AU2003303934A1

AU2003303934A1 - Novel pectinases and uses thereof

Info

Publication number: AU2003303934A1
Application number: AU2003303934A
Authority: AU
Inventors: Richard Albang; Luppo Edens; Armin Ehrenreich; Ulrike Folkers; Andreas Fritz; Beatrix Gerhard; Oliver Heinrich; Sylvia Hopper; Hilmar Ilgenfritz; Wolfram Kemmner; Erik Kimpel; Sabine Klugbauer; Sascha Losko; Dieter Maier; Fabio Spreafico; Alexander Stock; Pamela Tan; Albertus Alard Van Dijik; Christian Wagner
Original assignee: DSM IP Assets BV
Current assignee: DSM IP Assets BV
Priority date: 2002-05-30
Filing date: 2003-05-28
Publication date: 2004-09-09
Also published as: WO2004074468A1; EP1507857A1

Description

WO 2004/074468 PCT/EP2003/005726 1 NOVEL PECTINASES AND USES THEREOF Field of the invention 5 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 10 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 15 its activity and/or level of expression. Background of the invention Pectin polymers are important constituents of plant primary cell 20 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 25 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. 30 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 35 CDTA-soluble pectic substances from soybean meal are composed of WO 2004/074468 PCT/EP2003/005726 2 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, 5 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 10 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 WO 2004/074468 PCT/EP2003/005726 3 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 5 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 10 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 15 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. 20 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 25 described. The DNA and deduced amino acid sequences of several pectinases WO 2004/074468 PCT/EP2003/005726 4 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 5 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. 10 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 oC. Also, at high temperatures the currently available enzymes are easily 15 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. 20 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 25 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 30 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 35 The invention provides for novel polynucleotides encoding novel pectinases. These pectinases were found to digest pectinases from various WO 2004/074468 PCT/EP2003/005726 5 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 5 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 10 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 15 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, 20 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: 25 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: 30 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 35 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, WO 2004/074468 PCT/EP2003/005726 6 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: I 96 The group consisting of sequences according to SEQ ID NO: 1, 5 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: 10 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: 15 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 20 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 25 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 30 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 35 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, WO 2004/074468 PCT/EP2003/005726 7 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 5 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 10 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, 15 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, 20 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 25 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 30 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 35 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 WO 2004/074468 PCT/EP2003/005726 8 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 5 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. 10 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. 15 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 20 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 25 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. 30 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 35 invention. The invention also relates to recombinantly produced host cells WO 2004/074468 PCT/EP2003/005726 9 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 5 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 10 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 15 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 20 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 25 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 30 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 35 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 WO 2004/074468 PCT/EP2003/005726 10 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 5 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 10 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 15 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 20 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. 25 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 30 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 35 Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Moreover, a nucleic acid molecule encompassing all or a portion WO 2004/074468 PCT/EP2003/005726 11 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. 5 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. 10 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 15 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 20 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 25 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 30 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. 35 An "isolated polynucleotide" or "isolated nucleic acid" is a DNA or RNA that is not immediately contiguous with both of the coding sequences with WO 2004/074468 PCT/EP2003/005726 12 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 5 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 10 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 15 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 20 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. 25 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. 30 Sequencing errors The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The 35 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.

WO 2004/074468 PCT/EP2003/005726 13 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 5 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 10 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 15 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. 20 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 25 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 30 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 35 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 WO 2004/074468 PCT/EP2003/005726 14 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 5 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. 10 Identity & homologv 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 15 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 20 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) 25 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 30 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.qcq.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 35 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 WO 2004/074468 PCT/EP2003/005726 15 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.,qcq.com), using a NWSgapdna.CMP matrix and 5 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 10 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 15 (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 20 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 25 http://www.ncbi.nlm.nih.gqov. Hybridisation As used herein, the term "hybridizing" is intended to describe 30 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 35 conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45 OC, followed by one or more washes in 1 X SSC, 0.1 % SDS at 50 oC, preferably at 55 0C, preferably at 60 'C and even more preferably at 65 oC.

