WO2004074468A1 - Novel pectinases and uses thereof - Google Patents

Novel pectinases and uses thereof Download PDF

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Publication number
WO2004074468A1
WO2004074468A1 PCT/EP2003/005726 EP0305726W WO2004074468A1 WO 2004074468 A1 WO2004074468 A1 WO 2004074468A1 EP 0305726 W EP0305726 W EP 0305726W WO 2004074468 A1 WO2004074468 A1 WO 2004074468A1
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WIPO (PCT)
Prior art keywords
seq
pec
polypeptide
group
polynucleotide
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PCT/EP2003/005726
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French (fr)
Inventor
Luppo Edens
Albertus Alard Van Dijk
Sylvia Hopper
Hilmar Ilgenfritz
Wolfram Kemmner
Sabine Klugbauer
Andreas Fritz
Dieter Maier
Pamela Tan
Fabio Spreafico
Alexander Stock
Erik Kimpel
Oliver Heinrich
Beatrix Gerhard
Armin Ehrenreich
Sascha Losko
Christian Wagner
Ulrike Folkers
Richard Albang
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Dsm Ip Assets B.V.
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Priority to AU2003303934A priority Critical patent/AU2003303934A1/en
Priority to EP03815949A priority patent/EP1507857A1/en
Publication of WO2004074468A1 publication Critical patent/WO2004074468A1/en

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    • 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)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
    • A23C11/00—Milk substitutes, e.g. coffee whitener compositions
    • A23C11/02—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins
    • A23C11/10—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins
    • A23C11/103—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins containing only proteins from pulses, oilseeds or nuts, e.g. nut milk
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/189—Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K30/00—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs
    • A23K30/10—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder
    • A23K30/15—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging
    • A23K30/18—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging using microorganisms or enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
    • A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/30—Removing undesirable substances, e.g. bitter substances
    • A23L11/33—Removing undesirable substances, e.g. bitter substances using enzymes; Enzymatic transformation of pulses or legumes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/60—Drinks from legumes, e.g. lupine drinks
    • A23L11/65—Soy drinks
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
    • A23L19/09—Mashed or comminuted products, e.g. pulp, purée, sauce, or products made therefrom, e.g. snacks
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof
    • 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 OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/06—Enzymes
    • 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)

Definitions

  • the invention relates to newly identified polynucleotide sequences comprising genes that encode novel pectinases isolated from Aspergillus niger.
  • the invention features the full length nucleotide sequences of the novel genes, the cDNA sequences comprising the full length coding sequences of the novel pectinases as well as the amino acid sequences of the full-length functional proteins and functional equivalents thereof.
  • the invention also relates to methods of using these enzymes in industrial processes and methods of diagnosing fungal infections. Also included in the invention are cells transformed with a polynucleotide according to the invention and cells wherein a pectinase according to the invention is genetically modified to enhance or reduce its activity and/or level of expression.
  • Pectin polymers are important constituents of plant primary cell walls. They are composed of chains of 1 ,4-linked alpha-D-galacturonic acid and methylated derivatives thereof. Enzymes that are able to degrade the above- defined pectin polymers are called pectinases. Degradation in this respect means that at least one sugar residue or estergroup has been removed from the pectin molecule.
  • Pectinases such as polygalacturonase, pectin methylesterase, pectin lyase or pectate lyase are important for the food and feed industry, such as in the art of fruit and vegetable processing such as fruit juice production or wine making, where their ability to catalyse the degradation of the backbone of the pectin polymer is utilised.
  • pectinases in particular pectin methyl esterase, is the firming of fruit and vegetables.
  • the seeds of leguminicaeae like soybeans and other types of peas also contain pectin. This pectin, however, is quite different from pectins described for fruits like apple berries etc.
  • soybea pectin A large part of soybea pectin is water- unsoluble.
  • soybea pectin consists mainly of xylogalacturonan and rhamnogalacturonan and contains only minor amounts of homogalacturonan.
  • the CDTA-soluble pectic substances from soybean meal are composed of rhamnogalacturonan and xylogalacturonan but not homogalacturonan. Due to these structural differences soybea pectin is much more difficult to degrade by pectinases as compared to fruit pectin.
  • pectin degrading enzymes An assortment of different pectin degrading enzymes is known to be present in various microorganisms such as Aspergillus niger.
  • beta-fucosidase 3.2.1.38 beta-apiosidase 3.2.1.x alpha-rhamnosidase 3.2.1.40 beta-rhamnosidase 3.2.1.43 alpha-arabinopyranosidase 3.2.1.x beta-glucuronidase 3.2.1.31
  • Pectin methylesterase catalyses the removal of methanol from pectin, resulting in the formation of pectic acid (polygalacturonic acid).
  • Pectate lyase cleaves glycosidic bonds in polygalacturonic acid by beta-elimination
  • pectin lyase cleaves the glycosidic bonds of highly methylated pectins by beta- elimination
  • polygalacturonase hydrolyses the glycosidic linkages in the polygalacturonic acid chain.
  • pectinases such as Rapidase Press ® are actually a mixture of enzymes, which, along with other enzymes such as cellulases, are used in the fruit industry to help extract, clarify and modify fruit juices.
  • Pectinases have already been cloned in a variety of different microorganisms. Molecular cloning of pectinases in fungi has also been described. The DNA and deduced amino acid sequences of several pectinases from Aspergillus oryzae, Aspergillus kawachii and Emericella nidulans are known. However, there is still a need for other pectinases with different properties so that feed, fruit and vegetable processing may be optimized.
  • One of the disadvantages of the currently available enzymes is that they have pH optima above pH 4.0. Many fruit juices are more acidic and a pH optimum below 3.8 would be desirable. Fruit juices may become as acidic as pH 2.0 and it is therefore an object of the present invention to provide pectinases with a more acidic pH optimum. Pectinases with a pH optimum below 3.8 would be especially advantageous. Fruit processing is also often performed at more extreme temperatures. Use of pectinases with lower temperature optima would improve yield and quality of the juice because more aromas would be released. The currently available enzymes have a very low efficiency at temperatures below 15 °C. Also, at high temperatures the currently available enzymes are easily inactivated. This is a disadvantage for those fruit juices that have to be pasteurized.
  • Enzymes according to the invention also provide a better temperature stability and may withstand more extreme temperatures in comparison with pectinases according to the prior art. Also, the specificity of conventional enzymes leaves to be desired. Fruit juice that needs to be filtrated after or during processing tends to clot the filters after some time. This is due to the inability of prior art enzymes to properly digest fruit polysaccharides which causes the fouling of the filters.
  • a further object is to provide naturally and recombinantly produced pectinases as well as recombinant strains producing these.
  • fusion polypeptides are part of the invention as well as methods of making and using the polynucleotides and polypeptides according to the invention. Summary of the invention
  • the invention provides for novel polynucleotides encoding novel pectinases. These pectinases were found to digest pectinases from various sources in a better way than conventional pectinases. Such better performance was especially evident when the pectinases according to the invention were used to prevent filter fouling in fruit juice applications. Also, pectinases according to the invention may advantageously be employed for improving the nutritional value of soybean extracts. Moreover, the availability of improved, isolated enzymes allows a cocktail of enzymes to be be prepared which is optimized for the digestion of pectin from a particular feed, fruit or juice.
  • the invention provides for polynucleotides having a nucleotide sequence that hybridises preferably under highly stringent conditions to a sequence selected from the group consisting of SEQ ID NO: 33 - 64 or from the group consisting of SEQ ID NO: 1 - 32.
  • sequences revealed herein are shown in the accompanying sequence listing. For clarity reasons, the group consisting of sequences according to SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9; SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19; SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29; SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ
  • SEQ ID NO: 1 The group consisting of sequences according to SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9; SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19; SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29; SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32 is hereinafter collectively termed the group consisting of SEQ ID NO: 1 - 32
  • SEQ ID NO: 33 The group consisting of sequences according to SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO:37, SEQ ID NO: 38, SEQ ID NO: 39; SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49; SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59; SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO: 64 is hereinafter collectively termed
  • SEQ ID NO: 65 The group consisting of sequences according to SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69; SEQ ID NO: 70, SEQ ID NO: 71 , SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79; SEQ ID NO: 80, SEQ ID NO: 81 , SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89; SEQ ID NO: 90, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID
  • SEQ ID NO: 6 The group consisting of sequences according to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9; SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19; SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29; SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32 is hereinafter collectively termed the group consisting of SEQ ID NO: 6 - 32
  • SEQ ID NO: 38 SEQ ID NO: 39; SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49; SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59; SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO: 64 is hereinafter collectively termed the group consisting of SEQ ID NO: 38 - 64.
  • SEQ ID NO: 70 The group consisting of sequences according to SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79; SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89; SEQ ID NO: 90, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95 and SEQ ID NO: 96 is hereinafter collectively termed the group consisting of SEQ ID NO: 70 - 96.
  • SEQ ID NO: 1 - 21 The group consisting of sequences according to SEQ ID NO:
  • SEQ ID NO: 34 SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO:37, SEQ ID NO: 38, SEQ ID NO: 39
  • SEQ ID NO: 40 SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49
  • SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO: 53 is hereinafter collectively termed the group consisting of SEQ ID NO: 33 - 53.
  • SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84 and SEQ ID NO: 85 is hereinafter collectively termed the group consisting of SEQ ID NO: 65 - 85.
  • the invention provides nucleic acids that are more than 40% such as about 60%, preferably 65%, more preferably 70%, even more preferably 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the sequences selected from the group consisting of SEQ ID NO: 33 - 64 or from the group consisting of SEQ ID NO: 1 - 32.
  • the invention provides for a polynucleotide comprising the coding sequence of the polynucleotides according to the invention, preferred is a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1 - 32.
  • the invention also relates to vectors comprising a polynucleotide sequence according to the invention and primers, probes and fragments that may be used to amplify or detect the DNA according to the invention.
  • a vector is provided wherein the polynucleotide sequence according to the invention is functionally linked with regulatory sequences suitable for expression of the encoded amino acid sequence in a suitable host cell, such as A. niger or A. oryzea.
  • the invention also provides methods for preparing polynucleotides and vectors according to the invention.
  • the invention also relates to recombinantly produced host cells that contain heterologous or homologous polynucleotides according to the invention.
  • the invention provides recombinant host cells wherein the expression of a pectinase according to the invention is significantly increased or wherein the activity of the pectinase is increased.
  • polypeptides according to the invention include the polypeptides encoded by the polynucleotides according to the invention. Especially preferred is a polypeptide selected from the group consisting of SEQ ID NO: 65 - 96 or functional equivalents thereof.
  • the present invention provides polynucleotides encoding a number of novel pectinases, collectively called PEC 1 - PEC 32, having an amino acid sequence selected from the group consisting of SEQ ID NO: 65 - 96 or functional equivalents thereof.
  • PEC1 - PEC 32 is hereinafter used to refer to any polypeptide with pectinase activity selected from the group consisiting of SEQ ID NO: 65 - 96.
  • the sequences of the genes encoding PEC 1 - PEC 32 were determined by sequencing genomic clones obtained from Aspergillus niger.
  • polynucleotides may be obtained from filamentous fungi, in particular from Aspergillus niger. More specifically, the invention relates to an isolated polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO: 33 - 64 or from the group consisting of SEQ ID NO: 1 - 32.
  • the invention also relates to an isolated polynucleotide encoding at least one functional domain of a polypeptide selected from the group consisting of SEQ ID NO: 65 - 96 or functional equivalents thereof.
  • a nucleic acid molecule of the present invention such as a nucleic acid molecule selected from the group consisting of SEQ ID NO: 33 - 64 or from the group consisting of SEQ ID NO: 1 - 32 or a functional equivalent thereof, may be isolated using standard molecular biology techniques and the sequence information provided herein. For example, using all or portion of a nucleic acid selected from the group consisting of SEQ ID NO: 33 - 64 or from the group consisting of SEQ ID NO: 1 - 32 as a hybridization probe, nucleic acid molecules according to the invention may be isolated using standard hybridization and cloning techniques (e. g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
  • nucleic acid molecule encompassing all or a portion of a nucleotide sequence selected from the group consisting of SEQ ID NO: 33 - 64 or from the group consisting of SEQ ID NO: 1 - 32 may be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence information contained herein.
  • a nucleic acid according to the invention may be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid thus amplified may be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to or hybridisable to nucleotide sequences according to the invention may be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • an isolated nucleic acid molecule of the invention comprises the nucleotide sequence selected from the group consisting of SEQ ID NO: 1 - 32.
  • the sequences of the cDNAs selected from the group consisting of SEQ ID NO: 1 - 32 correspond to the coding region of the A. niger PEC 1 - PEC 32 pectinases.
  • Pectinases PEC 1 - PEC 32 correspond to polypeptides selected from the group consisting of SEQ ID NO: 65 - 96.
  • a nucleic acid molecule which is complementary to another nucleotide sequence is one which is sufficiently complementary to the other nucleotide sequence to such an extent that it may hybridize to the other nucleotide sequence thereby forming a stable duplex.
  • One aspect of the invention pertains to isolated nucleic acid molecules that encode a polypeptide of the invention or a functional equivalent thereof such as a biologically active fragment or domain, as well as nucleic acid molecules sufficient for use as hybridisation probes to identify nucleic acid molecules encoding a polypeptide of the invention and fragments of such nucleic acid molecules suitable for use as PCR primers for the amplification or mutation of nucleic acid molecules.
  • An "isolated polynucleotide” or “isolated nucleic acid” is a DNA or RNA that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived.
  • an isolated nucleic acid includes some or all of the 5' non-coding (e.g., promotor) sequences that are immediately contiguous to the coding sequence.
  • the term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences.
  • nucleic acid molecule As used herein, the terms “polynucleotide” or “nucleic acid molecule” are intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
  • the nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • the nucleic acid may be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides).
  • oligonucleotides may be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.
  • Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to a PEC 1 - PEC 32 nucleic acid molecule, e.g., the coding strand of a PEC 1 - PEC 32 nucleic acid molecule. Also included within the scope of the invention are the complement strands of the nucleic acid molecules described herein.
  • sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases.
  • the specific sequences disclosed herein may be readily used to isolate the complete gene from filamentous fungi, in particular A. niger which in turn may easily be subjected to further sequence analyses thereby identifying sequencing errors.
  • all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors.
  • Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule.
  • the actual sequence may be more precisely determined by other approaches including manual DNA sequencing methods well known in the art.
  • a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.
  • a nucleic acid molecule according to the invention may comprise only a portion or a fragment of the nucleic acid sequence selected from the group consisting of SEQ ID NO: 33 - 64 or from the group consisting of SEQ ID NO: 1 - 32, for example a fragment which may be used as a probe or primer or a fragment encoding a portion of a PEC 1 - PEC 32 protein.
  • the nucleotide sequence determined from the cloning of the PEC 1 - PEC 32 gene and cDNA allows for the generation of probes and primers designed for use in identifying and/or cloning other PEC 1 - PEC 32 family members, as well as PEC 1 - PEC 32 homologues from other species.
  • the probe/primer typically comprises substantially purified oligonucleotide which typically comprises a region of nucleotide sequence that hybridizes preferably under highly stringent conditions to at least about 12 or 15, preferably about 18 or 20, preferably about 22 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 or more consecutive nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NO: 33 - 64 or from the group consisting of SEQ ID NO: 1 - 32 or a functional equivalent thereof.
  • Probes based on the PEC 1 - PEC 32 nucleotide sequences may be used to detect transcripts or genomic PEC 1 - PEC 32 sequences encoding the same or homologous proteins for instance in other organisms.
  • the probe further comprises a label group attached thereto, e.g., the label group may be a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor.
  • the label group may be a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor.
  • Such probes may also be used as part of a diagnostic test kit for identifying cells which express a PEC 1 - PEC 32 protein.
  • the terms “homology” or “percent identity” are used interchangeably herein. For the purpose of this invention, it is defined here that in order to determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • % identity number of identical positions/total number of positions (i.e. overlapping positions) x 100).
  • the two sequences are the same length.
  • the skilled person will be aware of the fact that several different computer programms are available to determine the homology between two sequences. For instance, a comparison of sequences and determination of percent identity between two sequences may be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1 , 2, 3, 4, 5, or 6.
  • the percent identity two amino acid or nucleotide sequence is determined using the algorithm of E. Meyers and W.
  • the nucleic acid and protein sequences of the present invention may further be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences.
  • search may be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403—10.
  • Gapped BLAST may be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • hybridizing is intended to describe conditions for hybridization and washing under which nucleotide sequences at least about 50%, at least about 60%, at least about 70%, more preferably at least about 80%, even more preferably at least about 85% to 90%, more preferably at least 95%) homologous to each other typically remain hybridized to each other.
  • a preferred, non-limiting example of such hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45 °C, followed by one or more washes in 1 X SSC, 0.1 % SDS at 50 °C, preferably at 55 °C, preferably at 60 °C and even more preferably at 65 °C.
  • Highly stringent conditions include, for example, hybridizing at 68 °C in 5x SSC/5x Denhardt's solution / 1.0% SDS and washing in 0.2x SSC/0.1% SDS at room temperature. Alternatively, washing may be performed at 42 °C.
  • the skilled artisan will know which conditions to apply for stringent and highly stringent hybridisation conditions. Additional guidance regarding such conditions is readily available in the art, for example, in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; and Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology, (John Wiley & Sons, N.Y.).
  • cDNA libraries constructed from other organisms e.g. filamentous fungi, in particular from the species Aspergillus may be screened.
  • Homologous gene sequences may be isolated, for example, by performing PCR using two degenerate oligonucleotide primer pools designed on the basis of nucleotide sequences as taught herein.
  • the template for the reaction may be cDNA obtained by reverse transcription of mRNA prepared from strains known or suspected to express a polynucleotide according to the invention.
  • the PCR product may be subcloned and sequenced to ensure that the amplified sequences represent the sequences of a new PEC 1 - PEC 32 nucleic acid sequence, or a functional equivalent thereof.
  • the PCR fragment may then be used to isolate a full length cDNA clone by a variety of known methods.
