MXPA00005108A - Novel pectate lyases - Google Patents

Abstract

A novel group of pectate lyases comprising the amino acid sequence Asn Leu Asn Ser Arg Val Pro (NLNSRVP) belonging to Family 1 of polysaccharide lyases have good performance in industrial processes under neutral or alkaline conditions such as laundering and textile processing. The pectate lyase may be derivable from i(Bacillus) species.

Description

- - NEW PECTATO LIAS AS FIELD OF THE INVENTION The present invention relates to new microbial pectate lyases, more specifically to microbial enzymes that show pectate lyase activity as their main enzymatic activity in the neutral and alkaline pH ranges; to a method for producing such enzymes; and methods for using such enzymes in the textile processing, detergent and cellulose fiber processing industries.

BACKGROUND OF THE INVENTION Pectin polymers are important constituents of the cell walls of plants. The pectina is a hetero-poly with a main structure composed of alternative homogenous (soft regions) and ramnogalacturonano (regions with projections). The smooth or smooth regions are linear polymers of the acid to f a - D- ga 1 a c t uronico bound to 1.4. The galacturonic acid residues can be measured in the carboxyl group by several degrees, usually in a non-random manner with blocks of active 1-gauge acid that is completely methyl-esterified. .

Pectinases can be classified according to their preferential substrate, highly pectin 1-esterifi cation of pectins with a low me te 1 - esterification and acid po 1 i ga 1 act ionic (pectate), and its mechanism of reaction, beta-e 1 imion or hydrolysis. The pectinases can be mainly e ndo-a c t u a te r a s, cutting the polymer at random sites within. the chain to give a mixture of oligomers, or they can be exo -a c ua tors, attacking from one end of the polymer and producing monomers or dimers. Several activities of pectinases acting in the smooth or smooth regions of pectin are included in the classification of enzymes provided by the Enzyme Nomenclature (1992) such as pectate lyase (EC 4.2.2.2), pectin lyase (EC 4.2. 2.10), polygalacturonase (EC 3.2.1.15), exo- -polygalacturonase (EC 3.2.1.67), exo-polygalactide ato lyase (EC 4.2.2.9) and exo-poly-alpha-galacturonosidase (EC 3.2.1.82).

Pectate lyases have been cloned from different bacterial genera such as Erwinia, Pseudomonas, Klebsiella and Xanthomonas. Also from Bacillus subtilis (Nasser et al. (1993) FEBS 335: 319-326) and Bacillus sp. YA-14 (Kim et al. (1994) Biosci, Biotech, Biochem 58: 947-949) has described the cloning of a pectate lyase. The purification of pectate lyases with a maximum activity in the pH range of 8-10 produced by Bacillus pumilus has been reported.

(Dave and Vaughn (1971) J. Bacteriol. 108: 166-174), B. polymyxa (Nagel and Vaughn (1961) Arch.

Biochem. Biophys. 93: 34-352), B. stearothermophilus (Arbassi and Vaughn (1980) Can. J. Microbiol. 26: 377-384), Bacillus sp. (Hasegawa and Nagel (1966) J. Food Sci. 31: 838-845) and Bacillus sp. RK9 (Kelly and Fogarty (1978) Can. J.

Microbiol. 24: 116-1172), however, no publication has been found on the cloning of the genes encoding the -seeto lyase of these organisms. All pectate lyases described require divalent cations for maximum activity, calcium ions are the most stimulating.

WO 98/45393 describes the detergent compositions containing protopectinase with an important detergency against stains or mud.

In general, the microorganisms that produce pectinase show a wide range of degradation of pectin or modifying enzymes. Often microorganisms also produce cellulases and / or hemicellulases and complex multi-component enzyme preparations of such microorganisms can be difficult to optimize for various applications, even when they contain enzymes with a negative effect. Thus, it is an object of the present invention to provide a pectin degrading enzyme that exhibits only the desired effects for example in detergents or in different industrial processes.

BRIEF DESCRIPTION OF THE INVENTION The inventors have now found and identified several new enzymes that have the substantial activity of pectate lyase that perform excellent work in various industrial processes under neutral or alkaline conditions and have been successful in identifying the DNA sequences encoding such enzymes, these Enzymes form a new class of pectate lyases that have at least conserved regions with partially identical amino acid sequences.

Accordingly, in a first aspect this invention relates to a pectate lyase comprising a first amino acid sequence consisting of seven (7) amino acid residues having the following sequence: Asn Leu Asn Ser Arg Val Pro (NLNSRVP) . In additional embodiments, the pectate lyase can additionally maintain a second amino acid sequence consisting of six (6) amino acid residues - - selected from the group consisting of the Trp Val Asp His Asn Glu (WVDHNE) and Trp lie Asp His Asn sequences Glu (WIDHNE); and optionally also a third amino acid sequence consisting of three (3) amino acid residues having the following sequence: Ser Trp Asn (SWN).

The five pectate lyase DNA sequences of the invention are listed in the sequence listings as SEC. ID No. 1, 3, 5, 7 and 9, respectively, and the deduced amino acid sequences are listed in the sequence listings as SEC. ID No. 2, 4, 6, 8 and 10, respectively. It is believed that the new enzyme will be classified according to the Enzyme Nomenclature in the Enzyme Class EC 4.2.2.2. However, it should be noted that the enzyme of the invention also shows catalytic activity on pectin (which can be esterified) besides the activity on the pectate and the p 1 i g a 1 a c t ur on i do s is conventionally attributed to the enzymes belonging to the EC 4.2.2.2.

- - In a second aspect, the present invention relates to a pectate lyase which is i) a polypeptide produced by B acillusa ga ra dh aer in s, NCIMB 40482 or DSM 8721, or ii) a polypeptide comprising a sequence of amino acids as shown in positions 27-359 of the SEC. ID NO: 2, or iii) an analogue of the polypeptide defined in i) or ii) which is at least 45% homologous with said polypeptide, or iv) is derived from said polypeptide by substitution, removal or addition of one or more amino acids , with the proviso that the arginine at position 240, and optionally also the arginine at position 245, - are retained and the polypeptide derivative is at least 42% homologous with said polypeptide, or v) is immunologically reactive with polyclonal antibody generated against said polypeptide in purified form.

Within one aspect, the present invention provides an isolated olinucleotide molecule selected from the group consisting of (a) polynucleotide molecules that encode a polypeptide having pectate lyase activity and comprising a nucleotide sequence as shown in SEQ. ID NO: 1 from nucleotide 79 to nucleotide 1077; (b) homologous species of (a); (c) polynucleotide molecules that encode a polypeptide having pectate lyase activity that is at least 45% identical to the amino acid sequence of SEQ. ID NO: 2 of amino acid residue 27 to amino acid residue 359; (d) molecules complementary to (a), (b) or (c); and (e) degenerate nucleotide sequences of (a), (b), (c) or (d).

Plasmid pSJ1678 comprising the oligonucleotide molecule (the DNA sequence) encoding a pectate lyase of the present invention has been transformed into a strain of Es ch e ri chiacoli which was deposited by the inventors according to the Budapest Treaty in the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure in the Deutsche Sammiung von Mikroorganismen - -und Zellkulturen GmbH, Mascheroder Weg Ib, .D-38124 Braunschweig, Federal Republic of Germany, on September 25, 1997 under the number of deposit DSM 11788.

In a third aspect, the present invention relates to a pectate lyase which is i) a polypeptide produced by B acillusli ch enifo rm is, ATCC 14580, or ii) a polypeptide comprising an amino acid sequence as shown in the positions 28-341 of the SEC. ID NO: 4, or iii) an analogue of the polypeptide defined in i) or ii) which is at least 45% homologous with said polypeptide, or iv) is derived from said polypeptide by substitution, removal or addition of one or more amino acids , with the proviso that the arginine at position 233, and optionally also the arginine at position 238, is retained and the polypeptide derivative is at least 42% homologous with said polypeptide, or v) is immunologically reactive with a polyclonal antibody raised against said polypeptide in purified form.

- - Within one aspect, the present invention provides an isolated polynucleotide molecule selected from the group consisting of (a) polynucleotide molecules that encode a polypeptide having pectate lyase activity and comprising a nucleotide sequence as shown in SEQ. ID NO: 3 from nucleotide 82 to nucleotide 1026; (b) homologous species of (a); (c) polynucleotide molecules that encode a polypeptide having pectate lyase activity that is at least 45% identical to the amino acid sequence of SEQ. ID NO: 4 of amino acid residue 28 to amino acid residue 341; (d) molecules complementary to (a), (b) or (c); and (e) degenerate nucleotide sequences of (a), (b), (c) or (d).

Plasmid pSJ1678 comprising the polynucleotide molecule (the DNA sequence) encoding a pectate lyase of the present invention has been transformed into a strain of E s ch ri chiacoli which was deposited by the inventors according to the Treaty of Budapest at the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure at the Deutsche Sammiung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg Ib, D-38124 Braunschweig, Federal Republic of Germany, on September 25, 1997 under the number of deposit DSM 11789.

In a fourth aspect, the present invention relates to a pectate lyase which is a polypeptide produced by a species of B a c i l l s that has the 16S rDNA sequence of SEQ. ID NO: 14 or by a species of B a c i l l s that has a sequence homology of 16S rDNA to the SEC. ID NO: 14 greater than 97.3%; ii) a polypeptide comprising an amino acid sequence as shown in positions 181-509 of SEQ. ID NO: 6, or iii) an analogue of the polypeptide defined in i) or ii) which is at least 50% homologous with said polypeptide, or iv) is derived from said polypeptide by substitution, deletion or addition of one or - several amino acids, with the proviso that the arginine at position 390, and optionally also the arginine at position 395, is retained and the polypeptide derivative is at least 44% homologous with said polypeptide, or v) is immunologically reactive with a polyclonal antibody generated against said polypeptide in purified form.

Within one aspect, the present invention provides an isolated olinucleotide molecule selected from the group consisting of (a) polynucleotide molecules that encode a polypeptide having pectate lyase activity and comprising a nucleotide sequence as shown in SEQ. ID NO: 5 from nucleotide 541 to nucleotide 1530; (b) homologous species of (a); (c) polynucleotide molecules that encode a polypeptide having pectate lyase activity that is at least 50% identical to the amino acid sequence of SEQ. ID NO: 6 of amino acid residue 181 to amino acid residue 509; (d) molecules complementary to (a), (b) or (c); and (e) degenerate nucleotide sequences of (a), (b), (c) or (d).

Plasmid pSJ1678 comprising the polynucleotide molecule (the DNA sequence) encoding a pectate lyase of the present invention has been transformed into a strain of E s che ri chia coli which was deposited by the inventors according to the Budapest Treaty in the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure at the Deutsche Sammiung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg Ib, D-38124 Braunschweig, Federal Republic of Germany, September 8, 1998 under deposit number DSM 12403.

In a fifth aspect, the present invention relates to a pectate lyase which is i) a polypeptide produced by a species of Bacillus ha lodurans, preferably the species Bacillus sp. KJ59, DSM 12419, or ii) a polypeptide comprising an amino acid sequence as shown in positions 42-348 of SEQ. ID NO: 8, or iii) an analogue of the polypeptide defined in i) or ii) which is at least 45% homologous with said polypeptide, or iv) is derived from said polypeptide by substitution, deletion or addition of one or more amino acids , with the proviso that the arginine at position 240, and optionally also the arginine at "position 245, is retained and the derived polypeptide is at least 40% homologous with said polypeptide, or v) is immunologically reactive with a polyclonal antibody generated against said polypeptide in purified form.

The Ba c i l l u s sp. KJ59, which is believed to be a strain that belongs to or is closely related to the known species B acillusha 1 odu ra ns that was deposited by the inventors according to the Budapest Treaty in the International Recognition of the Deposit of Microorganisms for Purpose of the Patent Procedure in the Deutsche Sammiung von Mikroorganismen und -Zellkulturen GmbH, Mascheroder Weg Ib, D-38124 Braunschweig, Federal Republic of Germany, September 21, 1998 under the deposit number DSM 12419.

Within one aspect, the present invention provides an isolated polynucleotide molecule selected from the group consisting of (a) polynucleotide molecules that encode a polypeptide having pectate lyase activity and comprising a nucleotide sequence as shown in SEQ. ID NO: 7 from nucleotide 124 to nucleotide 1047; (b) homologous species of (a); (c) polynucleotide molecules that encode a polypeptide having pectate lyase activity that is at least 45% identical to the amino acid sequence of the SEC. ID NO of amino acid residue 42 to amino acid residue 348; (d) molecules complementary to (a), (b) or (c); and (e) degenerate nucleotide sequences of (a), (b), (c) or (d).

In a sixth aspect the present invention relates to a pectate lyase which is i) a polypeptide produced by a species of B a c i l l s that has the 16S rDNA sequence of SEQ. ID NO: 13 or by a species of B a c i l l s that has a sequence homology of 16S rDNA to the SEC. ID NO: 13 greater than 98.1%; ii) a polypeptide comprising an amino acid sequence as shown in positions 25-335 of SEQ. ID NO: 10, or iii) an analogue of the polypeptide defined in i) or ii) which is at least 45% homologous with said polypeptide, or iv) is derived from said polypeptide by substitution, removal or addition of one or more amino acids , provided that the arginine at position 227, and optionally also the arginine at position 232, is retained and the derived polypeptide is at least 41% homologous with said polypeptide, or v) is immunologically reactive with a polyclonal antibody raised against said polypeptide in purified form.

Within one aspect, the present invention provides an isolated polynucleotide molecule selected from the group consisting of (a) polynucleotide molecules that encode a polypeptide having pectate lyase activity and comprising a nucleotide sequence as shown in SEC. ID NO: 9 from nucleotide 73 to nucleotide 1008; (b) homologous species of (a); (c) polynucleotide molecules that encode a polypeptide having pectate lyase activity that is at least 45% identical to the amino acid sequence of SEQ. ID NO: 10 from amino acid residue 2.5 to amino acid residue 335; (d) compliant molecules a (a), (b) or (c); and (e) degenerate nucleotide sequences of (a), (b), (c) or (d).

The plasmid pSJ1678 comprising the polynucleotide molecule (the DNA sequence) encoding a pectate lyase of the present invention has been transformed into an E scheri chiacoli strain which was deposited by the inventors according to the Budapest Treaty in the Recognition International Deposit of Microorganisms - for the Purpose of the Patent Procedure at the Deutsche Sammiung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg Ib, D-38124 Braunschweig, Federal Republic of Germany, September 8, 1998 under deposit number DSM 12404.

Within another aspect of the invention there is provided an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment selected from the group consisting of a) polynucleotide molecules that encode a polypeptide having the pectate lyase activity which comprises a nucleotide sequence as shown in SEQ. ID NO: 1 from nucleotide 79 to nucleotide 1077, in SEQ. ID NO: 3 from nucleotide 82 to nucleotide 1026; in the SEC. ID NO: 5 from nucleotide 541 to nucleotide 1530; in the SEC. ID NO: 7 from nucleotide 124 to nucleotide 1047 or as shown in SEQ. ID NO: 9 from nucleotide 73 to nucleotide 1008; b) polynucleotide molecules that encode a polypeptide having the pectate lyase activity which is at least 50% identical to the amino acid sequence of SEC. ID NO: 2 of amino acid residue 27 to amino acid residue 359, of SEQ. ID NO: 4 of the amino acid residue 28 to the amino acid residue 341, of the SEC. ID NO: 6 of the amino acid residue 181 to the amino acid residue 509. of SEQ ID NO of amino acid residue 42 to amino acid residue 348 or to the amino acid sequence of SEQ. ID NO: 10 from amino acid residue 25 to amino acid residue 335, and (c) degenerate nucleotide sequences of (a) or (b); and a transcription terminator.

Within yet another aspect of the present invention there is provided a cultured cell into which an expression vector has been introduced as described above, wherein said cell expresses the polypeptide encoded by the DNA segment.

A further aspect of the present invention provides an isolated polypeptide having pectate lyase activity selected from the group consisting of a) polypeptide molecules having pectate lyase activity and comprising an amino acid sequence as shown in SEQ. ID NO: 2 from residue 27 to residue 359; b) polypeptide molecules having pectate lyase activity and which are at least 45% identical to the amino acids of SEC. ID NO: 2 of the amino acid residue 27 to the amino acid residue. 359; c) polypeptide molecules having pectate lyase activity and comprising an amino acid sequence - as shown in SEQ. ID NO: 4 from residue 28 to residue 241; d) molecules of polypeptides having the activity of pectate lyase and which are at least 45% identical to the amino acids of SEC. ID NO: 4 of amino acid residue 28 to amino acid residue 341; e) polypeptide molecules having pectate lyase activity and comprising an amino acid sequence as shown in SEQ. ID NO: 6 from residue 181 to residue 509; f) polypeptide molecules having pectate lyase activity and which are at least 50% identical to the amino acids of SEQ. ID NO: 6 of amino acid residue 181 to amino acid residue 509; g) polypeptide molecules having pectate lyase activity and comprising the amino acid sequence as shown in SEQ. ID NO: 8 from residue 42 to residue 348; h) polypeptide molecules having pectate lyase activity and which are at least 45% identical to the amino acids of SEQ. ID NO of the amino acid residue 42 to amino acid residue 348; i) polypeptide molecules having pectate lyase activity and comprising an amino acid sequence as shown in SEQ. ID NO: 10 from residue 25 to residue 335; k) polypeptide molecules having pectate lyase activity and which are at least 45% identical to the amino acids of SEQ. ID NO: 10 from amino acid residue 25 to amino acid residue 335; and 1) homologous species of a), b), c), d), e), f), g), h), i) and k).

- Within another aspect of the present invention there is provided a composition comprising a purified pectate lyase according to the invention in combination with other polypeptides having enzymatic activity.

Within another aspect of the present invention there are provided methods for producing a polypeptide according to the invention which comprises culturing a cell into which an expression vector has been introduced as described above, whereby said cell expresses a coded polypeptide for the DNA segment and the recovery of the polypeptide.

