WO1999041386A2 - Nouvelle endo-xylogalacturonase - Google Patents

Nouvelle endo-xylogalacturonase Download PDF

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Publication number
WO1999041386A2
WO1999041386A2 PCT/EP1999/000860 EP9900860W WO9941386A2 WO 1999041386 A2 WO1999041386 A2 WO 1999041386A2 EP 9900860 W EP9900860 W EP 9900860W WO 9941386 A2 WO9941386 A2 WO 9941386A2
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Prior art keywords
polypeptide
polynucleotide
sequence
endo
plant
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PCT/EP1999/000860
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English (en)
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WO1999041386A3 (fr
Inventor
Petrus Johannes Albertus Meeuwsen
Cecile Johanna Beatrix Van Der Vlugt-Bergmans
Jean Paul Vincken
Gerrit Beldman
Alphons Gerard Joseph Voragen
Margareta Adriana Herweijer
Albert Johannes Joseph Van Ooijen
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Dsm N.V.
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Application filed by Dsm N.V. filed Critical Dsm N.V.
Priority to BR9907831-7A priority Critical patent/BR9907831A/pt
Priority to AU27247/99A priority patent/AU2724799A/en
Priority to EP99907529A priority patent/EP1054978A2/fr
Publication of WO1999041386A2 publication Critical patent/WO1999041386A2/fr
Publication of WO1999041386A3 publication Critical patent/WO1999041386A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01171—Rhamnogalacturonan hydrolase (3.2.1.171), i.e. rhamnogalacturonase
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/70—Clarifying or fining of non-alcoholic beverages; Removing unwanted matter
    • A23L2/84—Clarifying or fining of non-alcoholic beverages; Removing unwanted matter using microorganisms or biological material, e.g. enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/06—Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
    • A23L29/206—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin
    • A23L29/231—Pectin; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01015—Polygalacturonase (3.2.1.15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01099—Arabinan endo-1,5-alpha-L-arabinosidase (3.2.1.99)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)

Definitions

  • the present invention relates to a novel endo-xylogalacturonase (XGH) and homologues thereof It further relates to the use of the endo-xylogalacturonase in a method of processing plant or pectin-containing material to produce fruit juice and other plant extracts
  • XGH endo-xylogalacturonase
  • Enzyme preparations are often used during the processing of plant materials, for example in the steps of extraction and liquefaction of fruit and fruit juice and their filtration and clarification
  • Commercial enzyme preparations contain a mixture of enzymes which degrade the pectin polymers which are a major component of plant cell walls
  • Such enzymes include pectin lyases, polygalacturonases, pectin esterases, celluloses, xyloglucanases, galactanases and arabinanases
  • Pectins occur in nature as constituents of higher plant cell walls They are found in primary cell wall lamella where they are embedded in between the cellulose fibrils
  • the composition of pectin is variable among plant species and moreover dependent on the age and the maturity of the fruit Among the richer sources of pectins are lemons and oranges, which can represent up to 30% of polysaccha ⁇ des present
  • pectin polymers are comprised of 'smooth' homogalacturonan regions and ramified 'hairy' regions
  • the 'smooth' regions consist of a linear homogalacturonan backbone
  • the 'hairy' regions of apples consists of three different subunits subunit I is xylogalacturonan (a galacturonan backbone heavily substituted with xylose), subunit II is a short section of a rhamnogalacturonan backbone, rich in relatively long arabinan, galactan and/or arabinogalactan side chains (the 'hairs'), and subunit III is a rhamnogalacturonan ohgomer, having a backbone 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 'hairy' regions intact
  • exo-galacturonase 42kDa, SDS-PAGE
  • This enzyme acts in an exo-fashion as it yields galacturonic acid or a disaccharide consisting of galacturonic acid and xylose
  • the enzyme was purified to near homogeneity (Fractions HTP2 and Q2) and partially characterized
  • this enzyme is not very specific for xylogalacturonan as it also acts on pectic acid
  • this enzyme is not able to digest the xylogalacturonan backbone in a random fashion, and therefore to date there are no known enzymes possessing endo-xylogalacturonase activity
  • the present invention has resulted from the isolation and characterization of a novel endo-xylogalacturonase and cDNA encoding it
  • the endo-xylogalacturonase cDNA sequence is set out in SEQ ID No 1
  • the amino acid sequence of the ORF from nucleotides 98 to 1315 is set out in SEQ ID No 2
  • an (e g isolated and/or purified) polypeptide possessing endo-xylogalacturonase activity there is also provided a polypeptide comprising an endo-xylogalacturonase, such as a polypeptide comprising the sequence set out in SEQ LD No. 2, or a polypeptide substantially homologous thereto, or a fragment of the polypeptide of SEQ LD No. 2 having at least 5 amino acids.
  • the polypeptide of the invention preferably has one or more of the following additional features, namely it:
  • (3) has optimum activity at a temperature of from 50 to 70°C; and/or (4) has a molecular weight (deglycosylated) of from 40 to 50 kDa.
  • Endo-xylogalacturonase activity is defined as the ability to cleave a galacturonic acid polymer (for example as found in pectin) which may be at least partially substituted with xylose at internal glycosidic bonds. The activity thus allows cleavage between adjacent galacturonan non-terminal units (where neither of such units is at the end of the polymer, which is in contrast to exo activity where the end unit would be cleaved). Preferably the cleavage occurs at a [galacturonic acid (1-4) galacturonic acid] linkage.
  • the polypeptide does not cleave terminal xylose residues from xylose substituted galacturonic acid residues, for example a [galacturonic acid (3-1) xylose] linkage.
  • the polypeptide may preferentially cleave in between two adjacent non-xylose substituted galacturonan units.
  • the substrate polymer may be from 40 to 80%o (e.g. xylose) substituted.
  • the two galacturonic acid residues between which the polypeptides of the invention cleave may both be (xylose) substituted, or only one may be (xylose) substituted or (preferably) neither may be (xylose) substituted.
  • the two galacturonic acid residues may both be methylated, or one may be methylated, or (preferably) neither may be methylated.
  • the polypeptide of the invention is obtainable from a microorganism which possesses a gene encoding an enzyme with endo-xylogalacturonase activity. More preferably the microorganism is a microbial organism, preferably fungal, and optimally a filamentous fungi.
