WO2017113024A1

WO2017113024A1 - Novel poligalacturonase that is active at low temperatures

Info

Publication number: WO2017113024A1
Application number: PCT/CL2015/050062
Authority: WO
Inventors: Mario Esteban CARRASCO TRONCOSO; Juan Manuel ROZAS ANDAUR; Pablo Alfonso VILLARREAL DÍAZ; Marcelo Enrique BAEZA CANCINO
Original assignee: Universidad De Chile; Innovacold S.A.
Priority date: 2015-12-30
Filing date: 2015-12-30
Publication date: 2017-07-06

Abstract

The invention relates to a nucleic acid molecule that codes for a poligalacturonase that is highly efficient at low temperatures between 5°C - 15°C, and the coded enzyme. The invention also relates to vectors, host cells, recombination methods for producing said enzyme, as well as compositions comprising the poligalacturonase. The invention further relates to the use of said enzyme in the degradation of pectin during the production of wine, beer or fruit juice and in the clarification of grape must or fruit juice.

Description

NOVEDOSA ACTIVE LOW TEMPERATURE POLYGALACTURONASE

1. Field of the invention

The present invention relates to an enzyme with highly efficient polygalacturonase activity at temperatures in a range between 5 ^Q C-15 ^Q C.

The present invention also relates to a liquid formulation or as a lyophilized powder comprising said enzyme and appropriate excipients, and having utility in fermentation processes related to agro-industry, specifically in fermentation processes of the wine industry, juice producing industries of fruits, beer industry and other industries that in their processes need to degrade pectin. The present invention is especially useful in clarification processes of grape must or fruit juice.

The present invention also relates to the synthetic nucleotide sequence encoding said enzyme and to the method for obtaining same by means of transformant strains of the yeast Pichia pastoris. As well as also, the present invention relates to the recombinant expression vector ppinka-HC (Invitrogen) which allows the appropriate transformation of the yeast Pichia pastoris.

2. Background

Pectin is an important component of the cell wall of plants, in general, giving them mainly the texture property of fruits and vegetables.

Pectinases are the enzymes that degrade the pectin, eliminating at least one residue of sugar or an ester group. Thus, pectinases catalyze the elimination of some functional group of pectin, hydrolyze specific types of binding, among other functions.

Pectinase polygalacturonase is important for the food industry, being especially useful in the production of fruit juices or in the winemaking, where its used for its ability to degrade pectin. In general, pectinases are used in the fruit industry to help extract, clarify and modify fruit juices.

In this sense, it is important to note that in the extraction processes of fruit juice, pectins agglomerate forming colloids that need to be clarified. The clarification is dependent on the type of fruit, and the flocculation of the suspended material and its separation by filtration, is only achieved, after using a suitable pectinase enzyme. However, said enzyme is generally due to inactivate by thermal treatment to avoid excessive degradation, and thereby, avoid unwanted changes in the taste, color and consistency of the final product.

Enzymes that degrade pectin (pectinases) are secreted, for example, by fungi and yeast, deducting their corresponding amino acid sequences as well as the nucleotide sequences that encode them. It is possible to name among the fungi that secrete said enzyme, Aspergillus niger, among several others. Also, a series of microorganisms, including bacteria and yeast, have been transformed to secrete pectinases with different properties, among which Saccharomyces cerevisiae can be mentioned.

Although there is still a need for the different pectinases already known with suitable for use in the production of fruit juices or wine making, where available commercial enzymes are effective at temperatures between 40-60 ^Q C properties, but they show a very diminished performance, in a temperature range as low as 5 ^Q C-15 ^Q C.

The use of pectinases that show activity at a low temperature range such as between 5 ^Q C-15 ^Q C, would improve the yield by decreasing the production times and with this, costs are reduced because less enzyme is added, and they could obtain in this way, fruit juices of good quality for human consumption, in a shorter time.

It is then an object of the present invention to provide such an isolated pectinase polygalacturonase (recombinant) enzyme which is capable of degrading pectin at a temperature in the range of 5-15 ^Q C, being very useful in the production of fruit juices or processing of wine, and that allows to clarify, for example, the grape must at temperatures as low as for example, 8 ^Q C (white wines) or 12 ^Q C (red wines).

In this way, the polygalacturonase enzyme according to the invention shows activity at lower temperatures than those currently commercially available or compared to those of the prior art. In prior art, there were efforts to obtain polygalacturonase. Accordingly, several methods have been developed to modify microorganisms that include yeasts such as Pichia pastoris (patent application ES200202566) or Saccharomyces cerevisiae (patent application ES200201596). But from such efforts, endopolygalacturonases that produce in large quantities have been achieved, and nothing is indicated or referred to addressing the problem of obtaining polygalacturonase that show activity at temperatures as low as 5 ^Q C and 12 ^Q C.

On the other hand, the publication Biosc. Biotechnol. Biochem., 69 (2), 419-421, 2005 by Nakagawa T. et al, teaches that from a psychrophilic basidiomycete yeast (that is, it grows at an optimum temperature under 20 ^Q C, and at most, at a maximum growth temperature of 22 ^Q C) identified as the PPY-1 strain of Cystofilobasidium capitatum, it is possible to obtain an active polygalacturonase even at temperatures as low as 0 ^Q C.

Meanwhile, the publication Letters in applied Microbiology 2004, 38, 383-387 by Nakagawa T. et al teaches that strains PPY-3, PPY-4, PPY-5 and PPY-6 Cystofilobasidium capitatum produce pectinase enzymes that show activity, for example, at 5 ^° C.

Likewise, the Letters in Applied Microbiology ISSN 0266-8254 publication by Sahay S et al teaches strain SPY1 1 of Cystofilobsidium capitatum and the PT1 strain of Rhodotorula mucilaginosa, which secrete polygalacturonase enzymes that are active at temperatures between 5 ^Q C and 12 ^Q C and that have 50-80% of activity at pH 3.5 (oenological conditions), which makes them an adequate alternative to be used in the production of wine and clarification of fruit juice.

The present invention discloses an enzyme with polygalacturonase activity different from that disclosed in Letters in Applied Microbiology ISSN 0266- 8254 of Sahay S et al., Which is highly efficient in a temperature range between 5 ^Q C-15 ^Q C.

3. Brief description of the invention

The present invention relates to an enzyme with polygalacturonase activity, highly efficient at temperatures in a range between 5 ^Q C-15 ^Q C, and consequently, useful in fermentation processes of the wine industry, specifically, in the clarification of the must of grape, a process that for example, can be carried out at low temperatures, 8 ° C - white wines elaboration, or at 12 ° C - elaboration of red wines, and that also requires the control of the enzymatic activity by means of moderate thermal treatments that inactivate the enzyme.

Thus, the present invention relates to a liquid formulation or as a lyophilized powder comprising said enzyme and appropriate excipients, and having utility in degrading pectin at a temperature between 5 ^Q C and 15 ^Q C. Similarly, the present invention is refers to a synthetic nucleotide sequence encoding said polygalacturonase enzyme or an amino acid sequence having at least about 55% sequence identity and, more preferably, at least 90% homology or identity and which, even more preferably, has at least 955 homology and which maintain the ability to be highly efficient at low temperatures, in particular in a temperature range between 5 ^Q C and 15 ^Q C. Said nucleotide sequence also comprises a variant nucleic acid encoding said enzyme which differs from the present enzyme in no more than about 55 amino acid substitutions and which, more preferably, has no more than 18 amino acid substitutions, and which maintains the same polygalacturonase activity at temperatures between 5 ^Q C and 15 ^Q C.

The present invention also relates to the method of obtaining the present enzyme by means of a transformant strain of the yeast Pichia pastoris. Accordingly, the present invention relates to a ppinka-HC recombinant expression vector (Invitrogen) that allows for the proper transformation of the yeast Pichia pastoris.

In particular, the present invention relates in a main aspect to a nucleic acid molecule comprising a polynucleotide having a DNA sequence encoding a polypeptide having characterized polygalacturonase activity in which the DNA sequence is selected from: a) DNA sequences comprising a nucleotide sequence of SEQ ID NO.:3; b) DNA sequences comprising a nucleotide sequence of SEQ ID NO: 5; c) DNA sequences comprising a nucleotide sequence of SEQ ID NO: 6; d) DNA sequences encoding the amino acid sequence SEQ ID NO: 1; e) DNA sequences encoding the amino acid sequence SEQ ID NO: 2; f) DNA sequences that hybridize under stringent conditions to one of the sequences according to a), b), c), d) or e); g) sequences having a sequence identity of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more with one of the DNA sequences according to a), b), c), d9) oe); h) allelic variant or homologous species of one of the DNA sequences according to a), b), c), d), e) f) or g) which differs from one of these sequence in no more than about 1 to about 165 substitutions, deletions, insertions, additions or mutations of nucleic acid, preferably in no more than about 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 1 0 , 1 to 1 1, 1 to 1 2, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19 or 1 to 20 substitutions, deletions, insertions, additions or nucleic acid mutations; j9 a fragment of any of the sequence according to a9 ai) encoding a polypeptide having polygalacturonase activity; ok) a complementary strand of any of the sequences according to a) aj).

Also in view is a polypeptide having polygalacturonase activity, characterized in that the polypeptide is selected from: a) a polypeptide that is encoded by the coding part of any DNA sequence as described above; b) a polypeptide comprising a sequence according to SEQ ID No: 1 or SEQ ID NO: 2, or c) a polypeptide that is derived from the sequence according to SEQ ID NO: 1 or 2, obtainable by substitution, addition or deletion of one or more amino acids of said SEQ ID NO: 1 or 2. In a preferred embodiment, the polypeptide is derived from the sequence according to SEQ ID NO: 1 or 2 by substitution of not more than about 55 amino acids in said SEQ ID NO: 1 or 2. In a still more preferred embodiment, the polypeptide is derived from the sequence according to SEQ ID NO: 1 or 2 by a substitution of not more than 18 amino acids in said SEQ ID NO: 1 or 2.

In a particularly preferred embodiment of the present invention said polypeptide is an enzyme with polygalacturonase activity, wherein said polygalacturonase activity is a high polygalacturonase activity in a temperature range of about 5 ^Q C to about 15 ^Q C, ie, the Enzyme polygalacturonase activity is present and is highly pronounced in a low temperature range of 5 to 1 5 ^QC . In a further preferred embodiment, said polypeptide or said nucleic acid can be isolated from the species Articulospora proliferata.

Also in view is a recombinant expression vector comprising the nucleic acid molecule as defined herein above.

In a preferred embodiment of the present invention, said recombinant expression vector comprises the nucleotide sequence of SEQ ID NO: 4,

5 or 6

In a further aspect, the present invention relates to a recombinant host cell characterized in that it has been transformed with the recombinant expression vector as defined hereinbefore.

In a preferred embodiment, said recombinant host cell is derived from an E. coli cell, a fungus of the family Aspergillus, Tricoderma or Neurospora, or a yeast from the family Kluveromyces, Saccharomyces, Schizosaccharomyces, Hansenula or Pichia. In a particularly preferred embodiment, said host cell is a Pichia pastoris cell.

The present invention relates in a further aspect to a method for the production of a polypeptide according to the invention as defined herein above, or which is encoded by the DNA sequence according to the invention as defined herein above, characterized in that it comprises: (i) culturing the recombinant host cell as mentioned above under conditions that allow the expression of said polypeptide, and (ii) recovering said polypeptide.

The present invention furthermore has in view in a different aspect a composition characterized in that it comprises the polypeptide of the invention as defined hereinbefore or obtained by the method of the present invention as mentioned hereinbefore. In a specific embodiment of the present invention, said composition comprises, essentially consists of or consists of a liquid formulation or a lyophilized powder.

In a further aspect, the present invention relates to the use of a polypeptide of the invention as defined hereinbefore or of a polypeptide of the invention obtained by a method as mentioned hereinbefore or of a composition as defined herein for degradation. of pectin in a beverage production process in which the degradation of pectin is desirable. In a particularly preferred embodiment, said process is in the production of wine, beer or fruit juice.

In another preferred embodiment said beverage production process in which the degradation is pectin is desirable to occur at a low temperature, for example, at a temperature below 25 ^Q C, more preferably at a temperature between 5 ^Q C and 20 ^Q C, more preferably at a temperature between 5 ^Q C and 15 ^Q C. In yet another preferred embodiment, said beverage production process in which the degradation of pectin is desirable is the clarification of grape must or fruit juice.

4. Brief description of Figures

Figure 1: Shows the fractionation of supernatants of induced cultures (YM-1% pectin (product Sigma-Aldrich P9135, commercial pectin, Pectin from citrus peel, CAS number 9000-69-5, EC number 232-553-0, number MDL MFCD00081838) with ammonium sulfate, visualized by SDS-PAGE and silver staining Lane 1: 60% fraction, Lane 2: 80% fraction, M: Molecular weight marker (Thermo Scientific) Figure 1 clearly shows an enrichment of certain proteins in the fractions of 60% of ammonium sulfate in relation to the 80% fraction.The fractionation is carried out in a culture of Articulospora proliferava. Figure 2: Shows an ion exchange chromatography. The total extracellular proteins enriched with 60% ammonium sulfate were loaded onto a DEAE sephadex column equilibrated in 20 mM Tris-HCl buffer, pH 8.0. The proteins were eluted using a gradient of NaCl from 0-1 M, and subsequently, they were loaded on an SDS-PAGE gel and stained with silver. The numbers 1 -41 indicate the fractions obtained and M, corresponds to the marker of molecular weight used. Enzymatic activity assays indicated that the samples enriched with the pectinase enzyme were fractions 10-20. The arrow indicates a size of approximately 40kDa.

Figure 3: They show an electrophoresis gel and quantification of purified RNA, and subsequently subjected to RNA-seq. Lanes A-D show different RNA extractions that presented different yields. In lane A, more RNA is observed but more degraded, whereas lane D shows good extraction performance but with much less degradation. RNA extractions were performed using the Ribopure Yeast RNA purification kit (Ambion).

