MXPA00007787A - Novel endo-xylogalacturonase - Google Patents

Abstract

Polypeptides possessing a novel activity, namely endo-xylogalacturonase activity, are disclosed. These polypeptides can degrade pectin found in plant extracts and plant materials, and in particular the"hairy"regions of pectin polymers. In particular, the polypeptides can cleave in a galacturonic acid polymer at internal glycocidic bonds. The novel enzyme XghA is disclosed, and its amino acid sequence and encoding DNA sequence given. This polypeptide was expressed in yeast cells and has been used to treat vegetable material, in particular soy and fruit juice in the preparation of edible foodstuffs.

Description

NEW ENDO-XILOGALACTÜRONASA Field of the Invention The present invention relates to a novel endo-xylogalacturonase (XGH) and homologs thereof. It also relates to the use of endo-xylogalacturonase in a method for processing plant material or containing pectin to produce fruit juice and other plant extracts.

Background of the Invention Enzyme preparations are often used during the processing of plant materials, for example in the stages of extraction and liquefaction of fruit and fruit juice and their filtration and clarification. Commercial enzyme preparations contain a mixture of enzymes that degrade the pectin polymers that are a major component of the cell walls of the plant. Such enzymes include pectin lyases, polygalacturonases, pectin esterases, celluloses, xyloglucanases, galactanases and arabinanases.

Pectins occur in nature as constituents of plant cell walls REF .: 122318 superiors. They are found in the foil of the primary cell wall where they fit between the cellulose fibrils. The composition of pectin is variable among plant species and also depends on the age and maturity of the fruit. Among the richest sources of pectins are lemons and oranges, which can represent up to 30% of the polysaccharides present.

The majority of the pectin polymers are comprised of 'smooth' homogalacturonan regions and branched 'filamentous' regions. The 'smooth' regions consist of a linear homogalacturonan structure. The 'filamentous' regions of apples consist of three different subunits: subunit I is of xylogalacturonan (a heavy galacturonan structure substituted with xylose); subunit II is a short section of a rhamnogalacturonan structure, rich in relatively long side chains of arabinano, galactan and / or arabinogalactan (the 'filaments'); and subunit III is an oligomer of ramnogalacturonan, which has a structure consisting of an alternating sequence of rhamnose and galacturonic acid residues.

Many of the well-known pectinases used in industrial food processing degrade only the 'smooth' part of the pectin polymer leaving the 'filamentous' regions intact. Consequently, for example, during the production of apple juice, the non-degraded portions of the pectin polymer cause production losses due to inefficient filtration as a result of fouling of the (ultra) filtration membrane.

Several enzymes have been reported that degrade parts of the 'filamentous' region, for example the rhamnogalacturonan regions of the structure (subunit III). These enzymes are referred to as rhamnogalact uronases (RGases), of which there are several types. However, hitherto the xylogalacturonan part of the 'filamentous' regions (subunit I) have been resistant to enzymatic digestion, and therefore in the enzymatic endo-digestion processes of the prior art xylogalacturonan is left as an inert carbohydrate.

Since xylogalacturonan has also been found in many other plants, e.g. legume plants such as soybean and pea enzymes, watermelons, grapes and pine pollen, to degrade this polymer would be useful for the processing of plant material.

An exo-galacturonase (42 kDa, SDS-PAGE) has been identified which is not hindered by the single unit xylose side chains and is capable of degrading xylogalacturonan using a soluble 'filamentous' pectic polysaccharide from soybean as a substrate. This enzyme acts in an exo manner as it produces galacturonic acid or a disaccharide consisting of galacturonic acid and xylose. The enzyme was purified to near homogeneity (fractions HTP2 and Q2) and was partially characterized. Contrary to known RGases (which do not degrade homogalacturonic acid) this enzyme is not very specific for xylogalacturonan as it acts on pectic acid. Furthermore, this enzyme is not capable of digesting the structure of xylogalacturonan in a random manner, and therefore to date, there are no known enzymes that possess endo-xylogalacturonase activity.

Brief Description of the Invention The present invention has resulted from the isolation and characterization of a new endo-xylogalacturonase and cDNA encoding it. The cDNA sequence of endo-xylogalacturonase is set forth in SEC. ID No. 1. The ORF amino acid sequence of nucleotides 98 to 1315 is set forth in SEC. ID No. 2 In a first aspect of the invention there is provided a polypeptide (e.g., isolated and / or purified) that possesses endo-xylogalacturonase activity. Also provided is a polypeptide comprising an endo-xylogalacturonase, such as a polypeptide comprising the sequence set forth in SEQ ID No. 2, or a polypeptide substantially homologous thereto, or a fragment of the polypeptide of SEQ ID No. 2 that has at least 5 amino acids.

The polypeptide of the invention preferably has one or more of the following additional characteristics, ie: 1) possesses endo-xylogalacturonase activity; (2) has an optimum pH range of 2.5 to 6; (3) has the optimal activity at a temperature of 50 to 70 ° C; I (4) has a molecular weight (γ-separated glyphs) of 40 to 50 kDa.

"Endo-xylogalacturonase activity" is defined as the ability to cut a galacturonic acid polymer (for example as found in pectin) that could be at least partially replaced with xylose in internal glycosidic linkages. In this way, the activity allows the cut between adjacent non-terminal galacturonan units (where none of the units is at the end of the polymer, which is the opposite of the exo activity, where the terminal unit would be cut off). Preferably, the cut is presented to a [galacturonic acid (1,4) galacturonic acid] junction. Preferably the polypeptide does not cleave the terminal xylose residues of the galacturonic acid residues substituted with xylose, for example a linkage [galacturonic acid (1,3) xylose]. The polypeptide could preferably be cut between two adjacent unsubstituted xylose galacturonan units. The polymer of the substrate could be from 40 to 80% (e.g., xylose) its substance.

The two galacturonic acid residues between which the polypeptides of the invention are cut, could both be replaced (xylose), or could only be replaced (xylose) or (preferably) could not be replaced (xylose). Alternatively or in addition to the two galacturonic acid residues, they could both be methylated, or one could be methylated, or (preferably) none could be methylated.

Preferably, the polypeptide of the invention is obtained from a microorganism possessing a gene encoding an enzyme with endo-xylogalacturonase activity. More preferably, the microorganism is a microbial organism, preferably fungal, and optimally a filamentous fungus. The organisms preferred in this way are of the genus Aspergillus, Trichoderma, Penicillum, Acremonium, Fusarium, Humicola, Neurospora, Mucor, Scytall idium, Mycel iophtora, Thielavia, Talaromyces, Thermomyces, Thermoascus, Chaetomium, Sporotrichum, Corynascus, Calcar isporiella or Mycelia. Optionally, the organism is of the species of the Aspergillus niger group (as defined by Raper and Fennell, The Genus Aspergillus, The Williams &Wilkins Company, Baltimore, pp. 293-344, 1965), which specifically includes, but is not limited to, to Aspergillus niger, Aspergillus awamori, Aspergillus tubigensis, Aspergillus aculeatus, Aspergillus foetidus, Aspergillus j aponicus or Aspergillus ficuum.

In a second aspect, the present invention provides a (e.g., isolated and / or purified) polynucleotide that encodes a polypeptide of the first aspect of the invention. For example, the present invention provides a polynucleotide that encodes an endo-xylogalacturonase, such as endo-xylogalacturonase whose amino acid sequence is set forth in SEQ ID No. 2. The present invention further provides a polynucleotide that encodes a polypeptide having a homology of the substantial amino acid sequence with respect to the amino acid sequence set forth in SEQ ID No. 2. A polynucleotide sequence of: (a) polynucleotides comprising the nucleotide sequence set forth in SEQ ID No. 1, or the complement thereof; (b) polynucleotides comprising a nucleotide sequence capable of hybridizing to the nucleotide sequence set forth in SEQ ID No. 1, or fragment thereof; (c) polynucleotides comprising a nucleotide sequence capable of hybridizing to the complement of the nucleotide sequence set forth in SEQ ID No. 1, or fragment thereof; and / or (d) polynucleotides comprising a polynucleotide sequence that is degenerate as a result of the genetic code with respect to the polynucleotides defined in (a), (b) or (c).

A polynucleotide of the invention also includes a polynucleotide that: to. encodes a polypeptide having endo-xylogalacturonase activity, the polynucleotide is: (1) the coding sequence of SEQ ID No. 1; (2) a sequence that selectively hybridizes to the complement of the sequence defined in (1); or 3) a sequence that is degenerate as a result of the genetic code with respect to a sequence defined in (1) or (2); or b. is a sequence complementary to a polynucleotide defined in (a).

The term "capable of hybridizing" means that the target polynucleotide of the invention can hybridize to the nucleic acid used as a probe (for example the nucleotide sequence set forth in SEQ ID No. 1, or a fragment thereof or the complement thereof). ) at a level significantly above the origin. The origin hybridization could be due to, for example, other polynucleotides, such as DNA, present in, for example, a genomic cDNA library that is screened. In this case, the origin involves a signal level generated by the interaction between the probe and a non-specific polynucleotide member of the library that is less than 10 folds, preferably less than 100 folds, as intense as the specific interaction observed with the white polynucleotide. The intensity of the interaction could be measured, for example, by radiolabeling the probe, e.g. with 32P. The appropriate conditions are described below.

Preferably, the polynucleotide of the invention is obtained from the same organism as the polypeptide, such as a fungus, in particular a fungus of the Aspergi genus.

The present invention also provides a polynucleotide probe comprising a fragment of at least 15 nucleotides of a polynucleotide of the invention as described above.

In a third aspect, the invention provides vectors comprising a polynucleotide of the invention, including cloning and expression vectors, and in a fourth aspect methods of growth, transformation or transfection of such vectors in an appropriate host cell, for example under the conditions in which the expression of a polypeptide is presented, or encoded by a sequence of the invention. There are provided in a fifth aspect, host cells comprising a polynucleotide or vector of the invention, wherein the polynucleotide is heterologous to the genome of the host cell. The term "heterologous for the host cell genome" means that the polynucleotide does not occur naturally in the genome of the host cell. Preferably, the host cell is a yeast cell, for example a yeast cell of the genus Kl uyveromyce s or Sa ccha romyces or a fungal cell, for example of the genus Aspergi l l us.

The polypeptides of the invention which possess endo-xylogalacturonase activity could be used in a sixth aspect to treat the plant material, which includes plant pulp and plant extracts. For example, it could be used to treat apple pulp and / or raw juice during the production of apple juice. Conveniently, the polypeptide of the invention is combined with suitable carriers or diluents including buffers, to produce a composition / enzyme preparation. Thus, the present invention provides in a seventh aspect a composition comprising a polypeptide of the invention. The composition may further comprise additional ingredients, such as one or more enzymes, for example pectinases, including endo-arabinanase and rhamnogalacturonase, cellulases and / or xyloglucanases.

The polypeptides and compositions of the invention could therefore be used in a method for processing plant materials to degrade or modify the pectin constituents of the cell walls of the plant material. Thus, in an eighth aspect, the present invention provides a method for degrading or modifying a cell wall of the plant, the method comprising contacting the cell wall of the plant with a polypeptide or composition of the invention.

The invention also provides a method for processing a plant material, the method comprising contacting the plant material with a polypeptide or composition of the invention to degrade or modify the pectin in the material of the plant. Preferably the material of the plant is a plant pulp or plant extract.