  • the amplified fragment may be labeled and used to screen a bacteriophage or cosmid cDNA library.
  • the labeled fragment may be used to screen a genomic library.
  • RNA may be isolated, following standard procedures, from an appropriate cellular or tissue source.
  • a reverse transcription reaction may be performed on the RNA using an oligonucleotide primer specific for the most 5' end of the amplified fragment for the priming of first strand synthesis.
  • RNA/DNA hybrid may then be "tailed" (e.g., with guanines) using a standard terminal transferase reaction, the hybrid may be digested with RNase H, and second strand synthesis may then be primed (e.g., with a poly-C primer).
  • second strand synthesis may then be primed (e.g., with a poly-C primer).
  • vectors preferably expression vectors, containing a nucleic acid encoding a PEC 1 - PEC 32 protein or a functional equivalent thereof.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • viral vector is another type of vector, wherein additional DNA segments may be ligated into the viral genome.
  • plasmid and "vector” may be used interchangeably herein as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • viral vectors e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
  • the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vector includes one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed.
  • operatively linked is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signal). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymologyl 85, Academic Press, San Diego, CA (1990).
  • Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in a certain host cell (e.g. tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors of the invention may be introduced into host cells to thereby produce proteins or peptides, encoded by nucleic acids as described herein (e.g. PEC 1 - PEC 32 proteins, mutant forms of PEC 1 - PEC 32 proteins, fragments, variants or functional equivalents thereof, fusion proteins, etc.).
  • Expression vectors useful in the present invention include chromosomal-, episomal- and virus-derived vectors e.g., vectors derived from bacterial plasmids, bacteriophage, yeast episome, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • vectors derived from bacterial plasmids, bacteriophage, yeast episome, yeast chromosomal elements viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses
  • vectors derived from combinations thereof such as those derived from plasmid and bacteriophage
  • Vector DNA may be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-percipitation, DEAE- dextran-mediated transfection, transduction, infection, lipofection, cationic lipidmediated transfection or electroporation.
  • Suitable methods for transforming or transfecting host cells may be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2 nd , ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Davis et al., Basic Methods in Molecular Biology (1986) and other laboratory manuals.
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methatrexate.
  • Nucleic acid encoding a selectable marker may be introduced into a host cell on the same vector as that encoding a PEC 1 - PEC 32 protein or may be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid may be identified by drug selection (e.g. cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • Fusion vectors add a number of amino acids to a protein encoded therein, e.g. to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • enzymes, and their cognate recognation sequences include Factor Xa, thrombin and enterokinase.
  • the expression vectors will preferably contain selectable markers. Such markers include dihydrofolate reductase or neomycin resistance for eukarotic cell culture and tetracyline or ampicilling resistance for culturing in E. coli and other bacteria. Representative examples of appropriate host include bacterial cells, such as E.
  • coli Streptomyces and Salmonella typhimurium
  • fungal cells such as yeast
  • insect cells such as Drosophila S2 and Spodoptera Sf9
  • animal cells such as CHO, COS and Bowes melanoma
  • plant cells Appropriate culture mediums and conditions for the above-described host cells are known in the art.
  • vectors preferred for use in bacteria are pQE70, pQE60 and PQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16A, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
  • preferred eukaryotic vectors are PWLNEO, pSV2CAT, pOG44, pZT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
  • Other suitable vectors will be readily apparent to the skilled artisan.
  • bacterial promotors for use in the present invention include E. coli lad and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR, PL promoters and the trp promoter, the HSVthymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus ("RSV”), and metallothionein promoters, such as the mouse metallothionein-l promoter.
  • retroviral LTRs such as those of the Rous sarcoma virus ("RSV")
  • metallothionein promoters such as the mouse metallothionein-l promoter.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type.
  • enhancers include the SV40 enhancer, which is located on the late side of the replication origin at bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • secretation signal may be incorporated into the expressed polypeptide.
  • the signals may be endogenous to the polypeptide or they may be heterologous signals.
  • the polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals but also additional heterologous functional regions.
  • a region of additional amino acids, particularly charged amino acids may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification or during subsequent handling and storage.
  • peptide moieties may be added to the polypeptide to facilitate purification.
  • the invention provides an isolated polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 65 - 96, an amino acid sequence obtainable by expressing a polynucleotide selected from the group consisting of SEQ ID NO: 33 - 64 in an appropriate host, as well as an amino acid sequence obtainable by expressing a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1 - 32 in an appropriate host. Also, a peptide or polypeptide comprising a functional equivalent of the above polypeptides is comprised within the present invention.
  • polypeptides are collectively comprised in the term "polypeptides according to the invention"
  • peptide and oligopeptide are considered synonymous (as is commonly recognized) and each term may be used interchangeably as the context requires to indicate a chain of at least two amino acids coupled by peptidyl linkages.
  • the word “polypeptide” is used herein for chains containing more than seven amino acid residues. All oligopeptide and polypeptide formulas or sequences herein are written from left to right and in the direction from amino terminus to carboxy terminus. The one-letter code of amino acids used herein is commonly known in the art and may be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2 nd , ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989)
  • isolated polypeptide or protein is intended a polypeptide or protein removed from its native environment.
  • recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the invention as are native or recombinant polypeptides which have been substantially purified by any suitable technique such as, for example, the single-step purification method disclosed in Smith and Johnson, Gene 67:31-40 (1988).
  • the PEC 1 - PEC 32 pectinase according to the invention may be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC”) is employed for purification.
  • Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells.
  • polypeptides of the present invention may be glycosylated or may be non-glycosylated.
  • polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
  • the invention also features nucleic acid fragments which encode the above biologically active fragments of the PEC 1 - PEC 32 protein.
  • a "PEC 1 - PEC 32 polypeptide” refers to a polypeptide having an amino acid sequence corresponding to PEC 1 - PEC 32
  • a “non-PEC 1 - PEC 32 polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the PEC 1 - PEC 32 protein, e.g., a protein which is different from the PEC 1 - PEC 32 protein and which is derived from the same or a different organism.
  • the PEC 1 - PEC 32 polypeptide may correspond to all or a portion of a PEC 1 - PEC 32 protein.
  • the fusion protein is a GST- PEC 1 - PEC 32 fusion protein in which the PEC 1 - PEC 32 sequences are fused to the C-terminus of the GST sequences.
  • Such fusion proteins may facilitate the purification of recombinant PEC 1 - PEC 32.
  • the fusion protein is a PEC 1 - PEC 32 protein containing a heterologous signal sequence at its N-terminus.
  • expression and/or secretion of PEC 1 - PEC 32 may be increased through use of a hetereologous signal sequence.
  • the gp67 secretory sequence of the baculovirus envelope protein may be used as a heterologous signal sequence (Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, 1992).
  • Other examples of eukaryotic heterologous signal sequences include the secretory sequences of melittin and human placental alkaline phosphatase (Stratagene; La Jolla, California).
  • useful prokarytic heterologous signal sequences include the phoA secretory signal (Sambrook et al., supra) and the protein A secretory signal (Pharmacia Biotech; Piscataway, New Jersey).
  • a signal sequence may be used to facilitate secretion and isolation of a protein or polypeptide of the invention.
  • Signal sequences are typically characterized by a core of hydrophobic amino acids which are generally cleaved from the mature protein during secretion in one or more cleavage events.
  • Such signal peptides contain processing sites that allow cleavage of the signal sequence from the mature proteins as they pass through the secretory pathway.
  • the signal sequence directs secretion of the protein, such as from a eukaryotic host into which the expression vector is transformed, and the signal sequence is subsequently or concurrently cleaved.
  • the protein may then be readily purified from the extracellular medium by art recognized methods.
  • the signal sequence may be linked to the protein of interest using a sequence which facilitates purification, such as with a GST domain.
  • the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide, which facilitates purification of the fused polypeptide.
  • the marker sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As described in Gentz et al, Proc. Natl. Acad. Sci.
  • PCR amplification of gene fragments may be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which may subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
  • anchor primers which give rise to complementary overhangs between two consecutive gene fragments which may subsequently be annealed and reamplified to generate a chimeric gene sequence
  • many expression vectors are commercially available that already encode a fusion moiety (e.g, a GST polypeptide).
  • a nucleic acid encoding a PEC 1 - PEC 32 polypeptide may be cloned into such an expression vector such that the fusion moiety is linked in-frame to the PEC 1 - PEC 32 polypeptide.
  • Functional equivalents and “functional variants” are used interchangeably herein.
  • Functional equivalents of PEC 1 - PEC 32 DNA are isolated DNA fragments that encode a polypeptide that exhibits a particular function of a PEC 1 - PEC 32 A. niger pectinase as defined herein.
  • a functional equivalent of a PEC 1 - PEC 32 polypeptide according to the invention is a polypeptide that exhibits at least one function of an A. niger pectinase as defined herein. Functional equivalents therefore also encompass biologically active fragments.
  • Functional protein or polypeptide equivalents may contain only conservative substitutions of one or more amino acids of a sequence selected from the group consisting of SEQ ID NO: 65 - 96 or substitutions, insertions or deletions of non-essential amino acids.
  • a non-essential amino acid is a residue that may be altered in a sequence selected from the group consisting of SEQ ID NO: 65 - 96 without substantially altering the biological function.
  • amino acid residues that are conserved within their own family among any of the PEC 1 - PEC 32 proteins of the present invention are predicted to be particularly unamenable to alteration.
  • substitution is intended to mean that a substitution in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • These families are known in the art and include amino acids with basic side chains (e.g. lysine, arginine and hystidine), acidic side chains (e.g.
  • aspartic acid glutamic acid
  • uncharged polar side chains e.g., glycine, asparagines, glutamine, serine, threonine, tyrosine, cysteine
  • non- polar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine tryptophan, histidine
  • Functional nucleic acid equivalents may typically contain silent mutations or mutations that do not alter the biological function of encoded polypeptide. Accordingly, the invention provides nucleic acid molecules encoding PEC 1 - PEC 32 proteins that contain changes in amino acid residues that are not essential for a particular biological activity. Such PEC 1 - PEC 32 proteins differ in amino acid sequence from a sequence selected from the group consisting of SEQ ID NO: 65 - 96 yet retain at least one biological activity.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises a substantially homologous amino acid sequence of at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%), 98%, 99% or more homologous to an amino acid sequence selected from the group consisting of SEQ ID NO: 65 - 96.
  • An isolated nucleic acid molecule encoding a PEC 1 - PEC 32 protein homologous to the protein selected from the group consisting of SEQ ID NO: 65 - 96 may be created by introducing one or more nucleotide substitutions, additions or deletions into the coding nucleotide sequences selected from the group consisting SEQ ID NO: 65 - 963 - 64 or from the group consisting of SEQ ID NO: 1 - 32 such that one or more amino acid substitutions, deletions or insertions are introduced into the encoded protein.
  • Such mutations may be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • orthologues of the A. niger PEC 1 - PEC 32 protein are proteins that may be isolated from other strains or species and possess a similar or identical biological activity. Such orthologues may readily be identified as comprising an amino acid sequence that is substantially homologous to a sequence selected from the group consisting of SEQ ID NO: 65 - 96.
  • substantially homologous refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., with similar side chain) amino acids or nucleotides to a second amino acid or nucleotide sequence such that the first and the second amino acid or nucleotide sequences have a common domain.
  • amino acid or nucleotide sequences which contain a common domain having about 60%, preferably 65%, more preferably 70%, even more preferably 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity or more are defined herein as sufficiently identical.
  • nucleic acids encoding other PEC 1 - PEC 32 family members which thus have a nucleotide sequence that differs from a sequence selected from the group consisting of SEQ ID NO: 33 - 64 or from the group consisting of SEQ ID NO: 1 - 32, are within the scope of the invention.
  • nucleic acids encoding PEC 1 - PEC 32 proteins from different species which thus may have a nucleotide sequence which differs from a sequence selected from the group consisting of SEQ ID NO: 33 - 64.or from the group consisting of SEQ ID NO: 1 - 32 are within the scope of the invention.
  • Nucleic acid molecules corresponding to variants (e.g. natural allelic variants) and homologues of the PEC 1 - PEC 32 DNA of the invention may be isolated based on their homology to the PEC 1 - PEC 32 nucleic acids disclosed herein using the cDNAs disclosed herein or a suitable fragment thereof, as a hybridisation probe according to standard hybridisation techniques preferably under highly stringent hybridisation conditions.
  • improved PEC 1 - PEC 32 proteins are provided.
  • Improved PEC 1 - PEC 32 proteins are proteins wherein at least one biological activity is improved. Such proteins may be obtained by randomly introducing mutations along all or part of the PEC 1 - PEC 32 coding sequence, such as by saturation mutagenesis, and the resulting mutants may be expressed recombinantly and screened for biological activity. For instance, the art provides for standard assays for measuring the enzymatic activity of pectinases and thus improved proteins may easily be selected.
  • the PEC 1 - PEC 32 protein has an amino acid sequence selected from the group consisting of SEQ ID NO: 65 - 96.
  • the PEC 1 - PEC 32 polypeptide is substantially homologous to the amino acid sequence selected from the group consisting of SEQ ID NO: 65 - 96 and retains at least one biological activity of a polypeptide selected from the group consisting of SEQ ID NO: 65 - 96, yet differs in amino acid sequence due to natural variation or mutagenesis as described above.
  • the PEC 1 - PEC 32 protein has an amino acid sequence encoded by an isolated nucleic acid fragment capable of hybridising to a nucleic acid selected from the group consisting of SEQ ID NO: 33 - 64 or from the group consisting of SEQ ID NO: 1 - 32, preferably under highly stringent hybridisation conditions.
  • the PEC 1 - PEC 32 protein is a protein which comprises an amino acid sequence at least about 60%), 65%, 70%, 75%, 80%, 85%o, 90%), 95%), 96%., 97%>, 98%, 99% or more homologous to the amino acid sequence selected from the group consisting of SEQ ID NO: 65 - 96 and retains at least one functional activity of a polypeptide selected from the group consisting of SEQ ID NO: 65 - 96.
  • Functional equivalents of a protein according to the invention may also be identified e.g. by screening combinatorial libraries of mutants, e.g. truncation mutants, of the protein of the invention for pectinase activity.
  • a variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level.
  • a variegated library of variants may be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential protein sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display).
  • libraries of fragments of the coding sequence of a polypeptide of the invention may be used to generate a variegated population of polypeptides for screening a subsequent selection of variants.
  • a library of coding sequence fragments may be generated by treating a double stranded PCR fragment of the coding sequence of interest with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which may include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector.
  • an expression library may be derived which encodes N-terminal and internal fragments of various sizes of the protein of interest.
  • REM Recursive ensemble mutagenesis
  • REM Recursive ensemble mutagenesis
  • a technique which enhances the frequency of functional mutants in the libraries may be used in combination with the screening assays to identify variants of a protein of the invention.
  • PEC 1 - PEC 32 gene sequence selected from the group consisting of SEQ ID NO: 33 - 64 it will be apparent for the person skilled in the art that DNA sequence polymorphisms that may lead to changes in the amino acid sequence of the PEC 1 - PEC 32 protein may exist within a given population. Such genetic polymorphisms may exist in cells from different populations or within a population due to natural allelic variation. Allelic variants may also include functional equivalents.
  • Fragments of a polynucleotide according to the invention may also comprise polynucleotides not encoding functional polypeptides. Such polynucleotides may function as probes or primers for a PCR reaction. Nucleic acids according to the invention irrespective of whether they encode functional or non-functional polypeptides, may be used as hybridization probes or polymerase chain reaction (PCR) primers. Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having a PEC 1 - PEC 32 activity include, inter alia, (1) isolating the gene encoding the PEC 1 - PEC 32 protein, or allelic variants thereof from a cDNA library e.g. from other organisms than A.
  • in situ hybridization e.g. FISH
  • metaphase chromosomal spreads to provide precise chromosomal location of the PEC 1 - PEC 32 gene as described in Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988);
  • Northern blot analysis for detecting expression of PEC 1 - PEC 32 mRNA in specific tissues and/or cells and 4) probes and primers that may be used as a diagnostic tool to analyse the presence of a nucleic acid hybridisable to the PEC 1 - PEC 32 probe in a given biological (e.g. tissue) sample.
  • Also encompassed by the invention is a method of obtaining a functional equivalent of a PEC 1 - PEC 32 gene or cDNA.
  • a method entails obtaining a labelled probe that includes an isolated nucleic acid which encodes all or a portion of the sequence selected from the group consisting of SEQ ID NO: 65 - 96 or a variant thereof; screening a nucleic acid fragment library with the labelled probe under conditions that allow hybridisation of the probe to nucleic acid fragments in the library, thereby forming nucleic acid duplexes, and preparing a full-length gene sequence from the nucleic acid fragments in any labelled duplex to obtain a gene related to the PEC 1 - PEC 32 gene.
  • a PEC 1 - PEC 32 nucleic acid of the invention is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%), 95%>, 96%o, 97% » 98%., 99%, or more homologous to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 33 - 64 , from the group consisting of SEQ ID NO: 1 - 32 or the complement thereof.
  • a PEC 1 - PEC 32 polypeptide according to the invention is at least 60%, 65%, 70%, 75%, 80%, 85%>, 90%>, 91%, 92%o, 93%, 94%, 95%, 96%, 97%, 98%, 99%>, or more homologous to an amino acid sequence selected from the group consisting of SEQ ID NO: 65 - 96.
  • a host cell may be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in a specific, desired fashion. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may facilitate optimal functioning of the protein.
  • Various host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products.
  • Appropriate cell lines or host systems familiar to those of skill in the art of molecular biology and/or microbiology may be chosen to ensure the desired and correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • Such host cells are well known in the art.
  • Host cells also include, but are not limited to, mammalian cell lines such as CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and choroid plexus cell lines.
  • mammalian cell lines such as CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and choroid plexus cell lines.
  • the polypeptides according to the invention may be produced by a stably-transfected cell line.