The new pectate lyase enzymes of the present invention are useful for the treatment of cellulosic material, especially cellulose-containing fibers, yarns, woven or non-woven fabrics, the treatment of pulps for the mechanical manufacture of recycled waste paper or paper, - -and to re-weave the fibers. The treatment can be carried out by processing the cellulosic material into a material that is ready for the manufacture of garments or for the manufacture of fabrics, for example, in the stage of desizing or washing with detergent; or during the industrial or home laundry of such fabrics or garments.

Accordingly, in additional aspects the present invention relates to a detergent composition comprising an enzyme having substantial pectate lyase activity; and to the use of the enzyme of the invention for the treatment of fibers containing cellulose, yarns, woven or non-woven fabrics.

Pectate lyases of the invention are very effective for use in an enzymatic washing process in the preparation of cellulosic material, for example, for the proper response in subsequent dyeing operations. In addition, it is contemplated that the detergent compositions comprising the new pectate lyases will be capable of removing or bleaching certain stains or soils present in the laundry, especially soils and stains resulting from food, plants and the like containing galactane oar ab inoga 1 actano It is also contemplated that the treatment with the detergent compositions comprising the new enzymes may prevent the binding or the binding of certain stains or dirt to the cellulosic material. The enzymes of the invention are also useful as ingredients in compositions for cleaning hard surfaces that have the effect of removing or assisting in the removal of certain stains or dirt from hard surfaces that need cleaning.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows a sequence alignment of the mature parts of the amino acid sequences of SEQ. ID NOS: 2, 4, 6, 8 and 10, the alignment numbered from 1 to 344. For example, position 223 of the - -numbering of the alignment corresponds to position 240 of the SÉC. ID NO: 2, to position 233 of the SEC. ID NO: 4, to position 390 of the SEC. ID NO: 6, to position 240 of the SEC. ID NO: 8 and to position 227 of the SEC. ID NO: 10 Figure 2 is a graphic illustration of the effect of pH and temperature on the removal of pectin from a cotton fabric using a thermostable pectate lyase. The pectin removal is expressed as a residual% pectin. The pectate lyase was applied to the fabric in a dosage of 100 ymol / min / kg of cloth.

Figure 3 is a graphic illustration of the effect of the dosage of the thermostable pectate lyase in the removal of the pectin from a cotton fabric. The elimination is expressed as a residual% of pectin, and the dosage as ymol / min / kg of fiber. The pectate lyase was applied to the cloth at a pH of 9 and at 80 ° C.

- DEFINITIONS Before discussing this invention in greater detail, the following terms will be defined first.

The term "ortholog" (or "homologous species") denotes a polypeptide or protein obtained from a species having homology to an analogous polypeptide or analogous protein of a different species.

The term "paralog" denotes a polypeptide or protein obtained from a given species that has homology to a polypeptide or protein other than that of the same. E.s p e c i e The term "expression vector" denotes a DNA molecule, linear or circular, comprising a segment encoding a polypeptide of interest operably linked to additional segments that provides its transcription. Such additional segments may include promoter and t-rimer sequences, and may optionally include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from viral DNA or plasmid, or may contain elements of both. The expression vector of the invention can be any expression vector that is conveniently subjected to recombinant DNA methods, and the choice of vector will often depend on the host cell into which the vector is to be introduced. Thus, the vector can be a vector of autonomous replication, that is, a vector that exists as an entity, such as a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the genome of the host cell and replicated together with the chromosomes into which it is to be integrated.

The term "recombinantly expressed" - "expressed re-combinantly" - is used herein in connection with an expression of a polypeptide or protein that is defined according to the standard definition in the art.The expression rec omomb inant eme nte of a protein it is generally carried out using an expression vector as described above.

The term "isolated", when applied to a polynucleotide molecule, denotes that the polynucleotide has been deleted from its natural genetic environment and therefore is free from other unwanted or foreign coding sequences, and is in a form suitable for used within the production systems of proteins generated by genetic engineering. Such isolated molecules are those that are separated from their natural environment and that include cDNAs and genomic clones. The isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include 5 'and 3' untranslated regions that occur naturally-such as promoters and terminators. The identification of associated regions will be apparent to someone of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316: 774-78 1985). The term "an associated polynucleotide" may alternatively be referred to as "a cloned polynucleotide".

When a protein / polypeptide is applied, the term "isolated" indicates that the protein is in a condition different from its natural environment. In a preferred form, the isolated protein is ubiquitously free of other proteins, particularly other homologous proteins (ie "homologous impurities"). (see below)). It is preferred to provide the protein in a form of greater purity of 40%, more preferably greater than a purity form of 60%.

Even more preferably it is preferred to provide the protein in a highly purified form, ie, greater than 80% pure, more preferably greater than 95% pure, and even more preferably greater than 99% pure, as determined by SDS-PAGE.

The term "isolated protein / polypeptide" may alternatively be referred to as "purified protein / polypeptide".

The term "homologous impurities" means any impurity (for example another polypeptide of that polypeptide of the invention) that originates from the homologous cells in which the polypeptide of the invention is originally obtained.

The term "obtained from" as used herein in relation to a specific microbial source means that the polynucleotide and / or the polypeptide produced by the specific source, or by a cell in which a gene has been inserted from the source.

The term "endogenous to" as used herein in relation to a specific microbial source means that a polypeptide is produced by the specific source due to the presence at the source of a native gene, i.e., a gene that has not been Inserted rec omomb inant emente within a cell of the source but occurs naturally.

The term "operably linked", when referring to DNA segments, denotes that the segments are arranged to function together for indicated purposes, for example, that initiate transcription in the promoter and proceed through the coding segment up to the rmina dor.

The term "polynucleotide" denotes a single- or double-stranded polymer of bases from oxo r i b onu c 1 e or t i b e r t e r t e r t e s ted read from the 5 'end to the 3' end. The polynucleotides include RNA and DNA, and can be isolated from natural sources, synthesized in vi t r o, or prepared from a combination of natural or synthetic molecules.

The term "polynucleotide molecule complements" denotes polynucleotide molecules having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the 5 'sequence ATGCACGGG 3' is complementary to 5 'CCCGTGCAT 3'.

The term "degenerate nucleotide sequence" denotes a nucleotide sequence that includes one or more degenerate codons (compared to a reference polynucleotide molecule that encodes a polypeptide). Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (ie, GAU and GAC triplets that encode each Asp).

The term "promoter" denotes a portion of a gene that contains the DNA sequences that provide the - - linkage of the RNA polymerase and the initiation of Aa transcript. Promoter sequences are commonly, but not always, found in the non-coding regions of the genes.

The term "secretory signal sequence" denotes a DNA sequence encoding a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it will be synthesized. The larger peptide is commonly divided to remove the secretory peptide during transit through the secretory pathway.

The term "peetin" denotes pectate polygalacturonic acid, and pectin which can be esterified to a higher or lower degree.

The term "pectinase" denotes a pectinase enzyme defined according to the technique in which the pectinases are a group of enzymes that divide the glycosidic bonds - of the pectic substances, mainly poly (1,4-a-fa-D-gal). act ur onido and its derivatives (see reference Sakai et al., Pectin, pectinase and prot ope ct ina se: production, properties and applications, pp 213-294 in: Advances in Applied Microbiology vol: 39, 1993).

Preferably a pectinase of the invention is a pectinase enzyme that catalyzes the random cleavage of the alpha-1, -glycosidic bonds in the pectic acid also called polygalacturonic acid by elimination as the class of enzymes polygalact urone to lyase (EC 4.2). .2.2) (PGL) also known as poly (1, 4-alf aD-ga 1 act ur oni do) lyase also known as pectate lyase.

DETAILED DESCRIPTION OF THE INVENTION HOW TO USE A SEQUENCE OF THE INVENTION TO OBTAIN OTHER SEQUENCES RELATED: The sequence information disclosed herein refers to a polynucleotide sequence encoding a pectate--liase of the invention that can be used as a tool to identify other homologous pectate lyases. For example, the polymerase chain reaction (PCR) can be used to amplify the sequences encoding other homologous pectate lyases from a variety of microbial sources, in particular from different species of B a c i l l s.

POLYUCLEOTIDES: Within the preferred embodiments of the invention, an isolated polynucleotide of the invention will hybridize regions similar in size to SEQ. ID No. 1, 3, 5, 7 or 9, respectively, or a sequence complementary thereto, under at least medium severe conditions.

In particular, the polynucleotides of the invention will hybridize to a denatured double-stranded DNA probe comprising either the entire sequence (encoding the mature part of the polypeptide) shown in -the positions 79-1077 of SEQ. ID NO: 1, at positions 82-1026 of the SEC. ID NO: 3, at positions 541-1530 of the SEC. ID NO: 5, at positions 124-1047 of the SEC. ID NO: 7 or in positions 73-1008 of the SEC. ID NO: 9, or any probe comprising a subsequence of the SEC. ID NO: 1, 3, 5, 7 or 9, respectively, or any probe comprising a subsequence of the SEC. ID NO: 1, 3, 5, 7 or 9 having a length of at least 100 base pairs at least at least severity conditions, but preferably at high severity conditions as described in detail below. • Suitable experimental conditions to determine hybridization in the medium or high severe conditions between a nucleotide probe and an RNA or homologous DNA sequence involves a pre-selection of the filter containing the DNA or RNA fragments to hybridize in 5x SSC (Chloride of sodium diisocyte, Sambrook et al., 1989) for 10 min, and the inhibition of the filter in a 5 x SSC solution, Denhardt's solution 5 x - (Sambrook et al., 1989 ), 0.5% SDS and 100 μg / ml of denatured sonicated salmon sperm DNA (Sambrook et al., 1989), followed by hybridization in the same solution containing a concentration of 10 ng / ml of a 1-well probe - c eb a da (Feinberg, AP and Vogelstein, B. (1993) Anal. Biochem. 132: 6-13), labeled with 32P-dCTP (with a specific activity greater than 1 x 109 cpm / μg) for 12 hours for approx. 45 ° C. The filter is then washed twice for 30 minutes in 2 x SSC, 0.5% SDS at least 60 ° C (medium severity), even more preferably at least 65 ° C (medium / high severity), even more preferred ib at least 70 ° C (high severity), and even more at least 75 ° C (very high severity).

The molecules to which the oligonucleotide probe hybridizes under these conditions are detected using an x-ray film.

As previously observed the isolated polynucleotides of the present invention include DNA and RNA. Methods for isolating DNA and RNA are well known in the art. The genes encoding the DNA and RNA of interest can be cloned into the Gene Banks or DNA libraries by methods well known in the art.

Polypeptides encoding polynucleotides having pectate lyase activity of the invention are then identified and isolated by, for example, hybridization or PCR.

The present invention also provides counterpart polypeptides and polynucleotides of different bacterial strains (orthologs or paralogs). Of particular interest are the pectate lyase polypeptides from strains 1 c to 1 or f i 1 i c a s g r am-p o s i t i va s, which includes the species B a c i l l s s.

The homologues of a species of a polypeptide with pectate lyase activity of the invention can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques. For example, DNA can be cloned using the chromosomal DNA obtained from a cell type that expresses the protein. Suitable sources of DNA can be identified by Northern blotting with designated probes of the sequences described herein. A library or library is then prepared from the chromosomal DNA of a positive cell line. A DNA encoding a polypeptide having pectate lyase activity. of the invention can then be isolated by a variety of methods, such as by testing with probes with a partial or complete DNA or with one or more sof degenerate probes based on the described sequences. A DNA can also be cloned using the polymerase chain reaction; or PCR (Mullis, US Patent No. 4, 683,202), using the primers of the sequences described herein. Within an additional method, the DNA library can be used to transform or transfect host cells, and the expression of the DNA of interest can be detected with an antibody (monoclonal or polyclonal) generated against the cloned pectate lyase of B. licheniformis , ATCC 14580, the cloned pectate lyase of B. agaradhaerens, NCIMB 40482, or the cloned pectate lyase of Bacillus sp. AAI12 identified by its 16S rDNA sequence listed in SEC. ID NO: 14, or the cloned pectate lyase of Bacillus sp. KJ59 DSM 12419, or the cloned pectate lyase of Bacillus sp. 1534 identified by its 16S rDNA sequence listed in SEQ. ID NO: 13 all of which are expressed and purified as described in Materials and Methods and Examples, or by an activity test that relates to a polypeptide having pectate lyase activity. Similar techniques can also be applied to the isolation of genomic clones.

The polypeptide encoding part of the DNA sequence cloned in the plasmid pSJ1678 present in the Escherichia coli DSM 11789 and / or an analogous DNA sequence of the invention can be cloned from a strain of the bacterial species Bacillus licheniformis, preferably strain ATCC 14580, which produces the enzyme pectate lyase, or other related organism as described herein.

Similarly, the polypeptide encoding part of the DNA sequence cloned in the plasmid pSJl678 present in the Esche ichia coli DSM 11789 and / or an analogous DNA sequence of the invention can be cloned from a strain of the bacterial species Bacillus agaradhaerens as represented by strain type DSM 8721, which produces the enzyme pectate lyase, or other related organism as described herein. Also, the polypeptide encoding the part of the DNA sequence cloned in the plasmid pS Jl 678 present in the Escherichia coli DSM 12403 and 12404, respectively, and / or an analogous DNA sequence of the invention can be cloned from a strain of Bacillus sp.

- -AAI12, Bacillus sp. KJ59, DSM 12419, or Bacillus sp. 1534 which produces the enzyme pectate lyase, or other related organism as described herein.

Alternatively, the analogous sequence can be constructed based on the DNA sequence obtainable from the plasmid present in the Escherichia Coli DSM 11788, 11789, DSM 12403 and 12404, for example it can be a subsequence thereof, and / or by the introduction of nucleotide substitutions that do not give rise to another amino acid sequence of pectate lyase encoded by. the DNA sequence, but corresponding to the codon used of the host organism that is intended for the production of the enzyme, or by the introduction of the nucleotide substitutions which may give rise to a different amino acid sequence (ie a variant of the pectate lyase of the invention).

Based on the sequence information described herein, a full-length DNA sequence encoding a pectinase of the invention and comprising the DNA sequence shown in SEQ. ID No. 1, 3, 5, 7 or 9, respectively, can be cloned.

The cloning is performed by standard procedures known in the art such as by, the preparation of a genomic library from a Bacillus strain; plating of such a library on suitable substrate plates; the identification of a clone comprising a polynucleotide sequences of the invention by standard hybridization techniques using a probe based on SEQ. ID No. 1, 3, 5, 7 or 9, respectively; or by identifying a clone from for example Bacillus licheniformis ATCC 14580 or Bacillus agaradhaerens DSM 8721 or a highly related Bacillus genomic library by an inverse PCR strategy using primers based on the sequence information from the SEC. ID NO: 1, 5, 7 or 9, res ectively. It is done - reference to M.J. CPherson et al. ("PCR A Practical Approach" Information Press Ltd, Oxford England) for further details that relate to reverse PCR.

Based on the sequence information described here (SEQ ID Nos. 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10) it is routine to work for a person skilled in the art to isolate the Homologous olinucleotide sequences encoding the homologous pectinases of the invention by a similar strategy using genomic libraries of linked microbial organisms, in particular from genomic libraries of other Bacillus strains such as Bacillus subtilis.

Alternatively, the DNA encoding the pectate lyase of the invention can, in accordance with well-known procedures, conveniently be cloned from a suitable source, such as any of the organisms mentioned below, by the use of synthetic oligonucleotide probes prepared based on the DNA sequence obtainable from the plasmid present in Escherichia coli DSM 11788, DSM 11789, DSM 12403 or DSM 12404.

- - Accordingly, the polynucleotide molecules of the invention can be isolated from Es ch eri ch iacoli, DSM 11788, DSM 11789, DSM 12403 or DSM 12404, in which each of the plasmids obtained by cloning as described up is deposited. Also, the present invention relates to a substantially pure, isolated, biological culture of each of the strains Es ch e ri ch i a c o l i DSM 11788, DSM 11789, DSM 12403 and DSM 12404, respectively.

POLYPEPTIDES The amino acid sequence does not. 27-359 of the SEC. ID No. 2 is a mature pectate lyase sequence; positions 1-26 is a propeptide. The amino acid sequence does not. 28-341 of the SEC. ID No. 4 is a mature pectate lyase sequence; positions 1-27 is a propeptide. The amino acid sequence does not. 181-509 of the SEC. ID No. 6 is a mature pectate lyase sequence; positions 1-31 is a transit peptide; positions 32-86 is a first lectin domain; positions 87-134 is a second lectin domain; positions 135- - - 180 is a third lectin domain. The amino acid sequence does not. 42-348 of the SEC. ID No. 8 is a mature pectate lyase sequence; positions 1-141 is a propeptide. The amino acid sequence does not. 25-335 of the SEC. ID No. 10 is a mature pectate lyase sequence; positions 1-24 is a propeptide. It is believed that pectate lyases of the invention belong to family 1 of the polysaccharide lyases.

The present invention also provides the pectate lyase polypeptides that are substantially homologous to the mature polypeptides of SEQ. ID NO: 2, 4, 6, 8 and 10 and their species counterparts (paralogs or orthologs). The term "substantially homologous" is used herein to denote polypeptides having at least 45%, preferably at least 50%, preferably at least 60%, more preferably at least 70%, more preferably 85%, and even more preferably at least 90 %, sequence identity to the sequences shown in the SEC. ID NO: 2, 4, 6, 8 and 10, or their orthologs and paralogs. Such polypeptides will more preferably be at least 95% identical, and more preferably 98% or more identical to the sequences shown in SEQ. ID NO: 2, 4, 6, 8 and 10, or their orthologs or paralogs. The percentage of sequence identity is determined by conventional methods, by means of computer programs known in the art such as GAP provided in the GCG program package (Program Manual for the Wisconsin Pac age, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) as described in Needleman, SB and Wunsch, C.D. , (1970), Journal of Molecular Biology, 48, 443-453, which are hereby incorporated by reference in their entirety. The GAP is used with the following settings for polypeptide sequence comparison: GAP creation penalty of 3.0 and GAP extension penalty of 0.1.

The sequence identity of the polynucleotide molecules is determined by similar methods using GAP with the following settings for comparison of the DNA sequence: GAP creation penalty of 5.0 and GAP extension penalty of 0.3.