WO 2004/074468 PCT/EP2003/005726 16 Highly stringent conditions include, for example, hybridizing at 68 OC 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 oC. 5 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, 10 (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 15 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 20 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 25 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 30 - 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 35 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 WO 2004/074468 PCT/EP2003/005726 17 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 5 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 10 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 15 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. 20 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 25 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 30 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 35 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" WO 2004/074468 PCT/EP2003/005726 18 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. 5 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 10 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 15 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 Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of 20 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 25 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 30 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. coil, 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 35 Diego, CA (1990). Alternatively, the recombinant expression vector may be transcribed and translated in vitro, for example using T7 promoter regulatory WO 2004/074468 PCT/EP2003/005726 19 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, 5 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 10 promoter, such as the phage lambda PL promoter, the E. coil 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 15 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. 20 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 25 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 30 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 35 (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 WO 2004/074468 PCT/EP2003/005726 20 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 5 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 E col with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of 10 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 15 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. 20 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. coil and other bacteria. Representative examples of appropriate host include bacterial cells, such as E. coil, Streptomyces and Salmonella 25 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 30 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 35 Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan. Among known bacterial promotors for use in the present WO 2004/074468 PCT/EP2003/005726 21 invention include E. colilacl and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR, PL promoters and the trp promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus ("RSV"), and metallothionein 5 promoters, such as the mouse metallothionein-I 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 10 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 15 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 20 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 25 to the polypeptide to facilitate purification. Polypeptides according to the invention The invention provides an isolated polypeptide having an amino 30 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. 35 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" WO 2004/074468 PCT/EP2003/005726 22 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 5 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 10 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 15 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 20 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. 25 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 30 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 35 The invention also features biologically active fragments of the polypeptides according to the invention.

WO 2004/074468 PCT/EP2003/005726 23 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), 5 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 10 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 15 encode the above biologically active fragments of the PEC 1 - PEC 32 protein. Fusion proteins The proteins of the present invention or functional equivalents 20 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 25 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 30 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 35 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 WO 2004/074468 PCT/EP2003/005726 24 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 5 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 10 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 15 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). 20 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 25 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 30 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 35 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.