  • a number of vectors suitable for stable transfection of mammalian cells are available to the public, methods for constructing such cell lines are also publicly known, e.g., in Ausubel et al. (supra).
  • the invention further features antibodies, such as monoclonal or polyclonal antibodies, that specifically bind PEC 1 - PEC 32 proteins according to the invention.
  • antibody or “monoclonal antibody” (Mab) is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab') 2 fragments) which are capable of specifically binding to PEC 1 - PEC 32 protein.
  • Fab and F(ab') 2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Thus, these fragments are preferred.
  • the antibodies of the present invention may be prepared by any of a variety of methods. For example, cells expressing the PEC 1 - PEC 32 protein or an antigenic fragment thereof may be administered to an animal in order to induce the production of sera containing polyclonal antibodies.
  • a preparation of PEC 1 - PEC 32 protein is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.
  • the antibodies of the present invention are monoclonal antibodies (or PEC 1 - PEC 32 protein binding fragments thereof).
  • any suitable myeloma cell line may be employed in accordance with the present inventoin; however, it is preferably to employ the parent myeloma cell line (SP 2 O), available from the American Type Culture Collection, Rockville, Maryland.
  • SP 2 O myeloma cell line
  • the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastro-enterology 80:225-232 (1981)).
  • the hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the PEC 1 - PEC 32 protein antigen.
  • the polypeptides may be coupled to a carrier protein, such as KLH, as described in Ausubel et al., supra, mixed with an adjuvant, and injected into a host mammal.
  • various host animals may be immunized by injection of a polypeptide of interest.
  • suitable host animals include rabbits, mice, guinea pigs, and rats.
  • Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), adjuvant mineral gels such as aluminum hydroxide, surface actve substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
  • Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of the immunized animals.
  • Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD, and any subclass thereof.
  • the hybridomas producing the mAbs of this invention may be cultivated in vitro or in vivo. Once produced, polyclonal or monoclonal antibodies are tested for specific recognition of a PEC 1 - PEC 32 polypeptide or functional equivalent thereof in an immunoassay, such as a Western blot or immunoprecipitation analysis using standard techniques, e.g., as described in Ausubel et al., supra.
  • Antibodies that specifically bind to PEC 1 - PEC 32 proteins or functional equivalents thereof are useful in the invention. For example, such antibodies may be used in an immunoassay to detect PEC 1 - PEC 32 in pathogenic or non- pathogenic strains of Aspergillus (e.g., in Aspergillus extracts).
  • the antisera are checked for their ability to immunoprecipitate a recombinant PEC 1 - PEC 32 polypeptide or functional equivalents thereof whereas unrelated proteins may serve as a control for the specificity of the immune reaction.
  • techniques decribed for the production of single chain antibodies (U.S. Patent 4,946,778 and 4,704,692) may be adapted to produce single chain antibodies against a PEC 1 - PEC 32 polypeptide or functional equivalents thereof.
  • Kits for generating and screening phage display libraries are commercially available e.g. from Pharmacia.
  • examples of methods and reagents particularly amenable for use in generating and screening antibody display library may be found in, for example, U.S. Patent No.
  • PEC 1 - PEC 32 polypeptides of functional equivalents thereof may be used, for example, to detect expression of a PEC 1 - PEC 32 gene or a functional equivalent thereof e.g. in another strain of Aspergillus.
  • PEC 1 - PEC 32 polypeptide may be readily detected in conventional immunoassays of Aspergillus cells or extracts. Examples of suitable assays include, without limitation, Western blotting, ELISAs, radioimmune assays, and the like.
  • an antibody recognizes and binds a particular antigen, e.g., a PEC 1 - PEC 32 polypeptide, but does not substantially recognize and bind other unrelated molecules in a sample.
  • Antibodies may be purified, for example, by affinity chromatography methods in which the polypeptide antigen is immobilized on a resin.
  • An antibody directed against a polypeptide of the invention may be used to isolate the polypeptide by standard techniques, such as affinity chromatography or immunoprecipitation. Moreover, such an antibody may be used to detect the protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the polypeptide.
  • the antibodies may also be used diagnostically to monitor protein levels in cells or tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen or in the diagnosis of Aspergillosis..
  • Detection may be facilitated by coupling the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive materials include 125 l, 131 l, 35 S or 3 H.
  • Preferred epitopes encompassed by the antigenic peptide are regions that are located on the surface of the protein, e.g., hydrophilic regions. Hydrophobicity plots of the proteins of the invention may be used to identify hydrophilic regions.
  • the antigenic peptide of a protein of the invention comprises at least 7 (preferably 10, 15, 20, or 30) contiguous amino acid residues of the amino acid sequense of a sequence selected from the group consisting of SEQ ID NO: 65 - 96 and encompasses an epitope of the protein such that an antibody raised against the peptide forms a specific immune complex with the protein.
  • Preferred epitopes encompassed by the antigenic peptide are regions of PEC 1 - PEC 32 that are located on the surface of the protein, e.g., hydrophilic regions, hydrophobic regions, alpha regions, beta regions, coil regions, turn regions and flexible regions.
  • Immunoassavs Qualitative or quantitative determination of a polypeptide according to the present invention in a biological sample may occur using any art- known method.
  • Antibody-based techniques provide special advantages for assaying specific polypeptide levels in a biological sample. In these, the specific recognition is provided by the primary antibody (polyclonal or monoclonal) but the secondary detection system may utilize fluorescent, enzyme, or other conjugated secondary antibodies. As a result, an immunocomplex is obtained.
  • the invention provides a method for diagnosing whether a certain organism is infected with Aspergillus comprising the steps of:
  • Tissues may also be extracted, e.g., with urea and neutral detergent, for the liberation of protein for Western-blot or dot/slot assay. This technique may also be applied to body fluids.
  • PEC 1 - PEC 32-specific monoclonal antibodies may be used both as an immunoabsorbent and as an enzyme-labeled probe to detect and quantify the PEC 1 - PEC 32 protein.
  • the amount of PEC 1 - PEC 32 protein present in the sample may be calculated by reference to the amount present in a standard preparation using a linear regression computer algorithm.
  • two distinct specific monoclonal antibodies may be used to detect PEC 1 - PEC 32 protein in a biological fluid. In this assay, one of the antibodies is used as the immuno-absorbent and the other as the enzyme-labeled probe.
  • the above techniques may be conducted essentially as a "one- step” or “two-step” assay.
  • the "one-step” assay involves contacting PEC 1 - PEC 32 protein with immobilized antibody and, without washing, contacting the mixture with the labeled antibody.
  • the "two-step” assay involves washing before contacting the mixture with the labeled antibody.
  • Other conventional methods may also be employed as suitable. It is usually desirable to immobilize one component of the assay system on a support, thereby allowing other components of the system to be brought into contact with the component and readily removed from the sample.
  • Suitable enzyme labels include, for example, those from the oxidase group, which catalyze the production of hydrogen peroxide by reacting with substrate. Activity of an oxidase label may be assayed by measuring the concentration of hydrogen peroxide formed by the enzyme-labelled antibody/substrate reaction.
  • the microtitre plates may be coated with a PEC 1 - PEC 32 polypeptide by adding the polypeptide in a solution (typically, at a concentration of 0.05 to 1 mg/ml in a volume of 1-100 ul) to each well, and incubating the plates at room temperature to 37 °C for 0.1 to 36 hours.
  • Polypeptides that are not bound to the plate may be removed by shaking the excess solution from the plate, and then washing the plate (once or repeatedly) with water or a buffer.
  • the polypeptide is contained in water or a buffer. The plate is then washed with a buffer that lacks the bound polypeptide.
  • the plates are blocked with a protein that is unrelated to the bound polypeptide.
  • a protein that is unrelated to the bound polypeptide 300 ul of bovine serum albumin (BSA) at a concentration of 2 mg/ml in Tris-HCl is suitable.
  • BSA bovine serum albumin
  • Suitable substrates include those substrates that contain a defined cross-linking chemistry (e.g., plastic substrates, such as polystyrene, styrene, or polypropylene substrates from Corning Costar Corp. (Cambridge, MA), for example) .
  • a beaded particle e.g., beaded agarose or beaded sepharose, may be used as the substrate.
  • Suitable antigens include enzymes (e.g., horse radish peroxidase, alkaline phosphatase, and a-galactosidase) and non-enzymatic polypeptides (e.g., serum proteins, such as BSA and globulins, and milk proteins, such as caseins).
  • enzymes e.g., horse radish peroxidase, alkaline phosphatase, and a-galactosidase
  • non-enzymatic polypeptides e.g., serum proteins, such as BSA and globulins, and milk proteins, such as caseins.
  • the invention provides a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide of the invention.
  • the epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide of the invention.
  • An "immunogenic epitope" is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen. These immunogenic epitopes are believed to be confined to a few loci on the molecule.
  • a region of a protein molecule to which an antibody may bind is defined as an "antigenic epitope.”
  • the number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes. See, for instance, Geysen, H. M. et al., Proc. Natl. Acad. Sci. USA 81 :3998-4002 (1984).
  • Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, may be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals. Peptides that are extremely hydrophobic and those of six or fewer residues generally are ineffective at inducing antibodies that bind to the mimicked protein; longer, soluble peptides, especially those containing proline residues, usually are effective. Sutcliffe et al., supra, at 661.
  • 18 of 20 peptides designed according to these guidelines containing 8- 39 residues covering 75% of the sequence of the influenza virus hemagglutinin HAI polypeptide chain, induced antibodies that reacted with the HA1 protein or intact virus; and 12/12 peptides from the MuLV polymerase and 18/18 from the rabies glycoprotein induced antibodies that precipitated the respective proteins.
  • Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention.
  • a high proportion of hybridomas obtained by fusion of spleen cells from donors immunized with an antigen epitope-bearing peptide generally secrete antibody reactive with the native protein.
  • the antibodies raised by antigenic epitope bearing peptides or polypeptides are useful to detect the mimicked protein, and antibodies to different peptides may be used for tracking the fate of various regions of a protein precursor which undergoes posttranslation processing.
  • the peptides and anti-peptide antibodies may be used in a variety of qualitative or quantitative assays for the mimicked protein, for instance in competition assays since it has been shown that even short peptides (e.g., about 9 amino acids) may bind and displace the larger peptides in immunoprecipitation assays. See, for instance, Wilson, I A et al., Cell 37:767-778 at 777 (1984).
  • the anti-peptide antibodies of the invention also are useful for purification of the mimicked protein, for instance, by adsorption chromatography using methods well known in the art.
  • Antigenic epitope-bearing peptides and polypeptides of the invention designed according to the above guidelines preferably contain a sequence of at least seven, more preferably at least nine and most preferably between about 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention.
  • peptides or polypeptides comprising a larger portion of an amino acid sequence of a polypeptide of the invention, containing about 30 to about 50 amino acids, or any length up to and including the entire amino acid sequence of a polypeptide of the invention also are considered epitope-bearing peptides or polypeptides of the invention and also are useful for inducing antibodies that react with the mimicked protein.
  • the amino acid sequence of the epitope-bearing peptide is selected to provide substantial solubility in aqueous solvents (i.e., the sequence includes relatively hydrophilic residues and highly hydrophobic sequences are preferably avoided); and sequences containing proline residues are particularly preferred.
  • the epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means for making peptides or polypeptides including recombinant means using nucleic acid molecules of the invention.
  • a short epitope-bearing amino acid sequence may be fused to a larger polypeptide which acts as a carrier during recombinant production and purification, as well as during immunization to produce anti-peptide antibodies.
  • Epitope-bearing peptides also may be synthesized using known methods of chemical synthesis. For instance, Houghten has described a simple method for synthesis of large numbers of peptides, such as 10-20 mg of 248 different 13 residue peptides representing single amino acid variants of a segment of the HAI polypeptide which were prepared and characterized (by ELISA-type binding studies) in less than four weeks. Houghten, R. A., Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985). This "Simultaneous Multiple Peptide Synthesis
  • Epitope-bearing peptides and polypeptides of the invention are used to induce antibodies according to methods well known in the art. See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow, M. et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle, F.J. et al., J. Gen. Virol. 66:2347-2354 (1985).
  • animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling of the peptide to a macromolecular carrier, such as keyhole limpet hemocyanin (KLH) or tetanus toxoid.
  • KLH keyhole limpet hemocyanin
  • peptides containing cysteine may be coupled to carrier using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carrier using a more general linking agent such as glutaraldehyde.
  • Animals such as rabbits, rats and mice are immunized with either free or carriercoupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 ug peptide or carrier protein and Freund's adjuvant. Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which may be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface.
  • the titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
  • U.S. Patent No. 5,194,392 to Geysen (1990) describes a general method of detecting or determining the sequence of monomers (amino acids or other compounds) which is a topological equivalent of the epitope (i.e., a "mimotope") which is complementary to a particular paratope (antigen binding site) of an antibody of interest. More generally, U.S. Patent No. 4,433,092 to
  • Geysen (1989) describes a method of detecting or determining a sequence of monomers which is a topographical equivalent of a ligand which is complementary to the ligand binding site of a particular receptor of interest.
  • U.S. Patent No. 5,480,971 to Houghten, R. A. et al. (1996) on Peralkylated Oligopeptide Mixtures discloses linear C1-C7-alkyl peralkylated oligopeptides and sets and libraries of such peptides, as well as methods for using such oligopeptide sets and libraries for determining the sequence of a peralkylated oligopeptide that preferentially binds to an acceptor molecule of interest.
  • non-peptide analogs of the epitope-bearing peptides of the invention also may be made routinely by these methods.
  • Pectins and pectinases play an important role in the food industry. Pectinases are used in the production of fruit and vegetable juices and purees, and in the extraction of useful components, like arome compounds, or even pharmaceuticals, from plant materials.
  • pectinases One of the main applications of pectinases is in the production of clear fruit and vegetable juice.
  • the production of juices from fruit and vegetables typically involves, after an optional washing step, grinding, crushing or otherwise destroying the integrity of the fruit or vegetables thus obtaining a fruit or vegetable pulp.
  • the pulp may be treated with enzymes to decrease soluble pectin, a process generally referred to as maceration.
  • maceration the pulp is ready for pressing, leading to a juice fraction and a residue fraction, the latter being referred to as the pomace.
  • the juice obtained after pressing is usually pasteurised, optionally with recovery of the aroma, which may then be added back at the end of the process.
  • the pasteurised juice is enzymatically depectinized with the aid of pectinases, optionally pre- concentrated, filtrated, optionally ultra-filtrated and concentrated to obtain a clear concentrate or juice which is ready for shipping and/or blending to obtain a clear apple juice for the consumer market
  • pectinases are useful in all production processes in which the integrity of the cell wall should be destroyed in order to release materials from the inside of the cells, or from the cell walls. Pectinases may also be used to destroy the connection between cells without affecting the integrity of the separate cells. When cells should remain intact, e.g.
  • PEC 1 - PEC 32 pectinases may advantageously be used to release the structural connection between the cells.
  • PEC 1 - PEC 32 pectinases are also able to decrease the water- binding and hence the viscosity of pectin-containing slurries, e.g. fruit or vegetable pulps, which improves the pressability/processability of the pulp.
  • PEC 1 - PEC 32 pectinases may also be used in the retting of flax (to release the cellulose fibres from the plant tissue) and the scouring of cotton (to increase the water absorbance and dye uptake of the cotton fibres).
  • Plant and pectin-containing materials include plant pulp, parts of plants and plant extracts.
  • an extract from a plant material is any substance which may 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).
  • Fruit juice that needs to be filtrated after or during processing tends to clot the filters after some time.
  • PEC 1 - PEC 32 pectinases may conveniently be produced in microorganisms.
  • Microbial pectinases are available from a variety of sources; Bacillus spec, are a common source of bacterial enzymes, whereas fungal enzymes are commonly produced in Aspergillus spec.
  • pectinases that are obtained by recombinant DNA techniques.
  • Such recombinant enzymes have a number of advantages over their traditionally purified counterparts.
  • Recombinant enzymes may be produced at a low cost price, high yield, free from contaminating agents like bacteria or viruses but also free from bacterial toxins or contaminating other enzyme activities.
  • the polypeptides of the invention may be used to treat plant material including plant pulp and plant extracts. For example, 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. Conveniently the polypeptide of the invention is combined with suitable (solid or liquid) carriers or diluents including buffers to produce a composition or enzyme preparation.
  • 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 r dry form.
  • the product is made as a composition which will optionally include, for example, a stabilising buffer and/or preservative.
  • 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.
  • pectinases such as an endo-arabinanase, rhamnogalacturonases, and/or polygalacturonase.
  • 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.
  • 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
  • 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 material will typically be carried out for a 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 may 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 may be improved.
  • a polypeptide of the invention may be added to raw juice obtained from pressing or liquefying the plant pulp. Treatment of raw juice may be carried out in a similar manner to the plant pulp in respect of dosage, temperature and holding time. Again, other enzymes than pectinases may be included.
  • a 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. Due to the highly specific action on pectins the polypeptides of the invention may also be used to prepare pectins with modified chara dsristics, e.g. modified gelation capacities for specific applications.
  • the polypeptides of the invention may be added to animal feeds rich in pectin, 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 pectins in the feed in vivo.
  • Polypeptides according to the invention have low pH optima and are capable of releasing important nutrients in such acidic environments as the stomach of an animal. The invention thus also provides (e.g.
  • PEC 1 - PEC 32 pectinases may also be advantageously used during the production of milk substitutes (or replacers) from soy bean. These milk substitutes may 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 according to the invention may be added to the slurry, either before or during the processing, enabling processing at a higher concentration (typically 40 to 50%) dry solids.
  • Addition of pectinases according to the invention to a soy suspension as described in WO 95/29598 results in a decrease in waterbinding and a concommitant decrease in viscosity of the slurry.
  • the enzyme may also be used in the preparation of savoury product(s), e.g. from soy bean.
  • the invention also relates to the use of PEC 1 - PEC 32 pectinases in a selected number of industrial and pharmaceutical processes.