Figure 4: Shows a graphic representation of the enzyme polygalacturonase, where the protein domains of the pectinase enzyme are observed. Also, the structural motifs that were identified through the InterproScan program (Available in the Geneious program) are shown, the most important being the amino acid residues 223-236, which define the catalytic site of polygalacturonase enzymes.

Figure 5: Shows a graphic representation of the gene that codes for the pectinase enzyme. It shows the structure of introns and exons of the gene that codes for the pectinase enzyme, and a schematization of the structure of the gene that codes for the enzyme polygalacturonase. The exons (celestial) are regions that code for the enzyme, while the introns (black) are regions eliminated during the "splicing" process of the mRNA. The complete mRNA sequence of the enzyme has a length of 1325 base pairs (bp) and would be composed of 5 exons.

Figure 6: Shows recombinant pectinases obtained from transformant clones of the yeast Pichica pastoris. In this figure, a protein gel is shown in which the recombinant pectinases obtained from the 4 transformant clones obtained (lanes 1-4) were separated. In lane 5, a protein sample was loaded, which was obtained without induction with methanol (negative control). In lane 6, another protein was loaded as a positive control. Lane M corresponds to a protein marker of known weight, with the objective of estimating the size of the pectinase enzyme. The clone N ^Q 1 was the one that expressed the highest amounts of the recombinant protein (40kDa).

Figure 7: Shows a purified pectinase enzyme. From the protein gel, the recombinant pectinase (polygalacturonase) enzyme of the present invention obtained after the two purification steps was separated. Two protein bands are observed (lane N ^Q 2), which differ only in that the upper band is glycosylated.

Figures 8A and 8B: Shows the evaluation of the polygalacturonase activity of the purified enzyme under laboratory and semi-industrial conditions. Figure 8A shows a DNS assay using a commercial pectin (Pectin from citrus peel, Sigma Aldrich). Polygalacturonase as both Antarctic commercial enzyme (Lafazym®, Laffort) with a solution of said commercial pectin (1 Omg / ml) at pH 3.0 was incubated for one hour at 37 ^Q C. Subsequently, 100μ! of the enzymatic reaction was assayed for enzymatic activity, by e! DNS method. Figure 8B shows a DNS assay, using white wine must as substrate. The samples were treated identically as in the case of the data of Figure 8A but the activity of the enzyme was evaluated. 15 ^Q C and on white wine must. The concentration of galacturonic acid released was quantified by a calibration curve previously performed. Figure 9: Shows the calibration curve performed with different known concentrations of galacturonic acid (2, 1 .5, 1, 0.5, 0.25 and 0.125 mg / ml) tested by the DNS method. It was incubated 1 00μ! of each solution of different concentrations of galacturonic acid with 10ΟμΙ of! DNS reagent and e! assay as described above.

Figure 10A and 10B, shows the quality control performed on the RNA samples that were sent to be sequenced (the induced sample and the control sample). Figure 1A shows the RNA concentration, the rRNA ratio in relation to the messengers and the RIN which is a measure of how complete this nucleic acid is (RIN> 7 is good for the analyzes), for the induced sample and control (10A and 10B, respectively).

Figure 11: Shows the clones sown on PDA plates (selective medium). The white colonies correspond to transformant colonies of the yeast Pichia pasoris, where white corresponds to the colonies that acquired the recombinant insert.

Figure 12: Alignment of the peptides obtained by mass spectrometry (VIFSGTTTFGYK and SGAVVQNQDDC) with respect to the complete amino acid sequence of the polygalacturonase enzyme of the invention. A 100% identity and coverage with respect to this sequence is observed.

Figure 13: Shows an electrophoresis in agarose gels in which the DNA fragments obtained after digestion of the ppinka-HC expression vector were separated with the restriction enzymes Mlyl and Kpnl (thermoscientific). In this figure, the linearized vector (largest fragment) and the pect fragment are displayed in lane 1. Lane M corresponds to a DNA standard of known weight, with the objective of estimating the size of the fragments. Figures 14A and 14B: Shows a pectinase activity assay performed on Y plates supplemented with 1% pectin and developed with hexadecyltrimethylammonium bromide (1%). Figure 14a shows a halo of positive hydrolysis while Figure 14b shows a yeast that did not exhibit positive pectinase activity (absence of halo).

Figure 15: Shows the evaluation of the polygalacturonase activity of the purified enzyme at 5 ^Q C. Figure 15 is a graphic representation of the data shown in table 4,

Figure 16: Shows the assays of polygalacturonase activity in plum pulp juice at 5 ^Q C of the purified enzyme.

5. Detailed description of the invention

In accordance with the above, the present invention relates in a main aspect to a nucleic acid molecule comprising a polynucleotide having a DNA sequence encoding a polypeptide having polygalacturonase activity and said polygalacturonase enzyme itself, as well as the proper uses of it, compositions that understand it etc. The inventors have, in particular, found that the enzyme claimed herein is highly active at low temperatures, for example, in a range of about 5 ^Q C to 15 ^Q C.

Although the present invention will be described with respect to particular embodiments, this description is not constructed in a limiting sense.

Before describing the exemplary embodiments in detail of the present invention, important definitions for the understanding of the invention are given.

As used in the specification and the accompanying claims, the singular forms of "a" also include the respective plurals unless the context dictates otherwise. In the context of the present invention, the terms "about" and "about" indicate a careful interval that a person skilled in the art will understand that secures the technical effect of the characteristic in question. The term typically indicates a deviation from the indicated numerical value of ± 20%, preferably ± 15%, more preferably ± 10%, and even more preferably ± 5%. In certain aspects the term "about" may also refer to a value, which is greater or less in several integers, preferably in, 4, 3, 2 or 1 compared to the starting value.

It should be understood that the term "understanding" is not limiting. For the purposes of the present invention, the term "consists of" is considered a referred embodiment of the term "comprising of". If from now on a group is defined to comprise at least a certain number of embodiments, this means that it also encompasses a group that preferably consists of these embodiments only.

If the term "comprising" is used in the context of sequences, in particular nucleotide sequences, the term may not only refer to the sequence mentioned in the listed sequence but also the complementary sequence thereof, unless the context indicates otherwise .

In addition, the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)" or "(i)", "(ü) "," (iii) "," (iv) "," (v) "," (vi) ", (vii)", etc. and similar in the description and in the claims are used to distinguish between similar elements and not necessarily to describe a sequence or chronological order It should be understood that the terms are used interchangeably under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in sequences other than those described or illustrated herein.

In case of the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)" or "(i)", "(ü ) "," (iii) "," (iv) "," (v) "," (vi) ", (vii)", etc. refers to stages of a method or use there is no coherence of time or intervals of time between stages, that is, the stages can be conducted simultaneously or there can be time intervals of seconds, minutes, hours, days, weeks, months or even years between such stages, unless another meaning is indicated to the one described or illustrated before or after.

It should be understood that this invention is not limited to a methodology, protocol, protein, bacterium, vector, reagent, etc. particular described here and this may vary. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only, and it is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless otherwise indicated, the terms used here are those described in "A multilingual glossary of biotechnological terms (IUAPC Recommendations)", Leuenberger, HGW, Nagel, B. and Kólbi, H. eds (199), Helvética Chimica Acta , CH-4010 Basel, Switzerland. The term "polynucleotide" as used herein refers to a molecule having, comprising or consisting of a nucleic acid sequence. A polynucleotide according to the present invention may comprise or consist of an entire sequence encoding or a portion of a sequence encoding and / or may comprise an untranslated region, for example a 5 'UTR and / or a 3' UTR. A polynucleotide according to the present invention may also comprise or consist of portions of a flanking gene, ie, 5 'or 3' for the genomic sequence, preferably of sequences corresponding to SEQ ID NO: 3, 5 or 6. The present invention looks at the polynucleotide to be a DNA sequence or an RNA sequence, or any derivative thereof.

SEQ ID NO: 3 indicates a cDNA encoding the recombinant enzyme with polygalacturonase activity according to SEQ ID NO: 1. SEQ ID NO: 5 indicates a cDNA encoding the recombinant enzyme with polygalacturonase activity according to SEQ ID NO: 2.

SEQ ID NO: 6 indicates an optimized nucleotide sequence based on the use of the codon Pichia pastoris, that is, a recombinant nucleotide sequence optimized for the use of Pichia pastoris.

The term "polypeptide" as used herein refers to a molecule that has, comprises or consists of the amino acid sequence generated from a polynucleotide or nucleic acid according to the present invention as defined throughout the specification. Preferably, the term refers to a molecule having, comprising or consisting of the sequence SEQ ID NO: 1 or 2, a fragment thereof, a domain thereof, an epitope contained therein, a variant thereof, an allelic variant of the same, a homologous species thereof as well as a homologous sequence thereof any combination, fusion, etc. of it as defined here.

SEQ ID NO: 1 indicates the complete amino acid sequence of the present polygalacturonase as also shown in Figure 4 which comprises 369 amino acids. This sequence also comprises a signal sequence in the N terminal. E polypeptide has been obtained from Articulospora proliferata.

SEQ IDNO: 2 indicates the recombinant enzyme with polygalacturonase activity according to the present invention without the signal peptide. This corresponds to SEQ ID NO: 1 without the signal peptide as derived from Figure 4 and the sequence listing.

A "polynucleotide fragment" as used herein refers to a short polynucleotide that has, comprises or consists of a nucleic acid sequence of SEQ ID NOs: 3, 5 or 6 or the complementary strand thereof. A nucleotide fragment according to the present invention can be at least about 500 nt, preferably about 700 nt, more preferably 800 nt, still more preferably 900 nt in length. A The fragment may, in certain embodiments, also have smaller lengths such as about 10 nt, 20 nt, 30 nt, 40 nt, etc. Representative examples of polynucleotide fragments of the invention include, for example, fragments comprising or alternatively consisting of a sequence of about 1 -500, 50-550, 100-600, 150-650, etc. number of nucleotides at the end of SEQ ID. NOs: 3, 5 or 6 or for the complementary thread of the same. Alternatively, the polynucleotide fragments of the present invention may include for example fragments comprising or alternatively consisting of a sequence of about 1 -500, 501-1000 etc. number of nucleotides at the end of SEQ ID NO: 3, 5 or 6 or the complementary thread for them.

A "polypeptide fragment" as used herein refers to an amino acid sequence that is a portion of the sequence contained in the amino acid sequence SEQ ID NOs: 1 or 2. Fragments of polypeptide (or alternative protein) may be either "freestanding" or being comprised within a larger polypeptide for example in the form of a fusion protein. Representative examples of fragments of polypeptides of the invention include, for example, fragments comprising alternatively consist of, from about 1 -5, 6-10, 1 1 etc. amino acid numbers at the end of SEQ ID NOs: 1 or 2. Alternatively, the polypeptide fragments of the invention may include, for example, fragments comprising or alternatively consisting of a sequence of about 1-150, 151-300, 301 amino acid numbers at the end of SEQ ID NO: 1 or 2. In addition, the polypeptide fragments may be about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 or around 100, 200, or 300, etc. amino acids in length.

A polypeptide fragment according to the present invention may comprise a polypeptide domain, i.e., a functional domain or structure of a polypeptide, preferably any typical or three-dimensional secondary protein structure such as a helical portion, a beta sheet, a beta bridge, a return or link curve. The domains of the polypeptide of the invention can be derivatives of Fig. 3. Examples of such domains are a glycol-hydro-2 domain, pectin-lyase polygalacturonase and non-cytoplasmic domain. A further example is a region around the amino acid residues 223 to 236 of SEQ ID NO: 1 which defines the catalytic site of the polygalacturonase enzymes for example, a region comprising approximately amino acid residues 200 to 250, for example . 210 to 240, or 215 to 240, or any portion of SEQ ID NO: 1 comprising said region. A fragment as defined above may be in compliance comprised of one or more such domains. It is preferred that one or more of said domains perform the polygalacturonase activity of the enzyme of the invention.

A polypeptide or polypeptide fragment according to the present invention may further comprise a polypeptide epitope, i.e., a portion of a polypeptide having antigenic and / or immunogenic activity in an appropriate host organism, eg, an animal. The term "immunogenic epitope" as used herein indicates a portion of the protein that elutes an antibody response in an appropriate host organism, eg, an animal, as determined by any of the methods known in the art, e.g. , by methods for generating antibodies known to a person skilled in the art. Accordingly the antibodies obtained for example an antibody that binds to an epitope of the polypeptide of the invention and can be used to identify the enzyme or its use in industrial or semi-industrial processes.

The term "variant" of the present invention as used in the context of the present invention refers to a polynucleotide or polypeptide that differs of the polynucleotide or polypeptide of the present invention, for example, the polynucleotide SEQID NOs: 2, 5 or 6, or the polypeptide of SEQ ID NOs: 1 or 2, but retains the essential properties or where the encoded polypeptide retains essential properties, in a particularly preferred embodiment the properties of the polygalacturonase.

The term "polygalacturonase property" or "galacturonase activity" as used herein refers to the enzymatic activity of hydrolyzing alpha-1, 4 bonds. This activity refers in particular to the degradation of pectin or the pectin network, ie a pectinase activity. Said polygalacturonase activity can be tested with any test or assay known to the person skilled in the art or derivable from sources of suitable literature. Preferably, the activity is tested with a DNS test as described herein, particularly in the Examples. According to preferred embodiments of the present invention, said galacturonase activity occurs in a wide range of temperatures including low temperatures that start around 5 ° C. Accordingly, a polygalacturonase according to the present invention is capable of hydrolyzing the 1,4-glycosidic bonds between residues of galacturonic acid at low temperature of about 5 ° C, 6 ° C, 7 ° C, 8 ° C, 9 ° C, 10 ° C, 1 1 ° C, 12 ° C, 13 ° C, 14 ° C, 15 ° C, 16 ° C, 17 ° C, 18 ° C, 19 ° C, 20 ° C or more , preferably in a range of 5 ° C to 15 ° C, or 5 ° C to 10 ° C. In a specific embodiment a high enzymatic activity is shown by the present enzyme polygalacturonase at a temperature between 5 ° C and 15 ° C when compared to other polygalacturonase enzymes.