In particular, the degradation preferably comprises the endo-type cleavage of the xylogalacturonan subunits of a pectin component of the cell wall of the plant. The material of the plant is preferably a fruit or pulp or vegetable fruit or vegetable extract, for example apple pulp or apple juice.

The present invention further provides a processed plant material obtained by contacting a material of the plant with a polypeptide or composition of the invention. Preferably, the material of the processed plant is a fruit or vegetable juice, for example apple juice.

The present invention also provides a method for reducing the viscosity of a plant extract, the method comprising contacting the extract of the plant with a polypeptide or composition of the invention in an amount effective in the degradation of the pectins contained in the extract. of the plant.

The preferred specifications and features of one aspect of the invention are applicable to another, many, many aspects.

Detailed description of the invention TO . Polynucleotides The invention provides a polynucleotide that to. encodes a polypeptide having endo-xylogalacturonase activity, the polynucleotide is: (1) the coding sequence of SEQ ID No. 1; (2) a sequence that selectively hybridizes to the complement of the sequence defined in (1), or (3) a sequence that is degenerate as a result of the genetic code with respect to the nucleic acid sequence defined in (1) or (2); or b. is a sequence complementary to a polynucleotide defined in (a).

The polynucleotides of the invention also include variants of the coding sequence of SEQ ID NO: 1 having endo-xylogalacturonase activity. The variants could be formed by additions, substitutions and / or deletions. Such variants, in this way, could have the ability to internally cut a galacturonic acid polymer. Typically, a polynucleotide of the invention comprises a continuous sequence of nucleotides that is capable of hybridizing under selective conditions to the complement of the coding sequence of SEQ ID No. 1.

A polynucleotide of the invention and complement to the coding sequence of SEQ ID No. 1, can hybridize at a level significantly above the origin. The origin hybridization could be due to, for example, other cDNAs present in a cDNA library. The signal level generated by the interaction between a polynucleotide of the invention and the complement of the coding sequence of SEQ ID No. 1, is typically at least 10 folds, preferably at least 100 folds, as intense as the interactions among other polynucleotides and the coding sequence of SEQ ID No. 1. The intensity of the interaction could be measured, for example, by radiolabeling the probe, for example 32P. Selective hybridization could typically be achieved using conditions of low stringency (eg, 0.03 M sodium chloride and 0.03 M sodium citrate at about 40 ° C), medium severity (eg, 0.03 M sodium chloride and 0.03 M sodium citrate). M at about 50 ° C) or high severity (for example, 0.03 M sodium chloride and 0.03 M sodium citrate at about 60 ° C).A preferred polynucleotide that is capable of selectively hybridizing to complement the DNA sequence of SEQ ID No. 1 will generally have at least 50%, at least 60%, at least 70%, at least 80%, at least 90% , at least 95%, at least 98% or at least 99% identity of the sequence with respect to the coding sequence of SEQ ID No. 1, over a region of at least 20, preferably at least 30, for example at least 40, at least 60, or preferably at least 100 continuous nucleotides or more preferably over the total length of SEQ ID No. 1.

Any combination of the aforementioned degrees of identity sequence and minimum sizes could be used to define the polynucleotides of the invention, being preferred with the most severe combinations (i.e., the identity of the upper sequence over the longer lengths) . Thus, for example, a polynucleotide having at least 90% sequence identity over 25, preferably over 30 nucleotides, is preferred, as is a polynucleotide having at least 95% sequence identity over 40 nucleotides.

The coding sequence of SEQ ID NO: 1 could be modified by nucleotide substitutions, for example 1, 2 or 3 to 10, 25, 50 or 100 substitutions. The polynucleotide of SEQ ID No. 1 could alternatively or additionally be modified by one or more insertions and / or deletions (such as the same number mentioned for substitutions) and / or by an extension for one or both ends. The modified polynucleotide generally encodes a polypeptide having endo-xylogalacturonase activity. The degenerate substitution could be made and / or the substitutions could be made which would result in a conservative amino acid substitution when the modified sequence is translated, for example as shown in the Table on page 12 in the section relating to polypeptides.

The polynucleotides of the invention could comprise DNA or RNA. They could be single or double stranded. They could also be polynucleotides that include within these synthetic or modified nucleotides. A number of different types of modifications to polynucleotides are known in the art. These include a structure of methylphosphonate and phosphorothionate, and in addition to the acridine or polylysine chains at the 3 'and / or 5' ends of the molecule. For the purposes of the present invention, it is understood that the polynucleotides described herein could be modified by any method available in the art.

It is to be understood that those skilled in the art could perform, using routine techniques, nucleotide substitutions that do not affect the polynucleotide sequence encoded by the polynucleotides of the invention to reflect the codon used of any host organism in which the polypeptides of the invention they are going to express themselves The polynucleotides of the invention could be used as a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, an e.g. labeled with a developing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides could be cloned into vectors.

Such primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length. Typically, they will exist up to 40, 50, 60, 70, 100 or 150 nucleotides in length. The probes and fragments may be greater than 150 nucleotides in length, for example up to 200, 300, 400, 500, 600, 700 nucleotides in length, or even up to a few short nucleotides (such as 5 or 10 nucleotides) of the sequence of coding of SEQ ID No. 1.

Polynucleotides such as a DNA polynucleotide and primers according to the invention, could be produced recombinantly, synthetically, or by any means available to those skilled in the art. They could also be cloned by standard techniques. The polynucleotides are typically provided in isolated and / or purified form.

In general, the primers will be produced by synthetic means, which involve progressively making the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.

Long polynucleotides, in general, will be produced using the recombinant media, for example using a PCR cloning (polymerase chain reaction) techniques. This will involve making a pair of primers (eg of about 15-30 nucleotides) to a region of the endo-xylogalacturonase gene that is desired to be cloned, bringing the primers into contact with mRNA or cDNA obtained from a fungus, yeast, bacterial plant or cell prokaryotic, performing a polymerase chain reaction under the conditions that allow the amplification of the desired region, the isolation of the amplified fragment (eg by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. The primers could be designed to contain the recognition sites of the appropriate restriction enzyme, so that the amplified DNA can be cloned into an appropriate cloning vector.

Such techniques could be used to obtain all or part of the endo-xylogalacturonase sequence described herein. Genomic clones corresponding to the cDNA of SEQ ID No. 1 or the endo-xylogalacturonase gene containing, for example, introns and also the promoter regions are within the invention, and could also be obtained in an analogous manner (eg media recombinants, PRC, cloning techniques), starting with the genomic DNA of a fungus, yeast, bacterial plant or prokaryotic cell.

Although in general the techniques mentioned herein are well known in the art, reference could in particular be made to Sambrook et al. , Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al. , Current Protocols in Molecular Biology (1995), John Wiley & Sons, Inc Polynucleotides that do not have 100% identity with SEQ ID No. 1, but fall within the scope of the invention, can be obtained in a number of ways.

In this manner, variants of the endo-xylogalacturonase sequence described herein could be obtained for example, by probing the genomic DNA libraries made from a range of organisms, for example those discussed as sources of the polypeptides of the invention. In addition, other prokaryotic fungi, plants or homologs of endo-xylogalacturonase could be obtained and such homologs and fragments thereof will generally be able to hybridize to SEQ ID No. 1. Such sequences could be obtained by probing the DNA libraries or libraries of genomic DNA from other species, and probing such libraries with probes comprising all or part of SEQ ID No. 1 under conditions of medium to high severity (e.g., 0.03 M sodium chloride and sodium citrate). 0.03 M from 50 ° C to 60 ° C). Nucleic acid probes comprising all or part of SEQ ID No. 1 could be used to probe DNA libraries of other species, such as those described as sources for the polypeptides of the invention.

Homologous species could also be obtained using degenerate PCR which will use primers designed for the target sequences within the variants and homologs encoding the conserved amino acid sequences. The primers will contain one or more degenerate positions and will be used at the lower severity conditions than those used for the cloning of the sequences with the primers of the single sequences against the known sequences.

Alternatively, such polynucleotides could be obtained by site-directed mutagenesis of the endo-xylogalacturonase sequences or variants thereof. This could be useful where, for example, moderate codon changes are required for the sequences to optimize the codon preferences for a particular host cell, in which the polynucleotide sequences are being expressed. Other changes of the sequence could be desired to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides.

The invention includes double-stranded polynucleotides comprising a polynucleotide of the invention and its complement.

The polynucleotides or primers of the invention could carry a developing tag. Appropriate labels include radioisotopes such as 32P or 35S, enzyme labels, or other protein labels, such as biotin and DIG-hapten. Such labels could be added to the polynucleotides or primers of the invention and could be detected using the techniques known per se.

The present invention also provides polynucleotides that encode the polypeptides of the invention described below. Since such polynucleotides will be used as sequences for the recombinant production of polypeptides of the invention, it is not necessary for them to be able to hybridize to the sequence of SEQ ID No. 1, although in general, this will be desirable. Also, such polynucleotides could be labeled, used and made as described above if desired.

B. Polypeptides A polypeptide of the invention comprises the amino acid sequence set forth in SEQ ID NO: 2 or a substantially homologous sequence, or a fragment of the sequence and may have endo-xylogalacturonase activity. In general, the naturally occurring amino acid sequence shown in SEQ ID No. 2 is preferred.

In particular, the polypeptide of the invention could comprise: to. the polypeptide sequence of SEQ ID No. 2. b. a variant or homologous species of the same that occurs naturally; or a protein with at least 60, at least 70, at least 80, at least 90, at least 95, at least 98 or at least 99% identity of the sequence a (a) or (b).

A variant will be one that occurs naturally, for example in fungal, bacterial, yeast or plant cells and that can function in a manner substantially similar to the protein of SEQ ID No. 2, for example it has endo-xylogalacturonase activity. Similarly, a homologous species of the protein will be the equivalent protein that occurs naturally in other species and that can function as an endo-xylogalacturonase enzyme.

The homology of the variants and species can be obtained by following the procedures described herein for the production of the polypeptide of SEQ ID No. 2 and performing such methods in an appropriate cellular source, for example a bacterium, fungus or plant cell. It will also be possible to use a probe as defined above to probe libraries made of yeast cells, bacteria, fungi or plants to obtain clones that include the homology of the variants or species. The clones can be manipulated by conventional techniques to generate a polypeptide of the invention which can then be produced by recombinant or synthetic techniques known per se.

The polypeptide of the invention preferably has at least 60% sequence identity for the protein of SEQ ID NO: 2, more preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity over a region of at least 20, preferably at least 30, for example at least 40, at least 60, at least 100, 200 or 300 contiguous amino acids or over full length of SEQ ID No. 2.

The sequence of the polypeptide of SEQ ID No. 2 and of the homologous species and variants, in this way, can be modified to provide the polypeptides of the invention. The amino acid substitutions could be made, for example from 1, 2 or 3 to 10, 20 to 30 substitutions. The same number of eliminations could also be made. The modified polypeptide generally retains activity as an endo-xylogalacturonase.

The conserved substitutions could be made according to the following Table, wherein the amino acids in the same block in the second column and preferably in the same line in the third column could be substituted with each other.

The polypeptides of the invention also include fragments of the full-length polypeptides mentioned above and variants thereof, including fragments of the sequence set forth in SEQ ID No. 2. Such fragments typically maintain activity as endo-xylogalacturonase. The fragments could be at least 10, 15, 20, 30, 50, 100 or 200 amino acids in length.