  • the pectinase according to the invention features a number of significant advantages over the prior art enzymes. Depending on the specific application, these advantages may include aspects like lower production costs, higher specificity towards the substrate, less antigenic, less undesirable side activities, higher yields when produced in a suitable microorganism, more suitable pH and temperature ranges, better tastes of the final product as well as food grade and kosher aspects.
  • An important aspect of the pectinases according to the invention is that they cover a whole range of pH and temperature optima which are ideally suited for a variety of applications. For example many large scale processes benefit from relatively high processing temperatures of 50 degrees C or higher, e.g. to control the risks of microbial infections.
  • pectinases according to the invention comply with this demand but at the same time they are not that heat stable that they resist attempts to inactivate the enzyme by an additional heat treatment. The latter feature allows production routes that yield final products free of residual enzyme activity. Similarly many feed and food products have slightly acidic pH values so that pectinases with acidic or near neutral pH optima are preferred for their processing. A PEC 1 - PEC 32 pectinase complies with this requirement as well.
  • polypeptides of the invention may also be added to animal feeds rich in pectin or xylogalacturonan, to reduce anti-nutritional effects of plant pectins and consequently improve production performances and welfare of the animals, as well as reducing environmental pollution.
  • Advantagous is that these fungal derived polypeptides of the invention have generally lower pH optima and their activities are therefore (more) capable of hydrolysis in such acidic environments as the stomach of an animal. With that, anti-nutritional properties of the plant cell wall fractions are already reduced early on in the digestive tract of the animal.
  • PEC 1 - PEC 32 were expressed in A. niger by cloning of cDNAs acco ding to SEQ ID NO: 1 - 32.
  • the resulting enzyme preparations were subsequently purified by ultrafiltration and used in food and feed conversion as described in the examples. It was found that PEC 1 - PEC 32, when added to animal feed, were able to increase the nutritional value of the feed, as measured by an improved body weight gain in the range of 1 to 5%> and in an improvement of the feed conversion ration of 1.5 to 6%>.
  • PEC 1 - PEC 21 corresponding to polypeptides with SEQ ID NO: 65 - 85 were found especially useful.
  • DNA fragments having a sequence according to SEQ ID NO: 1 - 32 were cloned under the control of the glucoamylase promoter.
  • the Aspergillus niger phytase (phyA) gene described in international patent application WO 98/46772 was replaced with the pectinase genes described above, yielding the vectors pPECTI - 32 respectively.
  • the expression vectors pPECTI - 32 were introduced into Aspergillus niger CBS 646.97 (described in WO 98/46772). Using PCR, transformants containing pPECTI - 32 were selected.
  • An experime t was designed to establish whether PEC 1 - PEC 32 pectinases, expressed as described above, could improve the nutritional value of soybean-based feed.
  • the experiment was performed using male broilers (Ross 308) housed in floor pens. Directly after arrival from the hatchery, the animals were randomly distributed over 40 pens, each pen containing 20 broilers. Eight pens were allocated to each treatment. The pens were set up in an artificially heated, illuminated and ventilated broiler house, applying conditions similar to those found in practice. Animals were vaccinated according to the normal vaccination program. The experiment was performed till day 35 of age.
  • Table 1 Feed composition and contents of main nutrients of diet 1.
  • Rapidase Press improved performance (treatment 2 compared to treatment 1 ), especially the growth of the animals. Additional supplementation with any enzyme chosen from PEC 1 - PEC 32, (treatments 3 - 5) further improved the performance of the animals, in particular supplementation with an enzyme chosen from PEC 1 - PEC 21 (corresponding to SEQ ID NO: 65 - 85) lead to a 1 - 4% gain in body weight and improved the feed conversion ratio with 1.5 - 6%.
  • Example 2 Example 2.
  • AME apparent metabolisable energy
  • Male broilers (Ross 308) of seven days of age were randomly distributed over 18 cages, each cage housing 8 broilers.
  • Six cages were allocated to each treatment.
  • the cages were set up in an artificially heated, illuminated and ventilated broiler house, using a three-tier cage system. Tiers were blocked over the treatments.
  • the room was illuminated 24 hours/day, but the light intensity was gradually decreased during the trial. Also the temperature was decreased gradually during the experiment, according to a practical schedule.
  • the humidity during the trial was kept at approximately 60%.
  • the treatments were: 1. basal diet (table 1 [example 1]), without enzyme was added (control), 2. addition of Rapidase Press, and 5. as diet 2, but with the addition of 25 ppm of additional pectinases PEC 1 - PEC 32.
  • the feeds were pelleted without the addition of steam in the process. Feed and water were offered ad libitum o the animals. Analytical procedures applied were similar to those typically applied in animal experimentation institutes, usually AOAC-methods.
  • Rapidase Press improved digestibility, especially of protein.
  • Addition of PEC 1 - PEC 32 had a large effect on energy metabolisability and on faecal digestibility of fat and protein, in particular PEC 1 - PEC 21 showed significant improvements in the order of 2 to 5%.
  • a next experiment was performed using mixed-sex broilers (Ross) fed a maize-soybean meal-based diet.
  • the animals were housed in floor pens. Directly after arrival from the hatchery, the animals were randomly distributed over 48 pens, each pen containing 24 broilers; eight pens (four pens of males and four pens of females) were allocated to each treatment.
  • the pens were set up in an artificially heated, illuminated and ventilated broiler house, applying conditions similar to those found in practice. Animals were vaccinated according to the normal vaccination program against Infectious Bronchitis and New Castle Disease. The experiment was performed up to day 42 of age.
  • Rapidase Press improved growth by 1.8% (treatment 2 compared to treatment 1).
  • a single enzyme selected from PEC 1 - 32 (treatment 3) showed a similar effect, but improved FCR as well (average 1.4%).
  • Supple entation with both enzymes, Rapidase Press and an enzyme selected from PEC 1 - PEC 32 in different doses (treatments 4 - 6) showed the best results.
  • PEC 1 - PEC 21 were found to be useful enzymes in this respect.
  • the treatments were:
  • Modified hairy regions (MHR) from apples were isolated as a filter retentate after treatment of apples with Rapidase Press, a commercially available pectinase preparation, and subsequently the MHR was saponified resulting in MHR-S.

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Abstract

The invention relates to newly identified polynucleotide sequences comprising genes that encode novel pectinases isolated from Aspergillus niger. The invention features the full length nucleotide sequence of the novel genes, the cDNA sequences comprising the full length coding sequence of the novel pectinases as well as the amino acid sequence of the full-length functional proteins and functional equivalents thereof. The invention also relates to methods of using these enzymes in industrial processes and methods of diagnosing fungal infections. Also included in the invention are cells transformed with a polynucleotide according to the invention and cells wherein a pectinase according to the invention is genetically modified to enhan e or reduce its activity and/or level of expression.

Description

NOVEL PECTINASES AND USES THEREOF
Field of the invention
The invention relates to newly identified polynucleotide sequences comprising genes that encode novel pectinases isolated from Aspergillus niger. The invention features the full length nucleotide sequences of the novel genes, the cDNA sequences comprising the full length coding sequences of the novel pectinases as well as the amino acid sequences of the full-length functional proteins and functional equivalents thereof. The invention also relates to methods of using these enzymes in industrial processes and methods of diagnosing fungal infections. Also included in the invention are cells transformed with a polynucleotide according to the invention and cells wherein a pectinase according to the invention is genetically modified to enhance or reduce its activity and/or level of expression.
Background of the invention
Pectin polymers are important constituents of plant primary cell walls. They are composed of chains of 1 ,4-linked alpha-D-galacturonic acid and methylated derivatives thereof. Enzymes that are able to degrade the above- defined pectin polymers are called pectinases. Degradation in this respect means that at least one sugar residue or estergroup has been removed from the pectin molecule. Pectinases such as polygalacturonase, pectin methylesterase, pectin lyase or pectate lyase are important for the food and feed industry, such as in the art of fruit and vegetable processing such as fruit juice production or wine making, where their ability to catalyse the degradation of the backbone of the pectin polymer is utilised. Another application of pectinases, in particular pectin methyl esterase, is the firming of fruit and vegetables. The seeds of leguminicaeae like soybeans and other types of peas also contain pectin. This pectin, however, is quite different from pectins described for fruits like apple berries etc. A large part of soybea pectin is water- unsoluble. In addition soybea pectin consists mainly of xylogalacturonan and rhamnogalacturonan and contains only minor amounts of homogalacturonan. The CDTA-soluble pectic substances from soybean meal are composed of rhamnogalacturonan and xylogalacturonan but not homogalacturonan. Due to these structural differences soybea pectin is much more difficult to degrade by pectinases as compared to fruit pectin. Even the use of technical multi-enzyme preparations was not sufficient to degrade soybea pectin structures (Huisman, MMH; Schols, HA; Voragen, AGJ, 1999, Enzymatic degradation of cell wall polysaccharides from soybean meal, Carbohydrate Polymers 38, 299-307, and: Ouhida, I; Perez, JF; Gasa, J; 2002, Soybean (glycine max) cell wall composition and availability to feed enzymes, J. Agric. Food Chem 50, 1933-1938).
An assortment of different pectin degrading enzymes is known to be present in various microorganisms such as Aspergillus niger.
The following is a non-exhaustive list of pectin-acting enzymes:
E.C.
Enzyme Number exopolygalacturonase 3.2.1.67 endopolygalacturonase 3.2.1.15 pectin lyase 4.2.2.10 pectate lyase 4.2.2.2 rhamnogalacturonan hydrolase 3.2.1.x rhamnogalacturonan lyase 4.2.2.x rhamnogalacturonan acetylesterase 3.1.1.x
RG rhamnohydrolase 3.2.1.x
RG galacturonohydrolase 3.2.1.x xylogalacturonan hydrolase 3.2.1.x pectin acetylesterase 3.1.1.x pectin methylesterase 3.1.1.11 endo-arabinase 3.2.1.99 beta-arabinofuranosidase 3.2.1.55 beta-1 ,4-galactanase 3.2.1.89 beta-1 ,3-galactanase 3.2.1.90 beta-galactosidase 3.2.1.23 alpha-galactosidase 3.2.1.22 feruloyl acetyl esterase 3.1.1.x alpha-fucosidase 3.2.1.51
(beta-fucosidase) 3.2.1.38 beta-apiosidase 3.2.1.x alpha-rhamnosidase 3.2.1.40 beta-rhamnosidase 3.2.1.43 alpha-arabinopyranosidase 3.2.1.x beta-glucuronidase 3.2.1.31
(alpha-glucuronidase) 3.2.1.139 beta-xylosidase 3.2.1.37
(alpha-xylosidase) 3.2.1.x
Pectin methylesterase catalyses the removal of methanol from pectin, resulting in the formation of pectic acid (polygalacturonic acid). Pectate lyase cleaves glycosidic bonds in polygalacturonic acid by beta-elimination, pectin lyase cleaves the glycosidic bonds of highly methylated pectins by beta- elimination, and polygalacturonase hydrolyses the glycosidic linkages in the polygalacturonic acid chain.
Commercially available pectinases such as Rapidase Press ® are actually a mixture of enzymes, which, along with other enzymes such as cellulases, are used in the fruit industry to help extract, clarify and modify fruit juices.
It is a disadvantage of these mixtures that they hardly ever contain the optimal mix of enzymes to treat a particular pectin containing composition, such as feed, fruit or juice. Optimal pectin degradation in fruit depends on many factors, including the kind of fruit to be treated, the season in which the fruit is harvested, the ripeness of the fruit, and many more. A well- known example is the release of unwanted methanol and methylated pectin by the action of pectinmethylesterases and/or exo-polygalacturonases when present in too high concentrations. It is therefore an object of the present invention to provide isolated (recombinant) pectinases that are better able to degrade pectins, for instance in food and feed applications.
Pectinases have already been cloned in a variety of different microorganisms. Molecular cloning of pectinases in fungi has also been described. The DNA and deduced amino acid sequences of several pectinases from Aspergillus oryzae, Aspergillus kawachii and Emericella nidulans are known. However, there is still a need for other pectinases with different properties so that feed, fruit and vegetable processing may be optimized.
One of the disadvantages of the currently available enzymes is that they have pH optima above pH 4.0. Many fruit juices are more acidic and a pH optimum below 3.8 would be desirable. Fruit juices may become as acidic as pH 2.0 and it is therefore an object of the present invention to provide pectinases with a more acidic pH optimum. Pectinases with a pH optimum below 3.8 would be especially advantageous. Fruit processing is also often performed at more extreme temperatures. Use of pectinases with lower temperature optima would improve yield and quality of the juice because more aromas would be released. The currently available enzymes have a very low efficiency at temperatures below 15 °C. Also, at high temperatures the currently available enzymes are easily inactivated. This is a disadvantage for those fruit juices that have to be pasteurized.
Enzymes according to the invention also provide a better temperature stability and may withstand more extreme temperatures in comparison with pectinases according to the prior art. Also, the specificity of conventional enzymes leaves to be desired. Fruit juice that needs to be filtrated after or during processing tends to clot the filters after some time. This is due to the inability of prior art enzymes to properly digest fruit polysaccharides which causes the fouling of the filters.
The present invention addresses at least one if not all of the above problems.
Object of the invention
It is an object of the invention to provide novel polynucleotides encoding novel pectinases with improved properties. A further object is to provide naturally and recombinantly produced pectinases as well as recombinant strains producing these. Also fusion polypeptides are part of the invention as well as methods of making and using the polynucleotides and polypeptides according to the invention. Summary of the invention
The invention provides for novel polynucleotides encoding novel pectinases. These pectinases were found to digest pectinases from various sources in a better way than conventional pectinases. Such better performance was especially evident when the pectinases according to the invention were used to prevent filter fouling in fruit juice applications. Also, pectinases according to the invention may advantageously be employed for improving the nutritional value of soybean extracts. Moreover, the availability of improved, isolated enzymes allows a cocktail of enzymes to be be prepared which is optimized for the digestion of pectin from a particular feed, fruit or juice.
More in particular, the invention provides for polynucleotides having a nucleotide sequence that hybridises preferably under highly stringent conditions to a sequence selected from the group consisting of SEQ ID NO: 33 - 64 or from the group consisting of SEQ ID NO: 1 - 32.
In one embodiment, the invention provides for an isolated polynucleotide comprising a nucleic acid sequence encoding a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO: 65 - 96 or functional equivalents thereof.
The sequences revealed herein are shown in the accompanying sequence listing. For clarity reasons, the group consisting of sequences according to SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9; SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19; SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29; SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO:37, SEQ ID NO: 38, SEQ ID NO: 39; SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49; SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59; SEQ ID NO: 60, SEQ ID NO: 61 , SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69; SEQ ID NO: 70, SEQ ID NO: 71 , SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79; SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89; SEQ ID NO: 90, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95 and SEQ ID NO: 96 is hereinafter collectively termed the group consisting of SEQ ID NO: 1 - 96
The group consisting of sequences according to SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9; SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19; SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29; SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32 is hereinafter collectively termed the group consisting of SEQ ID NO: 1 - 32
The group consisting of sequences according to SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO:37, SEQ ID NO: 38, SEQ ID NO: 39; SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49; SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59; SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO: 64 is hereinafter collectively termed the group consisting of SEQ ID NO: 33 - 64
The group consisting of sequences according to SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69; SEQ ID NO: 70, SEQ ID NO: 71 , SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79; SEQ ID NO: 80, SEQ ID NO: 81 , SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89; SEQ ID NO: 90, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95 and SEQ ID NO: 96 is hereinafter collectively termed the group consisting of SEQ ID NO: 65 - 96
The group consisting of sequences according to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9; SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19; SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29; SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32 is hereinafter collectively termed the group consisting of SEQ ID NO: 6 - 32
The group consisting of sequences according to SEQ ID NO: 38, SEQ ID NO: 39; SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49; SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59; SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO: 64 is hereinafter collectively termed the group consisting of SEQ ID NO: 38 - 64.
The group consisting of sequences according to SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79; SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89; SEQ ID NO: 90, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95 and SEQ ID NO: 96 is hereinafter collectively termed the group consisting of SEQ ID NO: 70 - 96.
The group consisting of sequences according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9; SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19; SEQ ID NO: 20 and SEQ ID NO: 21 is hereinafter collectively termed the group consisting of SEQ ID NO: 1 - 21 The group consisting of sequences according to SEQ ID NO:
33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO:37, SEQ ID NO: 38, SEQ ID NO: 39; SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49; SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO: 53 is hereinafter collectively termed the group consisting of SEQ ID NO: 33 - 53.
The group consisting of sequences according to SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69; SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79;
SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84 and SEQ ID NO: 85 is hereinafter collectively termed the group consisting of SEQ ID NO: 65 - 85.
The invention provides nucleic acids that are more than 40% such as about 60%, preferably 65%, more preferably 70%, even more preferably 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the sequences selected from the group consisting of SEQ ID NO: 33 - 64 or from the group consisting of SEQ ID NO: 1 - 32.
In a more preferred embodiment the invention provides for such an isolated polynucleotide obtainable from a filamentous fungus, in particular A. niger is preferred.
In a further preferred embodiment, the invention provides an isolated polynucleotide encoding at least one functional domain of a polypeptide selected from the group consisting of SEQ ID NO: 65 - 96 or functional equivalents thereof. In a preferred embodiment the invention provides a pectinase gene selected from the group consisting of SEQ ID NO: 33 - 64 . In another aspect the invention provides a polynucleotide, preferably a cDNA encoding an A. niger pectinase selected from the group consisting of SEQ ID NO: 65 - 96 or variants or fragments of that polypeptide. In a preferred embodiment the cDNA has a sequence selected from the group consisting of SEQ ID NO: 1 - 32 or functional equivalents thereof.
In an even further preferred embodiment, the invention provides for a polynucleotide comprising the coding sequence of the polynucleotides according to the invention, preferred is a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1 - 32.
The invention also relates to vectors comprising a polynucleotide sequence according to the invention and primers, probes and fragments that may be used to amplify or detect the DNA according to the invention. In a further preferred embodiment, a vector is provided wherein the polynucleotide sequence according to the invention is functionally linked with regulatory sequences suitable for expression of the encoded amino acid sequence in a suitable host cell, such as A. niger or A. oryzea. The invention also provides methods for preparing polynucleotides and vectors according to the invention.