In addition, a polygalacturonase according to the present invention is capable of hydrolyzing alpha-1, 4-glycosidic bonds between galacturonic acid residues at a low pH of from about 2 to about 5, for example, at a pH of 2, 2.5, 3, 3.5, 4, 4.5 or 5. In a specific embodiment the optimum pH of the polygalacturonase of the present invention in which the highest activity can be detected between pH 2.5 and 4.

In certain embodiments a variant according to the invention refers to a polynucleotide or polypeptide that differs from the polynucleotide or polypeptide of the present invention and retains essential properties thereof or where the encoded polypeptide retains essential properties such as polygalacturonase is active at low temperatures of about 5 to 25 ° C, such as 5 ° C to 20 ° C, 5 ° C to 15 ° or 5 ° C to 10 ° C, preferably at low temperatures of about 5 to 15 ° C. In further embodiments, the essential properties additionally include an activity at a pH of 2.5 and 4, more preferably at a pH of 3.5. The term "active" in this context refers to moderate to high activity, for example. An enzymatic activity at or near an optimum temperature and / or pH.

In general, the variants are, above all, closely similar, and, in many regions, identical to the polynucleotide or polypeptide of the present invention. Variants according to the present invention may contain alterations in the coding regions, non-coding regions or both. Especially preferred are the polynucleotide variants that contain alterations that produce silent substitutions, additions or deletions but do not alter the properties or activities of the polypeptide encoded polynucleotide variants. Also preferred are the nucleotide sequence variants, which are produced by silent substitutions due to the degeneracy of the genetic code. Polynucleotide variants can be produced for a variety of reasons, typically to optimize codon expression for a particular host, i.e., to change codons in the mRNA to those preferred by a host cell of bacteria, plant or fungus, preferably by a fungus host as Pichia pastorís, or another suitable fungus or expression system that does not include a fungus.

In addition, polypeptide variants in which 5-10, 1 -5 or 1-amino acids are substituted, deleted or added in any combination are preferred. In particular, by using protein engineering methods and recombinant DNA technology known to the person skilled in the art, variants can be generated to improve or alter the characteristics of the polypeptides of the present invention. For example, one more amino acids can be removed from the N or C terminal without substantial loss of biological function, in particular the polygalacturonase activity as defined hereinbefore.

Furthermore, even if amino acids are removed from the N or C terminals of the polypeptide, it results in a modification or loss of one or more biological functions, other biological activities can still be retained. For example, the ability of a deletion variant to induce and / or bind antibodies that recognize the polypeptide or peptide will likely be retained when at least most of the residues of the secreted form are removed from the N or C terminal. If a particular polypeptide It lacks terminal N or C residues of a protein retains such immunogenic activities and can be determined by standard methods known to the person skilled in the art.

In a further embodiment of the present invention, polynucleotide or polypeptide variants can include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as to have a small effect on the activity of the protein or encoded polypeptide. For example, the guide related to how to make substitutions of phenotypically silent amino acids is provided in Bowie et al., 1990, Science 247: 1306- 1310. Variants can be produced via site-directed mutagenesis or alanine scanning mutagenesis, that is, the introduction of a single alanine mutations in each residue in the molecule. The mutant molecules that result can often be tested for biological activity, for example, based on a DNS assay as described herein. Typically, buried amino acid residues (within the tertiary structure of the protein) require non-polar side chains, while few features of the surface side chains are generally conserved. In addition, tolerated preservative amino acid substitutions involve the replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and lie; in replacement of the Ser and Thr residues; the replacement of acidic residues Asp and Glu; the replacement of the amide residues Asn and Gln, the replacement of the basic residues Lys, Arg, and His; the replacement of the aromatic residues Phe, Tyr, and Trp, and the replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.

In addition to the preservative amino acid substitution, the variants of the present invention may include substitutions with one or more non-conserved amino acid residues, where the substituted amino acid residues may or may not be encoded by the genetic code, or substitutions with one or more amino acid residues having a substituent group, or a fusion of the mature polypeptide with another compound such as a compound to increase the stability and / or solubility of the polypeptide (eg, polyethylene glycol), or a fusion of the polypeptide with amino acids additional, such as, for example, an IgG Fe fusion region peptide, or leader or secretory sequence, or a sequence that facilitates purification.

Particularly preferred are variant polynucleotide sequences that differ from the sequence SEQ NO: 3, 5 and 6, or any derivative thereof as defined herein, by no more than about 1 to 165 substitutions, deletions, insertions, additions or mutations of nucleic acid, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, about 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 1 10, 1 15, 120, 125, 130, 135, 140, 145, 150, 155, 160 or about 165 substitutions, deletions, insertions, additions or mutations of nucleic acid. Further preferred are the variants of polynucleotide sequences that differ from the sequence SEQ NO: 3, 5 and 6 by no more than about 1 to 54 substitutions, deletions, insertions, additions or mutations of nucleic acid, more preferably by no more than 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 1 1, 1 to 12, 1 to 13, 1 to 14, 1 a 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19 or 1 to 20 substitutions, deletions, insertions, additions or mutations of nucleic acid. The term "nucleic acid mutation" as used herein refers to any modification to the nucleic acid other than a substitution, deletion, insertion or addition.

In a further preferred embodiment the present invention looks to variants of peptide or polypeptide sequences that differ from the sequence SEQ ID NO: 1 or 2, or any derivative as defined herein, by substitution, addition or deletion of one or more amino acids of said amino acids SEQ ID NO: 1 or 2. The term "plus" means that any number of substitutions, additions or deletions for the reference sequence without compromising, for example. Reduce or essentially change, the polygalacturonase activity in a given host environment as defined herein, preferably the property of polygalacturonase as being active at low temperatures of about 5 to 25 ° C, such as 5 ° C to 20 ° C, ° C at 15 ° or 5 ° C to 10 ° C which can be tested with any suitable test or assay known to the person skilled in the art in particular a DNS assay as described herein. Also preferred are variants that show a substitution, deletion or addition of no more than about 55 amino acids in SEQ ID NO: 1 or 2, for example, a substitution, deletion or addition of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or 55 amino acids in SEQ ID NO: 1 or 2. Such variants, while comprising an amino acid sequence are still functional as polygalacturonase as they are defined herein, preferably being active, for example, highly active, at low temperatures of about 5 to 25 ° C, such as 5 ° C to 20 ° C, 5 ° C to 15 ° or 5 ° C to 10 ° C . In a further preferred embodiment, the variants show a substitution, deletion or addition of no more than about 18 amino acids in SEQ ID NO: 1 or 2.

In a preferred embodiment, the present invention relates to polypeptide variants containing amino acid substitutions, amino acids charged with another charged or neutral amino acid, which can produce proteins with improved characteristics, such as less aggregation.

The term "allelic variant" as used herein refers to variants that occur naturally, that is, to one of several alternative forms of a gene occupying a given locus on a chromosome of an organism. These allelic variants can vary in either the polynucleotide and / or polypeptide level and are included in the present invention. In a specific embodiment of the present invention, the term also includes variants that do not occur naturally, it can for example be produced by mutagenesis or targeted synthesis techniques. In a further specific embodiment the term refers to a naturally occurring allele of the SEQID polynucleotide NOs: 3, 5, or 6, or the polypeptide of SEQ ID NOs: 1 or 2 in the form of a small nucleotide polymorphism, preferably in the form of SNPs.

In a further embodiment of the present invention, homologous species of the polynucleotide or polypeptide sequences are also contemplated. The term "homologous species" of the present invention as used in the context of the present invention refers to polypeptide sequences or polynucleotides which show a high degree of identity to the polynucleotide or polypeptide of the present invention, for example, to the polynucleotide SEQ ID NOs: 3.5 or 6, or to the polypeptide of SEQ ID NOs: 1 or 2 and which are derivatives of a species different, for example, from a species of fungus, yeast, bacteria, plant or animal, preferably a species of basidiomycete fungus.

For a nucleic acid having a nucleotide sequence or DNA sequence is at least, for example, about 85% "identical" to a nucleotide sequence or DNA sequence of the present invention, or showing an "identity of at least about 85". % ", this is intended that the nucleotide sequence of the nucleic acid is identical to the reference sequence except that the nucleotide sequence can include up to 15 mutation points per 100 nucleotides of the reference nucleotide sequence encoding the polypeptide. In other words, to obtain a nucleic acid having a nucleotide sequence of at least 85% identity to a reference nucleotide sequence up to 15% of the nucleotides in the reference sequence can be deleted or replaced with another nucleotide or a number of nucleotides Up to 15% of the total nucleotides in the reference sequence can be inserted into the reference sequence. The query sequence can be a whole sequence or any fragment as described here.

In certain embodiments of the invention, DNA sequences of the invention may have an identity sequence of at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98% or 99% or more with one of the DNA sequences as mentioned above, for example. SEQ ID NO: 3, 5 or 6, or any derivative thereof any derivative thereof according to the present invention. If any particular nucleic acid molecule or DNA sequence is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identical to the nucleotide sequence or DNA sequence of the present invention, for example. SEQ ID NO: 3, 5 or 6, or any derivative thereof can be determined conventionally using known computer programs. A preferred method for determining the best match between a query sequence (the sequence of the present invention) and a subject sequence, also referred to a global sequence alignment can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. , 1990, Comp. App. Biosci. 6: 237-245. In a nucleotide sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared when converting U's to T's. The result of this global sequence guideline is a percentage of identity. The preferred parameters used in a FASTDB alignment of DNA sequences to calculate the percent identity are: Matrix = Unitary, k-tuple = 4, Mismatch Penalty = 1, Joining Penalty = 30, Randomization Group Length = 0, Cutoff Score = l, Gap Penalty = 5, Gap Size Penalty 0.05, Window Size = 500 or the length of the nucleotide sequence, whichever is smaller. If the subject sequence is less than the sequence queried by deletions 5 'or 3' deletions, not by internal deletions, a manual correction should be made to the results. This is because the FASTDB program does not have 5 'and 3' truncations of the subject sequence when the percent identity is calculated. For the subject sequences truncated at the 5 'or 3' ends, in relation to the query sequence, the identity percentage is corrected when calculating the number of bases of the query sequence that are 5 'and 3' of the subject sequence, which do not match / align, as a percentage of the total bases of the query sequence. Whether a nucleotide matches / aligns is determined by the results of the FASTDB alignment sequence. This percentage can then be subtracted from the percentage of identity, calculated by the previous FASTDB program using the specific parameters, to arrive at a final percentage identity score. This corrected score is what is already for the purposes of the present invention. Only bases outside the 5 'and 3' bases of the subject sequence as displayed by the FASTDB alignment that do not match / align with the query sequence are calculated for the purposes of manually adjusting the percentage identity score.

In certain embodiments of the invention, the polypeptide sequence of the invention can have identity of at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% or more with one of the polypeptide sequences as described above, for example, SEQ ID NO: 1 or 2, or any derivative thereof according to the present invention.

For a polypeptide having amino acid sequence at least, for example, 85% "identical" to a query amino acid sequence of the present invention, it is intended that the amino acid sequence of the subject polypeptide be identical to the query sequence except that the subject polypeptide sequence may include up to 15 amino acid alterations per 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence of at least 85% identity to an amino acid query sequence, up to 15% of the amino acid residues in the subject sequence can be inserted, deleted, (indels ) or substituted with another amino acid. These alterations of the reference sequence can occur at the ammonium or carboxy terminal positions of the reference amino acid sequence or anywhere between these terminal positions, interspersed either between individual residues in the reference sequence or in one or more groups contiguous within the sequence of reference or in one or more contiguous groups within the reference sequence. If any particular polypeptide is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98% or 99% identical to, for example, an amino acid sequence of the present invention can be determined conventionally by using known computer programs. A preferred method for determining the best match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computational program based on the algorithm of Brutlag et al. ., 1990, Comp. App. Biosci. 6: 237-245. In the amino acid sequence an amino acid sequence alignment sequence query and subject are both amino acid sequences. The result of this global sequence alignment is given by the percentage of identity. Preferred parameters used in the FASTDB alignment of amino acids are: Matrix = PAM 0, k-tuple = 2, Mismatch Penalty = l, Joining Penalty = 20, Randomization Group Length = 0, Cutoff Score = 1, Window Size = sequence length, Gap Penalty = 5, Gap Size Penalty = 0.05, Window Size = 500 or the length of the subject amino acid sequence whichever is shorter. If the subject sequence is shorter than the query sequence is due to the deletions of the N- or C- terminals, not by the internal deletions, a manual correction must be made for the results. This is because the FASTDB program does not count for truncations of the N- and C- terminal of the subject sequence when calculating the global identity percentage. For subject sequences truncated at terminals N- and C-, in relation to the query sequence, the identity percentage is corrected when calculating the number of residues of the query sequence, the percentage of identity is corrected when calculating the number of residues of the query sequence that are terminal N- and C- of the query sequence that does not match / align with a corresponding subject residue as a percentage of the total bases of the query sequence. If a residue aligns / matches it can be determined by the results of the FASTDB sequence alignment. This percentage is then subtracted from the percentage of identity, calculated by the previous FASTDB program using the specific parameters to arrive at a final identity percentage score. This final identity percentage score is that used for the purposes of the present invention. Only residues for the N- and C- terminals of the subject sequence that do not match / align with the query sequence are considered for the purposes of manually adjusting the percentage identity score. That is, only query residue positions outside the N- and C- terminal residues furthest from the subject sequence. For example, a 90 amino acid residue is aligned with a query sequence of 100 residues to determine sequence identity. The deletion occurs at the N-terminal of the subject sequence and then, the FASTDB alignment does not show a match / alignment of the first 10 residues of the N- terminal. The 10 unattached represent 10% of the sequence (number of residues in the terminal N- and C- do not match / total number of residues in the query sequence) so 10% is subtracted from the identity percentage score calculated by the FASTDB program . If the remaining 90 residues match perfectly with the final percentage identity it would be 90%. In another example, a subject sequence of 90 residues is compared to a query sequence having 100 residues. This moment the deletions are internal deletions so there are residues in the terminal N- or C- of the subject sequence that do not coincide / alienate with the query. In this case, the identity percentage is calculated by FASTDB and is not corrected manually. Only residual positions outside the terminal ends N- and C- of the subject sequence as displayed by the FASTDB alignment that does not match / align with the query sequence are manually corrected. No other manual corrections are made for the purposes of the present invention.