The polypeptides of the invention could be in a substantially isolated form. It will be understood that the polypeptide could be mixed with carriers or diluents that will not interfere with the intended purpose of the polypeptide and still be considered as substantially isolated. A polypeptide of the invention could also be a substantially purified form, in which case in general, it will comprise the polypeptide in a preparation in which more than 50%, e.g. more than 80%, 90%, 95%, 98% or 99% by weight of the polypeptide in the preparation is a polypeptide of the invention. The polypeptides of the invention could be chemically modified, for example modified by post-translation. For example, they could be glycosylated (one or more times) or comprise one or more modified amino acid residues.

They could be modified, for example, by the addition of histidine residues or a T7 tag to aid their identification or purification or by the addition of a signal sequence to promote their secretion from a cell, as discussed below.

The polypeptides of the invention, if necessary, can be produced by hetic means, although they will usually be made recombinantly as described below.

Particularly preferred polypeptides of the invention include a polypeptide that spans from 19 to 406 amino acids of the amino acid sequence set forth in SEQ ID No. 2, since it lacks the N-terminal signal peptide, which consists of 1 to 18 amino acids of the amino acid sequence of SEQ ID No. 2. The polypeptides and fragments thereof could contain amino acid alterations as defined above.

The use of yeast host cells and fungi is expected to provide such post-translational modifications (eg, proteolytic processing, myristylation, glycosylation, truncation and phosphorylation of tyrosine, serine or threonine) as may be required to confer optimal biological activity in the recombinant expression products of the invention.

C. Recombinant aspects.

The polynucleotides of the invention can be incorporated into a recombinant replicable vector, for example a cloning or expression vector. The vector could be used to replicate the nucleic acid in a compatible host cell. Thus, in a further embodiment, the invention provides a method for making polynucleotides of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions that allow the vector replication. The vector could be recovered from the host cell. Appropriate host cells are subsequently described in conjunction with the expression vectors.

Expression Vectors.

Preferably, a polynucleotide of the invention in a vector is operably linked to a regulatory sequence that is capable of providing expression of the coding sequence by the host cell, i.e. the vector is an expression vector. The term "operably linked" refers to a juxtaposition, wherein the described components are in a relationship that allows them to function in their intended manner. A regulatory sequence such as a promoter, enhancer or other regulation signal of the expression "operably linked" to a coding sequence is placed, such that the expression of the coding sequence is reached under the condition compatible with the sequences of control.

The vectors could be for example, plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of the polynucleotide and optionally a regulation of the promoter.

The DNA sequence encoding the polypeptide is preferably introduced into an appropriate host as part of an expression construct, in which the DNA sequence is operably linked to the expression signals that are capable of directing the expression of the DNA sequence. in the host cells. For the transformation of the appropriate host with the expression construct, transformation procedures are appropriate, which are known to those skilled in the art3,4. The expression construct can be used for the transformation of the host as part of a vector carrying a selected marker, or the expression construct is a co-transformed construct as a separate molecule together with the vector carrying a selected marker. The vectors could contain one or more selected marker genes.

Preferred selected markers3'4 include, but are not limited to e.g. Versatile marker genes that can be used for the transformation of most fungi and filamentous yeasts such as acetamidase genes or cDNAs (the amdS genes or cDNAs of A. nidulans, A. oryzae or A. niger), or genes that provide resistance to antibiotics such as resistance to G418, hygromycin, phleomycin or benomyl (ben A). Alternatively, more specific selection markers can be used, such as auxotrophic markers that require the corresponding mutant host strains: e.g. URA3 (from S. cerevisiae or analogous genes from other yeasts), pyrG. { of A. nidulans or A. niger) or argB (of A. nidulans or A. niger). In a more preferred embodiment, the selection marker is removed from the introduction of the transformed host cell of the expression construct according to the methods described in EP-A-0 635 574, to obtain the transformed host cells capable of producing the polypeptide that are free of the selection marker genes.

Other markers include ATP synthetase, subunit 9 (oliC), orotidine-5'-phosphate decarboxylase (pyrA), the bacterial resistance gene G418 (this could be used in yeast, but not fungi), the ampicillin resistance gene. { E. col i), the neomycin resistance gene . { Ba ci l l us) and the E gene. col i n i dA, which codes for β-glucuronidase (GUS). The vectors could be used i n vi t ro, for example for the production of RNA or used to transfer or transform a host cell.

For most fungi and filamentous yeasts, the expression construct is preferably integrated into the genome of the host cell to obtain stable transformations. However, for certain yeasts, also suitable episomal vector systems are available, in which the expression construct can be incorporated for high level and stable expression, examples of which include vectors derived from the plasmids 2μ and pKDl of Sa cch a romyces and fCluy erojnyces, respectively. In the case that the expression constructs are integrated into the genome of the host cells, the constructs are integrated into the random loci in the genome, or at the predetermined target loci using the homologous recombination, in such case, the target loci comprises preferably a highly expressed gene. A highly expressed gene is defined herein as a gene whose mRNA can constitute at least 0.05% (w / w) of the total cellular mRNA, e.g. under induced conditions, or alternatively, a gene whose gene product can constitute at least 1% (w / w) of the total cellular protein, or, in the case of a product of the secreted gene, can be secreted at a level of at least 0.1 g / 1. A number of examples of the appropriate highly expressed genes are provided hereinafter.

An expression construct for a given host cell will usually contain the following elements operably linked to each other in a consecutive order from the 5 'end to the 3' end, relative to the coding strand of the sequence encoding the polypeptide of the first aspect: (1) a promoter sequence capable of directing the transcription of the DNA sequence encoding the polypeptide in the given host cell, (2) optionally, a signal sequence capable of directing the secretion of the polypeptide from a given host cell into the culture medium, (3) the DNA sequence encoding a mature and preferably active form of the polypeptide, and also preferably ( 4) a transcription termination region (terminator) capable of terminating transcription downstream of the DNA sequence encoding the polypeptide.

Improved expression of the polynucleotide encoding the polypeptide of the invention could also be achieved by the selection of the heterologous regulatory regions, e.g. the promoter, leader and secretory terminator regions, which serve to increase the expression and, if desired, the secretion levels of the protein of interest of the chosen expression host and / or provide the inducible control of the expression of the polypeptide of the invention In addition to the promoter native to the gene encoding the polypeptide of the invention, other promoters could be used to direct the expression of the polypeptide of the invention. The promoter could be selected for efficiency to direct expression of the polypeptide of the invention in the desired expression host.

A variety of promoters3,4 can be used to be able to direct transcription in the host cells of the invention. Preferably the promoter sequence is derived from a highly expressed gene as previously defined. Examples of preferred highly expressed genes, of which the promoters are preferentially derived and / or which are comprised in the preferred target loci for the integration of the expression constructs, include, but are not limited to genes encoding glycolytic enzymes such as t riosa-phosphate isomerases (TPl), glyceraldehyde-phosphate dehydrogenases (GAPDH), phosphoglycerate kinases (PGK), pyruvate kinases (PYK), alcohol dehydrogenases (ADH), as well as genes encoding amylases, glucoamides, xylanases, cellobiohydrolases, β- galactosidases, alcohol oxidases (methanol), elongation factors and ribosomal proteins. Specific examples of the appropriate highly expressed genes include e.g. the LAC4 gene of Kluyveromyces sp., the methanol oxidase genes. { AOX and MOX) of Hansenula and Pichia, respectively, the glucoamylase genes. { glaA) of A. niger and A. awamori, the gene of A. oryzae TAKA-amylase, the gene A. nidulans gpdA and the genes of celobiohydrolase T. reesei.

Examples of strong and / or inducible constitutive promoters that are preferred for use in fungal expression hosts are those obtained from the fungal genes for the xylanase promoters. { xlnA), phytase, ATP synthetase, subunit 9. { ol iC), triose phosphate isomerase. { tpi), alcohol dehydrogenase (AdhA), α-amylase (amy), amyloglucosidase (AG - of the glaA gene), acetamidase. { amdS) and glyceraldehyde-3-phosphate dehydrogenase. { gpd).

Examples of strong yeast promoters are those obtained from the genes for alcohol dehydrogenase, lactase, 3-phosphoglycerate kinase and triosephos phato isomerase.

Examples of strong bacterial promoters are the α-amylase and SP02 promoters, as well as promoters of the extracellular protease genes.

Host Cells and Expression.

Preferably, the polypeptide is produced as a secreted protein, in which case the DNA sequence encoding a mature form of the polypeptide in the expression construct is operably linked to a DNA sequence encoding the signal sequence. Preferably, the signal sequence is native (homologous) with respect to the DNA sequence encoding the polypeptide. Alternatively, the signal sequence is foreign (heterologous) with respect to the DNA sequence encoding the polypeptide, in which case the signal sequence is preferably endogenous to the host cell in which the DNA sequence is expressed. Examples of the appropriate signal sequences for the yeast host cells are the signal sequences derived from fator or yeast genes. Similarly, a suitable signal sequence for filamentous fungal host cells is e.g. a signal sequence derived from a filamentous (gluco) amylase gene, e.g. the gene of A. n i ger gl aA. This could be used in combination with the amyloglucosidase (AG) promoter itself, as well as in combination with other promoters. The hybrid signal sequences could also be used with the context of the present invention.

The preferred heterologous secretion leader sequences are those that originate from the fungal amyloglucosidase (AG) gene (glaA - both versions of 18 and 24 amino acids eg from Aspergi llus), the factor a gene (yeasts eg Sa ccha romyces and Kl uyveromyces ) or the α-amylase gene. { Ba ci l l us).

Downstream of the DNA sequence encoding the polypeptide, the expression construct preferably contains a 3 'untranslated region containing one or more transcription termination sites, also referred to as a terminator. The origin of the terminator is less critical. The terminator, e.g. it can be native to the DNA sequence encoding the polypeptide. However, a yeast terminator is preferably used in the yeast host cells and a filamentous fungal terminator is used in the fungal filamentous host cells. More preferably, the terminator is endogenous to the host cell in which the DNA sequence encoding the polypeptide is expressed.

In a further aspect, the invention provides a process for preparing the polypeptides according to the invention, which comprises culturing a transformed or transferred host cell with an expression vector as described above, under the conditions to provide for expression by the vector of a coding sequence that encodes the polypeptides, and recover the expressed polypeptides.

A further aspect of the invention, in this manner provides the host cells transformed or transferred with or comprising a polynucleotide or vector of the invention. Preferably, the polynucleotide is carried in a vector for the replication and expression of the polynucleotide. The cells will be chosen to be compatible with the vector and for example, they could be prokaryotic (for example bacterial), fungal, yeast or plant cells.

Depending on the nature of the polynucleotide encoding the polypeptide of the invention, and / or the desire for further processing of the expressed protein, eukaryotic hosts such as yeast and fungi may be preferred. In general, yeast cells are preferred over fungal cells, because they are easier to handle. However, some proteins are poorly secreted from yeast, or in some cases not properly processed (e.g. hyperglycosylation in yeast). In these cases, a host organism of fungus should be selected.

A heterologous host could also be chosen, wherein the polypeptide of the invention is produced in a form that is substantially free of other enzymes that degrade pectin. This could be achieved by choosing a host that does not normally produce such enzymes, such as Kl uyveromyces l a c t i s.

The invention encompasses the processes for the production of the polypeptide of the invention by means of the recombinant expression of a DNA sequence encoding the polypeptide. For this purpose, the DNA sequence of the invention can be used for the amplification of the gene and / or the exchange of expression signals, such as promoters, secretion signal sequences, to allow economical production of the polypeptide in a host cell homologous or heterologo appropriate. A homologous host cell is defined herein as a host cell that is of the same species or that is a variant within the same species as the species from which the DNA sequence is derived.