The invention also relates to recombinantly produced host cells that contain heterologous or homologous polynucleotides according to the invention.
In another embodiment, the invention provides recombinant host cells wherein the expression of a pectinase according to the invention is significantly increased or wherein the activity of the pectinase is increased.
In another embodiment, the invention provides recombinant host cells wherein the expression of a pectinase according to the invention is significantly decreased or wherein the activity of the pectinase is decreased. Such a host is especially useful if the polypeptide expresses an unwanted activity in an otherwise useful mixture of enzyme activities.
In another embodiment the invention provides for a recombinantly produced host cell that contains heterologous or homologous DNA according to the invention and wherein the cell is capable of producing a functional pectinase according to the invention, preferably a cell capable of over- expressing the pectinase according to the invention, for example an Aspergillus strain comprising an increased copy number of a gene or cDNA according to the invention.
In yet another aspect of the invention, a purified polypeptide is provided. The polypeptides according to the invention include the polypeptides encoded by the polynucleotides according to the invention. Especially preferred is a polypeptide selected from the group consisting of SEQ ID NO: 65 - 96 or functional equivalents thereof.
Fusion proteins comprising a polypeptide according to the invention are also within the scope of the invention. The invention also provides methods of making the polypeptides according to the invention.
The invention also relates to the use of the pectinase according to the invention in any industrial process as described herein
Detailed description of the invention
Polynucleotides
The present invention provides polynucleotides encoding a number of novel pectinases, collectively called PEC 1 - PEC 32, having an amino acid sequence selected from the group consisting of SEQ ID NO: 65 - 96 or functional equivalents thereof. The term PEC1 - PEC 32 is hereinafter used to refer to any polypeptide with pectinase activity selected from the group consisiting of SEQ ID NO: 65 - 96. The sequences of the genes encoding PEC 1 - PEC 32 were determined by sequencing genomic clones obtained from Aspergillus niger. The invention provides polynucleotide sequences comprising the genes encoding the PEC 1 - PEC 32 pectinases as well as their complete cDNA sequences. Accordingly, the invention relates to an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 33 - 64 or from the group consisting of SEQ ID NO: 1 - 32 or functional equivalents thereof.
More in particular, the invention relates to an isolated polynucleotide hybridisable under stringent conditions, preferably under highly stringent conditions, to a polynucleotide selected from the group consisting of SEQ ID NO: 33 - 64 or from the group consisting of SEQ ID NO: 1 - 32.
Advantageously, such polynucleotides may be obtained from filamentous fungi, in particular from Aspergillus niger. More specifically, the invention relates to an isolated polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO: 33 - 64 or from the group consisting of SEQ ID NO: 1 - 32.
The invention also relates to an isolated polynucleotide encoding at least one functional domain of a polypeptide selected from the group consisting of SEQ ID NO: 65 - 96 or functional equivalents thereof.
As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules which may be isolated from chromosomal DNA, which include an open reading frame encoding a protein, e.g. an A. niger pectinase. A gene may include coding sequences, non-coding sequences, introns and regulatory sequences. Moreover, a gene refers to an isolated nucleic acid molecule as defined herein. A nucleic acid molecule of the present invention, such as a nucleic acid molecule selected from the group consisting of SEQ ID NO: 33 - 64 or from the group consisting of SEQ ID NO: 1 - 32 or a functional equivalent thereof, may be isolated using standard molecular biology techniques and the sequence information provided herein. For example, using all or portion of a nucleic acid selected from the group consisting of SEQ ID NO: 33 - 64 or from the group consisting of SEQ ID NO: 1 - 32 as a hybridization probe, nucleic acid molecules according to the invention may be isolated using standard hybridization and cloning techniques (e. g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
Moreover, a nucleic acid molecule encompassing all or a portion of a nucleotide sequence selected from the group consisting of SEQ ID NO: 33 - 64 or from the group consisting of SEQ ID NO: 1 - 32 may be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence information contained herein. A nucleic acid according to the invention may be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid thus amplified may be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to or hybridisable to nucleotide sequences according to the invention may be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
In a preferred embodiment, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence selected from the group consisting of SEQ ID NO: 1 - 32. The sequences of the cDNAs selected from the group consisting of SEQ ID NO: 1 - 32 correspond to the coding region of the A. niger PEC 1 - PEC 32 pectinases. Pectinases PEC 1 - PEC 32 correspond to polypeptides selected from the group consisting of SEQ ID NO: 65 - 96.
In another preferred embodiment, an isolated nucleic acid molecule according to the invention comprises a nucleic acid molecule which is the complement of a nucleotide sequence selected from the group consisting of SEQ ID NO: 33 - 64 or from the group consisting of SEQ ID NO: 1 - 32 or a functional equivalent of these nucleotide sequences.
A nucleic acid molecule which is complementary to another nucleotide sequence is one which is sufficiently complementary to the other nucleotide sequence to such an extent that it may hybridize to the other nucleotide sequence thereby forming a stable duplex.
One aspect of the invention pertains to isolated nucleic acid molecules that encode a polypeptide of the invention or a functional equivalent thereof such as a biologically active fragment or domain, as well as nucleic acid molecules sufficient for use as hybridisation probes to identify nucleic acid molecules encoding a polypeptide of the invention and fragments of such nucleic acid molecules suitable for use as PCR primers for the amplification or mutation of nucleic acid molecules. An "isolated polynucleotide" or "isolated nucleic acid" is a DNA or RNA that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived. Thus, in one embodiment, an isolated nucleic acid includes some or all of the 5' non-coding (e.g., promotor) sequences that are immediately contiguous to the coding sequence. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding an additional polypeptide that is substantially free of cellular material, viral material, or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized). Moreover, an "isolated nucleic acid fragment" is a nucleic acid fragment that is not naturally occurring as a fragment and would not be found in the natural state.
As used herein, the terms "polynucleotide" or "nucleic acid molecule" are intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA. The nucleic acid may be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides may be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases. Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to a PEC 1 - PEC 32 nucleic acid molecule, e.g., the coding strand of a PEC 1 - PEC 32 nucleic acid molecule. Also included within the scope of the invention are the complement strands of the nucleic acid molecules described herein.
Sequencing errors
The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The specific sequences disclosed herein may be readily used to isolate the complete gene from filamentous fungi, in particular A. niger which in turn may easily be subjected to further sequence analyses thereby identifying sequencing errors. Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence may be more precisely determined by other approaches including manual DNA sequencing methods well known in the art. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.
The person skilled in the art is capable of identifying such erroneously identified bases and knows how to correct for such errors.
Nucleic acid fragments, probes and primers
A nucleic acid molecule according to the invention may comprise only a portion or a fragment of the nucleic acid sequence selected from the group consisting of SEQ ID NO: 33 - 64 or from the group consisting of SEQ ID NO: 1 - 32, for example a fragment which may be used as a probe or primer or a fragment encoding a portion of a PEC 1 - PEC 32 protein. The nucleotide sequence determined from the cloning of the PEC 1 - PEC 32 gene and cDNA allows for the generation of probes and primers designed for use in identifying and/or cloning other PEC 1 - PEC 32 family members, as well as PEC 1 - PEC 32 homologues from other species. The probe/primer typically comprises substantially purified oligonucleotide which typically comprises a region of nucleotide sequence that hybridizes preferably under highly stringent conditions to at least about 12 or 15, preferably about 18 or 20, preferably about 22 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 or more consecutive nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NO: 33 - 64 or from the group consisting of SEQ ID NO: 1 - 32 or a functional equivalent thereof.
Probes based on the PEC 1 - PEC 32 nucleotide sequences may be used to detect transcripts or genomic PEC 1 - PEC 32 sequences encoding the same or homologous proteins for instance in other organisms. In preferred embodiments, the probe further comprises a label group attached thereto, e.g., the label group may be a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor. Such probes may also be used as part of a diagnostic test kit for identifying cells which express a PEC 1 - PEC 32 protein.
Identity & homology
The terms "homology" or "percent identity" are used interchangeably herein. For the purpose of this invention, it is defined here that in order to determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions/total number of positions (i.e. overlapping positions) x 100). Preferably, the two sequences are the same length.
The skilled person will be aware of the fact that several different computer programms are available to determine the homology between two sequences. For instance, a comparison of sequences and determination of percent identity between two sequences may be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.
In yet another embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1 , 2, 3, 4, 5, or 6. In another embodiment, the percent identity two amino acid or nucleotide sequence is determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989) which has been incorporated into the ALIGN program (version 2.0) (available at: http://vega.igh.cnrs.fr/bin/align-guess.cgi) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
The nucleic acid and protein sequences of the present invention may further be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences. Such searches may be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403—10. BLAST nucleotide searches may be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to PEC 1 - PEC 32 nucleic acid molecules of the invention. BLAST protein searches may be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to PEC 1 - PEC 32 protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST may be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) may be used. See http://www.ncbi.nlm.nih.gov.
Hybridisation
As used herein, the term "hybridizing" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least about 50%, at least about 60%, at least about 70%, more preferably at least about 80%, even more preferably at least about 85% to 90%, more preferably at least 95%) homologous to each other typically remain hybridized to each other. A preferred, non-limiting example of such hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45 °C, followed by one or more washes in 1 X SSC, 0.1 % SDS at 50 °C, preferably at 55 °C, preferably at 60 °C and even more preferably at 65 °C. Highly stringent conditions include, for example, hybridizing at 68 °C in 5x SSC/5x Denhardt's solution / 1.0% SDS and washing in 0.2x SSC/0.1% SDS at room temperature. Alternatively, washing may be performed at 42 °C. The skilled artisan will know which conditions to apply for stringent and highly stringent hybridisation conditions. Additional guidance regarding such conditions is readily available in the art, for example, in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; and Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology, (John Wiley & Sons, N.Y.).
Of course, a polynucleotide which hybridizes only to a poly A sequence (such as the 3' terminal poly(A) tract of mRNAs), or to a complementary stretch of T (or U) resides, would not be included in a polynucleotide of the invention used to specifically hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-standed cDNA clone).
Obtaining full length DNA from other organisms
In a typical approach, cDNA libraries constructed from other organisms, e.g. filamentous fungi, in particular from the species Aspergillus may be screened.
For example, Aspergillus strains may be screened for homologous PEC 1 - PEC 32 polynucleotides by Northern blot analysis. Upon detection of transcripts homologous to polynucleotides according to the invention, cDNA libraries may be constructed from RNA isolated from the appropriate strain, utilizing standard techniques well known to those of skill in the art. Alternatively, a total genomic DNA library may be screened using a probe hybridisable to a PEC 1 - PEC 32 polynucleotide according to the invention.
Homologous gene sequences may be isolated, for example, by performing PCR using two degenerate oligonucleotide primer pools designed on the basis of nucleotide sequences as taught herein.
The template for the reaction may be cDNA obtained by reverse transcription of mRNA prepared from strains known or suspected to express a polynucleotide according to the invention. The PCR product may be subcloned and sequenced to ensure that the amplified sequences represent the sequences of a new PEC 1 - PEC 32 nucleic acid sequence, or a functional equivalent thereof.
The PCR fragment may then be used to isolate a full length cDNA clone by a variety of known methods. For example, the amplified fragment may be labeled and used to screen a bacteriophage or cosmid cDNA library. Alternatively, the labeled fragment may be used to screen a genomic library.
PCR technology may also be used to isolate full length cDNA sequences from other organisms. For example, RNA may be isolated, following standard procedures, from an appropriate cellular or tissue source. A reverse transcription reaction may be performed on the RNA using an oligonucleotide primer specific for the most 5' end of the amplified fragment for the priming of first strand synthesis.
The resulting RNA/DNA hybrid may then be "tailed" (e.g., with guanines) using a standard terminal transferase reaction, the hybrid may be digested with RNase H, and second strand synthesis may then be primed (e.g., with a poly-C primer). Thus, cDNA sequences upstream of the amplified fragment may easily be isolated. For a review of useful cloning strategies, see e.g., Sambrook et al., supra; and Ausubel et al., supra.
Vectors
Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a PEC 1 - PEC 32 protein or a functional equivalent thereof. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non- episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. The terms "plasmid" and "vector" may be used interchangeably herein as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vector includes one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operatively linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signal). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymologyl 85, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in a certain host cell (e.g. tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention may be introduced into host cells to thereby produce proteins or peptides, encoded by nucleic acids as described herein (e.g. PEC 1 - PEC 32 proteins, mutant forms of PEC 1 - PEC 32 proteins, fragments, variants or functional equivalents thereof, fusion proteins, etc.).
The recombinant expression vectors of the invention may be designed for expression of PEC 1 - PEC 32 proteins in prokaryotic or eukaryotic cells. For example, PEC 1 - PEC 32 proteins may be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Alternatively, the recombinant expression vector may be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression vectors useful in the present invention include chromosomal-, episomal- and virus-derived vectors e.g., vectors derived from bacterial plasmids, bacteriophage, yeast episome, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
The DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled person. In a specific embodiment, promoters are preferred that are capable of directing a high expression level of pectinases in filamentous fungi. Such promoters are known in the art. The expression constructs may contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will include a translation initiating AUG at the beginning and a termination codon appropriately positioned at the end of the polypeptide to be translated. Vector DNA may be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-percipitation, DEAE- dextran-mediated transfection, transduction, infection, lipofection, cationic lipidmediated transfection or electroporation. Suitable methods for transforming or transfecting host cells may be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2nd, ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Davis et al., Basic Methods in Molecular Biology (1986) and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methatrexate. Nucleic acid encoding a selectable marker may be introduced into a host cell on the same vector as that encoding a PEC 1 - PEC 32 protein or may be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid may be identified by drug selection (e.g. cells that have incorporated the selectable marker gene will survive, while the other cells die).
Expression of proteins in prokaryotes is often carried out in £ coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, e.g. to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognation sequences, include Factor Xa, thrombin and enterokinase. As indicated, the expression vectors will preferably contain selectable markers. Such markers include dihydrofolate reductase or neomycin resistance for eukarotic cell culture and tetracyline or ampicilling resistance for culturing in E. coli and other bacteria. Representative examples of appropriate host include bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS and Bowes melanoma; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
Among vectors preferred for use in bacteria are pQE70, pQE60 and PQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16A, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Among preferred eukaryotic vectors are PWLNEO, pSV2CAT, pOG44, pZT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.
Among known bacterial promotors for use in the present invention include E. coli lad and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR, PL promoters and the trp promoter, the HSVthymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus ("RSV"), and metallothionein promoters, such as the mouse metallothionein-l promoter.
Transcription by higher eukaryotes of a DNA encoding a polypeptide according to the present invention may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretation signal may be incorporated into the expressed polypeptide. The signals may be endogenous to the polypeptide or they may be heterologous signals.
The polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals but also additional heterologous functional regions. Thus, for instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification.
Polypeptides according to the invention
The invention provides an isolated polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 65 - 96, an amino acid sequence obtainable by expressing a polynucleotide selected from the group consisting of SEQ ID NO: 33 - 64 in an appropriate host, as well as an amino acid sequence obtainable by expressing a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1 - 32 in an appropriate host. Also, a peptide or polypeptide comprising a functional equivalent of the above polypeptides is comprised within the present invention. The above polypeptides are collectively comprised in the term "polypeptides according to the invention" The terms "peptide" and "oligopeptide" are considered synonymous (as is commonly recognized) and each term may be used interchangeably as the context requires to indicate a chain of at least two amino acids coupled by peptidyl linkages. The word "polypeptide" is used herein for chains containing more than seven amino acid residues. All oligopeptide and polypeptide formulas or sequences herein are written from left to right and in the direction from amino terminus to carboxy terminus. The one-letter code of amino acids used herein is commonly known in the art and may be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2nd, ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989)
By "isolated" polypeptide or protein is intended a polypeptide or protein removed from its native environment. For example, recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the invention as are native or recombinant polypeptides which have been substantially purified by any suitable technique such as, for example, the single-step purification method disclosed in Smith and Johnson, Gene 67:31-40 (1988).
The PEC 1 - PEC 32 pectinase according to the invention may be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification. Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
Protein fragments
The invention also features biologically active fragments of the polypeptides according to the invention. Biologically active fragments of a polypeptide of the invention include polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of a PEC 1 - PEC 32 protein (e.g., an amino acid sequence selected from the group consisting of SEQ ID NO: 65 - 96), which include fewer amino acids than the full length protein, and exhibit at least one biological activity of the corresponding full-length protein. Typically, biologically active fragments comprise a domain or motif with at least one activity of the PEC 1 - PEC 32 protein. A biologically active fragment of a protein of the invention may be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length. Moreover, other biologically active portions, in which other regions of the protein are deleted, may be prepared by recombinant techniques and evaluated for one or more of the biological activities of the native form of a polypeptide of the invention.
The invention also features nucleic acid fragments which encode the above biologically active fragments of the PEC 1 - PEC 32 protein.
Fusion proteins
The proteins of the present invention or functional equivalents thereof, e.g., biologically active portions thereof, may be operatively linked to a non-PEC 1 - PEC 32 polypeptide (e.g., heterologous amino acid sequences) to form fusion proteins. As used herein, a PEC 1 - PEC 32 "chimeric protein" or "fusion protein" comprises a PEC 1 - PEC 32 polypeptide operatively linked to a non-PEC 1 - PEC 32 polypeptide. A "PEC 1 - PEC 32 polypeptide" refers to a polypeptide having an amino acid sequence corresponding to PEC 1 - PEC 32, whereas a "non-PEC 1 - PEC 32 polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the PEC 1 - PEC 32 protein, e.g., a protein which is different from the PEC 1 - PEC 32 protein and which is derived from the same or a different organism. Within a PEC 1 - PEC 32 fusion protein the PEC 1 - PEC 32 polypeptide may correspond to all or a portion of a PEC 1 - PEC 32 protein. In a preferred embodiment, a PEC 1 - PEC 32 fusion protein comprises at least one biologically active fragment of a PEC 1 - PEC 32 protein. In another preferred embodiment, a PEC 1 - PEC 32 fusion protein comprises at least two biologically active portions of a PEC 1 - PEC 32 protein. Within the fusion protein, the term "operatively linked" is intended to indicate that the PEC 1 - PEC 32 polypeptide and the non-PEC 1 - PEC 32 polypeptide are fused in-frame to each other. The non-PEC 1 - PEC 32 polypeptide may be fused to the N-terminus or C-terminus of the PEC 1 - PEC 32 polypeptide.