The present invention also relates in one aspect to DNA sequences or nucleic acids capable of hybridizing under stringent hybridization conditions to the DNA sequences of the invention or nucleic acids as defined above, preferably to the nucleotide sequences SEQ ID NOs: 3 , 5, or 6, or the nucleotide sequence encoding the acid sequence of SEQ ID NO: 1 or 2. The term "stringent hybridization conditions" refers to an overnight incubation at 42 ° C in a solution that comprises 50% formamide 5x SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 g / m), denaturated, cut salmon sperm DNA , followed by washing in filters in 0.1 x SSC at around 65 ° C. Also contemplated are nucleic acid molecules that hybridize to the polynucleotides of the present invention under less stringent hybridization conditions. Changes in the requirement for hybridization and signal detection are primarily accompanied by manipulation of the formamide concentration (lower percentages of formamide result in decreased requirement); salt conditions, or temperature. For example, lower requirement conditions include an overnight incubation at 37 ° C ie a solution comprising 6X SSPE (20X SSPE = 3M NaCl, 0.2M NaH ₂ PO ₄ , 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 μg / ml DNA blocking salmon sperm; followed by washes at 50 ° C with 1 X SSPE, 0.1% SDS. In addition, to achieve even lesser requirement, washes are made following strict hybridization can be conducted at higher salt concentrations (for example, with 5X SSC). Additional variations in the above conditions can be accompanied through the inclusion and / or replacement of blocking reagents alternatives used to suppress the backup in hybridization experiments. Typical block reagents to be used in the context of the present invention include Denhardt's reagents, BLOTTO, heparin or denatured salmon sperm DNA. The inclusion of specific blocking reagents may require modifications of the above hybridization conditions, due to compatibility problems.

In certain embodiments a nucleic acid molecule or a polypeptide according to the present invention is a recombinant, non-natural molecule. It can be derived from an isolated nucleic acid molecule or polypeptide by a modification, for example, a variation as defined above, for example, substitution, deletion, addition, etc. of one or more nucleotide or amino acid residues. Also in view are fusions with other proteins or sequences that enclose such proteins. Such fusion partners may also comprise functionalities or may constitute tags for purification or secretion signals etc. Also in view are the synthetic nucleic acid molecules in which the nucleic acid molecule sequence derived from Articulospora proliferata has been reconstructed again, for example, based on oligonucleotides. Also, said sequence may comprise a modification or variation compared to the original isolated sequences.

In a further preferred embodiment, the use of codon in a sequence encoding a polygalacturonase as described above can be adapted to any suitable host organism. Accordingly, the use of codon (ie, the frequency of use of 3 nucleotides in an ORF) or use of dicodon (ie the frequency of use of 6 nucleotides in an ORF) can be adapted in view of the frequency of use of codon or dicodon in a target organism to the appropriate or preferred codon or dicodon in said target organism. The use of codon / dicodon can be adapted such that either the codons or more preferred dicodones of said target organism are employed, or 70%, 75%, 80%, 85%, 90%, 95%, or 97% of the most preferred codons or dicodies of said target organism are used, with the permanence of codons or dicodons being the second most preferred codons / dicodones or, in another embodiment, the most preferred third codons / dicodones of said organism. The corresponding frequency of use of an objective organism would be known to the person skilled in the art or can be calculated based on a genomic database and expression data derived from adequate literature sources. A preferred example of an adapted codon use is the sequence SEQ ID NO: 6 comprising a sequence having adapted the codon usage in Pichia pastoris.

In additional embodiments, the present invention contemplates the provision of a nucleic acid molecule according to the invention which includes variants thereof as defined above or the polypeptide according to the invention including variants thereof as defined above isolated from the Articulospora proliferata species. Also note that said nucleic acid molecules or polypeptides can be isolated from organisms of the same family, ie another Articulospora species. Further provided is a nucleic acid molecule according to the invention which includes variants thereof as defined above, or a polypeptide according to the invention which includes variants thereof as mentioned before they are isolated from fungal or bacterial species additional, for example, basidiomycetes or yeasts.

In another aspect of the present invention relates to a vector comprising the nucleic acid molecule as defined hereinbefore, preferably the nucleic acid molecule has the nucleotide sequence sequence of SEQ ID NO: 4, 5 or 6 or fragments or variants thereof as defined hereinbefore or a nucleic acid molecule that represents a fusion of said nucleotide sequence with other sequences or fragments thereof or fusion constructs that include other activities, etc. a suitable vector according to the present invention can be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication deficient. In the latter case, viral spread would generally only occur in complementing the host cells. Preferred are recombinant expression vectors for expression in microorganisms such as bacteria or yeast.

The polynucleotides according to the present invention can be attached to a vector containing a marker selected for propagation in a host. In addition, the polynucleotide insert can be operably linked to an appropriate promoter, such as the PL lambda phage promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and retroviral LTRs promoters or the promoter. induced by methanol AOX1. Other suitable promoters are known to the person skilled in the art. The expression constructs may also contain sites for the initiation of transcription, termination, and, in the transcribed region, a ribosome binding site. In particular, the specific initiation of signals may be required for the efficient translation of inserted coding sequence. These signals include the ATG initiation codon and adjacent sequence. In addition, the initiation codon is very typically in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translation control signals and initiation codons can have a variety of origins, both natural and synthetic. The efficiency of expression can be further enhanced by the inclusion of appropriate transcription elements etc. In a particularly preferred embodiment the encoding portion of the transcripts expressed by the constructs will preferably include a translation of the initiation codon at the beginning and end of the codon (UAA, UGA or UAG) appropriately positioned at the end of the translated polypeptide.

Expression vectors will preferably include at least one selectable marker suitable for the host cell in which it is intended to be used. Such labels include, for example, resistance to dihydrofolate reductase, G418, hygromycin or neomycin with for eukaryotic cell cultures and genes resistant to tetracycline, kanamycin or ampicillin for cultures in E. coli and other bacteria. In addition, selection markers including herpes simplex virus thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase. Methods commonly known in the art of recombinant DNA technology can be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al., Eds, 2007, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York. Preferred vectors for use in bacteria include pQE70, pQE60 and pQE9, available from QIAGEN, Inc .; pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc .; pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc., and pET vectors available from Novagen, or pUC57 available from Gensecript. Among the preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTI and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Preferred expression vectors for use in yeast systems include pYES2, pYDI, pTEFI / Zeo, pYES2 / GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-SI, pPIC3.5K, pPIC9K, and PAO815. Particularly preferred are vectors of expression for Pichia such as pPINK-LC and in particular pPINK-HC available from ThermoFischer Scientific or ppinka-HC available from Invitrogen. Other suitable vectors are known to the person skilled in the art.

In a further embodiment of the present invention it relates to a method for making a recombinant host cell, comprising introducing the nucleic acid molecule or the vector according to the present invention into the host cell. The term "host cell" as used herein refers to a suitable host cell any known to the person skilled in the art. Representative examples of suitable host cells include bacterial cells, such as E. coli, or Streptomyces; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris); insect cells such as Drosophila melanogaster S2 and Spodoptera frugiperda Sf9 cells; and plant cells. Particularly preferred with E. coli host cells, as well as host fungal cells, in particular of the genus Aspergillus, Trichoderma, or Neurospora. Also preferred are yeast host cells of a yeast of the family Kluyveromyces, Saccharomyces, Schizosaccharomyces, Hansenula or Pichia. The most preferred are host cells Pichia pastoris.

In addition, the host cell can be chosen because it modulates the expression of the inserted sequences or modifies the processes of the gene product in the specific form desired. Such modifications (e.g., glycosylation) and processes (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have specific characteristics and mechanisms for the post-translational process and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure correct modification and processing of the expressed protein introduced.

In a particularly preferred embodiment, the yeast Pichia pastoris can be used to express a polypeptide according to the present invention. Pichia pastoris is a methylotrophic yeast, which can metabolize methanol as its sole carbon source. A major step in the route of methanol metabolization is the oxidation of methanol to formaldehyde using O ₂ . This reaction is catalyzed by the enzyme alcohol oxidase. To metabolize methanol as its sole carbon source, Pichia pastoris must generate high levels of alcohol oxidase due, in part, to the relatively low affinity of alcohol oxidase for O ₂ . Consequently, in a growth medium dependent on methanol as the main carbon source, the promoter region of one of the alcohol oxidase (AOX1) genes is highly active. In the presence of methanol, alcohol oxidase produced from the AOX1 gene comprises up to about 30% of the total soluble protein in Pichia pastoris. Thus, a heterologous coding sequence, such as, for example, a ppolinucleotide according to the present invention as defined hereinbefore, under the transcriptional regulation of all or part of the regulatory sequence AOX1 is expressed at exceptionally high levels in yeast Pichia grown in the presence of methanol. In one example, the plasmid vector pPIC9K can be used to express the DNA sequence encoding the polypeptide of the invention, as indicated herein, in a Pichia yeast system essentially as described in "Pichia Protocols: Methods in Molecular Biology, "(DR Higgins and J. Cregg, eds, The Humana Press, Totowa, NJ, 1998). This expression vector allows expression and secretion of the protein of the invention in strong AOX1 promoter virus bound to the alkaline phosphatase secretory signal peptide Pichia pastoris (PHO) (ie, leader) located upstream of a multiple cloning site. Various other yeast vectors could be used in place of pPIC9K, eg, the yeast vectors mentioned hereinbefore, as well as the proposed expression constructs appropriately provided unique signals for transcription, translation, secretion (if desired), and the like, including an AUG in a frame, as required. In a further example, the plasmid vector pPINK-HC available from ThermoFisher Scientific or ppinka-HC available from Invitrogen can be used to express DNA encoding the polypeptide of the invention.

The term "transforming an expression vector into a host cell" as used herein refers to any cell suitable for the introduction of nucleic acid or transformation techniques known to the person skilled in the art. For example, such an introduction can be driven by transfixion, eg, DEAE-dextran-mediated transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer , gene transfer mediated by microcell, lipid-mediated cationia transfection, spheroplasty fusion, etc. In addition, the introduction technique contemplated by the present invention includes contact with defective or attenuated retrovirals, bombardment of macroparticles, the use of covers with lipids or surface receptors or transfection agents, the use of encapsulation in liposomes, microparticles or microcapsules, by example when administering them linked to a peptide that is known to enter the nucleus or to administer it to a peptide that is known to enter the nucleus or when administered bound to a peptide that is known to enter the nucleus, or when administered bound to a binder and subject to receptor-mediated endocytosis, or electroporation. Additional details would be known to the person versed in the material or may be derived from appropriate sources of literature. In a further aspect the present invention relates to a recombinant host cell or obtainable according to the introduction techniques described above, ie transformed with the recombinant expression vector as mentioned hereinbefore. Preferably, such recombinant host cells contain a nucleic acid molecule or a vector according to the present invention. In a particularly preferred embodiment the recombinant host cells express a polypeptide encoded by the nucleic acid molecule according to the present invention. Typically, host cells differ from naturally occurring cells or host cells known for the presence of additional nucleic acid molecules or fragments thereof, which are present in natural contexts or in parent cells or cell lines. For example, recombinant host cells may comprise selection markers, duplicate or multimeric copies of naturally occurring or nucleic acid genes, meteorological elements such as promoter sequences, termination sequences, etc. or genetic identification tags. These elements can preferably be used for the characterization of recombinant host cells and to distinguish it from natural contexts and parent cells or cell lines.

The expression of introduced elements can be controlled by numerous standard genetic tests known to the person skilled in the art. For example, transcription of an introduced nucleic acid can be tested in Northern analysis tests and / or the presence of correspondingly translated polypeptides can be tested Western analysis or enzymatic tests for the functioning of polygalacturonase as described herein.

In another aspect the invention relates to a method for making or producing a polypeptide encoded by a nucleic acid molecule according to present invention as defined herein before comprising: (a) culturing the recombinant host cell as defined herein above as the encoded polypeptide is expressed; and (b) recovering said polypeptide. Method for cultivating recombinant host cells to express encoded polypeptides widely known in the art. For example, if an adjustable promoter is used, an induction agent can be added to the culture or the appropriate conditions, optimal for work can be set, for example, a specific temperature, pH, ionic concentration, etc. The term "recover" as used herein refers to any method suitable for the extraction and / or purification of polypeptides from cells, cell suspensions or cell cultures known to the person skilled in the art. Typical recovery methods include ammonium sulfate or ethanol precipitation, acid extraction, anionic or cation exchange chromatography, phospholcellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. Further details of how the polypeptide can be recovered and / or purified can be derived from the Examples, whose corresponding steps and purification techniques are referenced as specific embodiments of the present invention.