WO 2004/074468 PCT/EP2003/005726 25 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 5 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 10 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, 15 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 20 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. 25 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 30 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. 35 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 WO 2004/074468 PCT/EP2003/005726 26 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 5 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 10 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, 15 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 20 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 25 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 30 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 35 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, WO 2004/074468 PCT/EP2003/005726 27 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 5 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, 10 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 15 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 20 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 25 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 30 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 35 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 WO 2004/074468 PCT/EP2003/005726 28 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 5 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. 10 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 15 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 20 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 25 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 30 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 35 under highly stringent hybridisation conditions. Accordingly, the PEC 1 - PEC 32 protein is a protein which WO 2004/074468 PCT/EP2003/005726 29 comprises an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 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 5 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 10 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 15 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. 20 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 25 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 30 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 35 screening cDNA libraries for gene products having a selected property. The most widely used techniques, which are amenable to high through-put analysis, for WO 2004/074468 PCT/EP2003/005726 30 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 5 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). 10 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 15 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. 20 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 25 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 30 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 35 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 WO 2004/074468 PCT/EP2003/005726 31 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 5 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%, 97%, 98%, 99%, or more homologous to a nucleic acid 10 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%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homologous to an amino 15 acid sequence selected from the group consisting of SEQ ID NO: 65 - 96. Host cells In another embodiment, the invention features cells, e.g., 20 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 25 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., 30 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 35 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 WO 2004/074468 PCT/EP2003/005726 32 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. 5 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). 10 Antibodies The invention further features antibodies, such as monoclonal or polyclonal antibodies, that specifically bind PEC 1 - PEC 32 proteins according to the invention. 15 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 20 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 25 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. 30 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 35 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 WO 2004/074468 PCT/EP2003/005726 33 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 (SP 2 0), available from the American Type Culture Collection, Rockville, Maryland. After fusion, the 5 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 10 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 15 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. 20 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. 25 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 30 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, 35 by criteria such as high frequency of charged residues. For example, such fragments may be generated by standard techniques of PCR, and then cloned WO 2004/074468 PCT/EP2003/005726 34 into the pGEX expression vector (Ausubel et al., su ra). Fusion proteins may then be expressed in E. coil 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 5 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. 10 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. 15 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 20 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/Technology9:1370-1372; 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. 25 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 30 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. 35 Antibodies may be purified, for example, by affinity chromatography methods in which the polypeptide antigen is immobilized on a WO 2004/074468 PCT/EP2003/005726 35 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, 5 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 10 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 15 enzymes include horseradish peroxidase, alkaline phosphatase, f3-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 20 include luciferase, luciferin, and aequorin, and examples of suitable radioactive materials include 1251, 1311, 3 5 S 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 25 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 30 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. 35 Immunoassays WO 2004/074468 PCT/EP2003/005726 36 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. 5 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 10 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. 15 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 20 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 25 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 30 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 35 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 WO 2004/074468 PCT/EP2003/005726 37 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 5 concentration of hydrogen peroxide formed by the enzyme-labelled antibody/substrate reaction. Besides enzymes, other suitable labels include radioisotopes, such as iodine (1251, 1211), carbon ( 14 C), sulphur (35S), tritium ( 3 H), indium ( 11 2 1In), and technetium (99mTc), and fluorescent labels, such as fluorescein and rhodamine, 10 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 15 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 OC for 0.1 to 36 hours. Polypeptides that are not bound to the plate may be 20 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 25 example, 300 ul of bovine serum albumin (BSA) at a concentration of 2 mg/ml in Tris-HCI 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 30 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 35 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 WO 2004/074468 PCT/EP2003/005726 38 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 5 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 10 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 15 polypeptides (e.g., serum proteins, such as BSA and globulins, and milk proteins, such as caseins). Epitopes, antigens and immunoglens. 20 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 25 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 30 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 35 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 WO 2004/074468 PCT/EP2003/005726 39 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 5 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 10 HAl 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, 15 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 20 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 25 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. 30 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 35 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 WO 2004/074468 PCT/EP2003/005726 40 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 5 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 10 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 15 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 HAl polypeptide which were prepared and characterized (by ELISA-type binding studies) in less than four weeks. Houghten, R. A., Proc. Natl. Acad. Sci. 20 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 25 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 30 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 35 a macromolecular carrier, such as keyhole limpet hemocyanin (KLH) or tetanus toxoid. For instance, peptides containing cysteine may be coupled to carrier using WO 2004/074468 PCT/EP2003/005726 41 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 5 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 10 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., 15 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 20 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 25 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 30 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) 35 describes a general method of detecting or determining the sequence of monomers (amino acids or other compounds) which is a topological equivalent of WO 2004/074468 PCT/EP2003/005726 42 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 5 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 10 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. 15 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 20 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 25 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 30 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 35 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.

WO 2004/074468 PCT/EP2003/005726 43 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. 5 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 10 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 15 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, 20 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). 25 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. 30 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 35 that are obtained by recombinant DNA techniques. Such recombinant enzymes have a number of advantages over their traditionally purified counterparts.

WO 2004/074468 PCT/EP2003/005726 44 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 5 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 10 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 15 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 20 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 25 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 10-55OC, e.g. from 15 to 25 0 C, optimally about 20 0 C and one may use 10-300g, preferably 30-70g, optimally about 50g of 30 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 pure) or liquefied. Using the polypeptides of the invention processing parameters such as the yield of the extraction, viscosity of the extract 35 and/or quality of the extract may be improved. Alternatively, or in addition to the above, a polypeptide of the WO 2004/074468 PCT/EP2003/005726 45 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. 5 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 0 C for a time of from 1 minute to 1 hour, under conditions to partially or fully inactivate the polypeptides of the invention. 10 Due to the highly specific action on pectins the polypeptides of the invention may also be used to prepare pectins with modified chara eristics, 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 15 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 20 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 obtainablEby incubation and/or digestion with at least one enzyme according to the invention. PEC 1 - PEC 32 pectinases may also be advantageously used 25 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 30 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. 35 The enzyme may also be used in the preparation of savoury product(s), e.g. from soy bean.