  • Preferred organisms are thus of the genera Aspergillus, Trichoderma, Penicillium, Acremonium, Fusarium, Humicola, Neurospora, Mucor, Scytallidium, Myceliophtora, Thielavia, Talaromyces, Thermomyces, Thermoascus, Chaetomium, Sporotrichum, Corynascus, Calcarisporiella o ⁇ Mycelia
  • the organism is of the species from the Aspergillus niger group (as defined by Raper and Fennell, The Genus Aspergillus, The Williams & Wilkins Company, Baltimore, pp 293-344, 1965), specifically including but not limited to Aspergillus niger, Aspergillus awamori, Aspergillus tubigensis, Aspergillus aculeatus, Aspergillus foetidus, Aspergillus japonicus or Aspergillus ficuum
  • the present invention provides an (e g isolated and/or purified) polynucleotide encoding a polypeptide of the first aspect of the invention
  • the present invention provides a polynucleotide encoding an endo-xylogalacturonase, such an endo-xylogalacturonase whose amino acid sequence is set out in SEQ ID No 2
  • the present invention further provides a polynucleotide encoding a polypeptide having substantial amino acid sequence homology to the amino acid sequence set out in SEQ ED No 2
  • polynucleotides comprising a nucleotide sequence capable of hybridising to the nucleotide sequence set out in SEQ ID No 1 , or a fragment thereof,
  • polynucleotides comprising a nucleotide sequence capable of hybridising to the complement of the nucleotide sequence set out in SEQ LD No 1 , or a fragment thereof, and/or
  • polynucleotides comprising a polynucleotide sequence which is degenerate as a result of the genetic code to the polynucleotides defined in (a), (b) or (c)
  • a polynucleotide of the invention also includes a polynucleotide which a encodes a polypeptide having endo-xylogalacturonase activity, which polynucleotide is
  • 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 out in SEQ. LD No.1 , or a fragment thereof or the complement thereof) at a level significantly above background.
  • the background hybridization may occur because of, for example, other polynucleotides, such as DNA, present in, for example a cDNA/genomic library being screened.
  • background implies a level of signal generated by interaction between the probe and a non-specific polynucleotide member of the library which is less than 10 fold, preferably less than 100 fold, as intense as the specific interaction observed with the target polynucleotide.
  • the intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with 32 P. Suitable conditions are described later.
  • the polynucleotide of the invention is obtainable from the same organism as the polypeptide, such as a fungus, in particular a fungus of the genus Aspergillus.
  • the present invention also provides a polynucleotide probe which comprises a fragment of at least 15 nucleotides of a polynucleotide of the invention as described above.
  • the invention provides vectors comprising a polynucleotide of the invention, including cloning and expression vectors, and in a fourth aspect methods of growing, transforming or transfecting such vectors in a suitable host cell, for example under conditions in which expression of a polypeptide of, or encoded by a sequence of, the invention occurs.
  • host cells comprising a polynucleotide or vector of the invention wherein the polynucleotide is heterologous to the genome of the host cell.
  • heterologous to the genome of the host cell means that the polynucleotide does not naturally occur in the genome of the host cell.
  • the host cell is a yeast cell, for example a yeast cell of the genus Kluyveromyces or Saccharomyces or a fungal cell, for example of the genus Aspergillus.
  • the polypeptides of the invention which possess endo-xylogalacturonase activity may be used in a sixth aspect to treat plant material including plant pulp and plant extracts.
  • compositions 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 present invention provides a method of degrading or modifying a plant cell wall which method comprises contacting the plant cell wall with a polypeptide or composition of the invention
  • the invention also provides a method of processing a plant material which method comprises contacting the plant material with a polypeptide or composition of the invention to degrade or modify the pectin in the plant material
  • a polypeptide or composition of the invention to degrade or modify the pectin in the plant material
  • the plant material is a plant pulp or plant extract
  • the degradation preferably comprises endo-type cleaving of xylogalacturonan subunits of a pectin component of the plant cell wall
  • the plant material is preferably a fruit or vegetable pulp or fruit or vegetable extract, for example apple pulp or apple juice
  • the present invention further provides a processed plant material obtainable by contacting a plant material with a polypeptide or composition of the invention
  • the processed plant material is a fruit or vegetable juice, for example apple juice
  • the invention provides a polynucleotide which a encodes a polypeptide that has endo-xylogalacturonase activity, which polynucleotide is
  • polynucleotides of the invention also include variants of the coding sequence of
  • SEQ ID No 1 which have endo-xylogalacturonase activity
  • Variants may be formed by additions, substitutions and/or deletions Such variants may thus have the ability to cleave internally a galacturonic acid polymer
  • a polynucleotide of the invention comprises a continuous sequence of nucleotides which is capable of hybridizing under selective conditions to the complement of the coding sequence of SEQ LD No 1
  • a polynucleotide of the invention and complement of the coding sequence of SEQ LD No 1 can hybridize at a level significantly above background Background hybridization may occur, for example, because of other cDNA's 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 LD No 1 is typically at least 10-fold, preferably at least 100-fold, as intense as interactions between other polynucleotides and the coding sequence of SEQ LD No 1
  • the intensity of interaction may be measured, for example, by radiolabellmg the probe, for example with 32 P
  • Selective hybridization may typically be achieved using conditions of low stringency (for example, 0 03M sodium chloride and 0 03M sodium citrate at about 40 °C), medium stringency (for example,
  • a preferred polynucleotide is capable of selectively hybridizing to complement the DNA sequence of SEQ LD 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% sequence identity to the coding sequence of SEQ LD No 1 over a region of at least 20, preferably at least 30, for instance at least 40, at least 60, or preferably at least 100 continuous nucleotides or most preferably over the full length of SEQ ID No 1
  • polynucleotides of the invention Any combination of the above mentioned degrees of sequence identity and minimum sizes may be used to define polynucleotides of the invention with the more stringent combinations (that is to say higher sequence identity over longer lengths) being preferred
  • a polynucleotide which has least 90% sequence identity over 25, -o- preferably over 30 nucleotides is preferred, as is a polynucleotide which has at least 95% sequence identity over 40 nucleotides
  • the coding sequence of SEQ LD No 1 may be modified by nucleotide substitutions, for example from 1, 2 or 3 to 10, 25, 50 or 100 substitutions
  • the polynucleotide of SEQ ID No 1 may 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 to either or both ends
  • the modified polynucleotide in general encodes a polypeptide which has endo-xylogalacturonase activity Degenerate substitution may be made and/or substitutions may 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 concerning polypeptides
  • Polynucleotides of the invention may comprise DNA or RNA They may be single or double stranded They may also be polynucleotides which include within them synthetic or modified nucleotides
  • a number of different types of modifications to polynucleotides are known in the art These include a methylphosphonate and phosphorothioate backbones, and addition of ac ⁇ dine or polylysine chains at the 3' and/or 5' ends of the molecule
  • the polynucleotides described herein may be modified by any method available in the art It is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides of the invention to reflect the codon usage of any particular host organism in which the polypeptides of the invention are to be expressed
  • Polynucleotides of the invention may be used as a primer, e g a PCR primer, a primer for an alternative amplification reaction, a probe e g labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may 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 There will typically be up to 40, 50, 60, 70, 100 or 150 nucleotides in length Probes and fragments can be longer than 150 nucleotides in length, for example up to 200, 300, 400, 500, 600, 700 nucleotides in length, or even up to a few nucleotides (such as 5 or 10 nucleotides) short of the coding sequence of SEQ ID No 1 Polynucleotides such as a DNA polynucleotide and primers according to the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art.