The pectate lyases of the invention comprise the first unique amino acid sequence NLNSRVP, which is believed to be unique and therefore sufficient to identify any new pectate lyase belonging to this new pectate lyase group of the present invention which has excellent functionality in Industrial processes such as the treatment and washing of textiles are preferably derived from a microorganism, preferably from a bacterium, an archaea or a fungus, especially a bacterium such as a bacterium belonging to a Bacillus, preferably a strain of Alkalophilic bacillus which may be selected from the group consisting of the species B. 1 i chen ifo rm is, B. agaradhaerens, Bacillus halodurans and the species Bacillus 1534 and AAI12 identified by the rDNA sequence listed as SEC. ID Nos. 13 or 14, respectively, and any other Bacillus species that is highly related to any of these species based on the 16S rDNA sequence aligned as explained below, preferably the species that are at least 97%, even more preferably at least 98%, homologous to each of these species These highly related B acillus species are based on the phylogenetic relationships identified using the published, aligned 16S rDNA sequences available through the ARB sequence database (published March 4, 1997 available at http: // / www. biol. chemie. tu-muenchen. / pub / ARB / data /). Sequence analysis was performed using the ARB program package (Strunck, 0. and Ludwig, W. 1995. ARB - a software environment for sequence data, Department of Microbiology, - University of Munich, Munich, Germany. mikro.biologie.tu-muenchen.de, or available via www at: http: / www. Biol. chemie. tu-muenchen. de / p ub / ARB /). The alignment was based on the secondary structure, and was performed using the automatic alignment function (version 2.0) of the ARB lineup (ARB_EDIT4) which includes the manual evaluation.

- - The sequences of the sequence were established using the Phylip Distance Matrix option (with fault settings, that is, without corrections) integrated in the ARB program package, converting the distances as shown in the sequence. percent. In accordance with this, most species closely related to B. licheniformis, ATCC 14580, is B. subtilis; the species most closely related to Bacillus sp. KJ59, DSM 12419, is Bacillus halodurans, DSM 8718 (16S data X76442) which is so close that it is believed that strain KJ59 is a strain of this species; the most closely related species Bacillus sp. AAI12 is B. alkalophilus, DSM 485 (16S data X76436) showing a homology of 97.3%; and the species most closely related to Bacillus sp. 1534 is Bacillus sp. PNl, DSM 8714 (16S data X76438) showing a 16.1 homology of 98.1%.

The phylogenetic trees were calculated using the ARB program - using a similar maximum method (FastDnaML algorithm of GJ Olsen, H. Matsuda, R. Hagstrom, and R. Overbeek.) FastDNAml: a tool for construction of phylogenetic trees of DNA sequences using maximum likelihood Comput. Appl. Biosci. 10 (l): 41-84, 1994.) using the error settings (without filter and without weighing mask).

Substantially homologous proteins and polypeptides are characterized by having one or more their amino acid cleavages, deletions or additions. These changes are preferably of a minor nature, which is conservative with the amino acid substitutions (see Table 2) and other substitutions that do not significantly affect the doubling or activity of the protein or polypeptide; small eliminations, typically one of approximately 30 amino acids; and small extensions of amino- oca rb oxi 1-te rmina 1 are, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or a small extension that facilitates purification (a 'affinity'), such as the poly-histidine tract, protein A (Nilsson et al., EMBO J. 4: 1075, 1985; Nilsson et al., Methods Enzymol 198: 3, 1991. See, in general Ford et al., Protein Expression and Purification 2: 95-107, 1991, which is incorporated herein by reference.The affinity tags encoding the DNAs are available from commercial suppliers (eg, Pharmacia Biotech), Piscataway, NJ; England Biolabs, Beverly, MA).

However, although the changes described above are preferably of a minor nature, such changes may also be of a larger nature such as the fusion of larger polypeptides of up to 300 amino acids or more as both amino-like and carboxyl-terminal extensions to a pectate lyase polypeptide of the invention.

- - Table 1 Substitutions of Basic Conservative Amino Acids argmma lysine histidine Acids Glutamic Acid Aspartic Acid Polare Glutamine Asparagine Hydrophobic Leucine Isoleucine Val ina Aroma Ticas Phenylalanine Tryptophan Tyrosine Small glycine alanine serine Threonine Methionine In addition to the 20 standard amino acids, the non-standard amino acids (such as 4-hydroxyproline, 6-N-methyl lysine, 2-amino non-obu tic acid, isovaline and a-methyl-serine) can be replaced by residue of amino acids of a polypeptide according to the invention. A limited number of non-conservative amino acids, amino acids that Ao are encoded by the genetic code, and non-natural amino acids can be substituted for amino acid residues. The "non-natural amino acids" have been modified after the synthesis of the protein, and / or have a chemical structure in their side chains different from those of the standard amino acids. The non-natural amino acids can be synthesized chemically, or preferably, are commercially available, and include pipecolic acid, thiazolidine carboxylic acid, from shidr op 1, 3- and 4-methyl-1-na , and 3,3-dimethylproline.

The essential amino acids in the pectate lyase polypeptides of the present invention can be identified according to methods known in the art, such as site-directed mutagenesis or alanine-based mutagenesis (Cunningham and -Wells, Science 244: 1081-1085). , 1989). In the latter technique, unique mutations of alanine are introduced into each residue in the molecule, and the resulting mutant molecules are tested for their biological activity (ie pectate lyase activity) to identify the amino acid residues that are critical for the activity of the molecule. See also, Hilton et al., J. Biol. Chem. 271: 4699-4708, 1996. The active site. The active site of the enzyme or other biological interaction can also be determined by physical analysis of the structure, as determined by such techniques as by nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with the mutation of the amino acids of the putative contact site. See, for example, de Vos et al., Science 255: 306-312, 1992; Smith et al., J. Mol. Biol. 244: 899-904, 1992; Wlodaver et al. , FEBS Le 11. 309: 59-64, 1992. The identities of the essential amino acids can also be inferred from the analysis of the homologies with polypeptides that are related to a polypeptide according to the invention.

Amino acid substitutions can be made and tested using known methods of mutagenesis, recbomb ination and / or intermixing followed by a relevant selection procedure, such as those described Re i dha ar-01 s on and Sauer (Science 241: 53 -37, 1988), Bowie and Sauer (Proc. Nati, Acad. Sci. USA 8_6: 2152 -2156, 1989), W095 / 17413, or WO 95/22625. Briefly, these authors describe the methods to generate a simultaneous 1 or 2 randomization of two or more positions in a polypeptide, orec omb ination / entry of different mutations (W095 / 17413, W095 / 22625) , followed by the functional selection of a polypeptide, and then forming the sequence of the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include the phage sample (e.g., Lowman et al., Biochem 30: 10832-10837, 1991; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., Gene 46: 145, 1986; Ner et al., DNA 7: 127, 1988).

The mutli-ions and remembend methods are described above and can be combined with high-throughput automated screening methods to detect the activity of mutagenized polypeptides cloned in the host cells. The mutagenized DNA molecules encoding the active polypeptides can be recovered from the host cells and rapidly sequenced using the modern eq.uipo. These methods allow rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.

Using the methods discussed above, one of ordinary skill in the art can identify and / or prepare a variety of polypeptides that are substantially homologous to residues 27 to 359 of the SEC. ID NO: 2, to residues 28 to 341 of the SEC. ID NO: 4, to residues 181 to 509 of the SEC. ID NO: 6, to residues 42 to 348 of the SEC. ID NO: 8 and to residues 25 to 335 of the SEC. ID NO: 10 and retain the pectate lyase activity of the native type protein.

Such variants of the invention are the pectate lyases that have, in position 223 in relation to the numbering in the alignment of the sequence of Figure 1, the arginine residue of the amino acids. In a preferred embodiment, such variant can maintain an arginine conserved at position 228 relative to the numbering in the alignment of the sequence of Figure 1. Accordingly, the present invention relates to pectate lyases having a sequence of amino acids that is derived from any of the amino acid sequences SEC. ID NO: 2, 4, 6, 8 and 10 by elimination, replacement or addition of one or more amino acid residues (hereinafter referred to as a mutation) as long as the pectate lyase activity is not deactivated and the mutation remains the arginine at position 223 and optionally also the arginine at position 228 of the sequence numbering in the alignment of Figure 1. These positions correspond to arginine (R) at position 240 and at position 245 in SEC . ID NO: 2, to positions 233 and 238 in the SEC. ID NO: 4, to positions 390 and 395 in the SEC. ID NO: 6, to positions 240 and 245 in the SEC. ID NO: 8, and to positions 227 and 232 in the SEC. ID NO: 10 In addition to the arginines conserved above, the mutation preferably retains aspartic acid (D) at position 169 and / or aspartic acid (D) at position 173 and / or lysine (K) at position 193 of the numbering of the sequence in the alignment of Figure 1. These positions correspond to aspartic acid (D) at position 186 and at position 190 and lysine (K) at position -210 in SEC. ID NO: 2; to positions D180, D184 and K204 in SEC. ID NO: 4; to positions D336, D340 and K360 in SEC. ID NO: 6; to positions D187, D191 and K211 in SEC. ID NO: 8; and to positions D174, D178 and K198 in the SEC. ID NO: 10. In a further embodiment of the invention, mutants of the mature progenitor polypeptides of any of the sequences listed in SEQ. ID Nos.: 2, 4, 6, 8 and 10, the mutants are active pectate lyases that have the aspartic acids in the specified positions above replaced with a selected amino acid residue -from the group consisting of glutamic acid (E), serine (S) and threonine (T), ie the following mutations: D169E, D169S, D169T, D173E, D173S, D173T (the alignment numbering of Figure 1).

Also, the degree of mutation is not particularly limited with the condition that the arginine described above is retained at position 223. Preferably, there is a 40% or higher homology between such mutant variants of the native pectate lyase enzymes. or progenitors, calculated on any of the SEC. ID Nos.: 2, 4, 6, 8 and 10: the homology of 42% or higher exists between amino acid positions 39 and 359 of the SEC. ID NO: 2 and amino acid positions 46 and 341 of SEC. ID NO: 4; the homology of 44% or higher exists at amino acid positions 187 and 509 of the SEC. ID NO: 6; the homology of 40% or higher exists in amino acid positions 50 and 348 of the SEC. ID NO: 8; and the homology of 41% or higher exists in the positions of amino acids 40 and 335 of the SEC. ID NO: 10. Preferably, the homology is at least 45%, preferably at least 50%, more preferably at least 55%, more preferably at least 60%, even more preferably at least 70%, even more preferably at least 75% %, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more - preferably at least 95%, especially at least 98%.

The pectate lyase of the invention can, in addition to the core of the enzyme, comprise the domain catalytically, it also comprises a cellulose binding domain (CBD), the cellulose binding domain and the enzyme core (catalytically active domain) of the enzyme is linked operamente. The cellulose binding domain (CBD) can exist as an integral part of the encoded enzyme, or a CBD from another source can be introduced into the. enzyme that degrades lactinin thus creating an enzyme hybrid. In this context, the term "cellulose binding domain" is meant to be understood as defined by Peter Tomme et al. "Cellulose-Binding Domains: Classification and Properties" in "Enzymatic Degradation of Insoluble Carbohydrates", John N. Saddler and Michael H. Penner (Eds.), ACS Symposium Series, No. 618, 1996. This definition classifies more than 120 domains of cellulose binding in 10 families (IX), and demonstrates that CBDs are found in several enzymes such as cellulases, xylanases, mannanases, ar ab in of anurines, acetyl esterases and chitinases. CBDs are also found in algae, for example, the red alga P or rphyra p u rp u r e a as a non-hydrolytic polysaccharide binding protein, see Tomme et al. , op. c i t. However, most of the CBDs are cellulases and xylanases, the CBDs are found in the N and C terminals of the proteins and are internal. Enzyme hybrids known in the art, see for example WO 90/00609 and WO 95/16782, can be prepared by transforming a DNA construct comprising at least one DNA fragment encoding the domain into a host cell. binding to bound cellulose, with or without a linker, to a DNA sequence encoding the enzyme that degrades pectin and the growth of the host cell to express the fused gene. The enzyme hybrids can be described by the following formula: CBD-MR X wherein CBD is the N-terminal or C-terminal region of an amino acid sequence corresponding to at least the cellulose binding domain; MR is the middle region (the linker), and may be a bond, or a short linking group preferably from about 2 to about 100 carbon atoms, more preferably from 2 to 40 carbon atoms; or is preferably from about 2 to about 100 amino acids, more preferably from 2 to 40 amino acids; and X is an N-terminal or C-terminal region of the pectin that degrades the enzyme of the invention.

Preferably, the enzyme of the present invention has its maximum catalytic activity at a pH of at least 8, more preferably greater than 8.5, more preferably greater than 9, more preferably greater than 9.5, more preferably greater than 10, even more preferably greater than 10.5, especially greater than 11; and preferably the enzyme activity -maximum is obtained at a temperature of at least 50 ° C, more preferably at least 55 ° C.

PRODUCTION OF PROTEIN The polypeptides of the present invention, including full length proteins, fragments thereof and fusion proteins, can be produced in host cells by genetic engineering according to conventional techniques. Suitable host cells are those types of cells that can be transformed or transfected with exogenous DNA and grown in a culture, and include bacteria, fungal cells, and higher, cultured eukaryotic cells. Bacterial cells, particularly cultured cells of gram-positive organisms, are preferred. Gram-positive cells of the genus of Bacillus are especially preferred, such as Bacillus subtilis, Bacillus lentus, Bacillus brevis, Bacillus stearo thermophilus, Bacillus-a lkalophi lus, Bacillus amy 1 or 1 i f a c i e n s, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus thu r i ng i in s, Bacillus agaradhaerens or Bacillus licheniformis.

Techniques for the manipulation of cloned DNA molecules and exogenous introduction into a variety of host cells are described by Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY 1987; and (Bacillus-subtilis and other Bacteria Gr am- P o s i t i va s Sonensheim et al., 1993, American Society for Microbiology, Washington D.C.), which are incorporated herein by reference.

In general, a DNA sequence encoding a pectate lyase of the present invention is operably linked to other genetic elements required for its expression, generally including a transcription-promoter and a terminator within an expression vector. The vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain selectable systems markers can be provided in separate vectors, and replication of the exogenous DNA can be provided by the integration into the host cell genome. The selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through providers such as relays.

To direct a polypeptide in the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence, the prepro sequence or the pre sequence) is provided in the expression vector. The secretory signal sequence may be that of the polypeptide or it may be derived from another secreted or synthesized protein of n o vo. Numerous suitable secretory signal sequences are known in the art and are referred to (Bacillus subtilis and other Bacterias Gr am-P ositi va s, Sonensheim et al., 1993, American Society for Microbiology, Washington "DC; and Cutting, SM (eds.) "Molecular Biological Methods for Bacillus", John Wiley and Sons, 1990) for further description of the secretory signal sequences suitable especially for secretion in a Bacillus host cell.The secretory signal sequence binds to the DNA sequence in the open reading structure The secretory signal sequences are commonly positioned 5 'to the DNA sequence encoding the polypeptide of interest, although certain signal sequences can be positioned anywhere in the DNA sequence of interest (See, for example, Welch et al., U.S. Patent No. 5,037,743, Holland et al., U.S. Patent No. 5,143,830).

- Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells. A variety of suitable media, including defined media and complex media, are known in the art and generally include a source of carbon, a source of nitrogen, essential amino acids, vitamins and minerals. The media may also contain such components as growth factors or serum, as required. The growth medium will generally be selected for cells containing the exogenously added DNA by, for example, the selection or deficiency of drugs in an essential nutrient which is complemented by the selectable marker carried in the expression vector or co-transfected into the host cell.

The polypeptides of the present invention can also be produced by the fermentation of a native strain belonging to the genus Bacillus, preferably a strain which can be selected from the group consisting of the species Bacillus licheniformis, Bacillus agaradhaerens and Bacillus species. highly related in which all species are at least 95% homologous to Bacillus licheniformis based on aligned 16S rDNA sequences. Specific and highly preferred examples are Bacillus licheniformis, ATCC 14580, and Bacillus agaradhaerens, DSM 8721.

In addition, the polypeptides of the present invention can be produced by the fermentation of a mutant or a variant derived from the strain mentioned above. Such a mutant can be obtained using conventional mutagenesis by subjecting the strain in question to treatment with a mutagen (for example NTG (n-ethi 1-N-nitro-N-nitroso guan i di a)) or to ultraviolet radiation, for example as described in the Manual of Methods for General Bacteriology; ASM 1981, Chapter 13. This mutagenesis is done to stimulate the mutation of the strains. Following mutagenesis a selection for mutants that high pectinase yields may be possible using conventional plate assays or liquid assays.

- The fermentation can be carried out by culturing the strain under aerobic conditions in a nutrient medium containing carbon and nitrogen sources together with other essential nutrients, the medium being composed according to the principles of the known art. The medium can be a complex rich medium or a minimal medium. The nitrogen source may be of inorganic and / or organic nature. The sources of inorganic nitrogen nitrogen are nitrates and ammonium salts. Among the organic nitrogen sources there is a large number that is regularly used in fermentations. Examples are soybean meal, casein, corn, corn liquor, yeast extract, urea and albumin. The proper carbon sources are carbohydrates or materials that contain carbohydrates. Preferably the nutrient medium contains pectate, polygalacturonic acid and / or pectin esterified to a higher or lower degree as a carbon source and / or inducer of the production of pectinase. Alternatively, the medium contains a material rich in pectin such as soybean meal, apple pulp or lemon peel.

Since the Bacillus species of this invention are at least 1%, the culture is preferably conducted at alkaline pH values such as at least pH 8 or at least pH 9, which can be obtained by the addition of suitable buffers such as sodium carbonate or mixtures of sodium carbonate and sodium bicarbonate after the removal of the growth medium.

It is contemplated that fermentation of a native or mutant type strain in a suitable medium can result in a yield of at least 0.5 g of the pectinase protein per liter of culture broth or even at least 1 g / 1 or 2 g / l. .