The invention also relates to a composition comprising a polypeptide according to the invention, or obtained by a production method according to the invention. Said polypeptide can be provided as an isolated or basically purified protein in a composition. An isolated and purified polypeptide refers to a polypeptide that is isolated from its native environment and is present in a purified form for further use. An isolated polypeptide may be present in a purified form or may be present in a non-native environment such as in a transgenic or recombinant host cell as described above. By example, an isolated or purified or biologically active protein part thereof is basically free of additional cellular material or culture medium if it is produced according to recombinant techniques or is basically free of chemical precursors or other chemical compounds. A protein that is substantially free of cellular material can be comprised of less than about 70%, 50%, 30%, 20%, 10%, 5% (relative to dry weight) of contaminating protein. If the polypeptide according to the invention or a biologically active fragment thereof is recombinantly produced, the culture medium preferably comprises less than about 70%, 50%, 30%, 20%, 10% or 5% (relative to the weight dry) of chemical precursors or chemicals that do not look like protein. In further embodiments, polygalacturonase compositions of the present invention may be liquid or dry. Liquid compositions preferably comprise the polygalacturonase enzyme in a purified or enriched form. However, auxiliary agents such as a stabilizer and / or glycerol, sorbitol or monopropylene glycol, additives such as salts, sugar, preservatives, agents for adjusting the pH value and proteins can be added. Typical liquid compositions are aqueous or oily suspensions.

Dry compositions can be lyophilized, dry pulverized, granulated or extruded compositions, which can only comprise the enzyme. The dry compositions can be granulates which can easily be mixed with, for example, additional ingredients or components, or which can form a component of a premix. Preferably, the particle size of the enzyme granulate is compatible with the other component of the mixture. This allows safe and useful agents to incorporate enzymes into processed premixes etc. Dry compositions can also comprise other additives such as salts, particularly phosphate salts and their anhydrous forms, and stabilizers such as poly (vinyl pyrrolidone) etc. to regulate certain conditions such as the pH value in the application.

In a further embodiment the polygalacturonase compositions according to the invention additionally comprise an effective amount of one or more enzymes for beverage production or pectin degradation. Such additional enzymes may include one or more pectinases, one or more pectin lyases that clive the glycosidic linkages between the galacturonic residues or beta elimination, or one or more esterases, for example, a pectin methyl esterase diva the methyl group of the residues of galacturonic acid.

The polypeptide of the invention, ie the polygalacturonase as defined hereinbefore or a composition of the present invention as defined hereinbefore can be used for a variety of applications. Examples are applications in processes in which the degradation of pectin is relevant or desirable. Such processes include the production of beverages, such as the production of wine, beer or fruit juice. Wine production includes, for example, the production of red wine, white wine, rosé wine or champagne. The production of fruit juices include, for example, the production of cherry juice, orange juice, apple juice, pineapple juice, prune juice, tangerine juice, banana juice, melon juice and any other juice. , for example, exotic fruits or mixtures of fruits.

Furthermore, it looks at the use of the polypeptide of the invention for the clarification of wort or fruit juice, for example, as mentioned hereinabove. The must may be grape must, for example, a red or bank.

The polygalacturonase as defined hereinbefore or a composition of the present invention as defined herein may accordingly be placed on contact with pectin comprising elements to be treated and incubated at a suitable temperature until the degradation process has begun. It is particularly preferred that said contact be made at low temperatures of about 5 ° C to 20 ° C, preferably at a temperature between 5 ° C and 15 ° C. The use of the polypeptides or compositions may be adapted to the elements that are treated. For example, for the production of white wines a temperature of 8 ° C can be chosen, and for the production of red wines a temperature of 12 ° C can be chosen. The polygalacturonase as defined hereinbefore or a composition of the present invention as defined herein may also be used at different temperatures, for example, even at temperatures beyond 20 ° C or 25 ° C. In further embodiments, the polygalacturonase as defined hereinbefore or a composition of the present invention as defined herein may also be used at different pH values, for example, at a pH of 2.5, 3.0, 3.5 or 4.

In specific aspects, the present invention refers to an enzyme with polygalacturonase activity with high activity in a temperature range between 5 ^Q C-15 ^Q C. Therefore, it is useful in grape must clarification processes- This process can be carried out at low temperatures, for example, at a temperature of 8 ° C - production of white wines, or at a temperature of 12 ° C, in the production of red wines.

The present invention also relates to a liquid formulation or lyophilized powder comprising said enzyme and appropriate excipients, which is useful for degrading pectin at temperatures between 5-15 ^QC . It also refers to the synthetic nucleotide sequence encoding it or encoding a sequence of amino acids having at least about 85% identity, and more preferably, at least 90% homology or percentage identity, and even more preferably, at least 95% homology with the present enzyme, and which is also highly efficient at low temperatures, particularly at a temperature range between 5 ^Q C-15 ^Q C.

Said nucleotide sequence also comprises a variant nucleic acid encoding an enzyme which differs from the present enzyme by no more than about 55 amino acid substitutions, and more preferably, no more than 18 amino acid substitutions, and which maintains the same Polygalacturonase activity at temperatures between 5 ^Q C-15 ^Q C.

The present invention also relates to the method of obtaining the present enzyme by transforming strains of the yeast Pichia pastoris. As well as also, the present invention relates to the recombinant expression vector ppinka-HC (Invitrogen which allows the appropriate transformation of the yeast Pichia pastoris.

To obtain the enzyme with polygalacturonase activity of the present invention which is highly efficient at low temperatures, preferably at temperatures in the range of 5 ^Q C-15 ^Q C, the presence of extracellular pectinases in a collection of isolated yeasts and fungi was evaluated belonging to the genera Cryptococcus, Mrakia, Cysto f lobes id ium, Aureobasidium, Naganishia, Articulospora, Rhodotorula, Rhodosporidium, Leucosporidella, Dioszegia, Sporobolomyces, Udeniomyces and Candida ("Diversity and extracellular enzymatic activities of yeasts isolated from King George Island, the sub-Antartic region, "Carrasco et al BMC Microbiology 2012, 12: 251, http: www.biomedcentral.com/1471-22180/12/251). In the assay, each yeast colony was seeded in plates with pectin, and then incubated at the optimum temperature of each yeast, and subsequently incubated with hexadecyltrimethylammonium bromide (1%). This allowed to select colonies of yeasts and fungi that showed pectinase activity, which was evidenced by the presence of a hydrolysis halo, see figure 11. Thus, the fungus Articulospora proliferata was selected, which presented the highest pectinase enzymatic activity in comparison with the others, also standing out for being able to grow between 4 and 37 ^Q C, and showing the highest hydrolysis halo between 10 and 30 ^Q C .

Subsequently, cultures of the fungus Articulospora proliferata were prepared and its pectinase activity was analyzed. For the development of this analysis, a control culture containing 1% glucose, 0.3% yeast extract, 0.3% malt extract and 0.5% peptone was used, and a culture induced mainly of the same content as the control culture but replacing glucose 1% for a commercial pectin, 1%.

Then, the cultures in late exponential phase of growth were harvested by centrifugation and the supernatant was filtered. The proteins present in the cell-free supernatants were precipitated and then subjected to dialysis by membrane. The protein extracts were then assayed for pectinase activity using the 3,5-dinitrosalicylic acid (DNS) method.

This method is based on the reaction of! DNS with any reducing sugar. The galacturonic acid (a reducing sugar) is the product of degradation of the pectin, which when released reacts with the DNS, causing an increase in absorbance at 540nm. If this value is compared with respect to a previously constructed calibration curve, which has known concentrations of galacturonic acid, see Figure 9, and in this way, it is possible to determine the content of galacturonic acid, in a sample.

Thus, based on this method, a polygalacturonase enzyme activity protocol was developed where the protein extracts are incubated with a commercial pectin !, to then add DNS, and incubate the reactions at 100 ^Q C, and then cool. Finally, the absorbance of the samples was quantified to 540 nm, the concentration of galacturonic acid being estimated by the aforementioned calibration curve, see Figure 9.

As the protein extract obtained from the induced culture medium presented greater pectinase activity in relation to the control culture, such medium was used in the following stages of semi-purification and identification of the pectinase enzyme.

Then, the fungus Articulospora proliferata was grown in the medium of the induced culture, and the total extracellular proteins were precipitated using different percentages of ammonium sulfate. Under these conditions, there was an enrichment in certain proteins in the 60% fraction and this coincided with the presence of enzymatic activity of the samples, see Figure 1.

Due to the lower amount of proteins and the presence of polygalacturonase activity in this fraction, this sample was used for ion exchange chromatography, and thus, obtain the sufficiently pure enzyme for a mass spectrometric analysis.

Proteins were eluted from the chromatographic column using a gradient of 0-1 M sodium chloride. Each fraction obtained was evaluated for enzymatic activity (DNS method) and SDS-PAGE. The results obtained showed that the pectinase was a protein of approximately 40kDa, see Figure 2. This protein band was cut from the SDS-PAGE gel and sent to be analyzed by a peptide fingerprint service (Alphalyse) to determine the complete amino acid sequence of the protein. In this way, the results indicate that the identified protein corresponds to a polygalacturonase enzyme, being able to obtain only fragments of the amino acid sequence.

Sequenced then, the genome of the selected fungus {Articulospora proliferata) to obtain the complete nucleotide sequence of the producer gene of the polygalacturonase enzyme of the present invention, and its possible variants. For this, high quality and pure genomic DNA was obtained and the genome of said fungus was sequenced by means of a sequencer. Then, the obtained DNA was subjected to genome sequencing in Macrogen. In said sequencing, the DNA was fragmented into different sizes by sonication, generating two libraries. The first library that was generated was a "Pair-end" type with inserts of 180 base pairs and the second library corresponded to a multiplexed library with inserts sizes of 3 and 10 Kilobases. From both libraries, the genome of the mentioned fungus was assembled efficiently. From the assembly, Scaffolds containing the genome of this fungus were generated, identifying 1 1,302 genes, and a gene model was elaborated using the Augustus tool available in the Geneious program (Biomatters).

To validate this gene model, the transcriptome of Articulospora proliferata was sequenced by RNA-seq. For this, RNA was extracted from cultures of the fungus grown in the control culture and in the induced culture, both cultures were described in the previous paragraphs. For the extraction of RNA, the "cell pellets" obtained from late growth exponential phase cultures were collected by centrifugation, which were subsequently washed with distilled water, and then, the RNA extracted by the kit Ribopure Yeast RNA purif cation (Ambion).

The RNAs thus obtained were subjected to electrophoresis (see Figure 3) and their absorbance at 260 and 280 nm was determined, calculating the A260 / A280 ratios. Then, the RNAs that showed the lowest degradation and absorbance ratios 260/280 greater than 1.8, were subjected to RNA-seq in the company Macrogen, and in addition, the concentration of purified RNAs and total RNA was determined. The degradation state of the RNAs was determined essentially by means of a chip that separated them, and subsequently, a library of cDNAs was generated from these RNAs. This library was completely sequenced through the Ilumina Hi-seq2000 sequencer. Through this methodology, mRNAs were identified and, therefore, the proteins that were expressed in the induced condition (induced culture) and in the control condition (control culture). Then, the proteome of said fungus was analyzed by the blastp tool (NCBI) to identify the possible polygalacturonase enzyme it expresses.

After this analysis, the presence of polygalacturonase was observed in the sequenced genome, which was previously identified by mass spectrometry. An alignment was made between the amino acid sequences of the peptides obtained by this approach and the amino acid sequences of the proteome of the fungus, see Figure 12. In this way, it was possible to identify the complete amino acid sequence of the present polygalacturonase, which is indicated as SEQ ID No.:1 in the present sequence listing, and in Figure 4, it is represented graphically the complete amino acid sequence of 369 amino acids. With these results it can be concluded that the enzyme found corresponds to the enzyme that was being sought.

Then, from the information derived from the RNA-seq experiments and the sequencing of the genome, a final gene model was elaborated for the gene that codes for this enzyme (see Figure 5). Then, we searched for and identified the complete nucleotide sequence of the cDNA, see SEQ ID No.:5, which codes for said mature polygalacturonase (without export signal), using tools available in the Geneious program (Biomatters).

A nucleotide sequence of 1066 bp, which codes for polygalacturonase, was chemically synthesized by synthesis of Genescript oligonucleotides. For this, we designed the mature cDNA that would code for the present enzyme with polygalacturonase activity. First, the first 57 nucleotides were deleted from the original sequence, since they code for a peptide exported to the extracellular medium, a signal peptide that is necessary to export the enzyme to the extracellular medium. As the ppinka-HC vector that was used to express the present enzyme with polygalacturonase activity has the export signal of Saccharomyces cerivisiae alpha pheromone, it was not necessary to maintain the native signal. Then, the sequence of the cDNA that would code for the present enzyme with polygalacturonase activity was optimized, changing the use of native codons by the use of the codons of the yeast Pichia pastoris, in which this enzyme was later to be expressed. In this way, a DNA fragment was obtained which is called with the arbitrary name "pect" .. In this way, the sequence ID NO 5 corresponds to the original nucleotide sequence and the sequence ID NO 6 corresponds to the optimized nucleotide sequence by using codons of Pichia pastoris.

Also, two cutting sites for two restriction enzymes were added to this pect fragment. At the 5 'end, a first cut site was added for the restriction enzyme Mlyl, the nucleotide sequence GAGTCCATAG, which corresponds to a recognition site for the restriction enzyme Mlyl. Whereas at the 3 'end, a second cut-off site was added for the restriction enzyme Kpnl, the nucleotide sequence GGTACC, which corresponds to the recognition site for the restriction enzyme Kpnl.