For example, in one embodiment, the fusion protein is a GST- PEC 1 - PEC 32 fusion protein in which the PEC 1 - PEC 32 sequences are fused to the C-terminus of the GST sequences. Such fusion proteins may facilitate the purification of recombinant PEC 1 - PEC 32. In another embodiment, the fusion protein is a PEC 1 - PEC 32 protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian and Yeast host cells), expression and/or secretion of PEC 1 - PEC 32 may be increased through use of a hetereologous signal sequence.
In another example, the gp67 secretory sequence of the baculovirus envelope protein may be used as a heterologous signal sequence (Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, 1992). Other examples of eukaryotic heterologous signal sequences include the secretory sequences of melittin and human placental alkaline phosphatase (Stratagene; La Jolla, California). In yet another example, useful prokarytic heterologous signal sequences include the phoA secretory signal (Sambrook et al., supra) and the protein A secretory signal (Pharmacia Biotech; Piscataway, New Jersey). A signal sequence may be used to facilitate secretion and isolation of a protein or polypeptide of the invention. Signal sequences are typically characterized by a core of hydrophobic amino acids which are generally cleaved from the mature protein during secretion in one or more cleavage events. Such signal peptides contain processing sites that allow cleavage of the signal sequence from the mature proteins as they pass through the secretory pathway. The signal sequence directs secretion of the protein, such as from a eukaryotic host into which the expression vector is transformed, and the signal sequence is subsequently or concurrently cleaved. The protein may then be readily purified from the extracellular medium by art recognized methods. Alternatively, the signal sequence may be linked to the protein of interest using a sequence which facilitates purification, such as with a GST domain. Thus, for instance, the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide, which facilitates purification of the fused polypeptide. In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As described in Gentz et al, Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purificaton of the fusion protein. The HA tag is another peptide useful for purification which corresponds to an epitope derived of influenza hemaglutinin protein, which has been described by Wilson et al., Cell 37:767 (1984), for instance.
Preferably, a PEC 1 - PEC 32 chimeric or fusion protein according to the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene may be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments may be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which may subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g, a GST polypeptide). A nucleic acid encoding a PEC 1 - PEC 32 polypeptide may be cloned into such an expression vector such that the fusion moiety is linked in-frame to the PEC 1 - PEC 32 polypeptide.
Functional equivalents
The terms "functional equivalents" and "functional variants" are used interchangeably herein. Functional equivalents of PEC 1 - PEC 32 DNA are isolated DNA fragments that encode a polypeptide that exhibits a particular function of a PEC 1 - PEC 32 A. niger pectinase as defined herein. A functional equivalent of a PEC 1 - PEC 32 polypeptide according to the invention is a polypeptide that exhibits at least one function of an A. niger pectinase as defined herein. Functional equivalents therefore also encompass biologically active fragments. Functional protein or polypeptide equivalents may contain only conservative substitutions of one or more amino acids of a sequence selected from the group consisting of SEQ ID NO: 65 - 96 or substitutions, insertions or deletions of non-essential amino acids. Accordingly, a non-essential amino acid is a residue that may be altered in a sequence selected from the group consisting of SEQ ID NO: 65 - 96 without substantially altering the biological function. For example, amino acid residues that are conserved within their own family among any of the PEC 1 - PEC 32 proteins of the present invention, are predicted to be particularly unamenable to alteration.
The term "conservative substitution" is intended to mean that a substitution in which the amino acid residue is replaced with an amino acid residue having a similar side chain. These families are known in the art and include amino acids with basic side chains (e.g. lysine, arginine and hystidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagines, glutamine, serine, threonine, tyrosine, cysteine), non- polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine).
Functional nucleic acid equivalents may typically contain silent mutations or mutations that do not alter the biological function of encoded polypeptide. Accordingly, the invention provides nucleic acid molecules encoding PEC 1 - PEC 32 proteins that contain changes in amino acid residues that are not essential for a particular biological activity. Such PEC 1 - PEC 32 proteins differ in amino acid sequence from a sequence selected from the group consisting of SEQ ID NO: 65 - 96 yet retain at least one biological activity. In one embodiment the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises a substantially homologous amino acid sequence of at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%), 98%, 99% or more homologous to an amino acid sequence selected from the group consisting of SEQ ID NO: 65 - 96.
For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie, J.U. et al., Science 247:1306- 1310 (1990) wherein the authors indicate that there are two main approaches for studying the tolerance of an amino acid sequence to change. The first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection. The second approach uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene and selects or screens to identify sequences that maintain functionality. As the authors state, these studies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which changes are likely to be permissive at a certain position of the protein. For example, most buried amino acid residues require non-polar side chains, whereas few features of surface side chains are generally conserved. Other such phenotypically silent substitutions are described in Bowie et al, supra, and the references cited therein.
An isolated nucleic acid molecule encoding a PEC 1 - PEC 32 protein homologous to the protein selected from the group consisting of SEQ ID NO: 65 - 96 may be created by introducing one or more nucleotide substitutions, additions or deletions into the coding nucleotide sequences selected from the group consisting SEQ ID NO: 65 - 963 - 64 or from the group consisting of SEQ ID NO: 1 - 32 such that one or more amino acid substitutions, deletions or insertions are introduced into the encoded protein. Such mutations may be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
The term "functional equivalents" also encompasses orthologues of the A. niger PEC 1 - PEC 32 protein. Orthologues of the A. niger PEC 1 - PEC 32 protein are proteins that may be isolated from other strains or species and possess a similar or identical biological activity. Such orthologues may readily be identified as comprising an amino acid sequence that is substantially homologous to a sequence selected from the group consisting of SEQ ID NO: 65 - 96.
As defined herein, the term "substantially homologous" refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., with similar side chain) amino acids or nucleotides to a second amino acid or nucleotide sequence such that the first and the second amino acid or nucleotide sequences have a common domain. For example, amino acid or nucleotide sequences which contain a common domain having about 60%, preferably 65%, more preferably 70%, even more preferably 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity or more are defined herein as sufficiently identical.
Also, nucleic acids encoding other PEC 1 - PEC 32 family members, which thus have a nucleotide sequence that differs from a sequence selected from the group consisting of SEQ ID NO: 33 - 64 or from the group consisting of SEQ ID NO: 1 - 32, are within the scope of the invention. Moreover, nucleic acids encoding PEC 1 - PEC 32 proteins from different species which thus may have a nucleotide sequence which differs from a sequence selected from the group consisting of SEQ ID NO: 33 - 64.or from the group consisting of SEQ ID NO: 1 - 32 are within the scope of the invention.
Nucleic acid molecules corresponding to variants (e.g. natural allelic variants) and homologues of the PEC 1 - PEC 32 DNA of the invention may be isolated based on their homology to the PEC 1 - PEC 32 nucleic acids disclosed herein using the cDNAs disclosed herein or a suitable fragment thereof, as a hybridisation probe according to standard hybridisation techniques preferably under highly stringent hybridisation conditions. In addition to naturally occurring allelic variants of the PEC 1 -
PEC 32 sequence, the skilled person will recognise that changes may be introduced by mutation into the nucleotide sequences selected from the group consisting of SEQ ID NO: 33 - 64.or from the group consisting of SEQ ID NO: 1 - 32 thereby leading to changes in the amino acid sequence of the PEC 1 - PEC 32 protein without substantially altering the function of the PEC 1 - PEC 32 protein.
In another aspect of the invention, improved PEC 1 - PEC 32 proteins are provided. Improved PEC 1 - PEC 32 proteins are proteins wherein at least one biological activity is improved. Such proteins may be obtained by randomly introducing mutations along all or part of the PEC 1 - PEC 32 coding sequence, such as by saturation mutagenesis, and the resulting mutants may be expressed recombinantly and screened for biological activity. For instance, the art provides for standard assays for measuring the enzymatic activity of pectinases and thus improved proteins may easily be selected.
In a preferred embodiment the PEC 1 - PEC 32 protein has an amino acid sequence selected from the group consisting of SEQ ID NO: 65 - 96. In another embodiment, the PEC 1 - PEC 32 polypeptide is substantially homologous to the amino acid sequence selected from the group consisting of SEQ ID NO: 65 - 96 and retains at least one biological activity of a polypeptide selected from the group consisting of SEQ ID NO: 65 - 96, yet differs in amino acid sequence due to natural variation or mutagenesis as described above.
In a further preferred embodiment, the PEC 1 - PEC 32 protein has an amino acid sequence encoded by an isolated nucleic acid fragment capable of hybridising to a nucleic acid selected from the group consisting of SEQ ID NO: 33 - 64 or from the group consisting of SEQ ID NO: 1 - 32, preferably under highly stringent hybridisation conditions.
Accordingly, the PEC 1 - PEC 32 protein is a protein which comprises an amino acid sequence at least about 60%), 65%, 70%, 75%, 80%, 85%o, 90%), 95%), 96%., 97%>, 98%, 99% or more homologous to the amino acid sequence selected from the group consisting of SEQ ID NO: 65 - 96 and retains at least one functional activity of a polypeptide selected from the group consisting of SEQ ID NO: 65 - 96.
Functional equivalents of a protein according to the invention may also be identified e.g. by screening combinatorial libraries of mutants, e.g. truncation mutants, of the protein of the invention for pectinase activity. In one embodiment, a variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level. A variegated library of variants may be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential protein sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display). There are a variety of methods that may be used to produce libraries of potential variants of the polypeptides of the invention from a degenerate oligonucleotide sequence. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477).
In addition, libraries of fragments of the coding sequence of a polypeptide of the invention may be used to generate a variegated population of polypeptides for screening a subsequent selection of variants. For example, a library of coding sequence fragments may be generated by treating a double stranded PCR fragment of the coding sequence of interest with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which may include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library may be derived which encodes N-terminal and internal fragments of various sizes of the protein of interest.
Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations of truncation, and for screening cDNA libraries for gene products having a selected property. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a technique which enhances the frequency of functional mutants in the libraries, may be used in combination with the screening assays to identify variants of a protein of the invention (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331). In addition to the PEC 1 - PEC 32 gene sequence selected from the group consisting of SEQ ID NO: 33 - 64, it will be apparent for the person skilled in the art that DNA sequence polymorphisms that may lead to changes in the amino acid sequence of the PEC 1 - PEC 32 protein may exist within a given population. Such genetic polymorphisms may exist in cells from different populations or within a population due to natural allelic variation. Allelic variants may also include functional equivalents.
Fragments of a polynucleotide according to the invention may also comprise polynucleotides not encoding functional polypeptides. Such polynucleotides may function as probes or primers for a PCR reaction. Nucleic acids according to the invention irrespective of whether they encode functional or non-functional polypeptides, may be used as hybridization probes or polymerase chain reaction (PCR) primers. Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having a PEC 1 - PEC 32 activity include, inter alia, (1) isolating the gene encoding the PEC 1 - PEC 32 protein, or allelic variants thereof from a cDNA library e.g. from other organisms than A. niger; (2) in situ hybridization (e.g. FISH) to metaphase chromosomal spreads to provide precise chromosomal location of the PEC 1 - PEC 32 gene as described in Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988); (3) Northern blot analysis for detecting expression of PEC 1 - PEC 32 mRNA in specific tissues and/or cells and 4) probes and primers that may be used as a diagnostic tool to analyse the presence of a nucleic acid hybridisable to the PEC 1 - PEC 32 probe in a given biological (e.g. tissue) sample.
Also encompassed by the invention is a method of obtaining a functional equivalent of a PEC 1 - PEC 32 gene or cDNA. Such a method entails obtaining a labelled probe that includes an isolated nucleic acid which encodes all or a portion of the sequence selected from the group consisting of SEQ ID NO: 65 - 96 or a variant thereof; screening a nucleic acid fragment library with the labelled probe under conditions that allow hybridisation of the probe to nucleic acid fragments in the library, thereby forming nucleic acid duplexes, and preparing a full-length gene sequence from the nucleic acid fragments in any labelled duplex to obtain a gene related to the PEC 1 - PEC 32 gene.
In one embodiment, a PEC 1 - PEC 32 nucleic acid of the invention is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%), 95%>, 96%o, 97%», 98%., 99%, or more homologous to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 33 - 64 , from the group consisting of SEQ ID NO: 1 - 32 or the complement thereof.
In another preferred embodiment a PEC 1 - PEC 32 polypeptide according to the invention is at least 60%, 65%, 70%, 75%, 80%, 85%>, 90%>, 91%, 92%o, 93%, 94%, 95%, 96%, 97%, 98%, 99%>, or more homologous to an amino acid sequence selected from the group consisting of SEQ ID NO: 65 - 96.
Host cells
In another embodiment, the invention features cells, e.g., transformed host cells or recombinant host cells that contain a nucleic acid encompassed by the invention. A "transformed cell" or "recombinant cell" is a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a nucleic acid according to the invention. Both prokaryotic and eukaryotic cells are included, e.g., bacteria, fungi, yeast, and the like, especially preferred are cells from filamentous fungi, in particular Aspergillus niger.
A host cell may be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in a specific, desired fashion. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may facilitate optimal functioning of the protein.
Various host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems familiar to those of skill in the art of molecular biology and/or microbiology may be chosen to ensure the desired and correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such host cells are well known in the art.
Host cells also include, but are not limited to, mammalian cell lines such as CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and choroid plexus cell lines. If desired, the polypeptides according to the invention may be produced by a stably-transfected cell line. A number of vectors suitable for stable transfection of mammalian cells are available to the public, methods for constructing such cell lines are also publicly known, e.g., in Ausubel et al. (supra).
Antibodies
The invention further features antibodies, such as monoclonal or polyclonal antibodies, that specifically bind PEC 1 - PEC 32 proteins according to the invention. As used herein, the term "antibody" (Ab) or "monoclonal antibody" (Mab) is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab')2 fragments) which are capable of specifically binding to PEC 1 - PEC 32 protein. Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Thus, these fragments are preferred.
The antibodies of the present invention may be prepared by any of a variety of methods. For example, cells expressing the PEC 1 - PEC 32 protein or an antigenic fragment thereof may be administered to an animal in order to induce the production of sera containing polyclonal antibodies. In a preferred method, a preparation of PEC 1 - PEC 32 protein is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity. In the most preferred method, the antibodies of the present invention are monoclonal antibodies (or PEC 1 - PEC 32 protein binding fragments thereof). Such monoclonal antibodies may be prepared using hybridoma technology (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Hammerling et al., In: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)). In general, such procedures involve immunizing an animal (preferably a mouse) with a PEC 1 - PEC 32 protein antigen or, with a PEC 1 - PEC 32 protein expressing cell. The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present inventoin; however, it is preferably to employ the parent myeloma cell line (SP2O), available from the American Type Culture Collection, Rockville, Maryland. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastro-enterology 80:225-232 (1981)). The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the PEC 1 - PEC 32 protein antigen. In general, the polypeptides may be coupled to a carrier protein, such as KLH, as described in Ausubel et al., supra, mixed with an adjuvant, and injected into a host mammal.
In particular, various host animals may be immunized by injection of a polypeptide of interest. Examples of suitable host animals include rabbits, mice, guinea pigs, and rats. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), adjuvant mineral gels such as aluminum hydroxide, surface actve substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of the immunized animals.
Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD, and any subclass thereof. The hybridomas producing the mAbs of this invention may be cultivated in vitro or in vivo. Once produced, polyclonal or monoclonal antibodies are tested for specific recognition of a PEC 1 - PEC 32 polypeptide or functional equivalent thereof in an immunoassay, such as a Western blot or immunoprecipitation analysis using standard techniques, e.g., as described in Ausubel et al., supra. Antibodies that specifically bind to PEC 1 - PEC 32 proteins or functional equivalents thereof are useful in the invention. For example, such antibodies may be used in an immunoassay to detect PEC 1 - PEC 32 in pathogenic or non- pathogenic strains of Aspergillus (e.g., in Aspergillus extracts).
Preferably, antibodies of the invention are produced using fragments of the PEC 1 - PEC 32 polypeptides that appear likely to be antigenic, by criteria such as high frequency of charged residues. For example, such fragments may be generated by standard techniques of PCR, and then cloned into the pGEX expression vector (Ausubel et al., supra). Fusion proteins may then be expressed in E. coli and purified using a glutathione agarose affinity matrix as described in Ausubel, et al., supra. If desired, several (e.g., two or three) fusions may be generated for each protein, and each fusion may be injected into at least two rabbits. Antisera may be raised by injections in a series, typically including at least three booster injections. Typically, the antisera are checked for their ability to immunoprecipitate a recombinant PEC 1 - PEC 32 polypeptide or functional equivalents thereof whereas unrelated proteins may serve as a control for the specificity of the immune reaction. Alternatively, techniques decribed for the production of single chain antibodies (U.S. Patent 4,946,778 and 4,704,692) may be adapted to produce single chain antibodies against a PEC 1 - PEC 32 polypeptide or functional equivalents thereof. Kits for generating and screening phage display libraries are commercially available e.g. from Pharmacia. Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library may be found in, for example, U.S. Patent No. 5,223, 409; PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO 20791; PCT Publication No. WO 92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs et al. (1991) Bio Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246; 1275-1281 ; Griffiths et al. (1993) EMBO J. 12:725-734. Polyclonal and monoclonal antibodies that specifically bind PEC
1 - PEC 32 polypeptides of functional equivalents thereof may be used, for example, to detect expression of a PEC 1 - PEC 32 gene or a functional equivalent thereof e.g. in another strain of Aspergillus. For example, PEC 1 - PEC 32 polypeptide may be readily detected in conventional immunoassays of Aspergillus cells or extracts. Examples of suitable assays include, without limitation, Western blotting, ELISAs, radioimmune assays, and the like.