ISOLATION OF PROTEIN: When the expressed recombinant polypeptide is secreted the polypeptide can be purified from the culture medium. Preferably, the expression host cells are removed from the medium prior to purification of the polypeptide (for example by centrifugation).

When the expressed recombinant polypeptide is not secreted from the host cell, the host cell preferably breaks down and the polypeptide is released into an aqueous "extract" which is the first step of such purification techniques. Preferably the expression host cells are removed from the medium before the cells break (for example by centrifugation).

The breaking of the cells can be done by conventional techniques such as by digestion - by lysozyme or by forcing the cells through high pressure. See (Robert K. Scobes, Protein Purification, Second edition, Springer-Verlag) for an additional description of such cell disruption techniques.

Either the expressed recombinant polypeptides (or the chimeric polypeptides) are secreted or can not be purified using conventional and / or fractionation purification methods and the corresponding media.

The precipitation of ammonium sulfate and acid or extraction of chaotrope can be used to fractionate the samples. Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse phase high resolution liquid chromatography. The anion exchange media include derivatives of dextrans, agarose, cellulose, polyacrylamide, specialized silicas and the like. PEI, DEAE, QAE and Q derivatives are preferred with DEAE Rapid Flow Sepharose (Pharmacia, Piscataway, NJ) which is the one that is particularly preferred. Exemplary cryptographic means include those mediated with phenyl, butyl or octyl groups, such as Fen i 1-S ep harose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Mont gomeryvi 11 e, PA ), Oct i 1 - S epharo se (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like. The solid supports include - glass beads, silica-based resins, cellulosic resins, agarose beads, crosslinked agarose beads, polystyrene beads, crosslinked polyacrylamide resins and the like which are insoluble under the conditions in which they are going to use. These supports can be modified with reactive groups that allow the binding of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups, and / or carbohydrate moieties. Examples of the coupling chemistries include activation with cyanogen bromide, activation and hydrotransmission, activation by epoxide, activation by sulfhydryl, activation by hydrazide, and amino and carboxyl derivatives for the coupling chemistries. carbodiimide. These and other solid media are well known and widely used in the art, and are available from commercial suppliers.

The selection of a particular method is a routine design material and is partly due to the properties of the chosen support. See, for example, Affinity Chromatography: Principies- & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden 1988.

The polypeptides of the invention or fragments thereof can also be prepared through chemical synthesis. The polypeptides of the invention can be monomers or multimers; glycosylated or non-glycosylated; pegylated or non-pegylated; and may or may not include an initial methionine amino acid residue.

TRANSGENIC PLANTS The present invention also relates to a transgenic plant, plant parts or plant cells that can be transformed with a DNA sequence encoding the enzyme that degrades the pectin of the invention to express and produce this enzyme in recoverable amounts. The enzyme can be recovered from the plant or part of the plant. Alternatively, the plant or part of the plant containing the recombinant enzyme can be used as such.

The transgenic plant can be dicotyledonous or mo n o c o t i 1 e d e, for a dicotyledon or mon o c o t i 1 e short don. Examples of monocotyledonous plants are grasses, such as hay (bluegrass, Poa) forage grass such as fescue, lolium, temperate grass such as Agrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum and corn (cereal).

Examples of dicotyledonous plants are tobacco, legumes, such as lupins, potatoes, sugar beet, peas, beans and soybeans and cruciferae (family B rassicaceae), such as cauliflower, oilseed turnip and organism models closely of Arabidopsis thaliana.

Examples of plants are stems, tripe, leaves, roots, fruits, seeds, and tubers. In the present context, the tissues of plants, such as chloroplast, apoplast, mitochondria, vacuoles, peroxisomes, and cytoplasm are also considered as part of a plant. In addition, any plant cell, whatever the origin of the tissue, is considered part of a plant.

The progeny of such plants, parts of plants and plant cells are also included within the scope of the invention.

The transgenic plant or plant cell expressing the enzyme of the invention can be constructed according to methods known in the art. In summary, the plant or plant cell is constructed by incorporating one or more expression constructs encoding the enzyme of the invention in the host genome of the plant and propagating the resulting modified plant or the resulting plant cell in a transgenic plant or transgenic plant cell - Conveniently, the expression construct is a DNA construct comprising a gene encoding the enzyme of the invention in operable association with the appropriate regulatory sequences required for the expression of the gene in the plant or part of the chosen plant. In addition, the expression construct may comprise a selectable marker useful for identifying host cells within which the expression construct has been integrated and the DNA sequences necessary for the introduction of the construct into the plant in question (the latter depends of the method of introduction of the DNA that is going to be used).

The choice of regulatory sequences, such as promoter and terminator sequences and optionally the signal or transit sequences is determined, for example on the basis of when, where and how it is desired that the enzyme is to be expressed. For example, the expression of the gene encoding the enzyme of the -8 O -invention may be constitutive or inducible or it may be of a labile, tissue-specific or step-wise role and the product of the gene may be an objective directed at a specific tissue or part of plant such as seeds or leaves. Regulatory sequences are described, for example, in Tague et al, Plant, Phys., 86, 506, 1988.

For constitutive expression the 35S-CaMV promoter can be used (Franck et al., 1980, Cell 21: 285-294). Organ-specific promoters may for example be a promoter for storing bury-ing tissues such as seeds, tubers of potatoes and fruits (Edwards &Coruzzi, 1990. Annu. Rev. Genet. 24: 275-303 ), or from burnable, metabolic tissues, such as meristems (Ito et al., 1994. Plant Mol. Biol. 24: 86.3-878), a seed-specific promoter such as glutelin, prolamin, globulin or rice albumin promoter (Wu et al., Plant and Cell Physiology Vol. 39, No. 8 pp. 885-889 (1998)), a Vi ciafaba promoter of legumin B4 and the protein gene of the - - unknown seed of Vi cia fa ba described by Conrad U. et al, Journal of Plant Physiology Vol. 152, No. 6 pp. 708-711 (1998), a promoter of an oily seed protein from the seed (Chen et al., Plant and cell physiology vol 39, No. 9 pp. 935-941 (1998), the napA promoter of the protein of storage of Brassica napus, or any other seed-specific promoter known in the art, for example as described in WO 91/14772. In addition, the promoter may be a leaf-specific promoter such as the rice rbcs promoter or of the tomato (Kyozuka et al., Plant Physiology Vol. 102, No. 3 pp. 991-1000 (1993), the promoter of the adenine me ti 11 transferase gene of the clone virus (Mitra, A. and Higgins, DW, Plant Molecular Biology Vol. 26, No. 1 pp. 85-93 (1994), or the promoter of the rice aldP gene (Kagaya et al., Molecular and General Genetics Vol. 248, No. 6 pp. 668-674 (1995), or a wound-inducible promoter such as the potato pin2 promoter (Xu et al, Plant Molecular Biology Vol. 22, No. 4 pp. 573-588 - (1993) A promoter enhancer element can be used to improve a greater expression of the enzyme in the plant. for example, the promoter enhancer element can be an intron which is placed between the promoter and the nucleotide sequence encoding the enzyme. For example, Xu et al., Op c i t describes the use of the first intron of the actin 1 gene of rice to improve expression.

The selectable marker gene and any other part of the expression construct can be chosen from those available in the art.

The DNA construct is incorporated into the genome of the plant according to the conventional techniques known in the art, which include Agrobacterium-mediated transformation, virus-mediated transformation, micro-injection, particle bombardment, biolistic transformation, and electroporation (Gasser et al, Science 244, 1293; Potrykus, Bio / Techn., 8, 535, 1990; Shimamoto et al., Nature, 338, 274, 1989).

In the present, the transfer of genes mediated by A gr ob acteri um t urn efaci in ses the method of choice to generate transgenic dicotyledons (for review Hooykas &Schilperoort, 1992, Plant Mol. Biol. 19: 15-38) however, it can be used to transform mon ocoti 1 ed nene, although other transformation methods are generally preferred for these plants. At the present time, the method of choice for generating transgenic cogeneration monocytes is the bombardment of embryonic embryo developing particles (microscopic tungsten or gold particles coated with the transforming DNA).

(Christou, 1992. Plant J. 2: 275-281; Shimamoto, 1994. Curr Opin Opin Biotechnol 5: 158-162; Vasil et al., 1992. Bio / Technology 10: 667-674). An alternative method for the transformation of the monocytes is based on the transformation of the protoplasts as described by Omirulleh S, et al., Plant Molecular biology Vol. 21, No. 3 pp. 415-428 (1993).

After transformation, transformants that have incorporated the expression construct are selected and regenerated into whole plants according to methods well known in the art.

PREPARATION OF ENZYME In the present context, the term "enzyme preparation" is intended to indicate either a conventional enzymatic fermentation product, possibly isolated and purified, of a single species of a microorganism, such preparation usually comprising a number of different enzymatic activities; or a mixture of enzymes of non-specific substances, preferably enzymes derived from bacterial or fungal species by the use of conventional recombinant techniques, whose enzymes - have been fermented and possibly isolated and purified separately and which may originate from different species , preferably of fungal or bacterial species; or the fermentation product of a microorganism that acts as a host cell for the expression of a recombinant pectate lyase, but whose microorganism simultaneously produces other enzymes, for example pectin lyases, proteases, or cellulases, which occur naturally as products of the fermentation of the microorganisms, that is to say the complex of the enzyme conventionally produced by the microorganisms that occurs naturally.

The preparation of the pectate lyase of the invention can fur comprise one or more enzymes selected from the group consisting of proteases, cellulases (endo-β-1,4-glucanases), β-glucanases (endo-β-1, 3 (4 ) -glucanases), lipases, cutinases, peroxidases, laccases, amylases, glucoamylases, pectinases, reductases, oxidases, phenoloxidases, ligninases, - -pululanases, ar ab in anasas, hemicellulases, mannanases, xi 1 og 1 uc anaas, xylanases , pectin acetyl esterases, rhamn oga 1 actur on an actil esterasas, polygalacturonasas, rhanmnogalacturonasas, ga 1 actanasas, pectin liases, pectin metilesterasas, celobiohidrolasas, transglutaminasas; or mixtures thereof. In a preferred embodiment, one or more or all of the enzymes in the preparation is produced using recombinant techniques, ie the enzyme or enzymes is / are mono-component enzymes that are mixed with the other enzymes to form an enzyme preparation with the mixture of desired enzymes.

IMMUNE CROSS REACTIVITY Polyclonal antibodies (which are mon sp e c i f i c s for a given enzyme protein) are to be used in the determination of immunological cross-reactivity and can be prepared for use of a purified pectate lyase enzyme. More specifically, the antiserum against the pectate lyase of the invention can be generated by the immunization of rabbits (or other rodents) according to the procedure described by N. Axelsen et al. in: A Manual of Quantitative Immunoelect Rophores is, Blackwell Scientific Publications, 1973, Chapter 23, or A. Johnstone and R. Thorpe, Immun or ch emi s t r and in Practice, Blackwell Scientific Publications, 1982 (more specifically p 27-31). Purified immunoglobulins can be obtained from the antiserum, for example by saline precipitation ((NH) 2 SO4), followed by ion exchange chromatography and dialysis, for example in DEAE-Sephadex. The immunochemical characterization of the proteins can be done either by means of a double-diffusion analysis of Outcherlony (O. Outchterlony in: Handbook of Experimental Immunology (DM Weir, Ed.), Blackwell Scientific Publications, 1967, pp. 655-706), by cross-linked immunoelectrophoresis (N. Axelen et al., supra, Chapters 3 and 4), or by rocket immunoelectrophoresis (N. Axelsen et al., Chapter 2).

Use in the detergent industry and cleaning In additional aspects, the present invention relates to a detergent composition comprising the pectate lyase enzyme or the preparation of the enzyme of the invention, to a process for treatment by machines of fabrics comprising treating the fabrics during the washing cycle of a machine washing process with a washing solution comprising the enzyme pectate lyase or the preparation of the enzyme of the invention, and cleaning compositions, including laundry, cleaning of hard surfaces, personal cleansing and compositions of either 1 st / den to 1 st, comprising a pectate lyase enzyme or the preparation of the enzyme of the invention that provide a superior cleaning activity, ie a superior removal of the stains .

Without being bound to this theory, it is believed that the mannanase of the present invention is capable of effectively degrading or hydrolyzing any stain or dirt containing ga 1 actomannan and, accordingly, cleaning by laundry comprising such stains or soils .

The cleaning compositions of the invention will contain at least one additional detergent component. The precise nature of these additional components, and the levels of incorporation thereof will depend on the physical form of the composition, and the nature of the cleaning operation for which it is to be used.

The cleaning compositions of the present invention preferably also comprise a detergent ingredient selected from a selected surfactant, another enzyme, a consistency generator and / or a bleaching system.

The cleaning compositions according to the invention can be liquids, pastes, gels, sticks, tablets, sprays, foams, powders or granules. The granular compositions may also be in a "compact" form and the liquid compositions may also be in a "concentrated" form.

The compositions of the invention for example, can be formulated manually and for machine wash compositions, and the machine and hand washing detergent compositions include additive compositions for laundry and compositions suitable for use in the pretreatment of soiled fabrics, fabric softening and rinsing compositions can be used and added, and the compositions for use in hard surface cleaning operations at home, in general. Compositions containing such carbohydrates can also be formulated as cleaning products, contact lens cleaners, and health and beauty care products such as personal cleansing compositions and oral / dental care.

When formulations are formulated as compositions for use in manual dishwashing methods, the compositions of the invention preferably contain a surfactant and preferably other detergent compounds selected from organic polymeric compounds, stain removal improving agents, metal ion Group II, solvents, hydrotropes and additional enzymes.

When formulated as suitable compositions for use in a laundry machine laundry method, the compositions of the invention preferably contain both a surfactant and a consistency generating compound - and additionally one or more detergent components preferably selected from the organic polymeric compounds , bleaching agents, additional enzymes, stain suppressants, dispersants, dispersants, soap foam dispersants, stain suspensions and antiredeposition agents and corrosion inhibitors. The laundry compositions may also contain softening agents, as additional detergent components. Such carbohydrate-containing compositions can provide fabric cleaning, stain removal, maintenance of whiteness, softness, appearance of color, inhibition of color transfer and sanitization when formulated as laundry detergent compositions.

The compositions of the invention can also be used as detergent additive products in solid or liquid form. Such additive products are intended to supplement or extend the performance of conventional detergent compositions and can be added at any stage of the washing process.

If needed, the density of laundry detergent compositions are in the range of 400 to 1200 g / liter, preferably 500 to 950 g / liter of the composition measured at 20 ° C.

The "compact" form of the compositions of the present invention is best reflected by density and, in terms of the composition, by the amount of inorganic filler salt; the inorganic filler salts are conventional ingredients of the detergent compositions in powder form; in conventional detergent compositions, the filler salts are present in substantial amounts, typically 17-35% by weight of the total composition. In compact compositions, the filler salt is present in amounts not exceeding 15% of the total composition, preferably not exceeding 10%, more preferably - not exceeding 5% by weight of the composition. The inorganic filler salts, such as those of the present compositions, are selected from the alkali metal salts and metal salts at 1 to 1 and not at sulfate and chloride levels. A preferred filler salt is sodium sulfate.

The liquid detergent compositions according to the present invention can also be in a "concentrated form", in this case, the liquid detergent compositions according to the present invention will contain a lower amount of water, compared to conventional liquid detergents. . Typically the water content of the concentrated liquid detergent is preferably less than 40%, more preferably less than 30%, more preferably less than 20% by weight of the detergent composition.

The specific detergent compounds to be used herein are selected from the group. which consists of the specific compounds as described in WO 97/01629 which is incorporated herein by reference in its entirety.

The mannanase can be incorporated into the cleaning compositions according to the invention, preferably at a level of 0.0001% to 2%, more preferably from 0.0005% to 0.5%, more preferably from 0.001% to 0.1% of the pure enzyme per weight of the composition Cells that can be used in the present invention include both bacterial and fungal cellulases. Preferably they will have an optimum pH of between 5 and 12 and a specific activity above 50 CEVU / mg (Cellulose Viscosity Unit). Suitable cellulases are described in U.S. Patent No. 4,435,307, J61078384 and WO 96/06253 which describe fungal cellulases produced by Humicola insolens, Trichoderma, Thielavia and Sporotricum, respectively. EP 739 982 describes cellulases isolated from new species of Bacillus. Suitable cellulases are also described in GB-A-2075028; GB-A-2095275; DE-OS-22 47 832 and WO 95/26398.

Examples of such cellulases are cellulases produced by a strain of Humicola insolens (Humicola grísea var. Thermoidea), particularly the strain Humicola insolens, DSM 1800. Other suitable cellulases are cellulases originated from Humi co insolens having a molecular weight of about 50 kD, an isoelectric point of 5.5 and containing 415 amino acids; and an endo-beta-1,4-glucanase derived from approximately 43 derived from kD Humicola insolens, DSM 1800; a preferred cellulase has the amino acid sequence described in PCT Patent Application No. WO 91/17243. The cellulases also available are the EGIII cellulases of Trichoderma lon ibrachiatum described in WO 94/21801. Especially suitable cellulases are cellulases that have color care benefits. Examples of such cellulases are those cellulases described in WO 96/29397, E P-A-0495257, WO 91/17243, WO 91/17244 and WO 91/21801. Other cellulases suitable for the care of the fas and / or with cleaning properties are described in WO 96/34092, WO 96/17994 and WO 95/24471.

Said cellulases are normally incorporated in the detergent composition at levels from 0.0001% to 2% of the pure enzyme by weight of the detergent composition.

- - Preferred cellulases for the purpose of the present invention are alkaline cellulases, ie enzymes having at least 25%, more preferably at least 40% of their maximum activity at a pH ranging from 7 to 12. More preferred cellulases are enzymes which have their maximum activity at a pH ranging from 7 to 12. A preferred alkaline cellulase is the cellulase sold under the trade name Carezyme® by Novo Nordisk A / S.