Suitable host cells are preferably prokaryotic microorganisms, such as bacterial organisms or more preferably eukaryotic organisms, for example fungi, such as yeast or fungal cells or filamentous plants.

Bacteria of the genus Bacillus are very suitable as heterologous hosts, due to their ability to secrete proteins in the culture medium. Other suitable bacteria such as hosts are those of the genus S trep t omyces and Ps e udom ona s. A preferred yeast host cell for the expression of the DNA sequence encoding the polypeptide is of the genus Saccharomyces, Kluyveromyces, Hansenula, Pichia, Yarrowia and Schizosaccharomyces. More preferably a yeast host cell is selected from the group consisting of the species of Saccharomyces cerivisiae, Kluyveromyces lactis (also known as Kluyveromyces marxianus var lactis), Hansenula polymorpha, Pichia pastor is, Yarrowia lipolytica and Schizosaccharomyces pombe.

However, filamentous fungal host cells are more preferred for the expression of the DNA sequence encoding the polypeptide. Host cells of filamentous fungi are selected from the group consisting of the genus Aspergillus, Trichoderma, Fusarium, Penicillium, Acremonium, Neurospora, Thermoascus, Myceliophtora, Sporotrichum, Thielavia and Talaromyces. More preferably, a filamentous fungal host cell is of the species Aspergillus oyzae, Aspergillus sojac, Aspergillus nidulans, species of the Aspergillus niger group (as defined by Raper and Fennell, The Genus Aspergillus, The Williams & Wilkins Company, Baltimore, pp 293-344, 1965). These include, but are not limited to, Aspergillus niger, Aspergillus owamori, Aspergillus tuJbigensis, Aspergillus aculeatus, Aspergillus foetidus, Aspergillus nidulans, Aspergillus j aponicus, Aspergillus oryzae and Aspergillus ficuum, and also consist of the species Trichoderma reesei, Fusarium graminearum, Penicillum chrysogenum. , Acremonium alabanense, Neurospora crassa, Myceliophtora, thermophilum, Sporotrichum cellulophilum and Thielavia terrestris.

Examples of preferred expression hosts within the scope of the present invention are fungi, such as Aspergillus species (described in EP-A-184,443 and EP-A-284, 603) and Trichoderma species; bacteria such as Bacillus species (described in EP-A-134,048 and EP-A-253, 455), e.g. species of Bacillus subtilis, Bacillus 1 i c h e n i f o rm i s, Bacillus amyloliquefaciens, Pseudomonas; and yeasts such as Kluyveromyces species (described in EP-A-096,430 e.g. Kluyveromyces lactic and EP-A-301, 670) and species of Saccharomyces e.g. Saccharomyces cerivisiae.

Culture of host cells and recombinant production According to the present invention, the production of the polypeptide of the invention can be carried out by culturing microbial expression hosts, which have been transformed with one or more polynucleotides of the present invention, in a conventional nutrient fermentation medium.

The recombinant host cells according to the invention could be cultured using methods known in the art. For each combination of a host cell and a promoter, the culture condition is available, which are convenient for the expression of the DNA sequence encoding the polypeptide. After reaching the desired cell density or polypeptide titration, the culture is stopped and the polypeptide is recovered using known procedures.

The fermentation medium may comprise a known culture medium containing a carbon source (eg glucose, maltose, badges, etc.), a nitrogen source (eg ammonium sulfate, ammonium nitrate, ammonium chloride, etc.). , a source of organic nitrogen (eg yeast extract, malt extract, peptone, etc.) and sources of inorganic nutrients (eg phosphate, magnesium, potassium, zinc, iron, etc.). Optionally, an inducer (e.g. apple MHR, pectin or xylogalacturonan) could be included.

The selection of the appropriate medium could be based on the choice of expression host based on the regulatory requirements of the expression construct. Such means are well known to those skilled in the art. The medium, if desired, could contain additional components that favor transformed expression hosts over other potentially contaminating microorganisms.

The fermentation can be carried out for a period of 0.5-20 days in a batch, continuous or intermittent feeding process at a temperature in the range between 0 and 45 ° C and, for example, a pH between 2 and 10. The conditions preferred fermentation are a temperature in the range of between 20 and 37 ° C and / or a pH between 3 and 9. The appropriate conditions are usually selected based on the choice of the expression host and the protein to be expressed.

After fermentation, the cells can be removed from the fermentation broth by centrifugation or filtration. After removal of the cells, the polypeptide of the invention could then be recovered and, if desired, purified and isolated by conventional means.

D. Methods for processing plant materials or containing pectin.

Plant or pectin-containing materials include pulp from plants, parts of the plant and plant extracts. In the context of this invention an extract of a plant material is any substance that can be derived from the plant material by extraction (mechanical and / or chemical), processing or by other separation techniques. The extract could be juice, nectar, base or concentrates made from it. Plant material could comprise or be derived from vegetables, eg, carrots, celery, onions, legumes or leguminous plants (soybeans, peas) or fruit, eg, apple or seed fruits (apples, pears, quince, etc.), grapes , tomatoes, citrus fruits (orange, lemon, lime, tangerine), melons, plums, cherries, black currant, redcurrants, raspberries, strawberries, blueberries, pineapple and other tropical fruits, trees and parts of them (eg pollen from Pine tree) . According to this invention, apples and apple juice are especially preferred.

The polypeptides of the invention can thus be used to treat the plant material including plant pulp and plant extracts. For example, they can be used to treat apple pulp and / or raw juice during the production of apple juice. They could also be used to treat food products or ingredients of edible food products. Conveniently, the polypeptide of the invention is combined with suitable carriers or diluents (solids or liquids) including buffers to produce a composition or enzyme preparation.

The polypeptide is typically formulated stably in either liquid or dry form. Typically, the product is made as a composition that will optionally include, for example, a stabilizing buffer and / or preservative. The compositions could also include other enzymes capable of digesting the material or pectin of the plant, for example other pectinases such as an endo-arabinanase, rhamnogalacturonase and / or polygalactulonase. For certain applications, the immobilization of the enzyme in a solid matrix or the incorporation onto or in particles of solid carriers could be preferred. The composition could also include a variety of other enzymes that degrade the material of the plant, for example cellulases and other pectinases.

The polypeptides and compositions of the invention could therefore be used in a method to process plant material to degrade or modify the pectin constituents of the cell walls of the plant material2.

Typically, the polypeptides of the invention are used as a composition / enzyme preparation as described above. The composition in general will be added to the pulp of the plant obtained by, for example, mechanical processing, such as grinding or milling the material of the plant. Incubation of the composition with the plant will typically be carried out for a time of 10 minutes to 5 hours, such as 30 minutes to 2 hours, preferably for about 1 hour. The process temperature is preferably 10-55 ° C, e.g. from 15 to 25 ° C, optimally about 20 ° C and 10-300 g, preferably 30-70 g, optimally about 50 g of the enzyme can be used per ton of material to be treated. All the enzymes or their compositions used could be added sequentially or at the same time to the pulp of the plant. Depending on the composition of the preparation of the enzyme in the plant material, it could first be macerated (e.g., a puree) or liquefied. Using the polypeptides of the invention, processing parameters such as extraction yield, extract viscosity and / or extract quality can be improved.

Alternatively, or in addition to the foregoing, a polypeptide of the invention could be added to the raw juice obtained from pressing or liquefying the pulp of the plant. The treatment of the raw juice will be carried out in a similar way to the pulp of the plant with respect to the dosage, temperature and time of maintenance. Again, other enzymes such as those discussed previously could be included. Typical incubation conditions are as described in the previous paragraph. Once the crude juice has been incubated with the polypeptides of the invention, the juice is then centrifuged or flushed to produce the final product.

A composition comprising a polypeptide of the invention could also be used during the preparation of fruit or vegetable purees.

The final product of these processes is typically treated by heat at 85 ° C for a time of 1 minute to 1 hour, under the conditions to partially or completely inactivate the polypeptides of the invention.

Due to the highly specific action on pectins, the polypeptides of the invention could also be used to prepare the pectins with modified characteristics, e.g. Modified gelation capabilities for specific applications.

The polypeptides of the invention could also be added to animal feeds rich in pectin or xylogalacturonan e.g. food containing soy, to improve the cutting of the cell wall of the plant, leading to improved utilization of plant nutrients by the animal. The polypeptides of the invention could be added to feed or silage if pre-rinse or wet diets are preferred. Advantageously, the polypeptides of the invention could continue to degrade the xylogalaturonans in the food. The fungal-derived polypeptides of the invention in particular, generally have lower optimum pH and are capable of releasing important nutrients in such acidic environments as the stomach of an animal. The invention in this manner also contemplates food or food products (e.g., animal) comprising one or more polypeptides of the invention.

The polypeptides of the invention could also be used during the production of milk substitutes (or replacements) of soybeans. These milk substitutes can be consumed by humans and animals. A typical problem during the preparation of these milk substitutes is the high viscosity of the soybean suspension, resulting in the need for an undesirable dilution of the suspension to a dry solids concentration of 10 to 15%. An enzyme preparation containing a polypeptide of the invention can be added to, or during the processing of the suspension, allowing processing at a higher concentration (typically 40 to 50%) of dry solids. The enzyme could also be used in the preparation of flavoring products, e.g. of soy.

Tests for enzymes that degrade pectin.

The new tests and substrates described here have allowed the identification and confirmation of endo-xylogalacturonase activity. However, these tests can be used to detect other enzymes that degrade pectin, whether or not they have endo-xylogalacturonase activity.

The substrate that can be used for this test may comprise tragacanth gum which has been treated with a strong acid. A preferred acid is trifluoroacetic acid (TFA). The gum tragacanth could optionally be saponified and / or it could have been treated with an alkali, for example an alkali metal hydroxide, for example NaOH.

Another aspect of the invention relates to a test for identifying or detecting a polypeptide that is capable of degrading pectin. The activity could be an endo-xylogalacturonase or, it could be pectin lyase, polygalacturonase, esterase, cellulase, xyloglucanase, galactonase, arabinanase or rhamnogalacturonase. The test could include: to. providing as a substrate for a candidate compound (usually a polypeptide) the substrate described in the previous paragraph; Y b. contacting the substrate with the candidate compound, and detecting if any carbohydrate reduction occurs.

The amount of these reducing carbohydrates can be measured. If necessary, they can then be compared with the amount of carbohydrates produced in a control experiment, in the absence of the candidate compound.

The measurement could involve a BCA test. This could comprise measuring the amount of Cu (II) reduced to Cu (I) by the reduction of the carbohydrates present. This could be by contact with bicinchoninic acid (BCA), and determining the amount of the BCA-Cu (I) complex formed.

The invention will now be described with reference to the following Examples which are intended to be illustrative and not limiting. In the Figures that accompany the Examples: Figure 1 is a diagram of the hypothetical structure of the prevalent apple MHR population (modified filamentous region) having the highest molecular weight (subunit I is xylogalacturonan, subunit II is the structure rich in arabinano side chains, subunit III are oligomers of rhamnogalacturonase The distribution of acetyl groups does not occur, but the main parts are thought to be located within subunit III Key: GalA = galacturonic acid, rham = rhamnose, gal = galactose, xyl = xylose; ara = arabinose) Figure 2 is a map of the vector pCVlacK according to the invention (construction described in Example 1); Figure 3 is a graph illustrating a HPAEC of xylogalacturonan after degradation by xylogalacturonase (a polypeptide of the invention); Figure 4 is a graph illustrating a HPSEC of xylogalacturonan before and after degradation by a xylogalacturonase; Figure 5 is a graph illustrating a Maldi-ToF mass spectrum of the products of the complete degradation of xylogalacturonan by a xylogalacturonase; Figures 6A-G are graphs of the HPSEC elutions showing the degradation of MHR-S by endo-arabinanase, rhamnogalacturonase and xylogalacturonase, separately and in combination.