In this way, the nucleotite sequence that was synthesized is the sequence SEO ID No.:4, a cDNA of 1066 base pairs that codes for the enzyme with polygalacturonase activity of the invention but that is capable of being expressed in the yeast Pichia pastoris by being cloned into the appropriate expression vector. The resulting fragment (from now pect2) was cloned into the cloning vector pUC57, by Genscript. The resulting plasmid (from now pUC57-pect2) was subsequently transformed into electrocompetent cells of the E. coli DH5a bacterium by electroporation and following the widely known and published protocols, specifically, in Sambrook J and Rusell DW. 2001 Molecular cloning. A laboratory manual, Cold Spring Harbor Laboratory Press.

10 transformant clones were selected on LB-agar plates (tryptone 1%, yeast extract 0.5%, sodium chloride 0.5% and agar 1.5%) supplemented. The selection of clones was by resistance to ampicillin and then, it was confirmed by PCR. Subsequently, plasmidial DNA was obtained from clone liquid cultures using the GeneJet plasmid Miniprep kit (Thermoscientific). It is important to note that the selection of the clone is done freely since in a cloning stage, it is not relevant which clone is selected. Then, this plasmidial DNA was digested with the restriction enzymes Mlyl and Kpnl (Thermoscientific) according to the manufacturer's specifications, and subsequently, the DNA fragments obtained, the linearized vector and the pect fragment were visualized by means of agarose gels (buffer TAE 1%, agarose 1% and Safe view nuclei acid stain 1 X), see Figure 13.

The 1050 bp DNA fragment (pect) was purified from gels by the genclean method (Sambrook J and Rusell DW, 2001. Molecular cloning A laboratory manual, Cold Spring Harbor Laboratory Press). In parallel, the ppinka-HC expression vector (Invitrogen) was digested for yeast with the restriction enzymes Stul and Kpnl (Themoscientific) according to the manufacturer's specifications. Then, the pect fragment was ligated to the vector ppinka-HC, digested through the enzyme T4 DNA ligase (Thermoscientific), using the manufacturer's specifications. Competent cells of the E. coli Top10 bacteria (Invitrogen) were transformed by electroporation according to the supplier's specifications, with the ppinkaHC-pect vector obtained as described above.

Clones were selected that were able to grow in a minimal culture medium (2% glucose, 20% potato infusion and 1.5% agar), in the absence of adenine supplementation, which was confirmed by PCR, using as a template genomic DNA obtained from the transformant clones. In the PCR reactions, the Pectfwd (5 ^' - GCACCTACAGTCTCATCATTG-3 ^' ) and Pectrev (5 ^' -) starters were used.

GCAGGAAGCAGGGGATGGGAA-3 ^' ), which hybridize with the nucleotide sequence of the pect fragment. All PCR reactions were performed with the enzyme Taq DNA polymerase (Thermoscientific), according to the manufacturer's specifications.

Plasmid DNA was extracted from 4 clones by the GeneJet Plasmid Miniprep kit (Thermoscientific). Then, the obtained plasmidial DNA was digested at 37 ^Q C with the restriction enzyme Aflll (Thermoscientific), following the protocol suggested by the manufacturer. The linearized vector (ppinkaHC-pect) was purified from the digestion solution by the addition of 3M sodium acetate and ethane! 100% Then, the solution was centrifuged to collect the DNA, and then, it was washed with 80% ethanol. The DNA was allowed to dry at room temperature and then suspended in sterile water. Finally, the linearized vector (ppinkaHC-pect) to be transformed was subjected to membrane dialysis with water, using VSWP membranes of pore size 0.025μιη (Millipore, Merck), in order to eliminate the salts present in the solution.

In parallel, electrocompetent cells of the yeast Pichia pastoris, strain Pichiapink strain 1 flnvitrogenj, which planted with a sterile loop, and a culture of the strain Pichiapink strain 1 on a YPD plate (glucose 2%, extract of yeast 1% were prepared , peptone 2% and agar 2%), was incubated. Then, with One of the colonies formed was inoculated with a flask containing liquid YPD, and subsequently, inoculated with this culture, a flask containing in its interior, YPD liquid medium (DO6Q0 ^ 0.2).

This crop was grown, monitoring the optical density until reaching an OD600 between 1.3 and 1.5. Then, the cells were centrifuged and suspended in sterile ice water, and then subjected to centrifugation again after being suspended in sterile ice water. Said cells were centrifuged once more and suspended in ice sorbitol to be centrifuged and suspended again in sorbitol.

The cells were transformed with the linearized vector obtained (ppinkaHC- pect) as described above. For this, the electrocompetent cells of the strain Pichiapink strain were mixed with this linearized vector in an electroporation cuvette. The mixture was incubated on ice and subjected to an electrical pulse. Immediately after the pulse, cold YPDS liquid medium (glucose 2%, yeast extract 1%, sorbitol 18.2% and pepona 2%) was added to the cuvette and the transformed cells were incubated and then seeded in a selective medium (infusion). of dads 20%, glucose 2% and agar 1.5%) and incubate again.

Only the colonies of the transformed cells that acquire said linearized vector containing the gene coding for adenine, were able to grow in the selective medium, since the strain that was transformed, is not able to grow in a medium with absence of adenine (the PDA medium does not contain this requirement).

In addition, the parental strain, Pichiapink strain 1, has a reddish coloration since it is not capable of metabolizing a compound due to the absence of a gene of the biosynthetic adenine route. This corresponds to a second method of selection, since the transformant colonies acquired a white color unlike the parental strain, see figure 11. For these reasons, as a first selection method, 4 transformant white colonies were isolated, and PDA plates were replated.

Then, the 4 clones mentioned above were extracted genomic DNA using the Wizard Genomic DNA Purification kit (Promega) according to the manufacturer's specifications. Finally, these genomic DNAs were used as a template to perform PCR reactions following the same steps of the PCR reactions described above, where the presence of the pect fragment encoding the present enzyme with polygalacturonase activity, sequence SEQ ID No. :two. These reactions were performed with the Pectfwd primers (5 ^' -GCACCTACAGTCTCATCATTG-3 ^' ) and Pectrev (5 ^' - GCAGGAAGCAGGGGATGGGAA-3 ^' ) which are complementary to the pect fragment and which do not amplify any endogenous genes of the strain Pichiapink strain 1, Therefore, the positive presence of amplicons (the 4 clones showed amplification) was indicative of a successful transformation.

To demonstrate the expression of the enzyme polygalacturonase in transformed yeast Pichia pastoris, a colony was grown from each of the 4 clones in BMGY medium (yeast extract 1%, peptone 2%, potassium phosphate buffer 100mM pH 6.0, YNB 1 .34%, biotin 0.0004% and glycerol 1%), at 30 ^Q C and with agitation. Then, transformed Pichia pastoris yeast was collected by centrifugation, and suspended in BMMY medium (composition identical to the BMGY medium, where only glycerol is replaced by 0.5% methanol). The protein composition of the supernatant was analyzed by SDS-PAGE. The cell pellets obtained were lysed using a break solution (50 mM sodium phosphate pH 7.4, 1 mM PMSF, 1 mM EDTA and 5% glycerol) and vortex. This fraction of intracellular proteins and the cell-free supernatant of each clone were separated by SDS-PAGE and visualized through silver staining. With this analysis, the clone that expressed the highest concentration of the recombinant protein in the extracellular medium was determined, see Figure 6. As seen in Figure 6, clone number 1 shows a greater intensity of a band of protein in relation to clones 2, 3 and 4. It is for this reason that this clone was chosen as the recombinant strain for the purification of the enzyme. Therefore, subsequent trials were performed with this clone No.1.

In the purification of the polygalacturonase enzyme, the supernatant of the culture from clone number 1 was filtered, and using different cuts of ammonium sulfate (20, 40, 60 and 80%), the proteins present in this were precipitated. cell-free supernatant. And then, each of the protein fractions was dialyzed against a buffer to then quantify the protein content and determine the enzymatic activity by the DNS method, described above. It was determined that the 80% fraction presented the highest levels of enzymatic activity.

Then the proteins present in this fraction were separated by anion exchange chromatography, which was balanced with a buffer, and the elution of the proteins, was carried out through a gradient line! of NaCl (0.1 M). Each fraction was monitored at 280 nm to evaluate the protein content. The fractions that presented absorbance at 280 nm were analyzed for enzymatic activity by the DNS method.

In this way it was possible to identify the fractions that contained the enzyme polygalacturonase. The fractions containing the polygalacturonase enzyme were concentrated by centrifugation systems and the resulting proteins were separated, through gel filtration. For this, a high resolution chromatographic column was equilibrated with a 50mM phosphate buffer, 0.15M NaCl, pH 7.0, and then, the protein extract to be separated was loaded. The fractions obtained were monitored spectrophotometrically at 280 nm and each fraction that presented proteins was evaluated for enzymatic activity. In this way, it was possible to obtain the purified polygalacturonase enzyme with a level of purity above 95%, see Figure 7. To evaluate the efficiency of the present polygalacturonase enzyme under laboratory and semi-industrial conditions, a commercial pectin solution was mixed with a solution of the present purified polygalacturonase enzyme or with one of a commercial polygalacturonase enzyme. Assays were incubated at 37 ⁹ C and then tested, polygalacturonase enzyme activity by DNS method, the same enzyme activity assay described in the previous paragraph but mimicking industrial conditions was also carried out . For this, white wine must (pH 3.0) was incubated with a solution of the present purified polygalacturonase or with a solution of a commercial polygalacturonase enzyme. The assays were incubated at 15 ^g C and 5 ^{C '} C, and then, the solutions were assayed for the enzymatic activity polygalacturonase by the DNS method, see figure 15.

The following are examples of embodiments for the present invention as described above:

Examples: Obtaining a highly efficient polygalacturonase at temperatures between 5-15 ^e C

Example 1: Evaluation of the production of pectinase enzymes by biological material from Antarctica

The presence of extracellular pectinases was evaluated in a collection of biological material isolated from Antarctica, mainly selected from the group consisting of: Cryptococcus terricola, Cryptococcus gastrícus, Cryptococcus victoriase, Cryptococcus gilvescens, Mrakia robertii, Mrakia blollopis, Mrakia frida, Mrakia psychrophila, Rhodotorula glacialis, Rhodotorula laryngis, Dioszegia crocea, Dioszegia fristingensis (now, Articulosporta proliferata), Sporidiobolus salmonicolor, Leucosporidella creatinivora, Candida sake. In the test, each colony of the aforementioned biological material was seeded in YM-pectin plates (product Sigma-Aldrich P91 35, commercial pectin, CAS number 9000-69-5, EC number 232-553-0, MDL number MFCD00081 838) 1%, yeast extract 0.3%, malt extract 0.3%, peptone 0.5% and agar 1.5%, and then, incubated for 3 days, each colony to the optimal growth temperature of each yeast ("Diversity and extracellular enzymatic activities of yeasts isolated from King George Island, the sub-Antartic region", Carrasco et al BMC Microbiology 2012, 12: 251, http: www.biomedcentral.com/1471 - 2180/12/251), that is, 4-22 ^Q C for C. sake, Cr. Gastrícus, Cr. Gilvescens, Le. creatinivora, M. blollopis; 4-15 ^Q C for Cr. Victoriae, M. gelia, M. psychrophila, M. robertii, Rh. Glacialis and 4-30 ^Q C for Rh. laryngis, Sp. salmonicolor.

The plates were then incubated with 1% hexadecyltrimethylammonium bromide for 10 minutes. They were selected from the colonies that showed positive pectinase activity, which was evidenced by the presence of a hydrolysis halo, see figure 14. The fungus called Articulospora proliferata presented pectinase enzymatic activity and stood out above the others because: (1) it is capable of growing between 4 and 37 ^Q C, and (2) presented the largest hydrolysis rings between 10 and 30 ^Q C. This yeast was the one selected for future characterizations regarding the production of pectinase enzymes.

Example 2: Analysis of the pectinase activity of extracellular protein extracts obtained from Articulospora proliferata cultures.

Articulospora proliferata was grown in two liquid culture media: (1) control culture (glucose 1%, yeast extract 0.3%, malt extract 0.3% and peptone 0.5%) and (2) induced culture (identical to the previous one only) replaced 1% glucose by YM-pectin of Example 1, 1%). A culture of Articulospora proliferata in late exponential phase of growth (OD ₆ oonm = 15), was harvested by centrifugation at 5,000g for 10 minutes and the supernatant was filtered through 0.45μιη filters (Millipore, Merck). The proteins present in these cell-free supernatants were precipitated with 80% ammonium sulfate and then subjected to dialysis using 10kDa dialysis membranes (Sigma Aldrich) and 20mM Tris-HCl buffer pH 7.0. The protein extracts obtained from the control culture and the induced culture were assayed for pectinase activity using the DNS method, so that 50μΙ of protein extracts were incubated with 50μ! of YM-pectin, for one hour at 22 ^e C. Then, 10ΟμΙ of DNS was added, the reactions were incubated at 100 ³ C for 10min and then, they were cooled on ice for 5 minutes. Finally, the absorbance of the samples was quantified at 540nm, estimating the concentration of galacturonic acid, using the previously constructed calibration curve, see Figure 9. The protein extract obtained from the inducer medium showed higher pectinase activity in relation to the control culture. Therefore, this was the means used for the following stages.

Example 3: Semi-purification and identification of the pectinase enzyme from Articulospora proliferata.

Articulospora proliferata was grown in the inducer culture medium described above and total extracellular proteins were precipitated using different percentages of ammonium sulfate (40, 60 and 80%). Under these conditions, there was an enrichment in certain proteins in the 60% fraction and this coincided with the presence of enzymatic activity of the samples, see Figure 1. Due to the lower amount of proteins and the presence of polygalacturonase activity in this fraction, this sample was used for the following stages. Then, by means of ion exchange chromatography, the sufficiently pure enzyme was obtained for mass spectrometry analyzes. For this, a chromatographic column of diethylethylaminoethyl, DEAE-sephadex® (General Electrics) was equilibrated with 20 mM Tris-HCl buffer, pH 8.0 and 2 ml of the 60% fraction was loaded in this column. Proteins were eluted from the column using a gradient of 0-1 M sodium chloride. 141 fractions were selected and evaluated for enzymatic activity (DNS method) and SDS-PAGE, see table 1. The results obtained showed that the pectinase of interest is a protein of approximately 40kDa, see Figure 2. This protein band was cut from the SDS-PAGE gel and analyzed by the peptide fingerprint service (Alphalyse), in order to determine the complete amino acid sequence of the protein. The results indicate that the identified protein corresponds to an enzyme with polygalacturonase activity. However, it was not possible to obtain the complete amino acid sequence but only the following fragments: VIFSGTTTFGYK and SGAVVQNQDDC.