Amylases (a and / or ß) can be included for the elimination of carbohydrate-based stains. WO 94/02597, from Novo Nordisk A / S published on February 3, 1994, describes cleaning compositions incorporating mutant amylases. See also WO 95/10603, from Novo Nordisk A / S published on April 20, 1995. Other amylases known to be used in cleaning compositions include both a and β-amylases. Amylases a and β are known in the art and include those described in US Patents Nos. 5,003,257; EP 252,666; WO 91/0353; FR 2,676,456; EP 285,123; EP 525,610; EP 368,341; and in the British Patent specification no. 1,296,839 (Novo). Other suitable amylases are the amylases improved in their stability described in WO 94/18314, published on August 18, 1994 and WO 96/05295, Genencor, published on February 22, 1996 and the variants of amylases that have additional modification in the immediate generating amylase available from Novo Nordisk A / S, described in WO 95/10603, published in April 1995. Amylases described in EP 277 216, WO 95/26397 and WO 96/23873 (all from Novo) are also suitable. Nordisk).

Examples of commercial α-amylases products are Purafect Ox Am® from Genencor and Termamyl®, Ban®, Fungamyl® and Duramyl®, all available from Novo Nordisk A / S Denmark. WO 95/26397 describes other suitable amylases: the α-amylases characterized by having a specific activity at -25% higher than the specific activity of Termamyl® in a temperature range of 25 ° C to 55 ° C and a pH value in the range of 8 to 10, measured by the activity assay of Phadebas® α-amylase. The variants of the above enzymes described in WO 96/23873 (Novo Nordisk) are suitable. Other amylolytic enzymes with improved properties with respect to the activity level and the combination of thermostability and a higher activity level are described in WO 95/35382.

Preferred amylases for the purpose of the present invention are the amylases sold under the trade name Termamyl, Duramyl and Maxamyl and the α-amylase variants which demonstrate increased thermostability described as SEC. ID No. 2 in WO 96/23873.

Preferred amylases for specific applications are alkaline amylases, ie enzymes having an enzymatic activity of at least 10%, preferably at least 25%, more preferably at least 40% of their maximum activity at a pH ranging from 7 up to 12 The most preferred amylases are enzymes that have their maximum activity at a pH ranging from 7 to 12.

The amylolytic enzymes are incorporated in the detergent compositions of the present invention at a level from 0.0001% to 2%, preferably from 0.00018% to 0.06%, more preferably from 0.00024% to 0.048% pure enzyme per weight of the composition.

The term x i 1 or g 1 u c a a s s encompasses the family of enzymes described by Vincken and Voragen at Wageningen University [Vincken et al (1994) Plant Physiol., 104, 97-107] and they are able to degrade the 'Xyloglucan as described in Hayashi et al (1989) Plant Physiol. Plant Mol. Biol., 40, 139-168. Vincken et al demonstrated that the removal of cellulase xyloglucan coating from the cell wall of the isolated apple by a purified xyloglunase from Tr i ch or d rm a vi ri (endo-IV-glucanase). This enzyme improves the enzymatic degradation of cellulose embedded in cell walls and works in synergy with pectic enzymes. The fast LIQ + of Gist-Brocades contains an activity of x i 1 or g 1 u c a n a s a.

This xyoglycan is incorporated into cleaning compositions according to the invention preferably at a level from 0.0001% to 25, more preferably from 0.0005% to 0.5%, more preferably from 0.001% to 0.1% pure. by weight of the composition.

Preferred xi 1 og 1 uc anas for specific applications are alkaline xyglucans, ie enzymes having an enzymatic activity of at least 10%, preferably at least 25%, more preferably at least 40% of their maximum activity at a pH ranging from 7 to 12. The most preferred xi 1 or gl uc ains are the - -zymes having their maximum activity at a pH ranging from 7 to 12.

The aforementioned enzymes can be used of any suitable origin, such as of vegetable, animal, bacterial, fungal and yeast origin. The origin can also be mesophilic or extremophilic (p s i e f i 1 i co, ps i cr o t r o f i co, thermophilic, barophilic, 1 c a 1 or f i 1 c, acidophilic, halophilic, etc.). Purified or unpurified forms of these enzymes can also be used. At present, it is a common practice to modify native-type enzymes via genetic engineering techniques to utilize their efficiency performance in the cleaning compositions of the invention. For example, the variants can be designed in such a way that the compatibility of the enzyme to the ingredients that are commonly found in such compositions can be increased. Alternatively, the variant can be designed in such a way that the optimum pH, the chelating or bleached stability, the catalytic activity and the like, of the enzyme variant is suitable for a particular cleaning application.

In particular, attention should be focused on amino acids sensitive to oxidation in the case of bleaching stability and in surface charges for the compatibility of the surfactant. The isoelectric point of such enzymes can be modified by the substitution of some charged amino acids, for example an increase in the isoelectric point can help to improve compatibility with anionic surfactants. The stability of the enzymes can be further enhanced by the creation of for example bridges of additional salts and binding sites of reinforcing metal to increase the chelating stability.

- -Use in the textile and cellulosic fiber processing industry The pectate lyase of the present invention can be used in combination with other enzymes that degrade carbohydrates (eg arabinanase, xi 1 or 1 ucanase, pectinase) for fiber op bration or for fiber cleaning in combination with detergents Cotton fabrics consist of a primary or main cell wall layer containing pectin and a secondary layer containing mainly cellulose. Under the preparation of cotton or cotton refining parts the main cell walls will be eliminated. The present invention relates to any type of assistance during the refining of cotton by removing the main cell walls. 0 during the cleaning of the cotton to remove residual pectic substances and prevent the textiles from turning gray.

- - In the present context, the term "cellulosic material" indicates fibers, stitched and non-sewn fabrics, including knitted fabrics, woven fabrics, denims, yarns and towels, made of cotton, cotton blends or cellulosic fibers made by man or natural (for example, they originate from cellulosic fibers containing xylan such as wood pulp) or mixtures thereof. Examples of blends are mixtures of plain cotton and viscose with one or more accompanying materials such as wool, synthetic fibers (eg, polyamide fibers, acrylic fibers, polyester fibers, polyvinyl alcohol fibers, polyvinyl, polyvinylidene chloride fibers, polyurethane fibers, polyurea fibers, aramid fibers), and cellulose-containing fibers (eg rayon / vi scosa, ramie, hemp, flax fiber / flax, jute, acetate fibers of cellulose, lyocell).

The preparation of the present invention is useful in the cellulose fiber processing industry for the pretreatment or retting of hemp fibers, flax or flax fibers.

The processing of the cellulosic material for the textile industry, such as for example cotton fibers, into a material ready for the manufacture of garments involves several steps: spinning of the fibers into a yarn; the construction of woven or knitted fabrics of the threads and the subsequent preparation, dyeing and finishing operations. Woven articles are constructed by weaving a stuffed yarn between a series of wrapping yarns; the threads could be of two different types. Woven articles are constructed by forming a network of loops that intertwine from a continuous length of the yarn. Cellulosic fibers can also be used for non-woven fabrics.

The preparation process prepares the textile for the appropriate response in weaving operations. The sub-stages - involved in the preparation are a. 'Unplaned or despressed (for woven articles) using polymeric sizing such as for example the addition of cotton, CMC or PVA before interwoven to increase the fabric speed; This material must be removed before further processing; b. Purification or washing, the objective of which is to remove non-cellulosic materials from cotton fibers, especially the cuticle (mainly consisting of waxes) and the primary or primary cell wall (mainly consisting of pectin, protein and xyloglucan). A proper wax removal is necessary to obtain a high wettability, which is a measure to obtain a good dyeing. The elimination of primary cell walls, especially pectins - improves the elimination of waxes and ensures greater staining. In addition, this improves whiteness in the bleaching process. The main chemical used in washing or purification is sodium -hydroxide at high concentrations, up to 70 g / kg of cotton and at high temperatures, 80-95 ° C; and c. Bleached; purification is usually followed by bleaching using hydrogen peroxide as the oxidizing agent to obtain either a completely bleached (white) fabric or to ensure a clean shade from dyeing or dyeing.

A one-stage combined washing / bleaching process by industry is also used. Although the preparation processes are the most commonly used in the state of the 1st s; Washing, bleaching and dyeing operations can also be done at the stage of fiber or yarn preparation.

The processing regime can be either discontinuous or continuous with the fabric being in contact with the liquid process stream in a string or open width form. Continuous operations generally use a saturator by means of which an approximate equal weight of the chemical bath by weight of the fabric is applied to the cloth, followed by a residence heating chamber in which the chemical reaction takes place. A washing section then prepares the fabric for the next processing step. Non-continuous processing generally takes place in a processing bath whereby the fabric is contacted with approximately 8-15 times its weight in the chemical bath. After a period of reaction, the chemicals are drained, the fabrics are rinsed and the next chemical is applied. Intermittent intermittent fill processing involves a saturator whereby an equal weight of the chemical bath per fabric weight is applied to the fabric, followed by a residence period which in the case of cold intermittent filling could be one or more days.

Woven articles are prevalently made of fabrics - textiles. The interweaving process demands a "gluing" of the warp yarn to protect it from abrasion. Starch, polyvinyl alcohol (PVA), carboxymethyl cellulose, waxes and acrylic binders are examples of typical sizing chemicals used due to their availability and cost. The glueing can be removed after the interwoven process as the first stage in the preparation of woven articles. Glued or sizing fabrics either in the form of an open width or in the form of a rope are brought into contact with the processing liquid containing the desizing agents. . The desizing agent used depends on the type of sizing to be removed. For PVA sizing, oxidative or hot water processes are often used. The most common sizing agent for cotton fabrics is based on starch. Therefore more often, the woven cotton fabrics are uncoiled by a combination of hot water, the α-amylase enzyme hydrolyzes the starch and a wetting or surfactant agent. The cellulosic material is allowed to remain retained with the desizing chemicals during a sufficiently long "retention period" to complete the desizing. The retention period depends on the type of processing regime and temperature and can vary from 15 minutes to 2 hours, or in some cases, several days. Typically, the desizing chemicals are applied in a saturating bath which is generally in the range of about 15 ° C to about 55 ° C. The fabric is then maintained in the equipment such as a "J-box" which provides sufficient heat, usually between about 55 ° C and about 100 ° C, to improve the activity of the desizing agents. The chemicals, which include the removed desizing agents, are washed and removed from the fabrics after the end of the retention period.

To ensure high whiteness and good wettability and a resulting dyeing capacity, the sizing chemicals and other applied chemicals must be completely removed. It is generally believed that an efficient desizing is of crucial importance for the following preparation processes: purification and bleaching.

The purification process eliminates many of the non-cellulose compounds found naturally in cotton. In addition to natural non-cellulosic impurities, the scrubbing can remove dirt, stains and residual materials introduced during manufacturing such as lubricants for spinning, tapering or cutting. The purification process employs sodium hydroxide or related caustic agents such as sodium carbonate, potassium hydroxide or mixtures thereof. In general, a stable alkaline surfactant is added to the process to improve the solubilization of the hydrophobic compounds and / or prevent their redeposition in the fabric again. The treatment is usually at high temperature, 80 ° C - 100 ° C, using solutions - strongly alkaline, pH 13-14, of the depuration agent. Due to the non-specific nature of the chemical processes not only are the impurities but also the cellulose itself that is going to be attacked, generating damage in the resistance or other desirable properties of the fabric. The softness of cellulose fabrics is a function of the residual natural cotton waxes. The non-specific nature of the high temperature in the strongly alkaline depuration processes can not discriminate between the desirable natural cotton lubricants and the lubricants introduced during manufacturing. In addition, the conventional purification process can cause environmental problems due to its highly alkaline effluent from these processes. The purification stage prepares the fabric for optimal response in bleaching. An inadequately purified fabric will need a high level of chemical bleaching in the subsequent bleaching stages.

The bleaching stage discolours the natural pigments of the cotton and eliminates any trace component of the wood cotton, natural, residual, not completely removed during the ginning, carded or debugging or washing. The main process in use today is a bleaching with alkaline hydrogen peroxide. In many cases, especially when a very high whiteness is not necessary, bleaching can be combined with washing or scrubbing.

In the following examples it is shown that the depuration step can be carried out using the pectate lyase or the pectate lyase preparation of the present invention at a temperature of about 50 ° C 80 ° C and a pH of about 7-11, thus replacing Up to 1 percent of the highly caustic agents. An optimized enzymatic process ensures high pectin removal and complete wettability.

- -Degradation or modification of plant material The enzyme or the preparation of the enzyme according to the invention is preferably used as an agent for the degradation or modification of the cell walls of plants or any pectin-containing material that originates from the cell walls of plants due to the high degradation activity of the cell walls of the pectate lyase of the invention.

The pectate lyase of the present invention can be used alone in conjunction with other enzymes such as glucanases, pectinases and / or hemicellulases to improve the extraction of oil from oil-rich plant materials, such as soybean oil, soybean oil. olives olive, rapeseed oil from sunflower seed or sunflower oil.

The pectate lyase of the present invention can be used for the separation of components from the cellular materials of plants. Of particular interest is the preparation of plant materials rich in sugar starch in components of considerable commercial interest (such as sucrose from sugar beet or potato starch) and low-interest components (such as pulp or skin fractions). Also, of particular interest is the separation of oil-rich or protein-rich cultures into valuable protein and oil fractions and non-valuable shell. The separation process can be carried out by using methods known in the art.

The pectate lyase of the present invention can also be used in the preparation of fruit or vegetable juices to increase the yield, and in the enzymatic hydrolysis of various materials derived from the cell walls of plants or from waste materials, for example from the production of wines or juices, or residues from agriculture such as - vegetable skins or husks, bean pods, pulp from sugar beet, pulp from olives, potato pulp, and the like .

The material of the plant can be degraded to improve the different types of processing, facilitate the purification or extraction of other components than galactans such as the purification of pectins from citrus, improve the value of the feed, decrease the capacity of Water linkage, improve the water quality of wastewater plants, improve the conversion of plant materials for storage, etc.

By means of an enzyme preparation of the invention it is possible to regulate the consistency and appearance of the processed fruits or vegetables. It has been shown that the consistency and appearance is a product of the actual combination of enzymes used for processing, ie the - specificity of the enzymes with which the pectate lyase of the invention is combined. Examples include the production of clear juices, for example of apples, pears, or berries, stable juices with a higher cloudiness, for example, of apples, pears, berries, citrus fruits, or tomatoes; and purees, for example carrots and tomatoes.

The pectate lyase of the present invention can be used in the modification of the viscosity of the materials derived from the cell walls of plants. For example, pectate lyase can be used to reduce viscosity. feed that contains galactane and to promote the processing of viscous galactane-containing material. The viscosity reduction can be obtained by treating the galactane-containing plant material with an enzyme preparation of the invention under conditions suitable for partial or complete degradation of the galactane-containing material.

The pectate lyase of the present invention can be used, for example, in combination with other enzymes for the removal of pectin substances from the fibers of plants. This elimination is essential for example in the production of textile fibers or other cellulosic materials. For this purpose the plant fiber material is treated with an adequate amount of the pectate lyase of the invention under suitable conditions to obtain a complete or partial degradation of 1 pectic substances associated with the plant fiber material.

Additives for animal feed The pectate lyases of the present invention can be used for modification of the feeding of the animals and can exert their effect either in vitro (by modifying the components of the feed) or in vivo. Pectate lyase is particularly suitable for addition to animal feed compositions which contain high amounts of agaminogalactans and galactans, for example the feed containing materials from soybean plants, turnip, lupine, etc. When pectate lyase is added to the feed, it significantly improves the in vivo breaking of the material of the cell walls of the plants, so that a better utilization of the nutrients of the plants by the animals is achieved. Therefore, the proportion of growth and / or feed conversion ratio (i.e. the weight of the feed ingested in relation to the weight gain) of the animal is improved. For example, indigestible galactane is degraded by the pectate lyases, for example in combination with β-galactosidase, galactose or g a 1 to c t or 1 i g e rs that are digestible by animals and thus contributes to the energy available from the feed. Also, by degradation of galactane, pectate lyases can improve the digestion and the production of non-carbohydrate food constituents such as proteins, fats and minerals.

For a further description reference is made to PCT / DK 96/00443 and to an example of work herein.

Processing of wines and juices The enzyme or enzyme preparation of the invention can be used for the preparation and reduction of viscosity in vegetable or fruit juices, especially in apple or pear juices. This can be complemented by treating the juice or vegetable with an enzyme preparation of the invention in an amount effective to degrade the pectin-containing material contained in the fruit or vegetable juice.

The enzyme or enzyme preparation of the invention can be used in the treatment of fruit and vegetable pulps to improve the extraction capacity or the capacity of the pulp. For example, the preparation of enzymes to be used in the treatment of the pulp of apples and pears for the production of juice, and in the treatment of the pulps of grapes for the production of wine.

DETERMINATION OF THE CATALYTIC ACTIVITY OF THE PECTATO LIASA The viscosity test APSU APSU Units: The APSU unit test is a measurement of viscosity using the polygalacturonic acid substrate without the addition of calcium.

The sodium salt of polygalacturonic acid at 5% of the substrate (Sigma P-1879) is solubilized in 0.1 M glycine buffer pH 10. The 4 ml substrate is preincubated for 5 min. at 40 ° C. The enzyme is added (in a volume of 250 μl) and mixed for 10 sec. in a mixer at a maximum speed, it is then incubated for -20 min. at 40 ° C. For a double normal curve determination of a dilution of the enzyme concentration in the range of 5 APSU / ml up to above 100 APSU / ml with a minimum of 4 concentrations between 10 and 60 APSU per ml. The viscosity is measured using a MIVI 600 from the company Sofraser, 45700 Vi 11 emande ur, France. Viscosity is measured as mV after 10 sec For the calculation of the APSU units a standard enzyme dilution as described above was used to obtain a standard curve: APSU / ml MV 0.00 300 4.00 276 9.00 249 14.00 227 19.00 206 24.00 188 34.00 177 49.00 163 - 1 4 99.00 16 The GrafPad Prism program, which uses a non-linear adjustment with an exponential decay of a phase with a plateau, was used for calculations. The plateau plus the interval is the mV obtained without the enzyme. The plateau is the mV of more than 100 APSU and the average reduction in viscosity in both examples was found to be 12 APSU units with a standard error of 1.5 APSU.