  • polynucleotides may also be cloned by standard techniques.
  • the polynucleotides are typically provided in isolated and/or purified form.
  • primers will be produced by synthetic means, involving a step-wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.
  • Longer polynucleotides will generally be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. This will involve making a pair of primers (e.g. of about 15-30 nucleotides) to a region of the endo-xylogalacturonase gene which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from a fungal, yeast, bacterial plant or prokaryotic cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA.
  • the primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector.
  • Genomic clones corresponding to the cDNA of SEQ LD No. 1 or the endo-xylogalacturonase gene containing, for example, introns and promoter regions are within the invention also and may also be obtained in an analogous manner (e.g. recombinant means, PCR, cloning techniques), starting with genomic DNA from a fungal, yeast, bacterial plant or prokaryotic cell.
  • Polynucleotides which do not have 100%) identity with SEQ LD No. 1 but fall within the scope of the invention can be obtained in a number of ways.
  • variants of the endo-xylogalacturonase sequence described herein may be obtained for example by probing genomic DNA libraries made from a range of organisms, for example those discussed as sources of the polypeptides of the invention.
  • other fungal, plant or prokaryotic homologues of endo-xylogalacturonase may be obtained and such homologues and fragments thereof in general will be capable of hybridising to SEQ ID No. 1.
  • sequences may be obtained by probing cDNA libraries or genomic DNA libraries from other species, and probing such libraries with probes comprising all or part of SEQ ID. 1 under conditions of medium to high stringency (for example 0.03M sodium chloride and 0.03M sodium citrate at from 50 °C to 60 °C).
  • Nucleic acid probes comprising all or part of SEQ ID No. 1 may be used to probe cDNA libraries from other species, such as those described as sources for the polypeptides of the invention .
  • Species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences.
  • the primers will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.
  • polynucleotides may be obtained by site directed mutagenesis of the endo-xylogalacturonase sequences or variants thereof. This may be useful where for example silent codon changes are required to sequences to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order 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.
  • Polynucleotides or primers of the invention may carry a revealing label.
  • Suitable labels include radioisotopes such as 32 P or 35 S, enzyme labels, or other protein labels such as biotin and DIG-hapten. Such labels may be added to polynucleotides or primers of the invention and may be detected using by techniques known per se.
  • the present invention also provides polynucleotides encoding the polypeptides of the invention described below. Since such polynucleotides will be useful as sequences for recombinant production of polypeptides of the invention, it is not necessary for them to be capable of hybridising to the sequence of SEQ ID No. 1, although this will generally be desirable. Otherwise, such polynucleotides may be labelled, used, and made as described above if desired. B Polypeptides
  • a polypeptide of the invention comprises the amino acid sequence set out in SEQ LD No 2 or a substantially homologous sequence, or a fragment of either sequence and can have endo-xylogalacturonase activity
  • SEQ LD No 2 the naturally occurring amino acid sequence shown in SEQ LD No 2 is preferred
  • polypeptide of the invention may comprise a the polypeptide sequence of SEQ ID No 2, b a naturally occurring variant or species homologue thereof, or c 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% sequence identity to (a) or (b)
  • a variant will be one that occurs naturally, for example in fungal, bacteria, yeast or plant cells and which can function in a substantially similar manner to the protein of SEQ ID No 2, for example it has endo-xylogalacturonase activity
  • a species homologue of the protein will be the equivalent protein which occurs naturally in another species and which can function as an endo-xylogalacturonase enzyme
  • Variants and species homology can be obtained by following the procedures described herein for the production of the polypeptide of SEQ ID No 2 and performing such procedures on a suitable cell source, for example a bacterial, yeast, fungal or plant cell It will also be possible to use a probe as defined above to probe libraries made from yeast, bacterial, fungal or plant cells in order to obtain clones including the variants or species homology
  • a suitable cell source for example a bacterial, yeast, fungal or plant cell
  • a probe as defined above to probe libraries made from yeast, bacterial, fungal or plant cells in order to obtain clones including the variants or species homology
  • 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 to the protein of SEQ LD 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 thereto over a region of at least 20, preferably at least 30, for instance at least 40, at least 60, at least 100, 200 or 300 contiguous amino acids or over the full length of SEQ LD No 2
  • sequence of the polypeptide of SEQ ID No 2 and of variants and species homologues can thus be modified to provide polypeptides of the invention
  • Amino acid substitutions may be made, for example from 1, 2 or 3 to 10, 20 to 30 substitutions
  • the same number of deletions and insertions may also be made.
  • the modified polypeptide generally retains activity as an endo-xylogalacturonase.
  • Polypeptides of the invention also include fragments of the above mentioned full length polypeptides and of variants thereof, including fragments of the sequence set out in SEQ ID No. 2. Such fragments typically retain activity as an endo-xylogalacturonase. Fragments may be at least 10, 15, 20, 30, 50, 100 or 200 amino acids long.
  • Polypeptides of the invention may be in a substantially isolated form. It will be understood that the polypeptide may be mixed with carriers or diluents which will not interfere with the intended purpose of the polypeptide and still be regarded as substantially isolated.
  • a polypeptide of the invention may also be in a substantially purified form, in which case it will generally 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.
  • Polypeptides of the invention may be chemically modified, for example post-transnationally modified. For example, they may be glycosylated (one or more times) or comprise one or more modified amino acid residues.
  • They may be modified for example by the addition of histidine residues or a T7 tag to assist their identification or purification or by the addition of a signal sequence to promote their secretion from a cell, as discussed below.
  • Polypeptides of the invention can if necessary be produced by synthetic means although usually they will be made recombinantly as described below.
  • Particularly preferred polypeptides of the invention include a polypeptide consisting of amino acids 19 to 406 of the amino acid sequence set out in SED ID No 2 since this lacks the N-terminal signal peptide which consists of amino acids 1 to 18 of the amino acid sequence of SEQ ID No 2
  • the polypeptides and fragments thereof may contain amino acid alterations as defined above
  • yeast and fungal host cells are expected to provide for such post-translational modifications (e g proteolytic processing, myristilation, glycosylation, truncation, and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the invention
  • post-translational modifications e g proteolytic processing, myristilation, glycosylation, truncation, and tyrosine, serine or threonine phosphorylation
  • Polynucleotides of the invention can be incorporated into a recombinant replicable vector, for example a cloning or expression vector.
  • the vector may be used to replicate the nucleic acid in a compatible host cell
  • the invention provides a method of 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 which bring about replication of the vector The vector may be recovered from the host cell Suitable host cells are described below in connection with expression vectors
  • a polynucleotide of the invention in a vector is operably linked to a regulatory sequence which is capable of providing for the expression of the coding sequence by the host cell, i e the vector is an expression vector
  • operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner
  • a regulatory sequence such as a promoter, enhancer or other expression regulation signal "operably linked" to a coding sequence is positioned in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences
  • the vectors may 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 a suitable host as part of an expression construct in which the DNA sequence is operably linked to expression signals which are capable of directing expression of the DNA sequence in the host cells.