WO 2004/074468 PCT/EP2003/005726 46 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 5 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. 10 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 15 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 20 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 25 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, 30 The Netherlands). As a consequence, several digestive physiological parameters are altered (Johnson, I.T. and Gee, J.M. (1981). Effect of gel-forming gums on the intes Miu. R. (1984). Effect of dietary 35 supplements of guar gurrcor ellulose on intestinal cell proliferation, enzyme levels WO 2004/074468 PCT/EP2003/005726 47 as present in the expression vector pGBTOPFYT1 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 75:1238-1242, Smits, C.M.H., 5 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 10 (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) 15 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 20 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 25 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 30 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 35 phytase (phyA) gene described in international patent application WO 98/46772 was replaced with the WO 2004/074468 PCT/EP2003/005726 48 pectinase genes described above, yielding the vectors pPECT1- 32 respectively. The expression vectors pPECTI - 32 were introduced into Aspergillus niger CBS 646.97 (described in WO 98/46772). Using PCR, transformants containing pPECT1 - 32 were selected. 107 spores of selected transformants were inoculated 5 into shake flasks, containing 20 ml of liquid preculture medium containing per liter: 30 g maltose.H 2 0; 5 g yeast extract; 10 g hydrolyzed casein; 1 g KH 2

PO

4 ; 0.5 g MgSO 4 .7H 2 0; 0.03 g ZnC 2 ; 0.02 g CaC 2 ; 0.01 g MnSO 4 . 4H 2 0; 0.3 g FeSO 4 . 7H 2 0; 3 g Tween 80; 10 ml penicillin (5000 IU/ml)/Streptomycin (5000 UG/mI); pH 5.5. These cultures were grown at 34oC for 20-24 hours. 10 ml of this culture was 10 inoculated into 100 ml of A. nigerfermentation medium containing per liter: 70 g maltodextrines; 25 g hydrolyzed casein; 12.5 g yeast extract; 1 g KH 2

PO

4 ; 2 g

K

2

SO

4 ; 0.5 g MgSO 4 .7H 2 0; 0.03 g ZnC 2 ; 0.02 g CaC 2 ; 0.01 g MnSO 4 .4H 2 0; 0.3 g FeSO 4 .7H 2 0; 10 ml penicillin (5000 IU/ml)/Streptomycin (5000 UG/ml); adjusted to pH 5.6 with 4 N H 2

SO

4 . These cultures were grown at 34 0 C for about 6 days. 15 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 20 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 25 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 30 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. 35 Table 1: Feed composition and contents of main nutrients of diet 1.

WO 2004/074468 PCT/EP2003/005726 49 Ingredient Contents (%) Wheat 60 Soybean meal 29 Soy oil 1 Blended animal fat 6 Minerals, vitamins, amino acids 4 ME (MJ/kg) 12.8 Crude protein (%) 21 Crude fat (%) 9 The diet was not supplemented with an antibiotic growth promoter or a coccidiostat. The results of this trial are presented in table 2. 5 Table 2. Average Body Weight Gain and Feed Conversion Ratio of the broilers (day 1-35). Treatment BWG (g/bird) FCR (g/g) 1. Control 1957 1.613 2. Control + Rapidase Press 100 ppm 2019 1.613-1.601 3. Control + Rapidase Press + PEC 1 - 2019-2044 1.610-1.589 PEC 32, 1 ppm 4. Control + Rapidase Press + PEC 1 - 2037 -2051 1.594-1.578 PEC 32, 5 ppm 5. Control + Rapidase Press + PEC 1 - 2035-2055 1.589- 1.505 PEC 32, 25 ppm Rapidase Press improved performance (treatment 2 compared 10 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 15 1.5 - 6%.

