMEI activity was assayed as the ability to inhibit the activity of the pectin methylesterase enzyme from tomato fruits, 1 unit of inhibitor was defined as the minimum quantity of inhibitor able to completely inhibit a unit of the enzyme for 10 min.

Measurement of the activity of the two inhibitors was taken at three different pH levels, as reported in Table 1. In each condition, we used the minimum quantity of inhibitor able to fully inhibit enzyme activity (tomato seed PME) at pH 6.5.

Table 1. Inhibitory activity of recombinant PMEI against tomato PME in relation to pH. Inhibitory activity (%) pH 6.5 pH 7.5 ph 8.5

AtPMEI-1 100 100 100

AtPMEI-2 100 100 60 kiwi PMEI 100 75 0

In addition,, inhibitory activity was tested at different temperatures (Table 2).

Table 2. hihibitory activity of recombinant PMEI against tomato PME in relation to temperature. PMEI Inhibitory activity (%) * 50 °C 60 °C 70 °C 80 °C AtPMEI-1 100 89 42 15 AtPMEI-2 75 60 27 12 Actinidia 100 63 0 0 *1 unit of inhibitor was incubated in a sealed vial at each temperature for 10 min and then immersed in ice. Inhibitory activity was determined using an automatic titrator, as described above. Methyl-esterified lime pectin (0.5% solution in phosphate buffer 50mM pH 6.5) with a degree of methyl-esterification of 81% (E81 from Danisco Ingredients, Denmark) have been treated with tomato PME (3xl0^"9 M, 0JU per mL) for 3 hours at 30°C in the absence or in the presence of 10-fold molar excess of SynAcPMEI. After boiling for 5 minute buffer was adjusted to pH 5 with sodium acetate to reach the acidic pH optimum of fungal polygalacturonase. The pectin solution was successively digested for 1 hour with Fusarium moniliforme polygalacturonase (PG) 0J RGU/mL at 30°C. The average of degree of polymerization was determined by reducing end-group analysis using the PAHBAH procedure (York WS et al. 1985 Methods Enzymology 118:3-40). One activity unit (RGU) was defined as the amount of PG producing 1 microequivalent of reducing groups/min at 30°C with 1% (w/v) polygalacturonic acid as substrate. As observed in Table 3, while PG alone is not able to efficiently hydrolyze highly methylated pectins due to the known inability of the enzyme to cleave glycosilic bonds in the vicinity of a methylated galacturonic unit, a cooperative effect of PME and PG was observed resulting in the fragmentation of pectin in short oligomers. The presence of PMEI abolished the cooperative effect of PME and PG on pectin degradation resulting in the presence of longer undigested fragments in solution.

Table 3. Action of PMEI on the cooperative degradation of 81% methyl esterified lime pectins (E81).by tomato PME and F.moniliforme PG .

E81: 0.5% solution in phosphate buffer 50mMpH 6.5; PME from tomato 0.1 U /mL; PG from F.moniliforme (0J U/mL)

References - D. Castaldo, A. Lovoi, L. Quagliuolo, L. Servillo, C. Balestrieri, A. Giovane, Orange juices and concentrates stabilization by a proteic inhibitor of pectin methylesterase, j Food Sci 56 (1991) 1632-1634.

- C. Balestrieri, D. Castaldo, A. Giovane, L. Quagliuolo, L. Servillo, A glycoprotein inhibitor of pectin methylesterase in kiwi fruit (Actinidia chinensis), Eur. j. Biochem. 193 (1990) 183-187.

- A. Giovane, C. Balestrieri, L. Quagliuolo, D. Castaldo, L. Servillo, A glycoprotein inhibitor of pectin methylesterase in kiwi fruit. Purification by affinity chromatography and evidence of a ripening-related precursor, Eur. j. Biochem. 233 (1995) 926-929.

- The Arabidopsis Genome Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408 : 796-815.

- L. Camardella, V. Carratore, M.A. Ciardiello, L. Servillo, C. Balestrieri, A. Giovane, Kiwi protein inhibitor of pectin methylesterase amino-acid sequence and structural importance of two disulfide bridges, Eur. j. Biochem. 267 (2000) 4561-4565.

Claims

CLAIMS 1. A nucleic acid that encodes a protein or a portion thereof having pectin methylesterase inhibitory activity.

2. A nucleic acid according to claim 1 derived from plants or parts thereof of the Arabidopsis or Actinidia genera that encodes a protein or portion thereof that inhibits the pectin methylesterase.

3. A nucleic acid according to claim 2 that encodes a protein or portion thereof with pectin methylesterase inhibitory activity, said protein having essentially a sequence comprised in the group of the following amino acid sequences: Seq. Id. No.2, Seq. Id. No.4, Seq. Id. No.6.

4. A nucleic acid according to claim 3 encoding a protein or a portion thereof with pectin methylesterase inhibitory activity and having a sequence essentially comprised in one of the following nucleotide sequences: Seq. Id. NoJ, Seq. Id. No.3, Seq. Id. No.5.

5. A recombinant vector for the correct and efficient expression in recombinant organisms of the nucleic acid according to any of previous claims.

6. A vector according to claim 5 wherein the recombinant organisms are eukaryotes, more preferably yeasts, and even more preferably Pichia pastoris.

7. A process for the recombinant production of a protein or of a portion thereof that inhibits pectin methylesterase characterized by the phases of: a) insertion of the vector according to claim 5 or 6 in an appropriate organism to obtain transformed organisms; b) direction of the recombinant expression of the encoded protein in said transformed organisms; c) purification of the protein from the culture medium and/or from transformed organisms.

8. Use of the protein or a portion thereof that inhibits pectin methylesterase produced by the method according to claim 7 in the stabilization of foodstuff products.

9. Use of the protein or portion thereof that inhibits pectin methylesterase produced by the method according to claim 7 also in combination with other pectolytic enzymes in the preparation of pectins for foodstuffs.

10. A stabilized fruit or vegetable derivative containing the protein or a portion thereof that inhibits pectin methylesterase produced by the method according to claim 7.

11. A pectin composition to be used in the food industry containing the protein or a portion thereof that inhibits pectin methylesterase produced by the method according to claim 7.