  • transformation procedures are available which are well known to the skilled person 34 .
  • the expression construct can be used for transformation of the host as part of a vector carrying a selectable marker, or the expression construct is co-transformed as a separate molecule together with the vector carrying a selectable marker.
  • the vectors may contain one or more selectable marker genes.
  • Preferred selectable markers 3,4 include but are not limited to e.g. versatile marker genes that can be used for transformation of most filamentous fungi and yeasts such as acetamidase genes or cDNAs (the amdS genes or cDNAs from A.nidulans, A.oryzae, or A.niger), or genes providing resistance to antibiotics like G418, hygromycin, phleomycin or benomyl resistance (benA).
  • more specific selection markers can be used such as auxotrophic markers which require corresponding mutant host strains: e.g.
  • the selection marker is deleted from the transformed host cell after introduction of the expression construct in accordance with the methods described in EP-A-0 635 574, so as to obtain transformed host cells capable of producing the polypeptide which are free of selection marker genes.
  • markers include ATP synthetase, subunit 9 (oliC), orotidine-5'-phosphate- decarboxylase (pvrA), the bacterial G418 resistance gene (this may also be used in yeast, but not in fungi), the ampicillin resistance gene (E. coli), the neomycin resistance gene (Bacillus) and the E. coli uidA gene, coding for ⁇ -glucuronidase (GUS).
  • Vectors may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell.
  • the expression construct is preferably integrated in the genome of the host cell in order to obtain stable transformants.
  • suitable episomal vector systems are available into which the expression construct can be incorporated for stable and high level expression, examples thereof include vectors derived from the 2 ⁇ and pKDl plasmids of Saccharomyces and Kluyveromyces, respectively.
  • the expression constructs are integrated in the host cells genome, the constructs are either integrated at random loci in the genome, or at predetermined target loci using homologous recombination, in which case the target loci preferably comprise a highly expressed gene.
  • a highly expressed gene is herein defined as a gene whose mRNA can make up at least 0.05%) (w/w) of the total cellular mRNA, e.g. under induced conditions, or alternatively, a gene whose gene product can make up at least 1% (w/w) of the total cellular protein, or, in case of a secreted gene product, can be secreted to a level of at least 0.1 g/1.
  • suitable highly expressed genes is provided herein below.
  • 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 3 '-end relative to the coding strand of the sequence encoding the polypeptide of the first aspect: (1) a promoter sequence capable of directing transcription of the DNA sequence encoding the polypeptide in the given host cell, (2) optionally, a signal sequence capable of directing secretion of the polypeptide from the given host cell into the culture medium, (3) the DNA sequence encoding a mature and preferably active form of the polypeptide, and preferably also (4) a transcription termination region (terminator) capable of terminating transcription downstream of the DNA sequence encoding the polypeptide.
  • a promoter sequence capable of directing transcription of the DNA sequence encoding the polypeptide in the given host cell
  • a signal sequence capable of directing secretion of the polypeptide from the given host cell into the culture medium
  • the DNA sequence encoding a mature and preferably active form of the polypeptide and preferably also (4)
  • Enhanced expression of the polynucleotide encoding the polypeptide of the invention may also be achieved by the selection of heterologous regulatory regions, e.g. promoter, secretion leader and terminator regions, which serve to increase expression and, if desired, secretion levels of the protein of interest from the chosen expression host and/or to provide for the inducible control of the expression of the polypeptide of the invention.
  • heterologous regulatory regions e.g. promoter, secretion leader and terminator regions
  • promoters may be used to direct expression of the polypeptide of the invention.
  • the promoter may be selected for its efficiency in directing the expression of the polypeptide of the invention in the desired expression host.
  • promoters 3,4 can be used that are capable of directing transcription in the host cells of the invention.
  • the promoter sequence is derived from a highly expressed gene as previously defined.
  • preferred highly expressed genes from which promoters are preferably derived and/or which are comprised in preferred predetermined target loci for integration of expression constructs include but are not limited to genes encoding glycolytic enzymes such as triose-phosphate isomerases (TPI), glyceraldehyde-phosphate dehydrogenases (GAPDH), phosphoglycerate kinases (PGK), pyruvate kinases (PYK), alcohol dehydrogenases (ADH), as well as genes encoding amylases, glucoamylases, xylanases, cellobiohydrolases, ⁇ -galactosidases, alcohol (methanol) oxidases, elongation factors and ribosomal proteins.
  • TPI triose-phosphate isomerases
  • suitable highly expressed genes include e.g. the LAC4 gene from Kluyveromyces sp., the methanol oxidase genes (A OX and MOX) from Hansenula and Pichia, respectively, the glucoamylase (glaA) genes from A.niger and A.awamori, the A.oryzae TAKA-amylase gene, the A.nidulans gpdA gene and the T.reesei cellobiohydrolase genes.
  • LAC4 gene from Kluyveromyces sp.
  • a OX and MOX methanol oxidase genes
  • glaA glucoamylase
  • strong constitutive and/or inducible promoters which are preferred for use in fungal expression hosts are those which are obtainable from the fungal genes for xylanase (xlnA), phytase, ATP-synthetase, subunit 9 (oliC), triose phosphate isomerase (fpi), alcohol dehydrogenase (AdhA), ⁇ -amylase (amy), amyloglucosidase (AG - from the glaA gene), acetamidase (amdS) and glyceraldehyde-3 -phosphate dehydrogenase (gpd) promoters.
  • xylanase xylanase
  • phytase ATP-synthetase
  • oliC subunit 9
  • fpi triose phosphate isomerase
  • AdhA alcohol dehydrogenase
  • ⁇ -amylase ⁇ -amy
  • strong yeast promoters are those obtainable from the genes for alcohol dehydrogenase, lactase, 3 -phosphoglycerate kinase and triosephosphate isomerase.
  • strong bacterial promoters are the ⁇ -amylase and SP02 promoters as well as promoters from extracellular protease genes.
  • 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 a signal sequence.
  • the signal sequence is native (homologous) to the DNA sequence encoding the polypeptide.
  • the signal sequence is foreign (heterologous) 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.
  • suitable signal sequences for yeast host cells are the signal sequences derived from yeast ⁇ -factor genes.
  • a suitable signal sequence for filamentous fungal host cells is e.g.
  • a signal sequence derived from a filamentous fungal (gluco)amylase gene e.g. the A.niger glaA gene. This may be used in combination with the amyloglucosidase (AG) promoter itself, as well as in combination with other promoters.
  • AG amyloglucosidase
  • Hybrid signal sequences may also be used with the context of the present invention.
  • Preferred heterologous secretion leader sequences are those originating from the fungal amyloglucosidase (AG) gene (glaA - both 18 and 24 amino acid versions e.g. from Aspergillus), the ⁇ -factor gene (yeasts e.g. Saccharomyces and Kluyveromyces) or the ⁇ -amylase gene (Bacillus).