Table 1 :

Activity Activity Activity

FracciomAU enzymatic FracciomAU enzymatic FracciomAU enzymatic nes (DO280nm) (DNS) nes (DO280nm) (DNS) nes (DO280nm) (DNS)

1 -2.63 0.085 48 9.82 1, 627 95 5.04 0.096

2 -0.04 0.085 49 10.03 0.865 96 4.54 0.089

3 -0.07 0.085 50 10.12 0.682 97 4.08 0.096

4 -0.11 0.085 51 9.65 0.286 98 3.64 0.089

5 -0.13 0.085 52 8.83 0.292 99 3.31 0.096

6 -0.16 0.085 53 7.63 0.18 100 3.05 0.048

7 -0.19 0.085 54 6.72 0.154 101 4.89 0.05

8 -0.21 0.085 55 6.90 0.126 102 4.07 0.05

9 -0.22 0.085 56 8.54 0.1 1 103 3.89 0.052

10 -0.23 0.085 57 1 1, 03 0.098 104 4.17 0.051

1 1 -0.25 0.085 58 12.54 0.089 105 4.50 0.058 -0.26 0.085 59 13.06 0.094 106 4.31 0.051

-0.26 0.085 60 13.73 0.098 107 3.58 0.053

-0.28 0.085 61 15,07 0.633 108 2.78 0.048

-0.30 0.085 62. 17.32 0.102 109 2.19 0.05

-0.32 0.085 63 20.38 0.095 110 1.84 0.054

-0.32 0.085 64 24.06 0.099 111 1.64 0.051

-0.34 0.085 65 27.89 0.095 112 1.49 0.064

-0.35 0.085 66 31.38 0.109 113 1.37 0.15

-0.34 0.085 67 34.34 0.098 114 1.27 0.1

-0.36 0.085 68 36.68 0.102 115 1.18 0.093

-0.36 0.085 69 38.34 0.096 116 1.08 0.15

-0.37 0.085 70 39.30 0.094 117 1.00 0.051

-0.38 0.085 71 39.51 0.088 118 0.93 0.048

-0.38 0.085 72 39.21 0.098 119 0.87 0.05

-0.39 0.085 73 38.66 0.092 120 0.83 0.052

-0.39 0.085 74 37.44 0.097 121 0.85 0.055

-0.39 0.085 75 35.06 0.104 122 1.01 0.066

-0.39 0.085 76 32.04 0.092 123 1.23 0.059

-0.39 0.101 77 28.90 0.092 124 1.34 0.05

-0.40 0.089 78.25.94 0.093 125 1.23 0.05

-0.40 0.085 79 23.31 0.083 126 1.08 0.05

-0.39 0.092 80 21.00 0.105 127 0.99 0.05 34 -0.35 0.085 81 19.05 0.103 128 0.94 0.05

35 -0.18 0.088 82 17.28 0.089 129 0.81 0.05

36 0.12 0.101 83 15.56 0.094 130 0.63 0.05

37 0.52 0.097 84 13.90 0.089 131 0.49 0.05

38 1, 43 0.1 13 85 12.43 0.096 132 0.41 0.05

39 2.14 0.27 86 1 1, 30 0.089 133 0.36 0.05

40 4.94 1, 771 87 10.52 0.096 134 0.31 0.05

41 15.53 2.508 88 9.98 0.089 135 0.29 0.05

42 17,47 2,449 89 9,31 0,06 136 0,28 0,05

43 14.71 2,344 90 8.40 0.06 137 0.27 0.05

44 13.20 2.519 91 7.54 0.06 138 0.30 0.05

45 12.26 2.303 92 6.78 0.050 139 0.40 0.05

46 1 1, 20 2,201 93 6.1 1 0.05 140 0.55 0.05

47 10.19 1, 683 94 5.58 0.04 141 0.67 0.05

Sequenced, the genome of the fungus Articulospora proliferata in order to obtain the complete sequence of the gene and its possible variants.

Example 4: Obtaining the nucleotide and amino acid sequences of the enzyme polygalacturonase.

To obtain the nucleotide and amino acid sequences, the genome of Articulospora proliferata was sequenced using an Ilumina HiSeq2000 sequencer. For this, high-quality and pure genomic DNA was obtained from said fungus, using the Wizard® Genomic DNA Purification Kit (Promega). Then, 550μ9 of the DNA obtained was subjected to genome sequencing and the DNA was fragmented into different sizes by sonication, generating two libraries. The first library that was generated was a "Pair-end" type with inserts of 180 base pairs (bp) and the second corresponded to a multiplexed library with inserts sizes of 3 and 10 Kilobases. The use of both libraries, allowed to assemble the genome efficiently.

Thus, Scaffolds containing the genome of this fungus were generated, identifying 1 1,302 genes and a gene model was elaborated for each of them. To validate this gene model, the transcriptome of said gene was sequenced by RNA-seq. For this, RNA was extracted from cultures of said fungus grown in two experimental conditions: a control culture (glucose 1%, yeast extract 0.3%, malt extract 0.3% and peptone (0.5%) and a induced culture (identical to the previous one only that the glucose was replaced by pectin 1%).

The "cell pellets" obtained from late exponential growth phase cultures (OD ₆ oonm = 15), grown in both conditions (control culture and induced culture) were collected by centrifugation (5,000g per 10min). These cultures were washed with distilled water, and subsequently, the RNA from each condition was extracted. The RNAs obtained were subjected to electrophoresis, see Figure 3 and determination of their absorbance at 260 and 280 nm, respectively, see Table 2.

Table 2 Reasons A260 / A280> 1, 8 for samples of extracted RNAs, concentration of purified RNAs and amount of total RNA.

The RNAs that showed the lowest degradation (see Figure 3, lane D) and absorbance ratios 260/280 greater than 1.8, were subjected to RNA-seq. He determined, the degradation of the RNAs, and subsequently, those that presented less degradation were used to generate a library of cDNAs. This library was completely sequenced through an Ilumina HiSeq2000 sequencer. By means of this methodology, the mRNA was identified and, therefore, the polygalacturonase that is expressed in the condition of the induced culture and in the condition of the control culture.

With this information, the proteome of said fungus was analyzed by the blastp tool (NCBI) in order to identify the possible enzyme with expressed polygalacturonase activity. This analysis showed the presence of the enzyme with polygalacturonase activity in the sequenced genome, which was previously identified by mass spectrometry. An alignment was made between the amino acid sequences of the peptides obtained by this approach and the amino acid sequences of the polygalacturonase identified in the genome, see Figure 12.

In this way, it was possible to identify the complete amino acid sequence of the enzyme with polygalacturonase activity, which is indicated as SEQ ID No.:1 in the sequence listing, and in Figure 4, the complete amino acid sequence of 369 is graphically represented. amino acids. With these results, it can be concluded that the enzyme found corresponds to the enzyme that was being sought.

Then, from the information derived from the RNA-seq, the complete nucleotide sequence of the cDNA coding for said enzyme with mature polygalacturonase activity (without export signal), SEQ ID. 5 of the sequence listing, was searched using the program Geneious (Biomatters). In parallel, a final gene model was elaborated for the gene that codes for this enzyme, see figure N ^Q 5.

Example 5: Cloning of the gene coding for polygalacturonase. The 1 066 bp nucleotide sequence, SEQ ID NO.:4, which codes for the enzyme with polygalacturonase activity of the invention, was chemically synthesized by synthesis of Genescript oligonucleotides. For this, we designed the mature cDNA that encodes this enzyme, eliminating from the original sequence, the first 57 nucleotides, then, optimizing the sequence of the cDNA that would code for this enzyme with polygalacturonase activity, by changing the use of native codons by the use of the codons of the yeast Pichia pastoris, in which was to be expressed later, the present enzyme with polygalacturonase activity.

Also, two cutting sites for two restriction enzymes were added to this DNA fragment (pect2). At the 5 'end, a first cut site was added for the restriction enzyme Mlyl, the nucleotide sequence GAGTCCATAG, which corresponds to a recognition site for the restriction enzyme Mlyl. Whereas at the 3 'end, a second cut-off site was added for the restriction enzyme Kpnl, the nucleotide sequence GGTACC, which corresponds to the recognition site for the restriction enzyme Kpnl.

In this way, the nucleotide sequence that was synthesized is the sequence SEQ ID No.:4, a cDNA of 1066 base pairs which codes for the enzyme with original polygalacturonase activity but which is capable of being expressed when it is cloned into a vector of appropriate expression in the yeast Pichia pastoris. The resulting pect fragment was cloned into the cloning vector pUC57, by Genscript. The resulting plasmid pUC57-pect2 was subsequently transformed into electrocompetent cells of the bacterium E. coli DH5a by electroporation according to previously published protocols (Sammbrook and Russell, 2002).

Transformant clones were selected on LB-agar plates (tryptone 1%, yeast extract 0.5%, sodium chloride 0.5% and agar 1.5%) supplemented with 100 g / ml, according to its resistance to ampicillin, which was confirmed by PCR. Subsequently, plasmidial DNA was obtained from liquid cultures of transforming clone number 4 by the GeneJet plasmid Miniprep kit (Thermoscientific), which was later digested with the restriction enzymes Mlyl and Kpnl (Thermoscientific) according to the manufacturer's specifications, and In this way, the fragments obtained by means of agarose gels (shock absorber TAE 1%, agarose 1% and Safe view nuclei acid stain 1 X) were visualized, see Figure 1.

The 1050 bp DNA fragment (pect) was purified from gels by the genclean method (Sambrook J and Rusell DW, 2001. Molecular cloning A laboratory manual, Cold Spring Harbor Laboratory Press). In parallel, the ppinka-HC expression vector (Invitrogen) was digested for yeast with the restriction enzymes Stul and Kpnl (Themoscientific) according to the manufacturer's specifications. Then, the pect fragment was ligated to the vector ppinka-HC, digested through the enzyme T4 DNA ligase (Thermoscientific), using the manufacturer's specifications. Competent cells of the E. coli TopW bacterium (Invitrogen) were transformed by electroporation according to the supplier's specifications, with the ppinkaHC-pect vector obtained as described above.

Transformant clones were selected on LB-agar plates (tryptone 1%, yeast extract 0.5%, sodium chloride 0.5% and agar 1.5%) according to their ability to grow in a culture medium supplemented with ampicillin. The results were confirmed through PCR using genomic DNA obtained from these transformant clones as a template and verified by PCR reactions using the Pectfwd primers (5 ^' - GCACCTACAGTCTCATCATTG-3 ^' ) and Pectrev (5 ^' -

GCAGGAAGCAGGGGATGGGAA-3 ^' ), those that hybridize with the sequence nucleotide pect. The enzyme Taq DNA polymerase (Thermoscientific) was used in the PCR reactions, according to the manufacturer's specifications. Example 6: Obtaining transformant strains of the yeast Pichia pastoris.

Plasmid DNA was extracted from the 4 selected clones by the GeneJet Plasmid Miniprep kit (Thermoscientific). Then, 10μ of the plasmidial DNA (200μΙ) obtained was digested at 37 ^Q C for 3 hours with the restriction enzyme Aflll (Thermoscientific), following the protocol suggested by the manufacturer. The linearized vector (ppinkaHC-pect) was purified from the digestion solution by the addition of 20μ! of sodium acetate 3 and 500μΙ of 100% ethanol. Then, the solution was centrifuged at 14,000g for 10 minutes to collect the DNA, which was then washed with 400μΙ of 80% ethanol, and allowed to dry at room temperature and then suspended in 10μ! of sterile water. Finally, the DNA fragment to be transformed was dialyzed with water using VSWP membranes of 0.025μϊϊΐ pore size (Millipore, Merck) in order to eliminate the salts present in the solution.

Separately, electrocompetent cells of the yeast Pichia pastoris, strain Pichiapink strain 1 lnvitrogen) were prepared. For this, a sterile loop was planted, a culture of the strain Pichiapink strain 1 on a YPD plate (glucose 2%, yeast extract 1%, peptone 2% and agar 2%), and incubated at 30 ^to C 3 days. Then, with one of the colonies, a 125ml flask containing 10ml of YPD liquid medium was inoculated. At the end of the incubation period, a 1 L flask containing 10 Qmi of liquid medium YPD (DO600 = 0.2) was inoculated with this culture. This culture was grown for one day at 30 ^Q C, the optical density being monitored until reaching an OD800 between 1.3 and 1.5. Then, the cells were centrifuged at 1, 500g at 4 ^S C for 5 minutes and then, said cells were suspended in 250ml of sterile ice water to centrifuge again at 1, 500g at 4 ^e C for 5 minutes, but before Suspending them in 50ml of sterile ice water. Said cells were again centrifuged and suspended in 10 ml of ice cold 1 M sorbitol, and once again, they were centrifuged and suspended in 300 μΙ of 1 M sorbitol.