The lyase assay (at 235 nm .. For the determination of ß elimination in the assays, the measurement of the increase in absorbance at 235 nm was made without using the sodium salt of polygalacturonic acid at 0.1% of 1 substrate (Sigma P-1879) solubilized in 0.1 M glycine buffer pH 10. For the calculation of the catalytic ratio an increase of . 2 in Absorbency at 235 units per min. corresponds to the formation of 1 μmol of the unsaturated product (Nasuna and Starr - - (1966) J. Biol. Chem. Vol. 241 page 5298-5306; and Bartling, Wegener and Olsen (1995) Microbiology Vol. 141 page 873-881 ).

The steady state condition using a 0.5 ml cuvette with a 1 cm light path in an HP diode array spectrophotometer in a temperature controlled cuvette holder with continuous measurements of absorbance at 235 nm. For steady state a linear increment for at least 200 sec. it was used for the calculation of the proportion or speed. It is used to convert the μmol formation per min. of the product .

Test in Agar The pectate lyase activity can be measured by applying a test solution to 4 mm holes punched on agar plates (such as, for example, LB agar), which contains 0.7% w / v po 1 i ga 1 act ur ona to (Sigma P 1879). The plates are incubated - then for 6 hours at a particular temperature (such as, for example 75 ° C). The plates are allowed to drain in either (i) CaC12IM for 0.5 hours or (ii) alkyl trimethylammonium Br mixed at 1% (M , Sigma M-7635) for 1 hour. Both procedures cause the precipitation of p or 1 i g a 1 a c t u r o na t o within the agar. The activity of pectate lyase can be detected by the appearance of clear zones within a background of precipitated precipitate. The sensitivity of the assay is calibrated using dilutions of a standard preparation of pectate lyase.

Analysis of the final point - r an s 1 im a n a c y at 235 nm for the Pectato Liasas (high Calcium method: 1 mM Calcium in the final incubation mixture) In this method, the substrate and the enzyme are incubated for 20 min. at 37 ° C followed by measurements at 235 nm of the formation of double bonds. Finally, the degradation rate is calculated based on the molar extinction coefficient in terms of Trans Units.

Pr o c e d i m e n t o: 0.5 ml of enzyme dilution is mixed with 0.5 ml of the 2 * substrate solution. Substrate: Polygalacturonic acid from Sigma P-1879 lot 77H3784. 2X damper: Glycine 0.1M pH 10 + 2. 0 mmol CaCl 2 Detention Reagent: H3PO4 0.02 m Incubation temperature: 37 ° C Reaction time: 20 min. 'Coefficient of extinction of the t r a ns e 1 imi n a tion: 0.0052 μmol cm ~ -A The enzyme was diluted in ion-free water from 0.5 to 5 APSU per ml. The main value in duplicate 0.5 ml. Substrate at 2% w / v in 2x buffer is mixed with 0.5 ml of the diluted enzyme. Both were pre-incubated 5 min. in a water bath at 37 ° C. They were incubated for 20 min. The stop was made using ml of reagent-1-stop and mixing. The enzyme was mixed with the model and the stop reagent first and then the entire substrate was a in the same volume. Enzyme 0.5 ml Substrate 0.5 ml Detention 5 ml Total volume 6 ml Measurement of absorbance at 235 nm in a 1 cm cuvette.

The formation of trans the imine is calculated per min. using the extinction coefficient of 0.0052 μmol cm-1 Calculation: [(main most important) / 2 Model] 0.0052 * 6 * 2 * Enzyme dilution / 20 min./lOOO ml = μmol per min.

MATERIALS AND METHODS Strains and Organisms Donors Bacillus licheniformis, ATCC 14580, which comprises the DNA sequence encoding the pectate lyase presented in SEQ. ID NO: 3 Bacillus agaradhaerens, NCIMB 40482 or DSM 8721, which comprises the DNA sequence coding for pectate lyase presented in SEQ. ID NO: 1.

Bacillus sp. AAI12 comprising the DNA sequence encoding the pectate lyase presented in SEQ. ID NO: 5 Bacillus sp. KJ59, DSM 12419, which comprises the DNA sequence encoding the pectate lyase presented in SEQ. ID NO: 7 Bacillus sp. 1534 comprising the DNA sequence encoding pectate lyase presented in SEQ. ID NO: 9 E. coli DSM 12403 comprising the plasmid containing the DNA sequence encoding the pectate lyase of the invention presented in SEQ. ID NO: 5 - E. coli DSM 12404 comprising the plasmid containing the DNA sequence encoding the pectate lyase of the invention presented in SEQ. ID NO: 9 E. coli DSM 11789 comprising the plasmid containing the DNA sequence encoding the pectate lyase of the invention presented in SEQ. ID NO: 3 E. coli DSM 11788 comprising the plasmid containing the DNA sequence encoding the pectate lyase of the invention presented in SEQ. ID NO: 1.

B. subtilis PL2306. This strain is B. subtilis DN1885 with broken apr and npr genes (Diderichsen, B., Wedsted, U., Hedegaard, L., Jensen, BR, Sj0holm, C. (1990) Cloning of aldB, which encodes alpha-acetolactate of ca rb ox i 1 ase, an exoenzyme from Bacillus brevis, J. Bacteriol., 172, 4315-4321) was disrupted in the transcriptional unit of the cellulase gene of -1-Bacillus subtilis, resulting in cellulase negative cells. Disruption or disruption was performed essentially as described in (Eds. A.L. Sonenshein, J.A. Hoch and Richard Losick (1993) Bacillus subtilis and other Gram-Positive Bacteria, American Society for microbiology p.618). The competent cells were prepared and transformed as described by Yasbin, R.E. Wilson, G.A. and Young, F.E. (1975) Transformation and transfection in lysogenic strains of Bacillus subtilis: evidence for selective induction of prophage in competent cells. J. Bacteriol, 121: 296-304.

E. coli: SJ2 (Diderichsen, B., Wedsted, U., Hedegaard, L., Jensen, B.R., Sj0holm, C. (1990) Cloning of aldB, which encodes to lph a-a c e t o 1 a c t a t e r ca rb o i a, e exoenzyme from Bacillus brevis. J. Bacteriol., 172, 4315-4321). The eT rcompetitive cells were prepared and transformed using a Bio-Rad GenePulser ™ as recommended by the manufacturer.

- P 1 a smi two pBK-CAMV (Stratagene inc., La Jolla Ca.) PSJ1678 (see WO 94/19454 WHICH IS hereby incorporated by reference in its entirety). pMOL944: This plasmid is a derivative of pUBllO which contains essentially the elements that make the plasmid propagatable in Bacillus subtilis, the kanamycin resistance gene and which has a strong promoter and a cloned signal peptide of the amyL gene of B. The ATCC 14580. The signal peptide contains a Sacll site that makes it convenient to clone the DNA encoding the mature part of a protein fused to the signal peptide. This results in the expression of a Pre-protein that is directed towards the outside of the cell.

The plasmid was constructed by conventional genetic engineering techniques as briefly described in the following.

Construction of pMOL944: The pUBllO plasmid (McKenzie, T. et al., 1986, Plasmid 15: 93-103) was digested with the single restriction enzyme Ncil. An amplified PCR fragment of the amyL promoter encoded in the plasmid pDN1981 (P.L. J0rgensen et al., 1990, Gene, 96, p.37-41) was digested with Ncil and inserted into pUBllO digested with Ncil to give plasmid pS J2624. The two PCR primers used have the following sequences: # LWN5494 5 '-GTCGCCGGGGCGGCCGCTATCAATTGGTAACTGTATCTCAGC-3' # LWN5495 5'-GTCGCCCGGGAGCTCTGATCAGGTACCAAGTCTTGTCGACCTGC AGAATGAGGCAGCAAGAAGAT-3 ' Primer # LWN5494 inserts a Notl site in the plasmid The plasmid pSJ2624 is then digested with Sacl and Notl and a new PCR fragment amplified in the amyL promoter encoded in pDN1981 is digested with Sacl and Notl and this DNA fragment is inserted into the pS J2624 digested with Sacl-Notl to give the plasmid pSJ2670.

This cloning replaces the first cloning of the amyL promoter with the same promoter but in the opposite direction. The two primers used for the PCR amplification have the following sequences: # LWN5938 5'-GTCGGCGGCCGCTGATCACGTACCAAGCTTGTCGACC TGCAGAATGAGGCAGCAAGAAGAT-3 '# LWN5939 5'-GTCGGAGCTCTATCAATTGGTAACTGTATCTCAGC-3' . Plasmid pSJ2670 was sorted with the restriction enzymes PstI and Bcll and a fragment of. PCR amplified from a cloned DNA sequence encoding the alkaline SP722 amylase (described in International Patent Application published as WO 95/26397 which is incorporated herein by reference in its entirety) is digested with PstI and Bcll and inserted to give the plasmid pMOL944. The two primers used for the PCR amplification have the following sequence: # LWN7864 5'-AACAGCTGATCACGACTGATCTTTTAGCTTGGCAC-3 '# LWN7901 5'-AACTGCAGCCGCGGCACATCATAATGGGACAAATGGG-3' Primer # LWN7901 inserts a Sacll site into the plasmid.

General methods of molecular biology Unless otherwise mentioned, manipulations and transformations of DNA were performed using standard methods of molecular biology (Sambrook et al. (1989) Molecular Cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, NY Ausubel, FM et al (eds.) "Current protocols in Molecular Biology." John Wiley and Sons, 1995; Harwood, CR, and Cutting, SM (eds.) "Molecular Biological Methods for Bacillus." John Wiley and Sons, 1990).

Enzymes for DNA manipulations were used according to the specifications of the suppliers (for example restriction endonucleases, ligands, etc. can be obtained from New England Biolabs, Inc.).

Propagation of donor strains The strain Bacillus 1 i che n i fo rm i s ATCC 14580 was propagated in the liquid medium 3 as specified by ATCC (Collection of American Type Crops, USA). After 18 hours of incubation at 37 ° C and 300 rpm, the cells were harvested, and the genomic DNA was isolated by the method described below.

Bacillus a ga radha e re-n NCIMB No. 40482, Bacillus sp. AAI12, Bacillus sp. KJ59, DSM 12419, and Bacillus sp. 1534 were all grown in TY with the pH adjusted to about 9.7 by the addition of 50 ml of the Sodium Sesquicarbonate ÍM per 500 ml of TY. After 24 hours of incubation at 30 ° C and 300 rpm, the cells were harvested, and the genomic DNA was isolated by the method described below.

Preparation of genomic DNA Strains of Bacillus sp. described above as donor organisms were propagated in liquid medium as described above. Cells were harvested, genomic DNA was isolated by the method described by Pitcher et al. [Pitcher, D.G. , Saunders, N. A., Owen, R. J; Rapid extraction of bacterial genomic DNA with guanidium thiocyanate; Lett Appl Microbiol 1989 8 151-156].

Creation of lambda libraries of bacteriophage of Bacillus species tolerant to alkalis LambdaZAP, which expresses the cloning equipment with digested Ba Hl and dephosphorylated arms of Stratagene, was selected to allow us to select the plates as well as plates in indicator means. The Bacillus DNA was isolated by the method of Pitcher et al., 1989). The isolated DNA was partially digested with Sau3A and fractionated in size on a 1% DNA agarose gel. The DNA was excised from the agarose gene between 2 and 6 Kb and purified using the Qiaspin DNA fragment purification method (Qiagen GmBH). 100 ng of the fractionated DNA that was ligated with 1 ug of the arms of ZAPexpress des f o s for i side with BamHl (4 degrees overnight) was purified. The ligation reaction was directly packed with GigaPackII Gold according to the manufacturer's instructions (Stratagene). The phage libraries were titered with XLlblue mrf- (Stratagene).

Creation of banks • of plasmids derived from primary phage libraries The cleavage of phagemid banks from ZAPexpress alkaline Bacillus libraries: XLl-blue cells were prepared (Stratagene, La Jolla Ca.) and resuspended in 10 mM MgSO4 as recommended in the mass cleavage protocol in the ZAPexpress manual from Stratagene. 40,000 units of forming plates from each library were placed in Falcon 2059 tubes. Samples were incubated with 400 uls of XLl-blue cells and > 10 pfus / ml of the assistant phage Exassist M13 (Stratagene) at 37 ° C for 15 minutes. Six mis of NZY broth were added to each tube and then the tubes were shaken at 37 ° C for 2.5 hours. The samples were then heated at 65 ° C for 20 minutes to kill the E cells. c o l i and the bacteriophage lambda; the phagemid is resistant to heating. Samples were spun at 3000 g to remove cellular debris and decanted into clean Falcon 2059 tubes. Samples from the phagemid library were adjusted to 10% glycerol (cr i opr es erva ti vo) and titrated accordingly. to the Stratagene protocol. Essentially, 10 uls of the treated supernatant of the supernatant diluted to 1/10 were used to infect 200 uls of the XLOLR cells (cells in MgSO4 10 m). Samples were placed in the incubator at 37 ° C for 15 min. 50 uls of the 5X NZY broth were added to the samples and then stirred for 45 min. at 37 ° C. 100 uls of the sample were placed on LB kanamycin plates and incubated overnight. After the titration was obtained, for each library, 10,000 colony forming units were mixed with 400 uls of XLOLR cells and incubated at room temperature for 2 0 minutes without agitation. XLOLR cells were prepared as described in the ZAPexpress manual by Stratagene (Stratagene inc., La Jolla CA). After 20 minutes, 200 uls of the NZY 5X medium, 3 mis of the NZY IX medium were added to the samples. The s-e samples were shaken at 200 rpm, 37 ° C for 90 minutes. The glycerol was added to a final concentration of 10% and the cells were frozen in aliquots at 80 ° C until further use.

Selection of libraries The standard selection for pectinases on the LB agar plates was carried out as follows: The plasmid libraries in-1-E. coli XLOLR cells were plated on LB kanamycin at a density of 5000 colony-forming units per plates petri with a diameter of 140 mm. The plates were incubated overnight at 37 ° C and then allowed to stand with 1% pectin, 35% SD or 75% SD and 1% agarose. The plates were incubated overnight at 37 ° C before being placed in a 1% MTAB solution. After two hours it was removed and the positive recombinant clones were identified by a zone of clarity around the colony. The positive isolates of r e c on f i rma r on by labeling in fresh LB media and retesting.

Media TY (as described in Ausubel, F. M. et al. (Eds.) "Current protocols in Molecular Biology." John Wiley and Sons, 1995). Agar LB (as described in Ausubel, F. M. et al. (Eds.) "Current protocols in Molecular Biology." John Wiley and Sons, 1995). - 14 -LBPG is an LB agar supplemented with 0.5% Glucose and 0.05 M potassium phosphate, pH 7.0. BPX medium as described in EP 0 506 780 (WO 91/09129).

The following examples illustrate the invention.

EXAMPLE 1 Cloning, expression, purification and characterization of a pectate lyase from Bacillus agaradhaerens Isolation of the DNA sequence encoding the pectate lyase of the invention The DNA sequence comprising the DNA sequence shown in SEQ. ID No. 1 and coding for the pectate lyase of the invention can be obtained from the deposited E. coli organism, DSM 11788, by extraction of the plasmid DNA by methods known in the art (Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab. , Cold Spring Harbor, NY).

Construction of the genomic library of Bacillus agaradhaerens The genomic DNA of Bacillus agaradhaerens NCIMB 40482 was partially digested with the restriction enzyme Sau3 ?, and fractionated in size by electrophoresis on a 0.7% agarose gel. Fragments between 2 and 7 kb in size were isolated by DEAE-cellulose paper electrophoresis (Dretzen, G., Bellard, M., Sassone-Corsi, P., Chambon, P. (1981). recovery of DNA fragments from agarose and acrylamide gels Anal. Biochem., 112, 295-298).

The isolated DNA fragments were ligated to the plasmid DNA pSJ1678 digested with BamHI, and the ligation mixture was used to transform E. coli SJ2. Transformed cells from the Bacillus agaradhaerens genomic library NCIMB 40482 were plated on LB agar plates containing 10 μg / ml of Chloramphenicol and Polygalacturon t or Sodium (SIGMA P-1879). The cells plated were incubated for 16 hours at 37 ° C. The colonies were replicated on fresh LB agar plates containing 10 μg / ml of Chloramphenicol and 0.7% of Sodium Polygalacton or Sodium (SIGMA P-1879) these plates were incubated for 8 hours at 37 ° C. The original main plates were filled with 5 ml of 1 M CaC12, after 5 to 30 min. different dark haloes appeared around the putative Sodium Po l ga 1 a c t ur ona t that degrades the clones. The corresponding main plate clones were collected for further characterization. These clones were further characterized by preparing the plasmid DNA that was obtained overnight from TY cultures of liquids at 30 ° C of the E. coli clones and preparing the plasmid DNA using the Qiagen Qiaspin Prep Kit according to the manufacturer ( Qiagen, Germany).

The pectate lyase positive clone of the Bacillus agaradhaerens genomic library NCIMB 40482 was deposited as DSM 11788. After the first obtaining of the E plasmid. coli DSM 11788 was identified SEC. ID NO: 1 of the DNA encoding the pectate lyase of Bacillus agaradhaerens NCIMB No. 40482.

Identification of positive clones through activity After incubation on plates the colonies were replicated again in plates on a set of LB + 6 CAM agar plates and then - subsequently incubated at 37 ° C for approx. 20 hours. An upper layer containing the 1% HSB agarose, 0.7% polygalacturonic acid sodium salt in an appropriate buffer was poured into replica plates and incubated for approx. 20 hours at 40 ° C. After precipitation with MTA3 positive colonies of pectate lyase were identified by the appearance of a clear halo in the positions where positive clones of pectate lyase were present.

The cells of the pectate lyase positive colonies were distributed for a single isolation of the colony on agar, and a single colony producing the pectate lyase was selected for each of the identified pectate lyase producing colonies.

Characterization of positive clones From plates where lines or veins have been re-formed, positive pectinase clones were obtained as single colonies, and the plasmids were extracted using Qiagen Plasmid Prep as indicated by the manufacturer (Qiagen, Germany). The phenotypes were confirmed by the r e t r a n s f o rm ation of E. coli SJ2, and the plasmids were characterized by restriction digestions.