  • AG fungal amyloglucosidase
  • the expression construct 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 can e.g. be native to the DNA sequence encoding the polypeptide.
  • a yeast terminator is used in yeast host cells and a filamentous fungal terminator is used in filamentous fungal host cells. More preferably, the terminator is endo- genus to the host cell in which the DNA sequence encoding the polypeptide is expressed.
  • the invention provides a process for preparing polypeptides according to the invention which comprises cultivating a host cell transformed or transfected with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the polypeptides, and recovering the expressed polypeptides.
  • a further aspect of the invention thus provides host cells transformed or transfected with or comprising a polynucleotide or vector of the invention.
  • 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 said vector and may for example be prokaryotic (for example bacterial), fungal, yeast or plant cells.
  • eukaryotic hosts such as yeasts or fungi may be preferred.
  • yeast cells are preferred over fungal cells because they are easier to manipulate.
  • some proteins are either poorly secreted from yeasts, or in some cases are not processed properly (e.g. hyperglycosylation in yeast).
  • a fungal host organism should be selected.
  • a heterologous host may also be chosen wherein the polypeptide of the invention is produced in a form which is substantially free from other pectin-degrading enzymes. This may be achieved by choosing a host which does not normally produce such enzymes such as Kluyveromyces lactis.
  • the invention encompasses processes for the production of the polypeptide of the invention by means of recombinant expression of a DNA sequence encoding the polypeptide.
  • the DNA sequence of the invention can be used for gene amplification and/or exchange of expression signals, such as promoters, secretion signal sequences, in order to allow economic production of the polypeptide in a suitable homologous or heterologous host cell.
  • a homologous host cell is herein defined as a host cell which is of the same species or which 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 bacteria, or more preferably eukaryotic organisms, for example fungi, such as yeasts or filamentous fungi, or plant cells.
  • Bacteria from the genus Bacillus are very suitable as heterologous hosts because of their capability to secrete proteins into the culture medium.
  • Other bacteria suitable as hosts are those from the genera Streptomyces and Pseudomonas.
  • a preferred yeast host cell for the expression of the DNA sequence encoding the polypeptide is of the genera Saccharomyces, Kluyveromyces, Hansenula, Pichia, Yarrowia, and Schizosaccharomyces . More preferably a yeast host cell is selected from the group consisting of the species Saccharomyces cerevisiae, Kluyveromyces lactis (also known as Kluyveromyces marxianus var. lactis), Hansenula polymorpha, Pichia pastor is, Yarrowia lipolytica,md Schizosaccharomyces pombe .
  • filamentous fungal host cells are selected from the group consisting of the genera. Aspergillus, Trichoderma, Fusarium, Penicillium, Acremonium, Neurospora, Thermoascus, Myceliophtora, Sporotrichum, Thielavia, and Talaromyces.
  • a filamentous fungal host cell is of the species Aspergillus oyzae, Aspergillus sojae, Aspergillus nidulans, species from the Aspergillus niger Group (as defined by Raper and Fennell, The Genus Aspergillus, The Williams & Wilkins Company, Baltimore, pp 293-344, 1965).
  • Aspergillus niger include but are not limited to Aspergillus niger, Aspergillus awamori, Aspergillus tubigensis, Aspergillus aculeatus, Aspergillus foetidus, Aspergillus nidulans, Aspergillus japonicus, Aspergillus oryzae and Aspergillus ficuiim, and further consisting of the species Trichoderma reesei, Fusarium graminearum, Penicillium chrysogenum, Acremonium alabamense, Neurospora crassa, Myceliophtora thermophilum, Sporotrichum cellulophilum, and Thielavia terrestris.
  • Trichoderma reesei Fusarium graminearum
  • Penicillium chrysogenum Acremonium alabamense
  • Neurospora crassa Myceliophtora thermophilum
  • fungi such as Aspergillus species (described in EP-A-184,438 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. Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Pseudomonas species; and yeasts such as Kluyveromyces species (described in EP-A-096,430 e.g. Kluyveronmyces lactic and EP-A-301,670) and Saccharomyces species, e.g. Saccharomyces cerevisiae.
  • fungi such as Aspergillus species (described in EP-A-184,438 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. Bac
  • the production of the polypeptide of the invention can be effected by the culturing of 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 may be cultured using procedures known in the art. For each combination of a promoter and a host cell, culture condition are available which are conducive to the expression the DNA sequence encoding the polypeptide. After reaching the desired cell density or titre of the polypeptide the culture is stopped and the polypeptide is recovered using known procedures.
  • the fermentation medium can comprise a known culture medium containing a carbon source (e.g.
  • glucose, maltose, molasses, etc. a nitrogen source (e.g. ammonium sulphate, ammonium nitrate, ammonium chloride, etc.), an organic nitrogen source (e.g. yeast extract, malt extract, peptone, etc.) and inorganic nutrient sources (e.g. phosphate, magnesium, potassium, zinc, iron, etc.).
  • an inducer e.g. apple MHR, pectin or xylogalacturonan
  • an inducer e.g. apple MHR, pectin or xylogalacturonan
  • the selection of the appropriate medium may be based on the choice of expression host and/or based on the regulatory requirements of the expression construct. Such media are well-known to those skilled in the art.
  • the medium may, if desired, contain additional components favouring the transformed expression hosts over other potentially contaminating microorganisms.
  • the fermentation can be performed over a period of 0.5-20 days in a batch, continuous or fed-batch process suitably at a temperature in the range of between 0 and 45°C and, for example, a pH between 2 and 10.
  • Preferred fermentation conditions 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.
  • the cells can be removed from the fermentation broth by means of centrifugation or filtration. After removal of the cells, the polypeptide of the invention may then be recovered and, if desired, purified and isolated by conventional means.
  • Plant and pectin-containing materials include plant pulp, parts of plants and plant extracts.
  • an extract from a plant material is any substance which can be derived from plant material by extraction (mechanical and/or chemical), processing or by other separation techniques.
  • the extract may be juice, nectar, base, or concentrates made thereof.
  • the plant material may comprise or be derived from vegetables, e.g., carrots, celery, onions, legumes or leguminous plants (soy, soybean, peas) or fruit, e.g., pome or seed fruit (apples, pears, quince etc.), grapes, tomatoes, citrus (orange, lemon, lime, mandarin), melons, prunes, cherries, black currants, redcurrants, raspberries, strawberries, cranberries, pineapple and other tropical fruits, trees and parts thereof (e.g. pollen, from pine trees).
  • apples and apple juice are especially preferred.
  • polypeptides of the invention can thus be used to treat plant material including plant pulp and plant extracts.
  • they may be used to treat apple pulp and/or raw juice during the production of apple juice. They may also be used to treat liquid or solid foodstuffs or edible foodstuff ingredients.
  • the polypeptide of the invention is combined with suitable (solid or liquid) carriers or diluents including buffers to produce a composition or enzyme preparation.