WO 2004/074468 PCT/EP2003/005726 50 Example 2. In this experiment the apparent metabolisable energy (AME) 5 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 10 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 15 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% 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]), 20 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 libitutn o the animals. Analytical procedures applied were similar to those typically applied in animal 25 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 30 fat and protein of the three experimental diets. Excreta of the male broilers were collected from day 24 - 28. Treatment AME (MJ/kg) Faecal digestibility (%) Fat Protein 1. Control 12.57 78.4 79.4 WO 2004/074468 PCT/EP2003/005726 51 2. Control + Rapidase Press 100 12.67 79.6 82.3 ppm 5. Control + Rapidase Press + 12.77-13.51 80.0-81.7 81.0-83.4 PEC 1 - PEC 32, 25 ppm 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 5 PEC 21 showed significant improvements in the order of 2 to 5%. Example 3. A next experiment was performed using mixed-sex broilers 10 (Ross) fed a maize-soybean meal-based diet. The animals were housed in floor pens. Dire ctly 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 15 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: 20 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 25 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 libitum t the animals. 30 WO 2004/074468 PCT/EP2003/005726 52 Table 4: Feed composition and contents of main nutrients of diet 2. Ingredient Contents (%) Maize 50.5 Soybean meal 31.7 Tapioca 6.0 Fish meal 1.0 Meat meal 2.1 Soy oil 2.1 Blended animal fat 3.0 Mono calcium phosphate 1.2 Limestone 0.45 Stenerol (coccidiostat) 0.05 Other minerals, vitamins, amino acids 1.9 ME (MJ/kg) 11.9 Crude protein (%) 21.5 Crude fat (%) 8.0 Digestible lysine (%) 1.06 Digestible methionine + cystine 0.80 The diet was not supplemented with an antibiotic growth promoter. The results of 5 this trial are presented (as averages over the period 1-42 days and over the sexes) in table 5.

WO 2004/074468 PCT/EP2003/005726 53 Table 5. Average BWG and FCR of the broilers (day 1-42). Treatment BWG (g/bird) FCR (g/g) 1 2166 1.811 2 2204 1.820 3 2170 -2210 1.776 - 1.803 4 2190 -2206 1.781 -1.806 5 2195 -2222 1.771 -1.823 6 2215 -2241 1.752 - 1.795 Rapidase Press improved growth by 1.8% (treatment 2 compared to treatment 1). 5 A single enzyme selected from PEC 1 - 32 (treatment 3) showed a similar effect, m 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. 10 Example 4. Also the diet tested in example 3 was submitted to a digestibility experiment. In this experiment the apparent metabolisable energy (AME) content 15 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 hetaspijpa brotiftelsaS~hinges,vHIumiittealtad ceatiltat llid RhaG' tagre 20 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 25 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 WO 2004/074468 PCT/EP2003/005726 54 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. 5 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 10 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 procedurs applie were similar to those typically applied in animal experimentation institutes, usually AOAC-methods. Methionine was determined 15 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 20 collected from day 24 - 28. Treatment AME (MJ/kg) Faecal digestibility (%) Fat Protein 1 12.2 80.5 81.3 2. 12.4 80.6 82.4 3 12.2-12.6 80.4 - 80.9 81.5 - 82.9 6 12.3 - 12.7 80.9 - 82.4 81.2 - 83.3 WO 2004/074468 PCT/EP2003/005726 55 Table 7. Apparent ileal digestibility coefficients (%) for lysine, methionine, and threonine. Digesta collected at day 29. Treatment Lysine Methionine Threonine 1 83.9 86.5 78.6 2 85.1 88.1 79.8 3 83.9 - 85.3 86.5-88.5 78.1 - 80.1 6 83.8 - 86.9 86.8 - 89.6 78.4 - 81.2 5 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. 10 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 15 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 20 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 25 for 17 hours at 300C. 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 when enzymes with different specificities within this group were combined. 30

Claims

16. 20) Purified antibodies reactive with a polypeptide according to claims 13 to 16. 25 21) Fusion protein comprising a polypeptide sequence according to claims 13 to 16.