Expression of Bacillus subtilis of the cloned genes encoding a pectate lyase The E. coli prep plasmid, DSM 11788, which contains the gene cloned into pSJ1678 (an E. coli / B. subtilis transfer vector), was used to transform B. subtilis PL2306. The competent cells were prepared and transformed as described by Yasbin et al. [Yasbin R E, Wilson G A & Young F E; Transformation and transfection in lysogenic strains of Bacillus subtilis: evidence for selective induction of prophage in competent cells; J Bacteriol 1975 121 296-304]. .

Ai s 1 am i on and Testing Transformants from ßacillus s btilis Transformed cells were plated on LB agar plates containing 6 mg / ml Chloramphenicol, 0.4% glucose and 10 mM KH2P04, and incubated at 37 ° C. C for 18 hours. Positive colonies of pectate lyase were identified as done before with E. coli - Each of the positive transformants was inoculated into -10 ml of the TY medium containing 6 mg / ml of Chloramphenicol. After 1 day of incubation at 37 ° C and shaking at 250 rpm, 50 μl of supernatant was removed. The pectate lyase activity was identified by adding 10 μl of supernatant to the holes made in the LB agar plates containing 0.7% Sodium Poly Spectium (Sigma, US).

After 16 hours of incubation at 37 ° C, the plates were allowed to drain in 1 M CaCl 2 for 5 to 30 min. Several hazy halos appeared where the supernatants contained the pectate lyase of a clone expressing the pectate lyase. One such clone was called MB464.

The cells were removed by centrifugation and the remaining supernatant was used as Az or as a source to purify the pectate lyase.

- - Purification and Characterization The transformant of B. s ub t i l i s obtained as described above was incubated in 100 ml of TY containing 6 mg / ml of Chloramphenicol. After incubation overnight at 37 ° C and shaking at 250 rpm, the culture was used as an inoculum in 1 L flasks containing 100 ml of the Complex Culture Medium (US 5,371,198, example 1 which is incorporated herein by reference). ). 1 ml of the culture was the inoculum volume, the cultures were incubated at 37 ° C and shaken at 250 rpm for 4 days.

The fermentation medium was adjusted to pH 7.5 with NaOH and flocculated using cationic flocculating agent C521 (10% solution) and 0.1% solution of the anionic agent A130: To 6500 ml of the fermentation medium 306 ml of C521 (10 ml) were added. %) simultaneously with 608 ml of A130 under stirring at room temperature. The flocculated material was separated by centrifugation using a centrifuge Sorval RC 3B at 10,000 rpm for 30 minutes. The supernatant was clarified using a Whatman No. F glass filter. In total, 7200 ml of clear solution was obtained.

The liquid was concentrated in 2 portions of 500 ml and 840 ml, respectively, using ultrafiltration with filtron with a molecular weight cut-off of 10 kDa.

The pH was adjusted to 5.3 using ethical aA acid, and the concentrate was applied to 200 ml of the S-Sepharose column. balanced with 50 M sodium acetate buffer, pH 5.3. The pectate lyase of the invention of the invention (B. agaradhaerens) was eluted using a linear gradient of 2 1 with 0.5 M NaCl as the final concentration. Pectate lyase from Bacillus subtilis will also bind to Sepharose at this pH but has a higher pl (7.6 versus 6.0). So that the cloned pectate lyase of the invention is eluted first, the fractions - were analyzed for the APSU units and for the reaction with the antiserum generated against the pectate lyase of Bacillus subtilis.

The pectate lyase from Bacillus aradhaerens was concentrated using an Amicon ultrafiltration cell with a GR61 membrane with a cut-off of 20 kDa.

A total of 90,000 APSU units were obtained. This sample was free of protease and the pectate lyase of Bacillus subtilis determined using antiserum generated against the pectate lyase of Bacillus s ub t i i s.

The pectate lyase enzyme of the invention could easily be seen in SDS-PAGE as a band with a molecular weight of 36 kDa. After the e1 ect of this band, the N-terminus was determined as: Ser-Asn-Gly-Pro-Gln-Gly-Tyr-Ala-Ser-Met-Asn-Gly-Gly-Thr.

This is in accordance with the amino acid sequence shown in SEC. ID No. 2 deduced from the DNA sequence shown in SEC. ID No. 1 with a pro sequence of 33 amino acids. The calculated molecular weight of the deduced sequence was 36 kDa and the calculated pl was 6. The molar extinction coefficient at 280 nm was 48,930.

The activity of ß-t r an s 1 imi na ci n n (using the lyase assay at 235 nm) at different pH values was determined as stable state kinetics at 40 ° C. The relative proportion was calculated as a percentage of the optimal activity, the following results were obtained: pH% of activity 6.5 0 7 5 7.5 8 8 21 8.5 32 38 9.5 39 10 52 - - 10.5 47 11 100 11.2 66 11.5 3 The pH profile was determined using the following buffers: pH 6.0: Na-MES 0. ÍM pH 6.5, 7.0 and 7.5: Na-MOPS 0. ÍM pH 8.0 & 8.5: Tris 0. ÍM pH 9.0, 9.5, 10.0 and 10.5: Na-glycine 0.1M pH 11-11.5: Na-Carbonate 0.1M MES is Acid 2 [N -Mo r f o 1 i n o] e t an s u 1 f or n i c o (SIGMA, No. M-8250). MOPS is Acid 3 - [N - Mo r f o 1 i n o] p r op a n s u 1 f or n i c o (SIGMA, No. M-1254). Tris (Merck No. 1.08382). Glycine (Merck). Sodium carbonate (Merck No. 6392).

Correspondingly, the activity relative to different temperatures was found (a p H 10): - t emp. C% of activity 40 69 50 100 55 97 60 65 71 Subcloning in B. subtilis The DNA sequence encoding the pectate lyase of the invention (SEQ ID NO: 1) was amplified with PCR using the PCR primer set consisting of these two oligonucleotides: Pecl.B. aga upper Sacll 5 '-CTG CAG CCG CGG CAG CTG CTT CAA ATC AGC CAA CTT C-3' Pecl. B. aga lower Notl 5'-GCG TTG AGA CGC GCG GCC GCT TTA CTC TGC ACA CAG GCA GAG C-3 ' The Sacll and Notl restriction sites were underlined.

- - Chromosomal DNA isolated from B. a ga r a dh a r in s NCIMB 40482 as described above was used as a template in a PCR reaction using the Amplitaq DNA Polymerase (Perkin Elmer) according to the manufacturer's instructions. The reaction with PCR was adjusted in a PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 M MgCl 2, 0.01% (w / v) gelatin containing 200 μM of each dNTP, 2.5 units of the polymerase AmpliTaq (Perkin-Elmer, Cetus, USA) and 100 pmol of each primer.

The PCR reaction was performed using a thermal cycler of DNA (Landgraf, Germany). An incubation at 94 ° C for 1 min. followed by thirty PCR cycles performed using a denaturation cycle profile at 94 ° C for 30 sec., rapid cooling at 60 ° C for 1 min., and extension at 72 ° C for 2 min. The aliquots of five μl of the amplification product were analyzed by - -electrophoresis in 0.7% agarose gels (NuSieve, FMC). The appearance of a 1.0 kb DNA fragment indicates adequate amplification of the gene segment.

Subcloning of the PCR fragment The forty-five μl aliquots of the PCR products generated as described above were purified using the QIAquick PCR purification kit (Qiagen, USA) according to the manufacturer's instructions. The purified DNA was eluted in 50 μl of 10 mM Tris-HCl, pH 8.5. 5 μg of pMOL944 and twenty-five μl of the purified PCR fragment was digested with Sacll and Notl, subjected to electrophoresis on 0.8% gelation low temperature agarose gels (SeaPlaque GTG, FMC), the relevant fragments were excised of the gels, and were purified using the QIAquick Gel Extraction Kit (Qiagen, USA) according to the manufacturer's instructions. The isolated PCR isolated DNA fragment was then ligated to the digested SacII-Notl and purified by pMOL944. The ligation was performed overnight at 16 ° C using 0.5 μg of the DNA fragment each, 1 U of the T4 DNA ligase and the T4 ligase buffer (Boehringer Mannheim, Germany).

The ligation mixture was used to transform B. s ub t i l i s PL2306 competent. The transformed cells were plated on LBPG-10 μg / ml Kanamycin plates. After 18 hours of incubation at 37 ° C several clones were layered on fresh agar plates and also grown in liquid TY cultures with 10 μg / ml kanamycin and incubated overnight at 37 ° C. . The following day, 1 ml of cells was used to isolate the plasmid from the cells using the Qiaprep Spin Plasmid Miniprep # 27106 Kit according to the manufacturer's recommendations for the B plasmid preparations. s ub t i l i s. This plasmid DNA was used as a template to sequence the DNA.

- - A clone containing the pectate lyase gene was maintained, this clone was called MB504.

The corresponding DNA from the mature part of the pectate lyase was characterized by DNA sequencing by the traces of a primer, using the Taq deoxy-terminal cycle sequencing kit (Perkin-Elmer, USA), the labeled fluorescent terminators and the appropriate oligonucleotides as primers.

The analysis of the sequence data was performed according to Devereux et al. (1984) Nucleic Acids Res. 12, 387-395. The cloned DNA sequence was expressed in B. s ub t i l i s and the protein that appeared in the supernatant corresponding to the mature protein was represented in the mature protein SEC. ID NO: 2 Purification and Characterization 5000 ml of the culture fluid of B transformant. s ub t i l i s obtained as - -described above (MB504) was flocculated using 125 ml of 10% C521 (cation) and 200 ml of 0.1% A130 (anion) at a pH of 7.5, followed by centrifugation and filtration. The clear supernatant was concentrated on a filtron UF membrane with a cut-off of 10 kDa to a final volume of 720 ml.

To obtain a highly pure enzyme, 40 ml were adjusted to pH 8.0 using NaOH and then applied to 50 ml of a Q-Sepharose column equilibrated with Tris HCl 25 m, pH 8.0. The pectate lyase was eluted from the column using a NaCl gradient. The eluted pectate lyase (total 150 ml) was concentrated using an Amicon ultrafiltration cell with a 10 kDa cutoff membrane. The concentrate was applied to a Superdex 200 column and a pure pectate lyase with a molecular weight of 38 kDa with an isoelectric point around 6.1 was obtained.

The pure enzyme was dialyzed against EDTA at pH 8.0 (20 mM tris pH 8.0), and at pH 10 - (20 M glycine pH 10) and was analyzed in circular Dichroism: no differences were observed in the spectra with and without EDTA The DSC of Differential Scanning Calorimetry of the 4 samples showed the enzyme more stable at pH 8.0 with a melting temperature of about 61 ° C in Tris pH 8.0 and 62 ° C after dialysis against EDTA '. At pH 10 the enzyme was fused at 59 ° C with and without EDTA.

The catalytic activity was inhibited by the presence of EDTA during incubation with substrate but the enzyme dialyzed against EDTA was still active if the EDTA was omitted during the incubation with the substrate. The divalent cations as Fe ++, Li ++, Mg ++, Cu ++, Mn ++ have no effect on catalytic activity.

Activity in detergents Using commercial detergents in place of the buffer and incubating for 20 minutes at 40 ° C with sodium salt of polygalacturonic acid (Sigma P-1879) followed by the determination of the reducing sugars, the enzyme was active in laundry detergent powder European commercially available Ariel Futur ™ with a relative activity of 37%, in commercially available Tide ™ laundry detergent for North American laundry with a relative activity of 58% and in commercially available US liquid detergent Tide ™ with a relative activity of 37% to the activity measured in the Glycine buffer. The concentration of the detergent was equal to the recommended concentration in the detergent packs for home use, and tap water was used having a German hardness of 18 degrees (detergent eur op eo / European co nd io ns) and a German hardness of 9 degrees (North American detergents / North American conditions).

- Immunological properties In the Danish company DAKO, the rabbit polyclonal serum was generated against the highly purified pectate lyase prepared as described above using conventional techniques. The serum formed a single precipitate on agarose gels with the pectate lyase of B. agaradhaerens of the invention.

EXAMPLE 2 Cloning, expression, purification and characterization of a pectate lyase from Bacillus licheniformis Construction of the genomic library of Bacillus licheniformis, ATCC 14580, was carried out as described in example 1 for B. agaradhaerens. The positive pectate lyase clone from the genomic library of the Bacillus licheniformis clone ATCC 14580 was deposited as DSM 11789. After a first step on the plasmid of E. coli -DSM 11789, SEC was identified. ID NO: 3 of the DNA encoding the pectate lyase of Bacillus licheniformis ATCC 14580.

Subcloning in Bacillus subtilis The DNA sequence encoding the pectate lyase (represented by the amino acid sequence SEQ ID NO: 4) of the invention was amplified with PCR using the PCR primer set consisting of these two oligonucleotides: Pecl. B. lich upper Sacll 5 '-CTA ACT GCA GCC GCG GCA GCT TCT GCC TTA AAC TCG GGC-3' Pecl. B. lich. lower Notl 5'-GCG TTG AGA CGC GCG GCC GCT GAA TGC CCC GGA CGT TTC ACC-3 'The restriction sites Sacll and Notl were underlined.

The chromosomal DNA isolated from B. licheniformis ATCC 14580 as described above was used as a template in - a PCR reaction performed as described in example 1. The appearance of a DNA fragment of size 1.0 kb indicated adequate amplification of the gene segment.

Subcloning of the PCR fragment was carried out as described in Example 1 except that the purified PCR fragment was digested with Sacll and NotlI. A clone containing the pectate lyase gene was maintained, this clone was called MB541.

The DNA corresponding to the mature part of the pectate lyase was characterized by DNA sequencing by primer screening, using the cyclodextrin 1 Taq sequencing kit (Perkin-Elmer, USA), the labeled fluorescent terminators and oligonucleotides. appropriate as primers.

The analysis of the sequence data was performed according to Devereux et al. (1984) Nucleic Acids Res. 12, 387-395. The sequence of the cloned DNA was expressed in B. s ub t i l i s and the protein that appeared in the supernatant corresponded to the mature protein represented in the SEC. ID NO: 4 Purification MB431 was grown in a 25 x 200 ml BPX medium with 10 μg / ml Kanamycin in 500 ml of two shake flasks with separators for 5 days at 37 ° C at 300 rpm, whereby 3500 ml of the broth was obtained of culture. The pH was adjusted to 5.0 using acetic acid and 100 ml of the cationic agent (C521J and 200 ml of the anionic agent (A130) was added during the agitation for flocculation.The flocculated material was separated by centrifugation using a Sorval RC 3B centrifuge. 10000 rpm for 30 min at 6 ° C. The resulting supernatant contained 370 APSU per ml in a total volume of 3600 ml.

The supernatant was clarified using Whatman GF / D and - C glass filters and finally concentrated on a UF filtron membrane with a cutoff of 10 kDa. The total volume of 2000 ml was adjusted to pH 8.5. 50 grams of DEAE A-50 Sephadex (Pharmacia) were diluted in 2000 ml of 50 mM Tris pH 8.5. The excess buffer was discharged and the clear concentrated enzyme solution was mixed with a suspension for 15 min. the enzyme was separated from the ion exchange material by suction in a Buchner funnel. The resulting solution was concentrated in a filtron with a cut-off of 10 kDa to a final volume of 800 ml.

To obtain a highly purified pectat-or lyase, a final step was performed using S-sepharose cation exchange chromatography. 50 ml of the 950 APSU solution per ml (see above) was adjusted to pH 5.0 using acetic acid. Applied to a 50 ml column containing S-Sepharose (Pharmacia) balanced with 50 mmol of a sodium acetate buffer pH 5.0. The pectate lyase was bound and eluted using a gradient of 0.5 M sodium chloride.

C raterization The pure enzyme gave a single band in SDS-PAGE of 35 kDa and an isoelectric point of around 6.1.

The concentration of the protein was determined using a molar extinction coefficient of 57750 (based on the amino acid composition deduced from the sequence).

Using the detection test the formation of cleavage by the formation of a double bond that can be measured at 235 nm, the following data was obtained 1. (conditions: pH 10; glycine buffer; no calcium; polygalacturonic acid Sigma P-1879 as substrate): 1 μmol per min. per mg. 2. (conditions: pH 10, glycine buffer, without calcium, DE 35 (pectin esterified at 35%) as substrate): 4 μmol per min. per mg.

The pure enzyme was dialyzed against EDTA at pH 8.0 (20 mM tris pH 8.0, and at pH 10 (20 mM glycine at pH 10) and the enzyme was analyzed in circular Dichroism, no differences were observed in the spectrum with and without EDTA.

The DSC of Exploration Calorimetry Differential of the 4 samples showed the enzyme more stable at pH 8.0 with a melting temperature of around 70 ° C in Tris pH 8.0 and 75 ° C after dialysis against EDTA. At pH 10 the enzyme was fused at 55 ° C with and without EDTA.

The catalytic activity of pectate lyase was inhibited by the presence of EDTA during substrate incubation but the enzyme dialyzed against EDTA was still active if EDTA was omitted during incubation with the substrate. The divalent cation such as Fe ++, Li ++, Mg ++, Cu ++, Mn ++ has no effect on the catalytic activity.

The activity of ß-tr an 1 imi nation (using the assay of liasa s 235 nm) at different pH values was determined as stable state kinetics at 40 ° C using the following buffers: pH 6.0: Na-Month 0.1M pH 6.5, 7.0 & 7.5: Na-MOPS 0.1M pH 8.0 & 8.5: Tris 0. ÍM pH 9.0, 9.5, 10.0 & 10.5: a-glycine 0.1M pH: 11-11.5: Na-carbonate 0.1M MES: From SIGMA number M-8250 (2 [N-Morpholino] ethane sulphonic acid). MOPS: From SIGMA, number M-1254 (Acid 3 - [N -Morfolino] propane sulfonic). Tris: De Merck No. 1.08382 Merck glycine and Merck sodium carbonate No. 6392.