  • the polypeptide is typically stably formulated either in liquid or dry form.
  • the product is made as a composition which will optionally include, for example, a stabilising buffer and/or preservative.
  • the compositions may also include other enzymes capable of digesting plant material or pectin, for example other pectinases such as an endo-arabinanase, rhamnogalacturonases, and/or polygalacturonase. For certain applications, immobilization of the enzyme on a solid matrix or incorporation on or into solid carrier particles may be preferred.
  • the composition may also include a variety of other plant material-degrading enzymes, for example cellulases and other pectinases.
  • the polypeptides and compositions of the invention may therefore be used in a method of processing plant material to degrade or modify the pectin constituents of the cell walls of the plant material 2 .
  • the polypeptides of the invention are used as a composition/ enzyme preparation as described above.
  • the composition will generally be added to plant pulp obtainable by, for example mechanical processing such as crushing or milling plant material. Incubation of the composition with the plant will typically be carried out for at time of from 10 minutes to 5 hours, such as 30 minutes to 2 hours, preferably for about 1 hour.
  • the processing temperature is preferably 10-55°C, e.g. from 15 to 25° C, optimally about 20°C and one can use 10-300g, preferably 30-70g, optimally about 50g of enzyme per ton of material to be treated. All the enzyme(s) or their compositions used may be added sequentially or at the same time to the plant pulp.
  • the plant material may first be macerated (e.g. to a puree) or liquefied.
  • processing parameters such as the yield of the extraction, viscosity of the extract and/or quality of the extract can be improved.
  • a polypeptide of the invention may be added to the raw juice obtained from pressing or liquefying the plant pulp. Treatment of the raw juice will be carried out in a similar manner to the plant pulp in respect of dosage, temperature and holding time. Again, other enzymes such as those discussed previously may be included. Typical incubation conditions are as described in the previous paragraph. Once the raw juice has been incubated with the polypeptides of the invention, the juice is then centrifuged or (ultra) filtered to produce the final product.
  • composition containing a polypeptide of the invention may also be used during the preparation of fruit or vegetable purees.
  • the end product of these processes is typically heat-treated at 85°C for a time of from 1 minute to 1 hour, under conditions to partially or fully inactivate the polypeptides of the invention.
  • polypeptides of the invention may also be used to prepare pectins with modified characteristics, e.g. modified gelation capacities for specific applications.
  • the polypeptides of the invention may also be added to animal feeds rich in pectin or xylogalacturonan, e.g. soy-containing food, to improve the breakdown of the plant cell wall leading to improved utilisation of the plant nutrients by the animal.
  • the polypeptides of the invention may be added to the feed or silage if pre-soaking or wet diets are preferred.
  • the polypeptides of the invention may continue to degrade xylogalacturonans in the feed in vivo.
  • Fungal derived polypeptides of the invention in particular generally have lower pH optima and are capable of releasing important nutrients in such acidic environments as the stomach of an animal.
  • the invention thus also contemplates (e.g. animal) feeds or foodstuffs comprising one or more polypeptides of the invention.
  • the polypeptides of the invention may also be used during the production of milk substitutes (or replacers) from soy bean. These milk substitutes can be consumed by both humans and animals. A typical problem during the preparation of these milk substitutes is the high viscosity of the soy bean slurry, resulting in the need for an undesirable dilution of the slurry to a concentration of dry solids of 10 to 15%.
  • An enzyme preparation containing a polypeptide of the invention can be added to, or during the processing of, the slurry, enabling processing at a higher concentration (typically 40 to 50%) dry solids.
  • the enzyme may also be used in the preparation of savoury product(s), e.g. from soy bean.
  • the novel assays and substrates described herein have allowed identification and confirmation of endo-xylogalacturonase activity. However, these assays can be used to detect other pectin degrading enzymes, whether or not they have endo-xylogalacturonase activity.
  • the substrate that can be used for this assay can comprise gum tragacanth which has been treated with a strong acid. A preferred acid is trifluoroactetic acid (TFA).
  • TFA trifluoroactetic acid
  • the gum tragacanth may be optionally saponified and/or it may have been treated with an alkali, for example an alkali metal hydroxide, for example NaOH.
  • Another aspect of the invention relates to an assay for identifying or detecting a polypeptide which is able to degrade pectin.
  • the activity may be an endo-xylogalacturonase or, may be pectin lyase, polygalacturonase, esterase, cellulase, xyloglucanase, galactonase, arabinanase or rahamnogalacturonase
  • the assay may comprise a providing, as a substrate for a candidate compound (usually a polypeptide) the substrate described in the previous paragraph, and b contacting the substrate with the candidate compound, and detecting whether any reducing carbohydrates are produced
  • the amount of these reducing carbohydrates can be measured If necessary, they can then be compared to the amount of the carbohydrates produced in a control experiment, in the absence of candidate compound
  • the measurement may involve a BCA assay This may comprise measuring the amount of Cu(II) reduced to Cu(I) by the reducing carbohydrates present This may be by contact with bicinchonimc acid (BCA), and determining the amount of BCA-Cu(I) complex formed
  • Figure 1 is a diagram of the hypothetical structure of the prevailing population of apple MHR (modified hairy region) having the highest molecular weight (subunit I is xylogalacturonan, subunit II is the backbone rich in arabinan side chains, subunit in is rhamnogalacturonase oligomers
  • subunit I is xylogalacturonan
  • subunit II is the backbone rich in arabinan side chains
  • subunit in rhamnogalacturonase oligomers
  • the distribution of acetyl groups is not presented but major parts are thought to be located within subunit III
  • 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 an HPAEC of xylogalacturonan after degradation by xylogalacturonase (a polypeptide of the invention)
  • Figure 4 is a graph illustrating an HP SEC of xylogalacturonan before and after degradation by a xylogalacturonase
  • Figure 5 is a graph illustrating a Maldi-ToF mass spectrum of the products of complete degradation of xylogalacturonan by a xylogalacturonase
  • Figures 6A-G are graphs of HPSEC elutions showing degradation of MHR-S by endo-arabinanase, rhamnogalacturonase and xylogalacturonase, separately and in combination,
  • Figures 7 and 8 are graphs of a HPSEC and HPAEC, respectively, elution patterns showing degradation of soy pectin by xylogalacturonase, and
  • Starting vector pGBHSA20 contains the promoter and terminator sequence of the lactase gene (lac4) o K lactis, a G418 selection marker and the E coli plasmid pTZ18r for propagation in this host
  • lac4 lactase gene
  • the K lactis KARSCEN cassette 17 was cloned in a unique Smal site of this vector
  • the resulting vector was named pCVlacK ( Figure 2)
  • the unique Hindlll and Xhol sites flanking the lac4 promoter and terminator, respectively, can be used as cloning sites for cDNA synthesized from Aspergillus tubigensis poly(A) RNA
  • Example 1 Isolation of polyfA RNA and cDNA synthesis
  • Aspergillus tubigensis conidia were inoculated in triplicate at a density of 10 6 spores/ml in 300 ml of medium containing (per liter) 6 g NaNO 3 , 0 5 g KC1, 1 5 g KH 2 PO 4 , 0 5 g MgSO 4 (pH6 5), 1 ml lOOOx Timberlake spore elements (per ml, 50mg EDTA, 22mg FeSO 4 7H 2 O, 5 mg MnCl 2 2H 2 O, 22mg ZnSO 4 7H 2 O, 1 6mg CuSO 4 5H 2 O, 1 7mg CoCl 2 6H 2 O, 1 5mg Na 2 MoO 4 2H 2 O, 1 lmg H BO 3 , adjusted to pH 6 5) and 10ml lOOx Timberlake vitamins (per ml, 0 2mg thiamine-HCl, 0 2mg ⁇ boflavin 0 2mg nic
  • RNAzol method (Cinna/Biotecx). Poly (A) RNA was isolated using QiagenTM oligotex columns (Westburg). Equal amounts of poly(A) RNA at time-points of 10, 16 and 24 hours were pooled.