These cells were used on the same day to be transformed with the linearized DNA fragment (ppinkaHC-pect) obtained in the previous stage. For this, 80μΙ of the electrocompetent cells of the strain Pichiapink sírain 1 obtained in the previous step were mixed in a 0.2μ electroporation cuvette with the 10μΙ of said linearized DNA fragment. The mixture was incubated on ice for 5 minutes and then, an electric pulse (200V, 25μF, 200Ω) was given on a GenePulserXcell electroporator (Biorad). Immediately after the pulse, 1 ml of cold YPDS liquid medium (glucose 2%, yeast extract 1%, sorbitol 18.2% and pepona 2%) was added to the cuvette and these eicctrocompetent cells were incubated at 30 ^e C for 2 hours . After incubation, seeded 300μΙ mixture on selective (dads infusion of 20% glucose and 2% agar 0.5% 1) medium and incubated at 30 ^Q C for 10 days.

In addition, the parental strain, Pichiapink sírain 1, which has a reddish coloration that differentiates it from the transformant colonies that acquire a white color, which was used as the first selection method, and allowed to isolate 4 transformant white colonies, which were re-sown on PDA plates, see figure 1 1.

Then, the genomic DNA of said clones was extracted, using the Wizard Genomic DNA Purification kit (Promega) according to the manufacturer's specifications. Said genomic DNA is then used as a template to carry out PCR reactions following the same steps of the PCR reactions described above, where the presence of the DNA fragment (pect) coding for polygalacturonase was checked. These reactions were carried out with the Pectfwd starters (5 ^' -GCACCTACAGTCTCATCATTG-3 ^' ) and Pectrev (5 ^' - GCAGGAAGCAGGGGATGGGAA-3 ^' ) which are complementary to the pect DNA fragment.

Example 7: Expression of the enzyme polygalacturonase in the yeast pichia pastoris.

A colony of each of the 4 previous clones was grown in 10ml of BMGY medium (yeast extract 1%, peptone 2%, buffer potassium phosphate 100mM pH 6.0, YNB 1.34%, biotin 0.0004% and glycerol 1%), for one day at 30 ^Q C and 300rpm. Then, the Pichia pastoris cells were collected by centrifugation at 1, 500g for 5 minutes, and suspended in BMMY medium (composition identical to the BMGY medium, only the glycerol was replaced by 0.5% methanol). Cultures were grown for 24 hours at 30 ^Q C and then, these cells were harvested and the protein composition of the supernatant was analyzed by SDS-PAGE.

The Pichia pastoris cell pellets obtained were lysed using a rupture solution (50mM sodium phosphate pH 7.4, 1mM PMSF, 1mM EDTA and 5% glycerol) and vortex. This fraction of intracellular proteins and the cell-free supernatant of each clone were separated by SDS-PAGE and visualized through silver staining.

With this analysis, the clone that expressed the highest concentration of the recombinant protein to the extracellular medium was determined, see Figure 6. As seen in Figure 6, clone number 1 presents a higher intensity of a protein band in relation to the clones 2, 3 and 4. It is for this reason that this clone was chosen as the recombinant strain for the purification of the enzyme. Therefore, subsequent trials were performed with this clone No.1

Example 8: Purification of the enzyme polygalacturonase.

One liter of culture supernatant was obtained from clone number 1, which better expresses the present enzyme with polygalacturonase activity, using growth conditions identical to the previous example. Then, this Supernatant was filtered through 0.45μηΊ filters (Millipore, Merck) with e! goal of eliminating any cellular debris. Subsequently, the proteins present in this cell-free supernatant were precipitated, using different cuts of ammonium sulfate (20, 40, 60 and 80%). Each of the protein fractions was dialysed against a 20mM Tris-HCl buffer using pore size 10kDa dialysis bags (Sigma Aldrich). Then, the protein content was quantified by the BCA Kit (Thermo Scientiic) and the enzymatic activity was determined by the DNS test. It was determined that the fraction 80% presented the highest levels of enzymatic activity.

The proteins present in this fraction were then separated by DEAE sephadex anion exchange chromatography (General Electrics) in an AKTA prime purification system (General Electrics). For this, the column was equilibrated with a buffer solution 20m Tris-HCI pH 8.0 and the elution of the proteins was carried out through a linear gradient of NaCl (0.1 M). Each fraction was monitored at 280 nm to evaluate the protein content. The fractions that presented absorbance at 280 nm were analyzed for enzymatic activity by the DNS method, see table 1

In this way, it was possible to identify the fractions that contained the enzyme with polygalacturonase activity. The positive fractions were concentrated by Amicons centrifugation systems of 10kDa (Millipore, Merck) and the resulting proteins were separated, through gel filtration. For this, a Superdex 75 10 / 300GL (General Electrics) column was equilibrated with a 50mM phosphate buffer, NaCl 0.15M, pH 7.0 and then, 500μΙ of the protein extract to be separated was loaded. The fractions obtained were monitored spectrophotometrically at 280 nm and each fraction that presented proteins was evaluated for enzymatic activity. In this way, the enzyme was obtained with purified poiigalacturonase activity with a purity level on e! 95%, see Figure 7,

Example 9: Evaluation of the poiigalacturonase enzyme in laboratory and semi-industrial conditions.

To evaluate the efficiency of the present enzyme in laboratory and semi-industrial conditions. First, 300μΙ of a 10mg / ml commercial pectin solution, the same commercial pectin as the previous examples, was mixed with 300μΙ of a 0.25mg / ml solution of the present purified poiigalacturonase enzyme or with 300μΙ of a 0.25mg / ml solution of a commercial poiigalacturonase enzyme (Lafazym®, Laffort) The assays were incubated for one hour at 37- ^? C and then, the solutions were tested for the enzymatic activity poiigalacturonasa by the DNS method.

In parallel, the same assay of enzymatic activity described in the previous paragraph but imitating industrial conditions was carried out. For this, 300μΙ of white wine must of Viña del Aromo (pH 3.0) were incubated with 300μΙ of a 0.25mg / ml solution of the present purified poiigalacturonase or with 300μΙ of a 0.25mg / ml solution of a commercial poiigalacturonase enzyme ( Lafazym®, Laffort). Assays were incubated for one hour at 15 C ^and then, the solutions were assayed for enzyme activity by the method poiigalacturonasa DNS.

As seen in Figure 8A, the present enzyme poiigalacturonase releases approximately double the galacturonic acid, compared to the commercial poiigalacturonase enzyme (Lafazym®, Laffort) tested.

These results were confirmed by assaying both enzymes under semi-industrial conditions, using white wine must as substrate and incubating the enzymatic reaction at 15 ⁹ C and 5 ^Q C, see Figures 8B and 15.

The results obtained are included in the following Tables 3 and 4 showing the pectinase activity quantified through the DNS method in laboratory and semi-industrial conditions. Table 3: Results of polygalacturonase enzymatic activity assays in laboratory conditions performed for one hour at 37 ^e C on commercial pectin (Sigma-Aldrich)

Concentraμιτιοΐββ Acid acid activity Specific enzyme

DO540nm galacturogalactuagregada ^ moles / mg

Enzyme (DNS) single (Mg / μΙ) * rónico ** (mg) enzyme)

Enzyme

commercial

(Lafazym®,

0.21

Laffort) 0.204 0.41 0.0125 16.8

Enzyme with

activity

polygalacturonase

the present

invention 0.533 0.94 0.48 0.0125 38.4

Table 4: Results of assays of enzymatic activity polygalacturonase in semi-industrial conditions, performed at 15 ^e C on commercial pectin (Sigma-Aldrich) for one hour on white wine wort

Concentration μιτιοΐθβ Acid acid activity Specific enzyme

DO540nm galacturonic galactuagregada ^ moles / mg

Enzyme (DNS) (Mg μΙΓ rónico ** (mg) enzyme)

Enzyme

commercial

(Lafazym®,

Laffort) 0, 151 0.32 0.16 0.0125 12.8 enzyme with

activity

Polygalacturonase from

the present

invention 0.338 0.62 0.32 0.0125 25.6

Table 5 summarizes the results of the pectinase activity assays evaluated by the DNS method using a commercial pectin (Pectin from citrus peel, Sigma Aldrich). Both Antarctic polygalacturonase and commercial enzyme (Lafazym®, Laffort) were incubated with a solution of this commercial pectin (10mg / ml) at pH 3.0 for one hour and two hours at 5 ^e C. Subsequently, 100μΙ of the enzymatic reaction was assayed for enzymatic activity (at each point), using the DNS method. In this table the galacturonic acid released by both enzymes is observed, the one released by the enzyme polygalacturonase of the invention being double that of the commercial enzyme.

Table 5: Results of the assays of enzymatic activity polygalacturonase in semi-industrial conditions, carried out at 5 ^e C on commercial pectin (Sigma-Aldrich) for one hour on white wine wort

1 hour 2 hours

Concentrad

on

galacturonat Concentration

DNS Enzymes 0 DNS galacturonate

Polygalacturone

sa of 0.24 0.47 0.283 0.54 invention

Lafazym 0.1 3 0.28 0.144 0.31

Example 10: Evaluation of the enzyme polygalacturonase in fruit juice

To evaluate the efficiency of the present enzyme in fruit juice, a commercial juice of plum pulp (Marca AFE) was purchased. This juice was chosen to perform the polygalacturonase activity tests, due to its low pH (around 3.0) and its high pectin content. First, 100μΙ of plum pulp juice (AFE Mark) was mixed with 100μΙ of a 0.25mg / ml solution of the present purified polygalacturonase enzyme or with 100μΙ of a 20mM Tris-HCl buffer solution, pH 8.0. The assays were incubated for one hour at 5 ^e C and then the concentration of gaiacturonic acid (by the action of polygalacturonase) was determined by the commercial kit D-glucuronic assay kit (Megazyme).

Claims

REVIVALS

one . A nucleic acid molecule comprising a polynucleotide having a DNA sequence encoding a polypeptide having polygalacturonase activity characterized in that the DNA sequence is selected from:

a) DNA sequences comprising a nucleotide sequence SEQ ID NO: 3;

b) DNA sequences comprising a nucleotide sequence SEQ ID NO: 5;

c) DNA sequences comprising a nucleotide sequence SEQ ID NO: 6;

d) DNA sequences encoding the amino acid sequence of SEQ ID NO: 1;

e) and sequences encoding the amino acid sequence SEQ ID NO: 2;

f) DNA sequences that hybridize under stringent conditions with one of the A DN sequences according to a), b), c), d) or e);

g) DNA sequences having a sequence identity of at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more with one of the DNA sequence according to a), b), c), d) oe); h) allelic variant or homologous species of a DNA sequence according to a), b), c), d), e), f) or g);

i) variant of one of the DNA sequences according to a), b), c), d), e), f) or g) which differs from one of these sequences in no more than about 1 to 165 substitutions, deletions, insertions, additions or mutations of nucleic acid, preferably no more than about 1 to 54 substitutions, deletions, insertions, additions or mutations of nucleic acid, more preferably no more than 1 to 3, 1 to 4, 1 to 5 , 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 a 10, 1 to 1 1, 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19 or 1 to 20 substitutions, deletions, insertions, additions or nucleic acid mutations;

j) a fragment of any of the sequences according to a) to i) that encodes an olipeptide having polygalacturonase activity; or

k) a strand complementary to any of the sequences according to a) to j).

2. A polypeptide having polygalacturonase activity characterized in that the polypeptide is selected from:

a) a polypeptide that is encoded by the coding part of any sequence according to claim 1;

b) a polypeptide comprising a sequence according to SEQ ID NO: 1 or SEQ ID NO: 2; or

c) a polypeptide derived from the sequence according to SEQ ID NO: 1 or 2, obtainable by substitution, addition or deletion of one or more amino acids of said SEQ ID NO: 1 or 2, preferably by substitution of no more than about 55 amino acids in said SEQ ID NO: 1 or 2, more preferably by substitution of no more than 18 amino acids in said SEQ ID NO: 1 or 2.

3. The nucleic acid molecule according to claim 1 or the polypeptide according to claim 2 characterized in that said polypeptide is an enzyme with polygalacturonase activity, wherein said polygalacturonase activity is a high polygalaturonase activity in a temperature range of about 5 ° C to around 15 ° C.

4. The nucleic acid molecule according to claim 3, or the polypeptide according to claim 2 or 3, characterized in that it can be isolated from the species Articulospora proliferata.

5. A recombinant expression vector, characterized in that it comprises the nucleic acid molecule according to claim 1 or 3.

6. The recombinant expression vector according to claim 5, characterized in that it comprises the nucleotide sequence of SEQ ID NO: 4, 5 or 6.

7. A host cell characterized in that it has been transformed with the recombinant expression vector according to claim 5 or 6.

8. The host cell according to claim 7, characterized in that it is derived from an E. coli cell, a fungus of the family Aspergillus, Trichoderma, or Neurospora, or from a yeast of the family Kluyveromyces, Saccharomyces, Schizosaccharomyces, Hansenula or Pichia, preferably Pichia pastorís.

9. A method for the production of a polypeptide according to claim 2, or encoded by a DNA sequence according to claim 1, characterized in that it comprises:

(i) culturing the host cell according to claim 7 or 8 under conditions that allow the expression of said polypeptide; Y

(ii) recovering said polypeptide.

10. A composition characterized in that it comprises the polypeptide according to claim 2, or is obtained by the method according to claim 9.

eleven . The composition according to claim 10 characterized in that it comprises or consists of a liquid formulation or a lyophilized powder.

12. Use of a polypeptide according to claim 2, or a polypeptide obtained by the method according to claim 9 or of a composition according to claim according to claim 10 or 1 characterized in that it serves for degradation of pectin in a Beverage production process in which the degradation of pectin is desirable, preferably in the production of wine, beer or fruit juice.

The use according to claim 12, characterized in that the degradation of pectin in a beverage production process in which pectin degradation is desirable occurs at a temperature between 5 ° C and 20 ° C, preferably at a temperature of between 5 ° C and 20 ° C. Temperature between 5 ° C and 15 ° C.

The use according to claim 12 or 13, characterized in that the degradation of pectin in a beverage production process in which the degradation of pectin is desirable, is the clarification of grape must or fruit juice.