Figures 7 and 8 are graphs of an HPSEC and HPAEC, respectively, elution patterns showing the degradation of soy pectin by xylogalacturonase; Figure 9 is a diagram showing the multiple alignment of the part of the PG, XghA and RHG sequences (the amino acids identical to the XghA sequence are replaced by a dot) and an introduction of spaces is indicated to obtain an optimal alignment (- ). The amino acids conserved in the whole plant are shaded, PG 's of fungi and prokaryotes. Key: Atub = A. tubigensis; Anig = A. niger; Aac = A. aculeatus.

EXAMPLE 1 Construction of a cDNA Expression Library of Aspersillus tubisens is Example 1.1: Construction of an expression vector The initiator vector pGBHSA20 (CBS 997.96) contains the promoter and terminator sequence of the lactase gene. { lac4) of K. lactis, a G418 selection marker and the E. coli plasmid pTZ18r for propagation in this host. The 17-chambered KARSCEN of K. lactis (a gift from Dr. A. A. Winkler, Dept. of Cell Biology and Genetics, Leiden University, The Netherlands) was cloned into a unique Smal site of this vector. The resulting vector was named pCVlacK (Figure 2). The unique ffindIII and Xh or l sites flanking the l a c 4 promoter and terminator, respectively, can be used as cloning sites for cDNAs synthesized from Aspergill's poly (A) RNAs.

Example 1.2: Isolation of RNA synthesis and poly (A) cDNA Aspergillus inoculans of conidia were inoculated in triplicate at a density of 106 spores / ml in 300 ml of medium containing (per liter): 6 g of NaN03, 0.5 g of KCl, 1.5 g of KH2P04, 0.5 g of MgSO4 (pH 6.5), 1 ml of Timberlake spore elements, lOOOx (per ml, 50 mg of EDTA, 22 mg of FeS04-7H20, 5 mg of MnCl2-2H20, 22 mg of ZnS04-7H20, 1.6 mg of CuSO) -5H20, 1.7 mg of CoCl2-6H20, 1.5 mg of Na2Mo04-2H20, 11 mg of H3B03, adjusted to pH 6.5) and 10 ml of Timberlake vitamins lOOx (per ml, 0.2 mg of thiamin-HCl, 0.2 mg of riboflavin, 0.2 mg of nicotinamide, 1 mg of pyridoxine-HC1; 0.02 mg of pantothenic acid, 0.4 μm of biotin, adjusted to pH 5 to 6), 1 g of yeast extract, 5 g of Soyoptim ™ (defatted soy flour, toast from Societe Industriele Olneux, France). The cultures were incubated on a rotary shaker at 28 ° C, 150 rpm. The mycelium of one culture was harvested 10 hours after inoculation, the mycelium of the other two cultures at 16 and 24 hours after inoculation. From 1 g rinsed and squeezed, RNA from the total mycelium was isolated by the RNAzol method (Cinna / Biotecx). Poly (A) RNA was isolated using oligotex Qiagen ™ columns (Westburg). Equal amounts of the poly (A) RNA at the 10, 16 and 24 hour points were poured. The cDNA was synthesized using the ZAP-cDNA synthesis kit (Stratagene ™) with the following modifications: the first synthesis of the strand was made with Superscript II reverse transcriptase (GibcoBRL). To 7.5 μg of poly (A) RNA, 2 μl of the linker primer and RNA-free water was added to a final volume of 28.5 μl. This mixture was incubated for 10 minutes at 70 ° C and cooled on ice. The following components were added. 10 μl of 5x first-strand buffer, 5 μl of 0.1 M DTT, 3 μl of first-strand methyl nucleotide mixture and 1 μl of RNAse block. This was incubated for 10 minutes at 25 ° C, followed by 2 minutes at 42 ° C. Subsequently, 2.5 μl of Superscript ™ II RT (200 U / μl) was added, mixed and incubated for 50 minutes at 42 ° C. A second ligation of the protocol was the ligation of a Hi n dI I I adapter instead of the EcoRI adapter.

The cDNA well was size-separated using a Sephacryl S-500 column. The first fraction eluted from the column did not contain any cDNAs, but the second and third fractions contained the largest cDNA. Subsequent fractions were assumed to contain relatively greater amounts of non-total cDNA length and were not of use for library construction. The cDNA from fractions 2 and 3 was ligated into the HindIII and Xh ol sites of the pClacK expression vector (see Figure 2) using the Clontech Ligation Express ™ kit. Each ligation mixture was transformed into two batches for the MRF 'XL-Blue cells of E. col i. The suspensions of the fourth transformation were plated on 32 agar plates (LB + 50 μg / ml ampicillin). After 16 hours of incubation at 37 ° C, 7366 transformations were obtained. The bacteria were collected by pouring 2.5 ml of LB medium into a plate and then scraping the cells; 0.5 ml of the cell suspension was added to glycerol and stored at -80 ° C, the remaining 2 ml were used for DNA isolation (Qiagen ™ Spin miniprep kit). In the case that lower number of transformations per plate was found, the 2.5 ml were transferred to a second, third or fourth plate. This produced 22 wells of approximately 325 individual transformations. Equal amounts of DNA from each well were combined for use in the transformation of K. l a c t i s.

Example 1.3: Transformation of the expression library in K. l a c t i s.

An overnight culture of K. lactis strain CBS 2359 grown in YPD (10 g / 1 yeast extract, 20 g / 1 Bacto-peptone, 20 g / 1 glucose at 30 ° X, diluted 3000, 600, 300 and 100 times in 150 ml of fresh YPD and incubated for 6 hours at 30 ° C, 160 rpm on a rotary shaker.The culture with an optical density of 0.7-1.0 was used to prepare the cells6 elect rocompet entities.

The elect rocompet ent cells were transformed with 1 μg of combined DNA from the E library. col i. Electroporation was performed using a Biorad Genepulser ™ with calibrations at 1.4 kV, 200 Ohm and 25 μF. Transformations were selected on double-layer YPD plates (YPD with 20 g / 1 of Bacto agar); the bottom layer contained 50 μg / ml of G418, the top layer was not selective. 660 μl of the transformation mixture was placed in plates on 80 double layer plates. The aliquots of 1.5 and 15 μL were taken with the pipette on the plates. Approximately 10,000 transformations were obtained.

EXAMPLE 2 Preparation of the substrate Example 2.1: Preparation of apples MHR-S The modified filamentous (MHR) regions of apples were isolated as a retentate from the filter after the treatment of the apples with pectinase, and subsequently the MHR was saponified resulting in MHR-S8.

Example 2.2: Synthesis of xyloqalacturonan of tragacanth gum g of tragacanth gum (Sigma, St. Louis, MO, USA) was suspended in 900 mL of distilled water cooled with ice. To this solution was added 10 mL of solution of M NaOH cooled with ice. After 24 h, saponified tragacanth gum (sGT) was extensively dialyzed against distilled water at 4 ° C, and concentrated under reduced pressure to 1 L. For moderate acid hydrolysis, 7.65 mL of trifluoroacetic acid (TFA) was added. to this solution of sGT. The final concentration of the TFA in the solution was 0.1 M. The sGT / TFA solution was heated to the boiling point in a microwave and subsequently incubated in a boiling water bath for 1 h. Finally, the hydrolyzate was dialyzed against distilled water at 4 ° C and dried by freezing. The procedure produced 2.61 g of the material (additionally referred to as xylogalacturonan).

Example 2.3: Characterization of xyloqalacturonan To determine the sugar composition of the original tragacanth gum as well as the xylogalacturonan, the samples were hydrolysed with 1M H2SO4 (100 ° C, 3 h) 8 and the neutral sugars were converted to their alditol acetates to quantify the individual sugars by gas chromatography (GC). The uronic acid content of the hydrolyzate was determined colorimetrically using m-hydroxy-phenyl lo8. The sugar composition in% mol of the original tragacanth gum (GT) in comparison with the xylogalacturonan (XG) is shown in Table 1 below.

Table 1 Moderate acid hydrolysis effectively removed the arabinosyl and fucosyl residues from this polysaccharide, while the GalA: Xyl ratio was more or less unchanged.

The degree of acetyl and methyl esterification of tragacanth gum was estimated by high pressure liquid chromatography (HPLC) 7. The degree of methylation and acetylation of tragacanth gum is approximately 75% and 20%, respectively (calculated as mol of methyl or acetyl groups per mole of GalA). The saponification of the gum removed all the methyl and acetyl groups. The molecular weight distribution of the polymers was carried out by high pressure size exclusion chromatography (HPSEC) in three columns Bio-Gel TSK (40XL, 30XL and 20XL) in series8. Moderate acid hydrolysis of the saponified tragacanth gum was performed with a decrease in molecular weight, although the resulting material still had a high molecular weight. Based on the elution profiles of HPSEC, xylogalacturonan has an estimated molecular mass of approximately 1,100 kDa, using the pullulan reference compounds.

Xylogalacturonan was effectively proven to be resistant to enzymatic degradation by all endo-polygalacturonase and rhamnogalacturonase.

EXAMPLE 3 Screening the library with a BCA test Example 3.1: Growth of the transformations and preparation of the enzyme.

Of approximately 10 000 K transformations. l a c t i s produced in Example 1.3, 3,500 individual colonies were selected and transferred to separate the wells from the multi-well plates. Transformations in the multi-well plates were grown for 48 hours at 30 ° C in medium 1 (for 500 mL of H20 (pH 6.0), mannitol, 10.00 g, NH4H2PO ", 1.50 g, KH2P04, 0.25, (NH4) S04, 0.50 g; CaCl, 2H20, 0.01 g, MgSO4-7H20, 0.15 g, trace elements of H3B03, 3.75 μg, CuS04-5H20, 40 μg, Kl, 75 μg, MnS04-4H20, 300 μg, NaMo04, 150 μg, ZnS04 -7H20, 300 μg; FeCl3-6H20, 200 μg) and Ca pantothenate vitamins, 500 μg; thiamine, 500 μg; myo-inositol, 500 μg; pyridoxine, 500 μg; nicotinic acid, 500 μg; biotin, 5 μg) containing 80 ng / mL of the antibiotic G418 and the 35 plates were stored as reserves of 15% glycerol.

These transformations were used to inoculate a new group of 35 multi-well plates containing 200 μL of the same medium with 80 ng / mL of G418 with a replica plate. The transformations of K. The plants were grown for two days at 30 ° C in an oven. The cells were precipitated by centrifugation at 3000 rpm in a Hermle ™ zk380 centrifuge.

Example 3.2: Degradation of the substrate μL of the supernatant from each well was carefully pipetted into a new multi-well plate, and 25 μL of a 0.2% solution of the substrate (either MHR-S or xylogalacturonan or sGT / TFA from Example 2.2) was added or 100 mM NaOAc buffer pH 5.0. After incubation overnight in an oven at 30 ° C, the increase was measured to reduce carbohydrates with the BCA test.