  • cDNA was synthesized using the ZAP-cDNA synthesis kit (StratageneTM) with the following modifications: the first-strand synthesis was done with Superscript LI reverse transcriptase (GibcoBRL).
  • the cDNA pool was size separated using a Sephacryl S-500 column.
  • the first fraction eluted from the column did not contain any cDNA but the second and third fraction contained the largest sized cDNA. Subsequent fractions were supposed to contain relatively higher amounts of non-full length cDNA and were of no use for construction of the library.
  • the cDNA of fractions 2 and 3 was ligated into theHwdi ⁇ and Xhol sites of expression vector pCVlacK (see Figure 2) using the Clontech Ligation ExpressTM kit. Each ligation mixture was transformed in two batches to electrocompetent E. coli XL-Blue MRF'cells. The four transformation suspensions were plated onto 32 agar plates (LB + 50 ⁇ g/ml ampicillin).
  • Example 1.3 Transformation of the expression library into K. lactis
  • K lactis strain CBS 2359 grown in YPD (10 g/1 yeast extract, 20 g/1 Bacto-peptone, 20 g/1 glucose) at 30°C was diluted 3000-, 600-, 300- and 100-fold in 150 ml of fresh YPD and incubated for 6 hours at 30°C, 160 rpm in a rotary shaker.
  • the culture with an optical density of 0 7-1 0 was used to prepare electrocompetent cells 6
  • Electrocompetent cells were transformed with 1 ⁇ g pooled DNA of the E. coli library
  • Electroporation was performed using a Biorad GenepulserTM with settings at 1 4 kV, 200 Ohm and 25 ⁇ F Transformants were selected on double layer YPD plates (YPD with 20 g/1 Bacto-agar) the bottom layer contained 50 ⁇ g/ml G418, the top layer was non-selective 660 ⁇ L of transformation mix was plated onto 80 double layer plates Aliquots of 1 5 and 15 ⁇ L were pipetted onto the plates About 10,000 transformants were obtained
  • Example 2 Preparation of MHR-S from apples Modified hairy regions (MHR) from apples were isolated as a filter retentate after treatment of apples with pectinase, and subsequently the MHR was saponified resulting in MHR-S 8
  • Example 2 3 Characterization of xylogalacturonan To determine the sugar composition both from the original gum tragacanth as well as of the xylogalacturonan, samples were hydrolyzed with 1 M H 2 SO 4 (100°C, 3 h) 8 and neutral sugars were converted to their alditol acetates in order to quantify the individual sugars by Gas Chromatography (GC). The uronic acid content of the hydrolysate was determined colorimetrically using m-hydroxybiphenyl 8 . The sugar composition in mol% of the original gum tragacanth (GT) in comparison with the xylogalacturonan (XG) is shown in Table 1 below.
  • the degree of acetyl and methyl esterification of gum tragacanth was estimated by High Pressure Liquid Chromatography (HPLC) 7 .
  • the degree of methylation and acetylation of gum tragacanth is approximately 75% and 20%, respectively (calculated as mol methyl or actyl groups per mol of GalA). Saponification of the gum removed all methyl and acetyl groups.
  • transformants were used to inoculate a new set of 35 multi-well plates containing 200 ⁇ L of the same medium with 80 ng/mL G418 with a replica plater
  • the K. lactis transformants were grown for two days at 30°C in a stove
  • the cells were precipitated by centrifugation at 3000 rpm in a HermleTM zk380 centrifuge
  • the BCA assay is based on the reduction of Cu(II) to Cu(f) by reducing carbohydrate mono- and oligomers
  • a complex is formed of bicinchoninic acid (BCA) and Cu(I) This complex produces an intense purple colour, which can be measured spectrophotometrically This colour increases with an increasing reducing carbohydrate concentration
  • the method used in this invention is a modification of a known method 9 but was used for screening purposes
  • Figure 9 shows the analysis of multiple alignments of Xgh to the PG's and to the RHG-A's
  • the multiple alignment shows four domains of conserved amino acids, which were first described for polygalacturonases of plant, fungal and bacterial origin 15
  • NXD, DD, HG and RXK (shaded in Figure 9, where X represents a variable amino acid)
  • Essential amino acids thought to be involved in the hydrolysis reaction are one of the three aspartic acid residues of domain I and LI and the histidine of domain III
  • These domains are fully conserved in XghA
  • the fourth domain contains amino acids that are involved in substrate binding
  • the argimne residue of this domain is a glycine residue in XghA
  • the domains are less conserved in the RHG sequences, as only two of the three aspartic acid residues are conserved and the histidine is replaced by g
  • Example 4 Southern blot analysis The copy number of theXghA gene was determined by southern blot analysis of genomic DNA of A tubigensis digested with several enzymes (results not shown) Hybridization under stringent (65 °C and 0.2 x SSC) and less stringent conditions (60°C and 1 x SSC) with a 1.0 kb Hin dill fragment of xghA clearly showed single hybridizing fragments. This demonstrates that the xghA gene is present as a single copy in the A. tubigensis genome.
  • K lactis transformants expressing the cDNA of endo-xylogalacturonase were transferred from multiwell plate glycerol stocks to reagent tubes: 10 ⁇ L 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 used to inoculate Erlenmeyer flasks containing 20 mL of this same medium supplemented with antibiotic. For larger scale production of the enzyme, these cultures were used to inoculate 500 mL of the same medium supplemented with antibiotic in 1 L Erlenmeyer flasks. Cells were grown at 30°C in a rotary incubator at 200 rpm for two days. Cultures were centrifuged to precipitate cells, the supernatant was used for the purification.