By "specifically binds" is meant that an antibody recognizes and binds a particular antigen, e.g., a PEC 1 - PEC 32 polypeptide, but does not substantially recognize and bind other unrelated molecules in a sample. Antibodies may be purified, for example, by affinity chromatography methods in which the polypeptide antigen is immobilized on a resin.
An antibody directed against a polypeptide of the invention (e.g., monoclonal antibody) may be used to isolate the polypeptide by standard techniques, such as affinity chromatography or immunoprecipitation. Moreover, such an antibody may be used to detect the protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the polypeptide. The antibodies may also be used diagnostically to monitor protein levels in cells or tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen or in the diagnosis of Aspergillosis..
Detection may be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive materials include 125l, 131l, 35S or 3H.
Preferred epitopes encompassed by the antigenic peptide are regions that are located on the surface of the protein, e.g., hydrophilic regions. Hydrophobicity plots of the proteins of the invention may be used to identify hydrophilic regions.
The antigenic peptide of a protein of the invention comprises at least 7 (preferably 10, 15, 20, or 30) contiguous amino acid residues of the amino acid sequense of a sequence selected from the group consisting of SEQ ID NO: 65 - 96 and encompasses an epitope of the protein such that an antibody raised against the peptide forms a specific immune complex with the protein.
Preferred epitopes encompassed by the antigenic peptide are regions of PEC 1 - PEC 32 that are located on the surface of the protein, e.g., hydrophilic regions, hydrophobic regions, alpha regions, beta regions, coil regions, turn regions and flexible regions.
Immunoassavs Qualitative or quantitative determination of a polypeptide according to the present invention in a biological sample may occur using any art- known method. Antibody-based techniques provide special advantages for assaying specific polypeptide levels in a biological sample. In these, the specific recognition is provided by the primary antibody (polyclonal or monoclonal) but the secondary detection system may utilize fluorescent, enzyme, or other conjugated secondary antibodies. As a result, an immunocomplex is obtained.
Accordingly, the invention provides a method for diagnosing whether a certain organism is infected with Aspergillus comprising the steps of:
• Isolating a biological sample from said organism suspected to be infected with Aspergillus,
• reacting said biological sample with an antibody according to the invention,
• determining whether immunecomplexes are formed. Tissues may also be extracted, e.g., with urea and neutral detergent, for the liberation of protein for Western-blot or dot/slot assay. This technique may also be applied to body fluids.
Other antibody-based methods useful for detecting PEC 1 - PEC 32 gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). For example, PEC 1 - PEC 32-specific monoclonal antibodies may be used both as an immunoabsorbent and as an enzyme-labeled probe to detect and quantify the PEC 1 - PEC 32 protein. The amount of PEC 1 - PEC 32 protein present in the sample may be calculated by reference to the amount present in a standard preparation using a linear regression computer algorithm. In another ELISA assay, two distinct specific monoclonal antibodies may be used to detect PEC 1 - PEC 32 protein in a biological fluid. In this assay, one of the antibodies is used as the immuno-absorbent and the other as the enzyme-labeled probe.
The above techniques may be conducted essentially as a "one- step" or "two-step" assay. The "one-step" assay involves contacting PEC 1 - PEC 32 protein with immobilized antibody and, without washing, contacting the mixture with the labeled antibody. The "two-step" assay involves washing before contacting the mixture with the labeled antibody. Other conventional methods may also be employed as suitable. It is usually desirable to immobilize one component of the assay system on a support, thereby allowing other components of the system to be brought into contact with the component and readily removed from the sample.
Suitable enzyme labels include, for example, those from the oxidase group, which catalyze the production of hydrogen peroxide by reacting with substrate. Activity of an oxidase label may be assayed by measuring the concentration of hydrogen peroxide formed by the enzyme-labelled antibody/substrate reaction.
Besides enzymes, other suitable labels include radioisotopes, such as iodine (125l, 12ll), carbon (1 C), sulphur (35S), tritium (3H), indium (112ln), and technetium (99mTc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.
Specific binding of a test compound to a PEC 1 - PEC 32 polypeptide may be detected, for example, in vitro by reversibly or irreversibly immobilizing the PEC 1 - PEC 32 polypeptide on a substrate, e.g., the surface of a well of a 96-well polystyrene microtitre plate. Methods for immobilizing polypeptides and other small molecules are well known in the art. For example, the microtitre plates may be coated with a PEC 1 - PEC 32 polypeptide by adding the polypeptide in a solution (typically, at a concentration of 0.05 to 1 mg/ml in a volume of 1-100 ul) to each well, and incubating the plates at room temperature to 37 °C for 0.1 to 36 hours. Polypeptides that are not bound to the plate may be removed by shaking the excess solution from the plate, and then washing the plate (once or repeatedly) with water or a buffer. Typically, the polypeptide is contained in water or a buffer. The plate is then washed with a buffer that lacks the bound polypeptide. To block the free protein-binding sites on the plates, the plates are blocked with a protein that is unrelated to the bound polypeptide. For example, 300 ul of bovine serum albumin (BSA) at a concentration of 2 mg/ml in Tris-HCl is suitable. Suitable substrates include those substrates that contain a defined cross-linking chemistry (e.g., plastic substrates, such as polystyrene, styrene, or polypropylene substrates from Corning Costar Corp. (Cambridge, MA), for example) . If desired, a beaded particle, e.g., beaded agarose or beaded sepharose, may be used as the substrate.
Binding of the test compound to the polypeptides according to the invention may be detected by any of a variety of artknown methods. For example, a specific antibody may be used in an immunoassay. If desired, the antibody may be labeled (e.g., fluorescently or with a radioisotope) and detected directly (see, e.g., West and McMahon, J. Cell Biol. 74:264, 1977). Alternatively, a second antibody may be used for detection (e.g., a labeled antibody that binds the Fc portion of an anti-AN97 antibody). In an alternative detection method, the PEC 1 - PEC 32 polypeptide is labeled, and the label is detected (e.g., by labeling aPEC 1 - PEC 32 polypeptide with a radioisotope, fluorophore, chromophore, or the like). In still another method, the PEC 1 - PEC 32 polypeptide is produced as a fusion protein with a protein that may be detected optically, e.g., green fluorescent protein (which may be detected under UV light). In an alternative method, the PEC 1 - PEC 32 polypeptide may be covalently attached to or fused with an enzyme having a detectable enzymatic activity, such as horse radish peroxidase, alkaline phosphatase, a-galactosidase, or glucose oxidase. Genes encoding all of these enzymes have been cloned and are readily available for use by those of skill in the art. If desired, the fusion protein may include an antigen, and such an antigen may be detected and measured with a polyclonal or monoclonal antibody using conventional methods. Suitable antigens include enzymes (e.g., horse radish peroxidase, alkaline phosphatase, and a-galactosidase) and non-enzymatic polypeptides (e.g., serum proteins, such as BSA and globulins, and milk proteins, such as caseins).
Epitopes, antigens and immunogens.
In another aspect, the invention provides a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide of the invention. The epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide of the invention. An "immunogenic epitope" is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen. These immunogenic epitopes are believed to be confined to a few loci on the molecule. On the other hand, a region of a protein molecule to which an antibody may bind is defined as an "antigenic epitope." The number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes. See, for instance, Geysen, H. M. et al., Proc. Natl. Acad. Sci. USA 81 :3998-4002 (1984).
As to the selection of peptides or polypeptides bearing an antigenic epitope (i.e., that contain a region of a protein molecule to which an antibody may bind), it is well known in that art that relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, for instance,
Sutcliffe, J. G. et al., Science 219:660-666 (1984). Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, may be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals. Peptides that are extremely hydrophobic and those of six or fewer residues generally are ineffective at inducing antibodies that bind to the mimicked protein; longer, soluble peptides, especially those containing proline residues, usually are effective. Sutcliffe et al., supra, at 661. For instance, 18 of 20 peptides designed according to these guidelines, containing 8- 39 residues covering 75% of the sequence of the influenza virus hemagglutinin HAI polypeptide chain, induced antibodies that reacted with the HA1 protein or intact virus; and 12/12 peptides from the MuLV polymerase and 18/18 from the rabies glycoprotein induced antibodies that precipitated the respective proteins.
Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention. Thus, a high proportion of hybridomas obtained by fusion of spleen cells from donors immunized with an antigen epitope-bearing peptide generally secrete antibody reactive with the native protein. Sutcliffe et al., supra, at 663. The antibodies raised by antigenic epitope bearing peptides or polypeptides are useful to detect the mimicked protein, and antibodies to different peptides may be used for tracking the fate of various regions of a protein precursor which undergoes posttranslation processing. The peptides and anti-peptide antibodies may be used in a variety of qualitative or quantitative assays for the mimicked protein, for instance in competition assays since it has been shown that even short peptides (e.g., about 9 amino acids) may bind and displace the larger peptides in immunoprecipitation assays. See, for instance, Wilson, I A et al., Cell 37:767-778 at 777 (1984). The anti-peptide antibodies of the invention also are useful for purification of the mimicked protein, for instance, by adsorption chromatography using methods well known in the art. Antigenic epitope-bearing peptides and polypeptides of the invention designed according to the above guidelines preferably contain a sequence of at least seven, more preferably at least nine and most preferably between about 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention. However, peptides or polypeptides comprising a larger portion of an amino acid sequence of a polypeptide of the invention, containing about 30 to about 50 amino acids, or any length up to and including the entire amino acid sequence of a polypeptide of the invention, also are considered epitope-bearing peptides or polypeptides of the invention and also are useful for inducing antibodies that react with the mimicked protein. Preferably, the amino acid sequence of the epitope-bearing peptide is selected to provide substantial solubility in aqueous solvents (i.e., the sequence includes relatively hydrophilic residues and highly hydrophobic sequences are preferably avoided); and sequences containing proline residues are particularly preferred.
The epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means for making peptides or polypeptides including recombinant means using nucleic acid molecules of the invention. For instance, a short epitope-bearing amino acid sequence may be fused to a larger polypeptide which acts as a carrier during recombinant production and purification, as well as during immunization to produce anti-peptide antibodies.
Epitope-bearing peptides also may be synthesized using known methods of chemical synthesis. For instance, Houghten has described a simple method for synthesis of large numbers of peptides, such as 10-20 mg of 248 different 13 residue peptides representing single amino acid variants of a segment of the HAI polypeptide which were prepared and characterized (by ELISA-type binding studies) in less than four weeks. Houghten, R. A., Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985). This "Simultaneous Multiple Peptide Synthesis
(SMPS)" process is further described in U.S. Patent No. 4,631 ,211 to Houghten et al. (1986). In this procedure the individual resins for the solid-phase synthesis of various peptides are contained in separate solvent-permeable packets, enabling the optimal use of the many identical repetitive steps involved in solid-phase methods.
A completely manual procedure allows 500-1000 or more syntheses to be conducted simultaneously. Houghten et al., supra, at 5134.
Epitope-bearing peptides and polypeptides of the invention are used to induce antibodies according to methods well known in the art. See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow, M. et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle, F.J. et al., J. Gen. Virol. 66:2347-2354 (1985).
Generally, animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling of the peptide to a macromolecular carrier, such as keyhole limpet hemocyanin (KLH) or tetanus toxoid. For instance, peptides containing cysteine may be coupled to carrier using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carrier using a more general linking agent such as glutaraldehyde.
Animals such as rabbits, rats and mice are immunized with either free or carriercoupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 ug peptide or carrier protein and Freund's adjuvant. Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which may be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface. The titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
Immunogenic epitope-bearing peptides of the invention, i.e., those parts of a protein that elicit an antibody response when the whole protein is the immunogen, are identified according to methods known in the art. For instance, Geysen et al., 1984, supra, discloses a procedure for rapid concurrent synthesis on solid supports of hundreds of peptides of sufficient purity to react in an enzyme-linked immunosorbent assay. Interaction of synthesized peptides with antibodies is then easily detected without removing them from the support. In this manner a peptide bearing an immunogenic epitope of a desired protein may be identified routinely by one of ordinary skill in the art. For instance, the immunologically important epitope in the coat protein of foot-and-mouth disease virus was located by Geysen et al. with a resolution of seven amino acids by synthesis of an overlapping set of all 208 possible hexapeptides covering the entire 213 amino acid sequence of the protein. Then, a complete replacement set of peptides in which all 20 amino acids were substituted in turn at every position within the epitope were synthesized, and the particular amino acids conferring specificity for the reaction with antibody were determined. Thus, peptide analogs of the epitope-bearing peptides of the invention may be made routinely by this method. U.S. Patent No. 4,708,781 to Geysen (1987) further describes this method of identifying a peptide bearing an immunogenic epitope of a desired protein.
Further still, U.S. Patent No. 5,194,392 to Geysen (1990) describes a general method of detecting or determining the sequence of monomers (amino acids or other compounds) which is a topological equivalent of the epitope (i.e., a "mimotope") which is complementary to a particular paratope (antigen binding site) of an antibody of interest. More generally, U.S. Patent No. 4,433,092 to
Geysen (1989) describes a method of detecting or determining a sequence of monomers which is a topographical equivalent of a ligand which is complementary to the ligand binding site of a particular receptor of interest. Similarly, U.S. Patent No. 5,480,971 to Houghten, R. A. et al. (1996) on Peralkylated Oligopeptide Mixtures discloses linear C1-C7-alkyl peralkylated oligopeptides and sets and libraries of such peptides, as well as methods for using such oligopeptide sets and libraries for determining the sequence of a peralkylated oligopeptide that preferentially binds to an acceptor molecule of interest. Thus, non-peptide analogs of the epitope-bearing peptides of the invention also may be made routinely by these methods.
Use of PEC 1 - PEC 32 pectinases in industrial processes
Pectins and pectinases play an important role in the food industry. Pectinases are used in the production of fruit and vegetable juices and purees, and in the extraction of useful components, like arome compounds, or even pharmaceuticals, from plant materials.
One of the main applications of pectinases is in the production of clear fruit and vegetable juice. The production of juices from fruit and vegetables typically involves, after an optional washing step, grinding, crushing or otherwise destroying the integrity of the fruit or vegetables thus obtaining a fruit or vegetable pulp. Subsequently, the pulp may be treated with enzymes to decrease soluble pectin, a process generally referred to as maceration. After maceration, the pulp is ready for pressing, leading to a juice fraction and a residue fraction, the latter being referred to as the pomace. The juice obtained after pressing is usually pasteurised, optionally with recovery of the aroma, which may then be added back at the end of the process. In order to obtain a clear concentrate, the pasteurised juice is enzymatically depectinized with the aid of pectinases, optionally pre- concentrated, filtrated, optionally ultra-filtrated and concentrated to obtain a clear concentrate or juice which is ready for shipping and/or blending to obtain a clear apple juice for the consumer market PEC 1 - PEC 32 pectinases are useful in all production processes in which the integrity of the cell wall should be destroyed in order to release materials from the inside of the cells, or from the cell walls. Pectinases may also be used to destroy the connection between cells without affecting the integrity of the separate cells. When cells should remain intact, e.g. in the preparation of potato puree, PEC 1 - PEC 32 pectinases may advantageously be used to release the structural connection between the cells. PEC 1 - PEC 32 pectinases are also able to decrease the water- binding and hence the viscosity of pectin-containing slurries, e.g. fruit or vegetable pulps, which improves the pressability/processability of the pulp.
PEC 1 - PEC 32 pectinases are able to clarify cloudy juices. Attacking the outer pectin layer of the cloud particles changes the charge of the particles, thus promoting the aggregation of the particles.
PEC 1 - PEC 32 pectinases may also be used in the retting of flax (to release the cellulose fibres from the plant tissue) and the scouring of cotton (to increase the water absorbance and dye uptake of the cotton fibres).
Plant and pectin-containing materials include plant pulp, parts of plants and plant extracts. In the context of this invention an extract from a plant material is any substance which may 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). Fruit juice that needs to be filtrated after or during processing tends to clot the filters after some time. This is due to the inability of prior art enzymes to properly digest fruit polysaccharides which causes the fouling of the filters. Use of the enzymes according to the invention prevents fouling of filters because of the unique properties of the PEC 1 - PEC 32 pectinase. PEC 1 - PEC 32 pectinases may conviently be produced in microorganisms. Microbial pectinases are available from a variety of sources; Bacillus spec, are a common source of bacterial enzymes, whereas fungal enzymes are commonly produced in Aspergillus spec.
In the above processes, it is advantageous to use pectinases that are obtained by recombinant DNA techniques. Such recombinant enzymes have a number of advantages over their traditionally purified counterparts. Recombinant enzymes may be produced at a low cost price, high yield, free from contaminating agents like bacteria or viruses but also free from bacterial toxins or contaminating other enzyme activities.
The polypeptides of the invention may be used to treat plant material including plant pulp and plant extracts. For example, 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. Conveniently 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 r dry form. Typically, 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
Typically, 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 material will typically be carried out for a 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 may 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. Depending on the composition of the enzyme preparation the plant material may first be macerated (e.g. to a puree) or liquefied. Using the polypeptides of the invention processing parameters such as the yield of the extraction, viscosity of the extract and/or quality of the extract may be improved.
Alternatively, or in addition to the above, a polypeptide of the invention may be added to raw juice obtained from pressing or liquefying the plant pulp. Treatment of raw juice may be carried out in a similar manner to the plant pulp in respect of dosage, temperature and holding time. Again, other enzymes than pectinases may be included. A 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. Due to the highly specific action on pectins the polypeptides of the invention may also be used to prepare pectins with modified chara dsristics, e.g. modified gelation capacities for specific applications.