Example 3.3: The BCA test The BCA test is based on the reduction of Cu (II) to Cu (I) by reducing monocarbohydrates and oligomers. A complex of bicinchoninic acid (BCA) and Cu (I) is formed. This complex produces an intense purple color, which can be measured spectrophotometrically. This heat increases with an increase in the reduction of carbohydrate concentration. The method used in this invention is a modification of a known method, 9 but it was used for screening purposes.

The procedure consisted in mixing 10 μL to reduce the sample containing the carbohydrate of Example 3.2 with 90 μL of water and 100 μL of BCA reagent together in a multi-well plate. The BCA reagent was made fresh every day by mixing two solutions, A and B, 1: 1 (v / v) together. Solution A consisted of 54.28 g of Na2CO3, 24.20 g of NaHCO3 and 1942 g of Na2BCA per liter of distilled water. Solution B consisted of 1248 g of CuSO4-5H20 and 1262 g of L-serine per liter of distilled water. The plate containing the sample, the reagent and the water were incubated for one hour in an oven at 80 ° C with a lid on the plate. After cooling the plate for 15 minutes, the absorbance at 550 nm was measured using a multi-well reading plate (SLT lab instruments, Austria, model EAR 400). The galactose test lines showed that the test was linear in the range of 0 to 125 μM galactose.

The transformations that occurred in the BCA plate in 0.1 absorbance units greater than the blank were verified for the degradation capacities of xylogalacturonan by growing them again and repeating the BCA test using xylogalacturonan as a substrate. Three transformations that produce xylogalacturonase were found.

EXAMPLE 4 Example 4.1: Characterization of cDNA encoding xylogalacturonase All the plasmid insertions of these three transformations were identical, as they were found after the analysis of the restriction patterns of these insertions. The transformation of K. l a c t i s 27E8, which exhibit xylogalacturonase activity, was used to isolate the expression plasmid pCVlacK by a glass beads method10. After the transformation and propagation of this plasmid in E. col i, the cDNA insert was deleted from pCVlacK with a digestion Hi ndl l l / Xh ol. This digestion released a fragment of 1.0 and 0.4 kb, due to an internal JíindlII site as it appeared from the nucleotide sequence afterwards. The DNA sequence of the cDNA insert was determined in both strands using the 5 'and 3' specific primers for the lac4 regulatory sequences, and the primers based on the cDNA sequence. The DNA sequence of the cDNA insert is presented in SEQ ID No. 1, together with the deduced amino acid sequence. Upstream of the ATG translation initiation codon, 20 nucleotides of the 51 non-translated sequence are present. Downstream of the TAA stop codon, 130 nucleotides of the untranslated sequence were found followed by the poly-A tail. The open reading frame of the 1218 nucleotides. { xghA) encodes a protein of 406 amino acids, presented in SEQ ID No. 2, named XghA. The potential cut-off site of the signal sequence, predicted according to rule (-3, -l) 11, is between position 18 and 19. ORF that starts with an ATG codon and ends with a TAA codon, from this way is predicted by a 5 'non-coding region of 20 base pairs and followed by a 3' non-coding region of 130 base pairs and a poly (A) end. The TCATCATGGC sequence that covers the ATG start codon resembles the contents of the sequence for initiation or translation in higher eukaryotes14. The xghA cDNA encodes an apparent signal sequence of 18 amino acids at the amino terminus, with a peptidase cleavage site between Ala18 and Ala19. Two potential N-glycosylation sites have been found in Asn178-Ser-Thr and Asn-Thr and Asn301-Val-Thr.

The comparison of the amino acid sequence to the proteins database showed homology for the polygalacturonase sequences of prokaryotes, fungi, plants and rhamnogalacturonase A and B from Aspergillus. A comparison of the amino acid sequence XghA has been made using the sequences from the EMBL results library. XghA showed 31 to 39% similarity for the similarity of endo-PG and 44% for exo-PG of A. tubigensis. The similarity of XghA to 2RHG-A was 30%. { A. niger) and 32%. { A. aculeatus) while the similarity to RGH-B of A. niger was only very limited.

Figure 9 shows the analysis of the multiple alignments of Xgh to the PG and to RHG-A. The multiple alignment shows four domains of the conserved amino acids, which were first described for the plant's polygalacturonase, fungi and bacterial origin15. When all the PG 's that were retrieved from a database search were aligned, only four small amino acid elongations are conserved: NXD, DD, HG and RXK (shaded in Figure 9, where X represents a variable amino acid) ). The essential amino acids that are thought to be involved in the hydrolysis reaction are one of the three aspartic acid residues of domain I and II and the histidine of domain III. These domains are completely conserved in the XghA. It is postulated that the four domains contain amino acids that are involved in the binding of the substrate. The arginine residue of this domain is a glycerin residue in XghA. The domains are less conserved in the RHG sequences, as two of the three aspartic acid residues are conserved and the histidine is replaced by glycine.

Example 4.2: Stain or transfer analysis The copy number of the Xh gA gene was determined by Southern blot analysis of A genomic DNA. t ubi gene if digested with several enzymes (results not shown).

Hybridization under severe conditions (65 ° C and 0.2 x SSC) and less severe conditions (60 ° C and 1 x SCC) with a 1.0 kb foldin fragment of the simple hybridization fragments clearly shown by xghA. This shows that the xghA gene is present as a single copy in the A genome. t ubi gen s i s.

EXAMPLE 5 Expression of the enzyme The transformations of K. lactis expressing the endo-xylogalacturonase cDNA were transferred from the glycerol stocks of the multi-well plate to the reagent tubes: 10 μL of glycerol stock was added to 1-2 mL of medium I (see Example 3.1) with 80 ng / mL of G418. These cultures were grown at 30 ° C in a rotary incubator at 200 rpm for two days and were used to inoculate Erlenmeyer flasks containing 20 mL of this same medium supplemented with the antibiotic. For the larger-scale production of the enzyme, these cultures were used to inoculate 500 mL of the same medium supplemented with the antibiotic in 1 L Erlenmeyer flasks. The cells were grown at 30 ° C in a rotary incubator at 200 rpm for two days . The cultures were centrifuged to precipitate the cells, the supernatant was used for purification This crude enzyme preparation (350 mL) was pre-concentrated on a Hitrap ™ Q ion exchange column (Pharmacia Biotech, Sweden) with a flow rate of 0.3 mL / min. Elution was performed in an FPLC system (Pharmacia Biotech, Sweden) with a saline gradient using 20 mM piperazine (pH 5.0) starting with the buffer (buffer A) and 0.5 M NaCl in 20 mM piperazine elution buffer (pH 5.0) (shock absorber B). The following gradient was used: for B at 10% in 1 minute, for B at 35% in 19 minutes, for B at 100% in 2 minutes and B at 100% for three more minutes. The activity was verified as described in Example 3 and the active fractions were poured. They were diluted three times with 20 mM piperazine buffer (pH 5.0) and applied on a MiniQ column (Pharmacia Biotech, Sweden). Elution was performed in a Smart system (Pharmacia Biotech, Sweden) with a linear pH gradient of 20 mM piperazine (pH 5.0) starting with the buffer 10 mM HCl a flow rate of 0.4 mL / min. Fractions of the activity were poured and investigated using SDS-PAGE. Due to the silver staining of the gel, a protein band with a molecular mass of approximately 60 kDa was found. The difference with the predicted MW of approximately 45 kDa, based on the DNA sequence (see Example 4) is thought to be due to the glycosylation of the protein.

EXAMPLE 6 Influences of pH and temperature on enzymatic activity The purified enzyme, obtained as described in Example 5, was used for the characterization of the enzyme. The measurements at t = 0 were used as targets. For the determination of pH stability, the purified enzyme was pre-incubated without substrate for one hour at a pH in the range of 2.5 to 8 in Mcllvaine buffers. After the enzyme was incubated with the substrate for two hours and the increase in the reduced sugars was determined as described in Example 3. The enzyme was stable over a pH range of 3 to 6.

For the determination of the pH and the optimum temperature, the purified enzyme was incubated with the substrate for two hours at a pH range of 2.5 to 8 or at a temperature in the range of 20 to 80 ° C. After this, the increase in the reduced sugars was determined as described in Example 3. The enzyme had an optimal activity at a temperature of 60 ° C and a pH of 3.0. The enzyme shows more than 50% of its activity in the pH range of 2.5 to 5.0. The activity at pH 2.5 was still 90% of the maximum value at pH 3.0. No lower pH 2.5 values were measured.

EXAMPLE 7 Mode of action of xylogalacturonase The degradation of xylogalacturonan (modified tragacanth gum, Example 2.2) by the supernatant of clone K. l a c t i s that produces xylogalacturonase was monitored by high-ion exchange chromatography (HPAEC) and high-throughput size exclusion chromatography (HPSEC).

The HPAEC was performed using a Dionex carbopack PAl column of a size of 4 x 250 ml. The elution was carried out with 0.1 M NaOH (solution A) and 1 M NaOAc in 0.1 NaOH (solution B). The following gradient was used: from 0 to 62% B in 50 minutes, for 100% B in 5 minutes, followed by 100% B in 5 minutes. The enzyme did not produce xylose (expected at a retention time of 5 minutes) or galacturonic acid (expected at a retention time of 15 minutes), not even after 8 hours of incubation. Only the oligomers were released, the smallest oligomer was found at a retention time of approximately 22 minutes: this was a dimer of xylose-galacturonic acid.

The HPSEC was made using three columns in series: Bio-Gel TSK 40 (300 x 7.5 mm, from Biorad), Bio-Gel TSK 30 XL (300 x 7.5 mm, from Biorad) and TSKGel G 2500 P XL (300 x 7.8 mm, from TosoHaas). Figure 4 shows that the high molecular weight fraction of xylogalacturonan (left in the Figure) was rapidly degraded (the top line (B) represents the polymer before degradation and the bottom line (A) represents the polymer after degradation ).

When the degradation products were monitored by Maldi-ToF mass spectroscopy, the products found were identified and are shown in Table 2.

Table 2 In this way, two products were formed when MHR-S was incubated with XghA. These products appeared even after a short incubation time.

These results show that xylogalacturonan is degraded by xylogalacturonase in an endo form. The profiles obtained were compared with those of a digested polygalacturonic acid, and it was clear that none of the MHR-S degradation products formed were oligomers of polygalacturonic acid. This demonstrates that XghA produces oligomers of galacturonic acid substituted with xylose.

Due to the incubation of the supernatant of the transformant of K. l a c t i s that produces xylogalacturonase with polygalacturonic acid, no degradation of this substrate was observed.

EXAMPLE 8 Complete degradation of MHR-S by xylogalacturonase in combination with other enzymes.

To study the degradation of MHR-S, 200 μL of a 0.3% MHR-S solution in 50 mM NaOAc pH 5.0, was incubated with 5 μL of the purified xylogalacturonase, 5 μL of endo-arabinanase12.5 μL of ramnogalacturonasa13, or with combinations of these enzymes, added sequentially or at the same time. MHR-S without the enzyme was used as a control.

The degradation of MHR-S was monitored with HPSEC, as described in Example 7. The results are shown in Figures 6 to 6G where the upper line (b) represents the control and the lower line (d) represents the incubation with the enzyme. The incubations were with: A: arabinanase; B: xylogalacturonase; C: rhamnogalacturonase; D: endo-a rabbi nana s and xylogalacturonase sequentially; E: combined endo-arabinanase and xylogalacturonase; F: endo-arabinanase and rhamnogalacturonase sequentially; and G: combined endo-arabinanase, rhamnogalacturonase and xylogalacturonase.