  • the crude enzyme preparation (350 mL) was preconcentrated on a HitrapTM Q ion-exchange column (Pharmacia Biotech, Sweden) with a flow rate of 0.3 mL/min. Elution was performed on a FPLC system (Pharmacia Biotech, Sweden) with a salt gradient using a 20 mM piperazine (pH 5.0) starting buffer (buffer A) and a 0.5 M NaCl in 20 mM piperazine (pH 5.0) elution buffer (buffer B). The following gradient was used: to 10% B in 1 minute, to 35% B in 19 minutes, to 100% B in 2 minutes and 100% B for three more minutes. Activity was checked as described in Example 3 and active fractions were pooled.
  • the purified enzyme was preincubated without substrate for one hour at a pH range from 2.5 to 8 in Mcllvaine buffers. Afterwards the enzyme was incubated with substrate for two hours and the increase in reducing sugars was determined as described in Example 3. The enzyme was stable over a pH range of 3 to 6.
  • the purified enzyme was incubated with substrate for two hours at a pH range from 2.5 to 8 or a temperature range from 20 to 80 °C. After this, the increase in reducing sugars was determined as described in Example 3.
  • the enzyme has an optimum activity at a temperature of 60°C and at 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. Values lower than pH 2.5 were not measured.
  • EXAMPLE 7 Mode of action of the xylogalacturonase
  • HPAEC high performance anion exchange chromatography
  • HPSEC high performance size exclusion chromatography
  • HPSEC was performed 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 picture) was rapidly degraded (the top line (B) represents the polymer before degradation and the bottom line (A) represents the polymer after degradation).
  • Figure 6B shows that xylogalacturonase was able to degrade MHR-S a small shift to lower molecular weight material can be observed Also the enzymes endo-arabinanase ( Figure 6A) and rhamnogalacturonase (Figure 6C) caused some shift in molecular weight However, somewhat better results are obtained by combining two different enzymes in one 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 striking combined addition of endo-arabinanase and xylogalacturonase was much more effective than with sequential addition Almost complete degradation of the high molecular weight material was possible when the three enzymes were
  • a solution of 0.5% in 50 mM acetate buffer pH 4.0 was incubated with a combination of three enzymes: endo-arabinanase, rhamnogalacturonase and xylogalacturonase (ea/rg/xgh), a combination of two enzymes: endo-arabinanase and rhamnoglacturonase (ea rg), and with xylogalacturonase (xgh) separately for 17 hours at 30 °C.
  • the solutions were filtrated using an Amicon device equipped with a 30 kD filter at a pressure of 2 bars. The increase in weight of the filtrate was followed over time. The results are shown in Figure 5.
  • Figure 7 shows the changes in the molecular weight distribution as measured by HPSEC: in the xylogalacturonase-treated material (curve b) the peak at approximately 20 min, representing the high molecular weight material, decreases to 70% of the value of the starting material (curve a).
  • Figure 8 the results of the HPAEC analysis is shown. Comparing the enzyme- treated material (curve b) with the blank (curve a) it can be seen that xylogalacturonase causes the release of the characteristic xylosyl galacturonic acid dimer (marked with an X) and of other unidentified oligomers (peaks to the right side of X), comparable with the peaks appearing in Figure 6 of Example 7.

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Abstract

L'invention concerne des polypeptides possédant une nouvelle activité, notamment une activité d'endo-xylogalacturonase. Ces polypeptides peuvent dégrader la pectine trouvée dans des extraits de plantes et matières végétales, et notamment dans les régions 'velues' de polymères de pectine. Ces polypeptides peuvent notamment cliver un polymère d'acide galacturonique, au niveau de liaisons glycocidiques internes. L'invention concerne une nouvelle enzyme XghA, la séquence aminée de celle-ci ainsi que la séquence d'ADN codant pour celle-ci. On a fait s'exprimer ce polypeptide dans ces cellules de levure et on l'a utilisé pour traiter une matière végétale, notamment du soja et des jus de fruits, dans la préparation de denrées alimentaires comestibles.
PCT/EP1999/000860 1998-02-10 1999-02-09 Nouvelle endo-xylogalacturonase WO1999041386A2 (fr)

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BR9907831-7A BR9907831A (pt) 1998-02-10 1999-02-09 Endo-xilogalacturonase
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WO2014170498A1 (fr) 2013-04-19 2014-10-23 Danmarks Tekniske Universitet Production enzymatique de polysaccharides à partir de gomme adragante

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CN102762104B (zh) * 2010-02-26 2014-08-06 诺维信公司 用于制备果干的酶预处理
EP2538793A4 (fr) * 2010-02-26 2014-07-23 Novozymes As Prétraitement enzymatique pour obtenir des fruits séchés
CN107916268B (zh) * 2017-09-30 2021-10-22 武汉轻工大学 聚半乳糖醛酸裂解酶基因、重组表达载体、菌株、聚半乳糖醛酸裂解酶及其制备方法
CN114292832A (zh) * 2022-01-27 2022-04-08 大连海洋大学 一种嗜热耐高温多聚半乳糖醛酸酶MlPG28B、编码基因及制备方法

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EP0421919A2 (fr) * 1989-09-02 1991-04-10 Ciba-Geigy Ag Système d'expression fongique encodant la polygalacturonase
WO1994014966A1 (fr) * 1992-12-24 1994-07-07 Gist-Brocades N.V. CLONAGE ET EXPRESSION DU GENE DE L'EXO-POLYGALACTURONASE DE L'$i(ASPERGILLUS)
WO1995034223A1 (fr) * 1994-06-15 1995-12-21 Novo Nordisk A/S Stabilite des extraits troubles

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Publication number Priority date Publication date Assignee Title
EP0421919A2 (fr) * 1989-09-02 1991-04-10 Ciba-Geigy Ag Système d'expression fongique encodant la polygalacturonase
WO1994014966A1 (fr) * 1992-12-24 1994-07-07 Gist-Brocades N.V. CLONAGE ET EXPRESSION DU GENE DE L'EXO-POLYGALACTURONASE DE L'$i(ASPERGILLUS)
WO1995034223A1 (fr) * 1994-06-15 1995-12-21 Novo Nordisk A/S Stabilite des extraits troubles

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CHEN W P ET AL.: "Purification and some properties of beta-1,3-xylanases from Aspergillus terreus A-07. Endo-1,3-beta-D-xylanase isolation and characterization" AGRICULTURAL AND BIOLOGICAL CHEMISTRY., vol. 50, no. 5, 1986, pages 1183-1194, XP002111869 TOKYO., JP ISSN: 0002-1369 *
RENARD C.M.G.C. ET AL.: "The xylose-rich pectins from pae hulls" INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES, vol. 21, no. 1-2, August 1997 (1997-08), pages 155-162, XP002112536 AMSTERDAM NL *
SCHOLS H A ET AL: "A xylogalacturonan subunit present in the modified hairy regions of apple pectin" CARBOHYDRATE RESEARCH, vol. 279, 27 December 1995 (1995-12-27), page 265-279 XP004018802 amsterdam nl ISSN: 0008-6215 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014170498A1 (fr) 2013-04-19 2014-10-23 Danmarks Tekniske Universitet Production enzymatique de polysaccharides à partir de gomme adragante

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