The polypeptides of the invention may be added to animal feeds rich in pectin, 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. Advantageously, the polypeptides of the invention may continue to degrade pectins in the feed in vivo. Polypeptides according to the invention have low pH optima and are capable of releasing important nutrients in such acidic environments as the stomach of an animal. The invention thus also provides (e.g. animal) feeds and/or foodstuffs comprising one or more polypeptides of the inv ntion, as well as feeds and/or foodstuffs obtainablceby incubation and/or digestion with at least one enzyme according to the invention. PEC 1 - PEC 32 pectinases may also be advantageously used during the production of milk substitutes (or replacers) from soy bean. These milk substitutes may 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 according to the invention may be added to the slurry, either before or during the processing, enabling processing at a higher concentration (typically 40 to 50%) dry solids. Addition of pectinases according to the invention to a soy suspension as described in WO 95/29598 results in a decrease in waterbinding and a concommitant decrease in viscosity of the slurry. The enzyme may also be used in the preparation of savoury product(s), e.g. from soy bean. The invention also relates to the use of PEC 1 - PEC 32 pectinases in a selected number of industrial and pharmaceutical processes. Despite the long term experience obtained with such processes, the pectinase according to the invention features a number of significant advantages over the prior art enzymes. Depending on the specific application, these advantages may include aspects like lower production costs, higher specificity towards the substrate, less antigenic, less undesirable side activities, higher yields when produced in a suitable microorganism, more suitable pH and temperature ranges, better tastes of the final product as well as food grade and kosher aspects. An important aspect of the pectinases according to the invention is that they cover a whole range of pH and temperature optima which are ideally suited for a variety of applications. For example many large scale processes benefit from relatively high processing temperatures of 50 degrees C or higher, e.g. to control the risks of microbial infections. Several pectinases according to the invention comply with this demand but at the same time they are not that heat stable that they resist attempts to inactivate the enzyme by an additional heat treatment. The latter feature allows production routes that yield final products free of residual enzyme activity. Similarly many feed and food products have slightly acidic pH values so that pectinases with acidic or near neutral pH optima are preferred for their processing. A PEC 1 - PEC 32 pectinase complies with this requirement as well.
The polypeptides of the invention may also be added to animal feeds rich in pectin or xylogalacturonan, to reduce anti-nutritional effects of plant pectins and consequently improve production performances and welfare of the animals, as well as reducing environmental pollution.
It has been shown that plant pectins increase digesta viscosity, fermentation and colonisation of bacterial proliferation in the gastro intestinal tract (Langhout, D.J. (1998). The role of the intestinal flora as affected by non-starch polysaccharides in broiler chicks. PhD thesis, Agricultural University Wageningen, The Netherlands). As a consequence, several digestive physiological parameters are altered (Johnson, I . and Gee, J.M. (1981). Effect of gel-forming gums on the intes Mu . R. (1984). Effect of dietary
supplements of guar gurrcor ellulose on intestinal cell proliferation, enzyme levels as present in the expression vector pGBTOPFYTI as and glucose transport in the rat. British Journal of Nutrition 52:477-487) resulting in reduced dietary nutrient, mineral and energy utilization (Langhout, D.J. and Schutte, J.B. (1996). Nutritional implications of pectins in chicks in relation to esterification and origin of pectins. Poultry Science 75:1236-1242, Smits, C.M.H., Veldman, A., Verkade, H.J. and Beynen, A.C. (1998). The inhibitory effect of carboxymethylcellulose with high viscosity on lipid absorption in broiler chickens coincides with reduced bile salt concentration and raised microbial numbers in the small intestine. Poultry Science 77:1534-1539). The magnitude efficiency of dietary nutrient utilization is reduced, the effectivity in production performances (e.g. efficiency of producing milk, eggs, wool and meat) declines. Moreover, at reduced dietary nutrient utilization by the animal, excretion of and environmental pollution with e.g. nitrogenous compounds and heavy metals is increased.
Advantagous is that these fungal derived polypeptides of the invention have generally lower pH optima and their activities are therefore (more) capable of hydrolysis in such acidic environments as the stomach of an animal. With that, anti-nutritional properties of the plant cell wall fractions are already reduced early on in the digestive tract of the animal.
The improved characteristics of pectinases according to the invention were established as described in the examples below. PEC 1 - PEC 32 were expressed in A. niger by cloning of cDNAs acco ding to SEQ ID NO: 1 - 32. The resulting enzyme preparations were subsequently purified by ultrafiltration and used in food and feed conversion as described in the examples. It was found that PEC 1 - PEC 32, when added to animal feed, were able to increase the nutritional value of the feed, as measured by an improved body weight gain in the range of 1 to 5%> and in an improvement of the feed conversion ration of 1.5 to 6%>. In particular, PEC 1 - PEC 21 corresponding to polypeptides with SEQ ID NO: 65 - 85 were found especially useful.
EXAMPLES
Example 1.
DNA fragments having a sequence according to SEQ ID NO: 1 - 32 were cloned under the control of the glucoamylase promoter. To this end, the Aspergillus niger phytase (phyA) gene described in international patent application WO 98/46772 was replaced with the pectinase genes described above, yielding the vectors pPECTI - 32 respectively. The expression vectors pPECTI - 32 were introduced into Aspergillus niger CBS 646.97 (described in WO 98/46772). Using PCR, transformants containing pPECTI - 32 were selected. 107 spores of selected transformants were inoculated into shake flasks, containing 20 ml of liquid preculture medium containing per liter: 30 g maltose.H2O; 5 g yeast extract; 10 g hydrolyzed casein; 1 g KH2PO4; 0.5 g MgSO4.7H2O; 0.03 g ZnCI2; 0.02 g CaCi2; 0.01 g MnSO4 . 4H2O; 0.3 g FeSO4 . 7H20; 3 g Tween 80; 10 ml penicillin (5000 IU/ml)/Streptomycin (5000 UG/ml); pH 5.5. These cultures were grown at 34°C for 20-24 hours. 10 ml of this culture was inoculated into 100 ml of A. niger fermentation medium containing per liter: 70 g maltodextrines; 25 g hydrolyzed casein; 12.5 g yeast extract; 1 g KH2PO4; 2 g K2SO4; 0.5 g MgSO4.7H2O; 0.03 g ZnCI2; 0.02 g CaCI2; 0.01 g MnSO4.4H2O; 0.3 g FeSO4.7H2O; 10 ml penicillin (5000 IU/ml)/Streptomycin (5000 UG/ml); adjusted to pH 5.6 with 4 N H2SO4. These cultures were grown at 34°C for about 6 days. Samples taken from the fermentation broth were centrifuged (10', 5.000 rpm in a swinging bucket centrifuge) and supernatants collected. The culture supernatants were concentrated by ultrafiltration and used as enzyme source.
An experime t was designed to establish whether PEC 1 - PEC 32 pectinases, expressed as described above, could improve the nutritional value of soybean-based feed. The experiment was performed using male broilers (Ross 308) housed in floor pens. Directly after arrival from the hatchery, the animals were randomly distributed over 40 pens, each pen containing 20 broilers. Eight pens were allocated to each treatment. The pens were set up in an artificially heated, illuminated and ventilated broiler house, applying conditions similar to those found in practice. Animals were vaccinated according to the normal vaccination program. The experiment was performed till day 35 of age.
Five treatments were included in this trial. To the basal diet (table 1), no enzyme was added (control). To diet 2 a multi-enzyme (pectinase) mixture (Rapidase Press ® DSM) was added in a concentration of 100 ppm. Diets 3, 4 and 5 additionally contained one single pectinase selected from the group consisting of SEQ ID NO: 65 - 95, in three different doses 1 , 5 and 25 ppm, respectively. The feeds were pelleted without the addition of steam in the process. Feed and water were offered ad libitum to the animals.
Table 1 : Feed composition and contents of main nutrients of diet 1.
Figure imgf000051_0001
The diet was not supplemented with an antibiotic growth promoter or a coccidiostat. The results of this trial are presented in table 2.
Table 2. Average Body Weight Gain and Feed Conversion Ratio of the broilers (day 1-35).
Figure imgf000051_0002
Rapidase Press improved performance (treatment 2 compared to treatment 1 ), especially the growth of the animals. Additional supplementation with any enzyme chosen from PEC 1 - PEC 32, (treatments 3 - 5) further improved the performance of the animals, in particular supplementation with an enzyme chosen from PEC 1 - PEC 21 (corresponding to SEQ ID NO: 65 - 85) lead to a 1 - 4% gain in body weight and improved the feed conversion ratio with 1.5 - 6%. Example 2.
In this experiment the apparent metabolisable energy (AME) content of the experimental diets was determined, as well as the apparent faecal digestibility of fat and protein. Male broilers (Ross 308) of seven days of age were randomly distributed over 18 cages, each cage housing 8 broilers. Six cages were allocated to each treatment. The cages were set up in an artificially heated, illuminated and ventilated broiler house, using a three-tier cage system. Tiers were blocked over the treatments. The room was illuminated 24 hours/day, but the light intensity was gradually decreased during the trial. Also the temperature was decreased gradually during the experiment, according to a practical schedule. The humidity during the trial was kept at approximately 60%. Animals were vaccinated according to the normal vaccination program against Infectious Bronchitis and New Castle Disease. The experiment was performed till day 28 of age. Excreta were collected from day 24 to day 28. To the diets 0.25%o chromic oxide was added as an indigestible marker, at the expense of wheat.
Three treatments were included in this trial: treatments 1, 2 and 5 from example 1. The treatments were: 1. basal diet (table 1 [example 1]), without enzyme was added (control), 2. addition of Rapidase Press, and 5. as diet 2, but with the addition of 25 ppm of additional pectinases PEC 1 - PEC 32.
The feeds were pelleted without the addition of steam in the process. Feed and water were offered ad libitum o the animals. Analytical procedures applied were similar to those typically applied in animal experimentation institutes, usually AOAC-methods.
The results of this trial are presented in table 3.
Table 3. AME (MJ/kg) content and apparent faecal digestibility coefficients (%) of fat and protein of the three experimental diets. Excreta of the male broilers were collected from day 24 - 28.
Figure imgf000052_0001
Figure imgf000053_0001
It may be concluded that Rapidase Press improved digestibility, especially of protein. Addition of PEC 1 - PEC 32 had a large effect on energy metabolisability and on faecal digestibility of fat and protein, in particular PEC 1 - PEC 21 showed significant improvements in the order of 2 to 5%.
Example 3.
A next experiment was performed using mixed-sex broilers (Ross) fed a maize-soybean meal-based diet. The animals were housed in floor pens. Directly after arrival from the hatchery, the animals were randomly distributed over 48 pens, each pen containing 24 broilers; eight pens (four pens of males and four pens of females) were allocated to each treatment. The pens were set up in an artificially heated, illuminated and ventilated broiler house, applying conditions similar to those found in practice. Animals were vaccinated according to the normal vaccination program against Infectious Bronchitis and New Castle Disease. The experiment was performed up to day 42 of age.
The following treatments were included in this trial: 1. Basal diet (table 4) without addition of enzyme (control)
2. Control + Rapidase Press 100 ppm
3. Diet 1 + PEC 1 - PEC 32, 25 ppm
4. Diet 2 + PEC 1 - PEC 32, 1 ppm
5. Diet 2 + PEC 1 - PEC 32, 5 ppm 6. Diet 2 + PEC 1 - PEC 32, 25 ppm
The feeds were pelleted without the addition of steam in the process. Feed and water were offered ad IMum t the animals. Table 4: Feed composition and contents of main nutrients of diet 2.
Figure imgf000054_0001
The diet was not supplemented with an antibiotic growth promoter. The results of this trial are presented (as averages over the period 1-42 days and over the sexes) in table 5.
Table 5. Average BWG and FCR of the broilers (day 1-42).
Figure imgf000055_0001
Rapidase Press improved growth by 1.8% (treatment 2 compared to treatment 1). A single enzyme selected from PEC 1 - 32 (treatment 3) showed a similar effect, but improved FCR as well (average 1.4%). Supple entation with both enzymes, Rapidase Press and an enzyme selected from PEC 1 - PEC 32 in different doses (treatments 4 - 6) showed the best results. In particular PEC 1 - PEC 21 were found to be useful enzymes in this respect.
Example 4.
Also the diet tested in example 3 was submitted to a digestibility experiment. In this experiment the apparent metabolisable energy (AME) content of the experimental diets was determined, as well as the apparent faecal digestibility of fat and protein. Also the apparent ileal digestibility of amino acids was determined. Male broilers (Ross) of nine days of age were used in this experiment. The animals were randomly distributed over 24 cages, each cage hetiBμn &bratt&reiaSjixft^eεl. to^
three-tier cage system. Tiers were blocked over the treatments. The room was illuminated 24 hours/day, but the light intensity was gradually decreased during the trial. Also the temperature was decreased gradually during the experiment, according to a practical schedule. The humidity during the trial was kept at approximately 60%. Animals were vaccinated according to the normal vaccination program against Infectious Bronchitis and New Castle Disease. The experiment was performed till day 29 of age. Excreta were collected from day 25 to day 29. At day 29, the animals were euthanized using T61, and the contents of the last 30 cm of the small intestine were collected. The digesta were pooled per cage, to form six samples per treatment. The diets were identical to those mentioned in table 4, but 0.25%> chromic oxide was added as an indigestible marker, at the expense of tapioca.
Four treatments were included in this trial: treatments 1, 2, 3 and 6 from example 3. The treatments were:
1. Basal diet (table 4) without addition of enzyme (control)
2. Control + Rapidase Press 100 ppm
3. Diet 1 + PEC 1 - PEC 32, 25 ppm
4. Diet 2 + PEC 1 - PEC 32, 25 ppm
The feeds were pelleted without the addition of steam in the process. Feed and water were offered ad libitum to the animals. Analytical procedunEfcs applie were similar to those typically applied in animal experimentation institutes, usually AOAC-methods. Methionine was determined after prior oxidation with performic acid. The results of this trial are presented in tables 6 (AME and faecal digestibility) and in table 7 (ileal amino acid digestibility).
Table 6. AME (MJ/kg) content and apparent faecal digestibility coefficients (%>) of fat and protein of the three experimental diets. Excreta of the male broilers were collected from day 24 - 28.
Figure imgf000056_0001
Table 7. Apparent ileal digestibility coefficients (%>) for lysine, methionine, and threonine. Digesta collected at day 29.
Figure imgf000057_0001
The enzymes according to the invention improved AME and digestibility of the different nutrients. In particular PEC 1 - PEC 21 were found to be suitable in the above application. Combination of PEC 1 - PEC 21 with a commercial pectinase preparartion was found to be the better treatment in this experiment, and seemed especially to have a synergistic effect on AME and fat digestibility.
EXAMPLE 5
Substrate preparation: Preparation of MHR-S from apples
Modified hairy regions (MHR) from apples were isolated as a filter retentate after treatment of apples with Rapidase Press, a commercially available pectinase preparation, and subsequently the MHR was saponified resulting in MHR-S.
EXAMPLE 6
Improvement of filtration rate by PEC 1 - PEC 32 and combinations thereof
Experiments were done to see if the pectinases according to the invention could improve filterfouling during filtration. Apple MHR-S prepared as described in Example 2.1 was used as a substrate. A solution of 0.5% in 50 mM acetate buffer pH 4.0 was incubated with each individual enzyme chosen from PEC 1 - PEC 32 for 17 hours at 30DC. 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. In particular PEC 6 - PEC 32 showed significant improvements in filter fouling. Further improvements were obtained w, en enzymes with different specificities within this group were combined.

Claims

1) An isolated polynucleotide hybridisable to a polynucleotide selected from the group consisting of SEQ ID NO: 33 - 64 or from the group consisting of SEQ ID NO: 1 - 32
2) An isolated polynucleotide according to claim 1 hybridisable under high stringency conditions to a polynucleotide selected from the group consisting of SEQ ID NO: 33 - 64 or from the group consisting of SEQ ID NO: 1 - 32
3) An isolated polynucleotide according to claims 1 or 2 obtainable from a filamentous fungus.
4) An isolated polynucleotide according to claim 3 obtainable from A. niger.
5) An isolated polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 65 - 96 or functional equivalents thereof.
6) An isolated polynucleotide encoding at least one functional domain of a polypeptide selected from the group consisting of SEQ ID NO: 65 - 96 or functional equivalents thereof.
7) An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 33 - 64 or from the group consisting of SEQ ID NO: 1 - 32 or functional equivalents thereof
8) An isolated polynucleotide selected from the group consisting of SEQ ID NO: 33 - 64 or from the group consisting of SEQ ID NO: 1 - 32.
9) A vector comprising a polynucleotide sequence according to claims 1 to 8.
10) A vector according to claim 9 wherein said polynucleotide sequence according to claims 1 to 8 is operatively linked with regulatory sequences suitable for expression of said polynucleotide sequence in a suitable host cell.
11) A vector according to claim 10 wherein said suitable host cell is a filamentous fungus 12) A method for manufacturing a polynucleotide according to claims 1 - 8 or a vector according to claims 9 to 11 comprising the steps of culturing a host cell transformed with said polynucleotide or said vector and isolating said polynucleotide or said vector from said host cell.
13) An isolated polypeptide selected from the group consisting of SEQ ID NO: 65 - 96 or functional equivalents thereof.
14) An isolated polypeptide according to claim 13 obtainable from Aspergillus niger
15) An isolated polypeptide obtainable by expressing a polynucleotide according to claims 1 to 8 or a vector according to claims 9 to 11 in an appropriate host cell, e.g. Aspergillus niger.
16) Recombinant pectinase comprising a functional domain of a PEC 1 - PEC 32 polypeptide.
17) A method for manufacturing a polypeptide according to claims 13 to 16 comprising the steps of transforming a suitable host cell with an isolated polynucleotide according to claims 1 to 8 or a vector according to claims 9 to 11 , culturing said cell under conditions allowing expression of said polynucleotide and optionally purifying the encoded polypeptide from said cell or culture medium.
18) A recombinant host cell comprising a polynucleotide according to claims 1 to 8 or a vector according to claims 9 to 11.
19) A recombinant host cell expressing a polypeptide according to claims 13 to 16.
20) Purified antibodies reactive with a polypeptide according to claims 13 to 16.
21) Fusion protein comprising a polypeptide sequence according to claims 13 to 16.
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