Figure 6B shows that xylogalacturonase was able to degrade MHR-S: a small change to lower molecular weight material can be observed. Also enzymes endo-arabinanase (Figure 6A) and rhamnogalacturonase (Figure 6C) caused some change in molecular weight. However, much better results are obtained by combining two different enzymes in an incubation (Figures 6D-endo-arabinanase and xylogalacturonase sequentially, 6E-endo-arabinanase and xylogalacturonase combined and 6F-endo-arabinanase and endo-rhamnogalacturonase sequentially). The difference between Figures 6D and 6E is remarkable: the combined addition of the endo-arabinanase and xylogalacturonase was much more effective than with the sequential addition. Almost all degradation of the high molecular weight material was possible when the three combined enzymes were added (Figure 6G).

EXAMPLE 9 Improvement of filtration rate by xylogalacturonase in combination with other enzymes.

The experiments were done to see if xylogalacturonase could prevent filtration during filter fouling. Apple MHR-S prepared as described in Example 2.1, was used as a substrate. A 0.5% solution in 50 mM acetate buffer pH 4.0 was incubated with a combination of the three enzymes: endo-arabinanase, rhamnogalacturonase and xylogalacturonase (ea / rg / xgh), a combination of two enzymes: endo-arabinanase and rhamnogalacturonase ( ea / rg) and with xylogalacturonase (xgh) separately for 17 hours at a pressure of 2 bar. The increase in filtrate weight was followed all the time. The results are shown in Figure 5.

EXAMPLE 10 Degradation of a pectic fraction of soy by xylogalacturonase An alkaline soluble fraction of soy flour, 1 MASS, rich in pectic substances was isolated and characterized16. A 0.25% solution of 1 MASS in 50 mM sodium acetate buffer pH 5, including 0.01% NaN3 was incubated with xylogalacturonase. The digestion obtained after 24 hours of incubation at 30 ° C was analyzed for the molecular weight distribution by HPSEC and the release of the oligomeric degradation product by HPAEC. The analyzes were performed as described in Example 7.

Figure 7 shows the changes in the molecular weight distribution as measured by HPSEC: in the material treated with xylogalacturonase (curve b) the peak at approximately 20 min, which represents the high molecular weight material, decreases to 70% of the value of the initiating material (curve a).

Figure 8 shows the results of the HPAEC analysis. Comparing the material treated with the enzyme (curve b) with the target (curve a) it can be observed that xylogalacturonase causes the release of the characteristic xylosyl galacturonic acid dimer (marked with an X) and the other unidentified oligomers (peaks next to it) right of X), comparable with the peaks shown in Figure 6 of Example 7.

REFERENCES 1. G. Beldman L.A.M. van den Broeck, H.A. Schols, M.J.F. Searle-van Leeuwen, K.M.J. van Laere and A.G.J. Voragen (1996) Biotechnology Letters 18: 707-712 2. C. Grassin and P. Fauquembergue (1996) Pectins and Pectinases, Progress in Biotechnology 14: 453-462 3. Goosen et al., 1992, "Transformation and Gene Manipulation in filamentous fungi: an overview" In: Handbook of Applied Mycology "Vol. 4:" Fungal Biotechnology ", D.K.

Romanos et al. Yeast 8: 423-488 (1992) Arora, R.P. Elander and K.G. Mukerji, eds., Marcel Deker In. , New York; pp 151-195 K.N. Faber, P. Haima, W. Harder, M. Veenhuis, G. AB (1994), Current Genetics 25: 305-310.

A.G.J. Voragen, H.A. Schols and W. Pilnik, (1986) Food Hydrocoll. 1: 65-70 HE HAS. Schols, M.A. Posthumus, A.G.J. Voragen (1990) Carbohydrate Research 206: 117-129 J.D. Fox, J.F. Robyt (1991) Analytical Biochemistry 195: 93-96 M.A. Sobanski and Dickinson (1995) Biotechnology Techniques 9: 225-230) G. von Heijne (1986) Nucleic Acids Research 14: 4683-4690) G. Beldman. M.J.F. Searle van Leeuwen, G.A. de Ruiter, H.A. Siliha, A.G.J. Voragen (1993) Carbohydrate Polymers 20: 159-168 13. H.A. Schols, C.C., J.M. Geraerds, M.J.F. Searle van Leeuwen, F.J.M. Kormelink, A.G.J. Voragen (1990) Carbohydrate Research 206: 105-115 14. M. Kozak, et al, The Scanning Model for Translation: an update, J. Cell. Biol. 108 (1989) 229-241.

. H.J.D. Bussink et al, Curr. Genet 19 (1991), 467-474 16. Huisman, M.M.H. et al (1998) Carbohydr. Polym. 37: 87-95 17. Winkler A. A. et al, PhD thesis, University of Leiden, The Netherlands (1998).

LIST OF SEQUENCES (1. GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Gist-brocades B.V. (B) ADDRESS: Water1 ingseweg 1 (C) CITY: Delft (E) COUNTRY: The Netherlands (F) ZIP CODE: 2611 XT (ii) TITLE OF THE INVENTION: New Endo-xylogalacturonase (iii) NUMBER OF SEQUENCES: 2 (iv) AVAILABLE COMPUTER FORM: (A) TYPE OF MEDIA: Flexible disk (B) COMPUTER: IBM PC Compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PROGRAMMING ELEMENTS: Patentln Relay # 1.0, Version # 1.25 (EPO) (v) CURRENT APPLICATION DATE: APPLICATION NUMBER: N / A (2) INFORMATION FOR SECTION ID NO: l: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1602 base pairs (B) TYPE: nucleic acid (C) HEBRA: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTICIPATION: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Aspergillus tubigensis (ix) FEATURE: (A) NAME / KEY: CDS (B) LOCATION: 98..1318 CTCGAG is the site Xhol (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: l: GCTTGTGTTT CTTAGGAGAA TTATTATTCT TTTGTTATGT TGCGCTTGTA GTTGGAAAAG 60 GTGAAGAGAC AAAGCTTGAA TTCCGAAATC GCTCATC ATG GCG CTA TAT CGT AAC 115 Met Ala Leu Tyr Arg Asn 1 5 CTC TAC CTT CTG GCC AGC CTT GGG CTA AGC AGT GCT GCT CCC TCC AAG 163 Leu Tyr Leu Leu Wing Ser Leu Gly Leu Ser Wing Wing Pro Ser Lys 10 15 20 GTC CAG CGA GCC CCG GAT TCT TCC ATT CAT GCT CGC GCT GTC TGT ACC 211 Val Gln Arg Ala Pro Asp Ser Ser lie His Ala Arg Ala Val Cys Thr 25 30 35 CCG ACC GCA GGA GGC GAT TCG TCC ACC GAC GAT GTC CCC GCC ATC ACC 259 Pro Thr Wing Gly Gly Asp Ser Ser Thr Asp Asp Val Pro Ala Lie Thr 40 45 50 GAG GCC CTC AGC TCG TGC GGA AAT GGT GGC ACC ATC GTC TTC CCC GAG 307 Glu Ala Leu Ser Ser Cys Gly Asn Gly Gly Thr lie Val Phe Pro Glu 55 60 65 70 GGC AGC ACC TAC TAC CTC AAC AGT GTG CTG GAC TTG GGC AGC TGC AGT 355 Gly Ser Thr Tyr Tyr Leu Asn Ser Val Leu Asp Leu Gly Ser Cys Ser 75 80 85 GAT TGC GAC ATC CAG GTG GAA GGT CTT CTG AAG TTC GCC AGC GAT ACC 403 Asp Cys Asp lie Gln Val Glu Glu Leu Leu Lys Phe Wing Ser Asp Thr 90 95 100 GAT TAC TGG AGC GGT CGC ACT GCC ATG ATC AGT GTT TCC AAT GTA GAT 451 Asp Tyr Trp Ser Gly Arg Thr Wing Met lie Ser Val Ser Asn Val Asp 105 110 115 GGT TTG AAG CTG CGC TCA TTG ACT GGA TCT GGT GTC ATT GAT GGC AAT 499 Gly Leu Lys Leu Arg Ser Leu Thr Gly Ser Gly Val lie Asp Gly Asn 120 125 130 GGC CAG GAT GCG TGG GAT CTC TTT GCT TCG GAC AGT AGT TAC TCA CGC 547 Gly Gln Asp Wing Trp Asp Leu Phe Wing Ser Asp Ser Ser Tyr Ser Arg 135 140 145 150 CCG ACG CTC TTG TAC ATC ACT GGC GGC AGC AAC CTA GAA ATC TCC GGG 595 Pro Thr Leu Leu Tyr lie Thr Gly Gly Ser Asn Leu Glu lie Ser Gly 155 160 165 CTG CGT CAA AAG AAT CCA CCT AAC GTG TTC AAC TCG GTC AAG GGT GGC 643 Leu Arg Gln Lys Asn Pro Pro Asn Val Phe Asn Ser Val Lys Gly Gly 170 175 180 GCC ACT AAT GTC GTC TTC TCC AAC CTG AAG ATG GAT GCC AAC TCC AAG 691 Wing Thr Asn Val Val Phe Ser Asn Leu Lys Met Asp Wing Asn Ser Lys 185 190 195 TCG GAC AAT CCG CCC AAG AAC ACT GAT GGG TTC GAC ATT GGC GAG AGT 739 Ser Asp Asn Pro Pro Lys Asn Thr Asp Gly Phe Asp lie Gly Glu Ser 200 205 210 ACC TAT GTG ACC ATC ACC GAG GTC ACC GTA GTC AAC GAT GAC GAC TGT 787 Thr Tyr Val Thr lie Thr Glu Val Thr Val Val Asn Asp Asp Asp Cys 215 220 225 230 GTC GCC TTC AAG CCC AGT TCC AAC TAC GTG ACÁ GTG GAC ACG ATC AGC 835 Val Wing Phe Lys Pro Ser Ser Asn Tyr Val Thr Val Asp Thr lie Ser 235 240 245 TGC ACC GGC TCC CAT GGA ATT TCC GTG GGA TCA TTA GGA AAG TCG AGC 883 Cys Thr Gly Ser His Gly lie Ser Val Gly Ser Leu Gly Lys Ser Ser 250 255 260 GAC GAC TCG GTC AAG AAC ATT TAT GTC ACG GGC GCA ACT ATG ATC AAC 931 Asp Asp Ser Val Lys Asn lie Tyr Val Thr Gly Ala Thr Met lie Asn 265 270 275 TCC ACC AAA GCC GCC GGG ATC AAG ACT TAT CCG AGT GGA GGC GAC CAC 979 Ser Thr Lys Wing Wing Gly lie Lys Thr Tyr Pro Ser Gly Gly Asp His 280 285 290 GGT ACC TCC ACG GTC AGC AAT GTG ACC TTC AAC GAT TTC ACT GTG GAC 1027 Gly Thr Ser Thr Val Ser Asn Val Thr Phe Asn Asp Phe Thr Val Asp 295 300 305 310 AAC TCC GAC TAT GCC TTC CAG ATC CAG AGC TGC TAT GGC GAG GAC GAT 1075 Asn Ser Asp Tyr Wing Phe Gln lie Gln Ser Cys Tyr Gly Glu Asp Asp 315 320 325 GAC TAT TGC GAG GAA AAC CCG GGC AAC GCC AAA CTG ACT GAT ATA GTC 1123 Asp Tyr Cys Glu Glu Asn Pro Gly Asn Wing Lys Leu Thr Asp lie Val 330 335 340 TTC AGT GGG ACA ACC AGT GAC AAG TAC GAT CCG GTC GTG 1171 Val Ser Ser Phe Ser Gly Thr Ser Asp Lys Tyr Asp Pro Val Val 345 350 355 GCC AAC CTC GAC TGC GGT GCG GAT GGA ACT TGT GGC ATC TCC ATC AGT 1219 Wing Asn Leu Asp Cys Gly Wing Asp Gly Thr Cys Gly lie Ser lie Ser 360 365 370 GGG TTC GAT GTC AAG GCG CCA TCG GGC AAG TCT GAA GTG TTG TGC GCC 1267 Gly Phe Asp Val Lys Wing Pro Ser Gly Lys Ser Glu Val Leu Lys Wing 375 380 385 390 AAC ACC CCG TCT GAT TTG GGC GTC ACT TGC ACT TCG GGG GCT TCG GGC 1315 Asn Thr Pro Ser Asp Leu Gly Val Thr Cys Thr Ser Gly Wing Ser Gly 395 400 405 TAAATAGCTT TGGCCGGGTT GCTTTCTGAA TCCACTGAGT GGAGGTCTTC TTCGGGTTTG 1375 ATATTTTGTA TGGTCGTGTG TATAGCAGAA TGTGACAATA GAATTAGTGA AATTGCCATT 1435 CTTTTCGAAA GACAAAAAAA AAAAAAAAAA AAAAAAAAAA ACTCGAGAAT TTATACTTAG_1495_ATAAGTATGT ACTTACAGGT ATATTTCTAT GAGATACTGA TGTATACATG CATGATAATA 1555 TTTAAACGGT TATTAGTGCC GATTGTCTTG TGCGATAATG ACGTTCC 1602 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 406 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: Met Ala Leu Tyr Arg Asn Leu Tyr Leu Leu Ala Ser Leu Gly Leu Ser 1 5 10 15 Be Ala Ala Pro Ser Lys Val Gln Arg Ala Pro Asp Ser Ser lie His 20 25 30 Wing Arg Wing Val Cys Thr Pro Thr Wing Gly Gly Asp Being Ser Thr Asp 35 40 45 Asp Val Pro Ala lie Thr Glu Ala Leu Ser Ser Cys Gly Asn Gly Gly 50 55 60 Thr lie Val Phe Pro Glu Gly Ser Thr Tyr Tyr Leu Asn Ser Val Leu 65 70 75 80 Asp Leu Gly Ser Cys Ser Asp Cys Asp lie Gln Val Glu Gly Leu Leu Lys Phe Wing Being Asp Thr Asp Tyr Trp Ser Gly Arg Thr Wing Met lie 100 105 110 Ser Val Ser Asn Val Asp Gly Leu Lys Leu Arg Ser Leu Thr Gly Ser 115 120 125 Gly Val lie Asp Gly Asn Gly Gln Asp Wing Trp Asp Leu Phe Wing Ser 130 135 140 Asp Ser Ser Tyr Ser Arg Pro Thr Leu Leu Tyr lie Thr Gly Gly Ser 145 150 155 160 Asn Leu Glu Lie Ser Gly Leu Arg Gln Lys Asn Pro Pro Asn Val Phe 165 170 175 Asn Ser Val Lys Gly Gly Ala Thr Asn Val Val Phe Ser Asn Leu Lys 180 185 190 Met Asp Wing Asn Ser Lys Ser Asp Asn Pro Pro Lys Asn Thr Asp Gly 195 200 205 Phe Asp lie Gly Glu Be Thr Tyr Val Thr lie Thr Glu Val Thr Val 210 215 220 Val Asn Asp Asp Asp Cys Val Wing Phe Lys Pro Ser Ser Asn Tyr Val 225 230 235 240 Thr Val Asp Thr lie Ser Cys Thr Gly Ser His Gly lie Ser Val Gly 245 250 255 Ser Leu Gly Lys Ser Ser Asp Asp Ser Val Lys Asn lie Tyr Val Thr 260 265 270 Gly Ala Thr Met Lie Asn Ser Thr Lys Ala Ala Gly lie Lys Thr Tyr 275 280 285 Pro Ser Gly Gly Asp His Gly Thr Ser Thr Val Ser Asn Val Thr Phe 290 295 300 Asn Asp Phe Thr Val Asp Asn Ser Asp Tyr Wing Phe Gln lie Gln Ser 305 310 315 320 Cys Tyr Gly Glu Asp Asp Asp Tyr Cys Glu Glu Asn Pro Gly Asn Ala 325 330 335 Lys Leu Thr Asp lie Val Val Ser Ser Phe Ser Gly Thr Thr Ser Asp 340 345 350 Lys Tyr Asp Pro Val Val Wing Asn Leu Asp Cys Gly Wing Asp Gly Thr 355 360 365 Cys Gly lie Ser lie Ser Gly Phe Asp Val Lys Ala Pro Ser Gly Lys 370 375 380 Ser Glu Val Leu Cys Wing Asn Thr Pro Ser Asp Leu Gly Val Thr Cys 385 390 395 400 Thr Ser Gly Ala Ser Gly 405 It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Having described the invention as above, the content of the following is claimed as property.

Claims (38)

1. A polypeptide, characterized in that it possesses endo-xylogalacturonase activity.

2. A polypeptide having endo-xylogalacturonase activity, characterized in that it is obtained from a fungus and possesses endo-xylogalacturonase activity.

3. A polypeptide according to claim 2, characterized in that the fungus is of the genus Aspergi l l u s.

4. A polypeptide according to any of the preceding claims, characterized in that it comprises the sequence set forth in SEQ ID NO: 2, or a sequence substantially homologous thereto, or a fragment of the sequence.

5. A polypeptide according to claim 4, characterized in that the fragment has at least 5 amino acids or the homologous sequence is at least 60% identical to SEQ ID NO: 2.

6. A polypeptide according to claim 5, characterized in that it comprises amino acids 19 to 406 of the amino acid sequence set forth in SEQ ID NO: 2.

7. A polynucleotide, characterized in that it encodes a polypeptide according to any of the preceding claims.

8. A polynucleotide, characterized in that it comprises (a) the polynucleotide sequence set forth in SEQ ID No. 1, or the complement thereof; (b) a polynucleotide sequence capable of hybridizing to the nucleotide sequence set forth in SEQ ID No. 1, or a fragment thereof; (c) a polynucleotide sequence capable of hybridizing to the complement of the polynucleotide sequence set forth in SEQ ID No. 1, or a fragment thereof; I (d) a polynucleotide sequence that is degenerate as a result of the genetic code with respect to the polynucleotides defined in (a), (b) or (c).

9. A polynucleotide according to claim 8, characterized in that: to. encodes a polypeptide having endo-xylogalacturonase activity, the polynucleotide is: (1) the coding sequence of SEQ ID No. 1; (2) a sequence that selectively hybridizes to the complement of the sequence defined in (1); or (3) a sequence that is degenerate as a result of the genetic code with respect to a sequence defined in (1) or (2); or b. is a sequence complementary to a polynucleotide defined in (a).

10. An isolated polynucleotide according to claim 7, 8 or 9, characterized in that it is obtained from a fungus.

11. A polynucleotide according to claim 10, characterized in that the fungus is of the genus Aspergi l l us.

12. A polynucleotide probe, characterized in that it comprises a fragment of at least 15 nucleotides of a polynucleotide as defined in any of claims 7 to 11.

13. A vector, characterized in that it comprises a polynucleotide as defined in any of claims 7 to 12.

14. An expression vector, characterized in that it comprises a polynucleotide as defined in any of claims 7 to 11 operably linked to one or more regulatory sequences capable of directing the expression of the polynucleotide in a host cell.

15. A host cell transformed or transferred with, characterized in that it comprises or incorporates a vector according to any of claims 13 to 14.

16. A host cell comprising or harboring a polynucleotide according to any of claims 7 to 11, characterized in that the polynucleotide is heterologous to the genome of the host cell.

17. A host cell according to claim 15 or claim 16, characterized in that it is a yeast cell.

18. A method for producing a polypeptide according to any of claims 1 to 6, characterized in that it comprises incubating or culturing a host cell according to any of claims 15 to 17 under conditions that allow the expression of the polynucleotide, and optionally purifying the polypeptide.

19. A host cell comprising or expressing a polypeptide according to any of claims 1 to 6, characterized in that it is heterologous to the host cell.

20. A composition, characterized in that it comprises a polypeptide according to any of claims 1 to 6.

21. A composition according to claim 2 0, characterized in that it also comprises a polypeptide having endo-arabinanase, rhamnogalacturonase or polygalacturonase activity.

22. A method for treating a plant material, characterized in that the method comprises contacting the material of the plant with a polypeptide according to any of claims 1 to 6 or a composition according to claim 20 or claim 21.

23. A method according to claim 22, characterized in that the treatment comprises degrading or modifying the pectin in the material of the plant.

24. A method according to claim 22, characterized in that it degrades or modifies the cell walls of the plant.

25. A method according to claim 22 or 23, characterized in that the treatment comprises the endo-type cleavage of the xylogalacturonan subunits of a pectin component of the material.

26. A method according to any of claims 22 to 24, characterized in that the material comprises a plant, plant pulp, plant extract or edible food material or ingredient thereof

27. A method according to claim 26, characterized in that the material is fruit or pulp, juice or vegetable extract.

28. A processed plant material, characterized in that it is obtained by contacting a plant material with a polypeptide according to any of claims 1 to 6 or a composition according to claim 20 or claim 21, or resulting from a method of according to any of claims 22 to 26.

29. A plant material processed according to claim 27, characterized in that it is a vegetable fruit or vegetable.

30. A method for reducing the viscosity of a plant material, characterized in that the method comprises contacting the material of the plant with a polypeptide according to any of claims 1 to 6 or a composition according to claim 20 or claim 21, in an amount and under the effective conditions to degrade the pectin contained in the material.

31. The use of a polypeptide according to any of claims 1 to 6 or a composition according to claim 20 or claim 21, characterized in that the method is used to treat the material of the plant.

32. The use of a polypeptide according to claim 31, characterized in that the treatment comprises the substituents of endo-type cleaving xylogalacturonan of the pectin in the material of the plant.

33. The use of a polypeptide according to any of claims 1 to 6 or a composition according to claim 20 or claim 21 in a method for processing pulp, juice or plant extract, characterized in that the method comprises incubating the pulp, juice or extract with the polypeptide or composition to at least partially degrade the pectin.

34. A food or food product for (animal), characterized in that it comprises a polypeptide according to any of claims 1 to 6.

35. A composition, characterized in that it comprises tragacanth gum (sGT) (optionally saponified) treated with a strong acid.

36. A test for identifying or detecting a polypeptide having pectin degrading activity, characterized in that the test comprises: providing as a substrate a candidate compound, (optionally saponified) gum tragacanth treated with a strong acid (sGT / TFA); Y b. contact the sGT / TFA with a candidate compound and detect if any carbohydrate reduction occurs.

37. A test according to claim 35, characterized in that the amount of carbohydrate reduction is measured and optionally compared with the amount of carbohydrates produced in a control in the absence of the candidate compound.

38. A test according to claim 35 or claim 36, characterized in that it comprises measuring the amount of Cu (II) reduced to Cu (I) by the carbohydrates, optionally by contact with bicinchoninic acid (BCA) and determining the amount of the complex of BCA-Cu (I) formed.