The relative activity (proportion) was calculated as a percentage of the optimal activity, the following results were obtained: - pH% of activity 6.5 1 7 5 7.5 4 4 8.5 4 9 6 9.5 23 10 100 10.5 n.d. 11 52 11.2 0 Correspondingly, the activity relative to different temperatures was found (a p H 10): temp. ° C% activity 40 65 50 87 55 87 60 100 65 90 - - Activity in detergents: Using commercial detergents in place of the buffer and incubating for 20 minutes at 40 ° C with sodium salt of polygalacturonic acid (Sigma P- 1879) followed by the determination of the reduction sugars, the enzyme was active in the European commercial powder detergent Ariel Futur with a relative activity of 4 4%, the North American commercial powder Tide 'with a relative activity of 51% and in the American commercial liquid detergent Tide with a relative activity of 30% to the activity measured in the Glycine buffer. The concentration of the detergent as recommended for use and running water with 18 degrees of German hardness under European conditions and 9 degrees under North American conditions).

Immunological properties: In the Danish company DAKO, rabbit polyclonal monoe spe ci i co serum against highly purified pectate lyase was generated using conventional techniques. The serum - formed a single precipitate in agarose gels with the pectate lyase of the invention and had only one arch of precipitation against the crude products of Bacillus licheniformis as Pulpzyme HC lot no.

CKF0054 or lot no. CKN00009 from Novo Nordisk A / S.

EXAMPLE 3 Cloning, expression, purification and characterization of a pectate lyase from Bacillus sp. KJ59, DSM 12419 Subcloning in B. subtilis- of the DNA sequence encoding the mature part of the pectate lyase (SEQ ID NO: 7).

Using the same procedure exactly as described in example 1, the DNA sequence encoding the mature part of the pectate lyase encoded in SEQ. ID NO: 7 was cloned and expressed in the expression system pMOL 944 / PL2306. The only difference was that the following two PCR primers were used and the genomic DNA was isolated from Ba c i l l u s sp. KJ59, DSM 12419, was also used: # 145375 5'-CAT TCT GCA GCC GCG GCA AAT ACG CCA AAT TTC AAC TTA CAA G-3 '# 145376 5'-CAG CAG TAG CGG CCG CTT ACG GTT GGA TGA CAC CAA CTC-3' The resulting B. subtilis clone expressing the pectate lyase was named MB888. The cloned DNA sequence was expressed in b. subtilis and the protein that appeared in the supernatant corresponded to the mature protein represented in the mature protein of the SEC. ID NO: 8 Purification The Bacillus subtilis transformed with this plasmid (MB888) was grown in a PS medium containing Kanamycin.

- - Flocculation was done using the cationic flocculating agent C521 (10% solution) and 0.1% solution of the anionic agent A130: 2000 ml of the fermentation medium was added with 2000 ml of ion-free water and had a pH of 6.0 then 80 ml of C521 (10%) simultaneously with 40 ml of A130 were added under stirring at room temperature. The flocculated material was separated by centrifugation using a Sorval RC 3B centrifuge at 10,000 rpm for 30 minutes. The supernatant was clarified using the Whatman No. F glass filter. In total, 40,000 ml of the clear solution containing 204,000 Trans Units was obtained.

The liquid was concentrated to 500 ml using ultrafiltration with filtron with a molecular weight cutoff of 10 kDa. The concentrate was treated with 5 grams of DEAE A-50 Sephadex equilibrated in 25 mM Tris pH 8.0 for 30 minutes and the pectate lyase was not bound and was filtered free of ion exchange materials. The filtrate was adjusted to pH 5.0 using HCl and applied to the S-Sepharose column equilibrated with 25 mM sodium acetate pH 5.0. The linked pectate lyase was eluted as a pure protein using a NaCl gradient.

Characterization The enzyme, pure had a molecular weight of 36 kDa and a pl of 8.98.

The optimum temperature at pH 10 is about 65 ° C.

.. Relative activity - is more than 50% activity at 40 ° C between pH 9 and 11.

Pectate lyase has a melting temperature of 74 ° C measured using DSC in a 0.1 M sodium acetate pH 6.0.

EXAMPLE 4 Cloning, expression, purification and characterization of a pectate lyase from Ba c i l l u s sp. 1534 Subcloning in B. subtilis of the DNA sequence encoding the hard part of the pectate lyase (SEQ ID NO: 9) Using the same procedure exactly as described above, the DNA sequence encoding the mature part of the pectate lyase (SEQ ID NO: 9) was cloned and expressed in the expression system pMOL 944 / PL2306. The only difference was that the following two PCR primers were used and the genomic DNA was isolated from Ba c i l l u s sp. 1534 was used: # 136558 5 '-CCT GCA GCC GCG GCA AAA GGT GAA AGC GAT TCC ACT ATG-3 '# 136559 5' -GTT GAG AAA AGC GGC CGC AAC GGA CAC TCG GCT TTA GAG-3 'The resulting B. subtilis clone expressing the pectate lyase was named MB746. The cloned DNA sequence was expressed in B. subtilis and the protein that appeared in the supernatant corresponded to the mature protein represented in the mature protein of the SEC. ID NO: 10 Purification The Bacillus subtilis transformed with this plasmid (MB746) was grown in a PS medium containing Kanamycin.

Flocculation was done using the cationic flocculating agent C521 (10% solution) and 0.1% solution of the anionic agent A130: 3700 ml of the fermentation medium was adjusted to pH 5.5 using HCl and then 37 ml of C521 was added ( 10%) simultaneously with 75 ml of A130 under stirring at room temperature. The flocculated material was separated by centrifugation using a centrifuge - Sorval RC 3B at 10,000 rpm for 30 minutes. The supernatant was clarified using the Whatman No. F glass filter. In total 40,000 ml of the clear solution containing 1,044,000 Trans Units were obtained.

The liquid was concentrated to 550 ml using ultrafiltration with filtron with a molecular weight cutoff of 10 kDa. This product was used for the application tests after stabilization using 50% MPG (batch # 9831).

The highly purified enzyme was obtained using anion chromatography (HPQ column at pH 8.0 using a 25 M tris buffer); the enzyme was eluted using a gradient of NaCl, the final purification step was size chromatography on a Superdex 200 column that ran on a sodium acetate buffer.

Characterization The pure enzyme had a molecular weight of 35 kDa and an isoelectric point of 6.2.

The optimum temperature at pH 10 is above 70 ° C.

The relative activity is more than 50% between pH 9 and 11 at a temperature of 40 ° C.

The N-terminus of the purified pectate lyase has the amino acid sequence KGESDSTMNA starting at the p-or s i c i n n 25 of the amino acid sequence of SEC. ID NO: 10 - - EXAMPLE 5 Cloning, expression, purification and characterization of a pectate lyase from B a c i l l s s. AAI12 Subcloning in B. subtiiis of the AD ^ sequence that encodes the mature part of the pectate lyase (SEQ ID NO: 5) Using the same procedure exactly as described in Example 1, the DNA sequence encoding the mature part of pectate lyase (SEQ ID NO: 5) was cloned and expressed in the expression system pMOL 944 / PL2306. The only difference was that the following two PCR primers were used and the genomic DNA was isolated from Ba c i l l u s sp. AAI12 was used: # 80501D1C12 5 '-CAT TCT GCA GCC GCG GCA GCA TCA TTT CAG TCT AAT AAA AAT TAT C-3 '# 80501D1B12 5' -GAC GAC GTA CAA GCG GCC GCG CTA CTG TAC AAC CCC TAC ACC-3 'The resulting B. subtilis clone expressing the pectate lyase was named MB644. The cloned DNA sequence was expressed in B. subtilis and the protein that appeared in the supernatant corresponded to the mature protein represented in the mature protein of the SEC. ID NO: 6 Purification and Characterization The Bacillus subtilis transformed with this plasmid (MB644) was grown in a PS medium containing Kanamycin. It was found that the purification of this enzyme contained 3 lectin binding domains in the N-t e r i nal EXAMPLE 6 Treatment with Pectate Liasa of the Cellulose Material: Effect of the Temperature in the Elimination of the Pectin and Humectability A 100% cotton woven fabric, Dehusked Test Cloth # 428U, representing a typical cellulosic material, was treated with an aqueous enzyme solution comprising the pectate lyase of B. l i ch e n i f o rm i s of Example 2, dosed at 9 APSU / g of cloth at pH 9 and at a liquor ratio of 15: 1. The treatment time was 2 hours and the temperature varied between 35-75 ° C. The fabric was rinsed well after treatment with the enzyme, dried and stained with Ruthenium Red. The retention of the dyeing was measured spectrophotometrically and is a measure of the residual pectin in the fiber. The percentage of residual pectin was calculated using the retention -of dyeing the starting material as a 100% residual pectin and that of the bleached cloth and completely chemically washed as 0%. The results are shown in Table 1. In addition, wettability (drop test - measurement of time in seconds after a drop of water has been absorbed by the fabric) was measured and compared to a control without enzyme. Results are shown in table 2.

- Table 1 of residual pectin) an alkaline wash typically leaves a residual pectin of 20-25% Table 2 - the wettability goal is typically < 5 seconds The beneficial effects of the increase in temperature are clearly seen in both responses.

EXAMPLE 7 Treatment with Liase Pectate from the Cellulose Material: Effect of pH on the Elimination of Pectin A 100% cotton woven fabric, - De-doled Test Fabric # 428 U, representing a typical cellulosic material, was treated with aqueous enzyme solution comprising the pectate lyase of B. l i ch in i f or rm i s of example 2, dosed at 9 APSU / g of fabric at 15: 1 liquor ratio. The treatment time was 2 hours and the temperature was 55 ° C. The pH varied between 8-11. The fabric was rinsed well after treatment with the enzyme, dried and then stained with Ruthenium Red. The retention of the dyeing was measured and is a measure of the residual pectin in the fiber. The percentage of res i-dual pectin was calculated using the retention of staining of the starting material as a residual pectin at 100% and that of the bleached cloth and completely chemically washed as 0%. The results are shown in Table 3.

Table 3 (% residual pectin) - - The optimum pH is found to be in approx. 9.5, but good activity is demonstrated in a broad alkaline range.

EXAMPLE 8 Use of the enzyme of the invention in detergents The purified enzyme obtained as described in example 2 (lot 9751) showed an improved cleaning performance when tested at a level of 1 ppm in a mini-wash test using a conventional commercial liquid detergent. The test was performed under conventional North American washing conditions.

EXAMPLE 9 Effects of carbohydrates on cotton textiles stained with banana or banana Médodo Three bananas or bananas were crushed and homogenized in an Ultra Turrax with 40 ml of water. Cotton Style 400 (T e s t f ab r i c s, - -Inc.) Was moistened in the solution, squeezed between two rollers and dried overnight.

The stained cotton fabric was washed in the commercial liquid detergent of Ariel Futur Liquid brand under European washing conditions, with an addition of 0.1 ppm, 0.2 ppm, 1 ppm and 10 ppm, respectively, of the pectate lyase of Example 2 to the detergent liquid and an addition of 10 ppm of the pectate lyase of example 1. The test was repeated.

Results Ariel liquid:% elimination of banana or banana stains (100% is the total elimination of the stain) Test A Test B Without enzyme 26% 32% 10 ppm of enzyme, ex. 2 59% 58% 1 ppm of enzyme, ex. 2 47% 48% 0.1 ppm of enzyme, ex. 2 35% 34% 10 ppm of enzyme, ex. 1 45% - -EJEMPLO 10 Construction and expression of the fusion protein between Pectate lyase and CBD The DNA sequence that encodes the CBD of the CipB gene of strain YS of C ost ri di um the rm ocell um (Poole DM; Morag E; Lamed R; Bayer EA; Hazlewood GP; Gilbert HJ (1992). Identification of the ce 11 u 1 ose -b indi ng domain of the cellulosome subunit Sl from Clostridium thermocellum YS, Fems Microbiology Letters Vol. 78, No. 2-3 pp. 181-186, was amplified with PCR using the PCR primer set consisting of the following two oligonucleotides: CIPCBD. upper PECL. I left 5 '-CGA CAA TGT CGA CAA TGT AAA ATC AAT CGT CAA GCA AAA TGC CGG AGT CGG CAA AAT CCA GCG CAG ACC GCC AAC ACC GAC CCC GAC TTC ACC GCC AAG CGC AAA TAC ACC GGT ATC AGG CAA TTT G-3 CIPCBD. lower otl 5 '-GCG TTG AGA CGC GCG GCC GCT ATA CCA CAC TGC CAC CGG GTT CTT TAC-3' The restriction sites Sacll and Notl were underlined.

The chromosomal DNA encoding the CBD can be obtained as described in Poole DM; Morag E; Lamed R; Bayer EA; Hazlewood GP; Gilbert HJ (1992). Identification of the c o 11 u 1 o s -b i n di n g domain of the cellulosome subunit Sl from Clostridium thermocellum YS, Fems Microbiology Letters Vol. 78, No. 2-3 pp. 131-186. A sample of DNA encoding CBD was used as a template in a PCR reaction performed as described in Example 1. The appearance of a DNA fragment of approximately 0.5 kb in size indicated adequate amplification of the gene segment. .

Subcloning of the PCR fragment Subcloning was carried out as described in Example 1 except that the purified PCR fragment was digested with Sacll and Notl. Several clones were analyzed by isolating the plasmid DNA from the culture broth overnight.

One such positive clone was layered several times on agar plates as previously used, this clone was called MB914. Clone MB914 was grown overnight in TY-10 μg / ml Kanamycin at 37 ° C, and the next day 1 ml of cells was used to isolate the plasmid from the cells using the Qiaprep Spin Plasmid Miniprep Kit # 27106 according to the manufacturer's recommendations for the preparations of the B. subtilis plasmid. This DNA was the DNA sequenced and revealed the DNA sequence corresponding to the fusion protein of: Pectate lyase-e n 1 a z a do r-cbd as depicted in SEC. ID NO: 11 and in the sequence of attached proteins SEC. ID NO: 12 Expression and detection ^ of the fusion protein P e c t a t o - 1 i a s a - cbd The MB914 was incubated for 20 hours in the TY medium at 37 ° C and 250 rpm. 1 ml of cell-free supernatant was mixed with 200 μl of 10% Avicel (Merck, Darmstadt, Germany) in Millipore H20. The mixture was left for an hour at incubation at 0 ° C. After this binding of the fusion protein of P e c t a t i 1 a a - Enl a z a do r - C B D to the Avicel, the Avicel with the bound protein was rotated 5 min. to 5000 g. The pellets were resuspended in 100 μl of SDS-page buffer, boiled at 95 ° C for 5 min., Spun at 5000 g for 5 min. and 25 μl were loaded on a NOVEX SDS-PAGE gel, Laemmli T r i s - G 1 i c a n a, 4-20% (Novex, USA). The samples were subjected to electrophoresis in Xcell ™ Mini Cells (NOVEX, USA) as recommended by the manufacturer, all subsequent handling of the gels including dyeing with comassie, bleaching and drying was performed as described by the manufacturer .

The appearance of a protein protein band of approx. 55 kDa, indicated expression in B. s ub t i 1 s of fusion - -P e c t a t 1 s a - In 1 a z a d r - CB D coded in -plasmic pMB914.

EXAMPLE 11 Treatment of Cotton Fabric with pectate lyase (SEQ ID NO: 10) The following experiments were conducted to evaluate the use of SEC pectate lyase. ID NO: 10 for refined textiles.

A. Materials 1) Fabric: Raw cotton satin fabric, woven for the army, of 428R quality (242 g / m2) was used. 2) Equipment: A Labo at (Mathis, Switzerland) was used at a liquor ratio of 12.5: 1 (12 g of cloth in 150 ml of a buffer / enzyme solution). 3) Pectate lyase: In Experiment 1, a pectate lyase corresponding to SEC. ID NO: 10, was used, formulated in a solution containing 0.02 M phosphate buffer and 0.4 g / L of a non-ionic surfactant (Tergitol 15-S-12 Union - Carbide). In Experiment 2, the pectate lyase of the SEC was used. ID NO: 4, formulated in a solution containing a 0.05 M phosphate / borate buffer, in 2.0 g / L of nonionic surfactant (Tergitol 15-S-12 from Union Carbide) and 1.0 g / L of humidifier (Sulfosuccinate of dioctyl).

B. Procedures and Results In Experiment 1, the test fabrics were contacted with the aqueous solution containing the pectate lyase for 15 minutes at temperatures between 60-80 ° C and pHs ranging from 7-11, after which the pectin was quantified residual.

Figure 3 below shows an outline of the percentage of residual pectin as a function of both pH and temperature, and Figure 3 shows the% residual pectin as a function of the dosage of the enzyme. The optimum pH for pectin removal was 9.2 and the optimum temperature was above 8Q ° C. In Example 2, the test fabrics were contacted with the aqueous solution containing the pectate lyase at 600 APSU / kg of cotton, squeezed in a roller system to give an 85% retained solution, and incubated for 60 minutes at temperatures between 40-70 ° C, after which the residual pectin was quantified. The% residual pectin as a function of temperature is shown in the following Table.

Temperature (° C) Residual Pectin (%) I 40 ° C 35% '55 ° C 28% 70c 40% LITERATURE Lever, M. (1972) A new reaction for colormetric of termination of carbohydrates. Anal. Biochem. 4 7, 273-279.

- N. C. Carpita and D. M G ib e a u t 1993) The Plant Journal 3: 1-30.

Diderichsen, B., Wedsted, U., Hedegaard, L., Jensen, BR, Sjoholm, C. (1990) Cloning of aldB, which encodes to lpha - to ce to 1 actate de ca rb ox i 1 ase, an exoenzyme from Bacillus brevis, J. Bacteriol., 172: 4315-4321.

SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: NOVO NORDISK A / S (B) STREET: Novo Alie (C) CITY: Bagsvaerd (E) COUNTRY: Denmark (F) POSTAL CODE (CP) ): DK-2880 (G) TELEPHONE: +45 44 44 88 88 (H) TELEFAX: +45 44 49 32 56 (ii) TITLE OF THE INVENTION: NEW PECTATO LIASAS (iii) NUMBER OF SEQUENCES: 14 (iv) FORM COMPUTER READER: (A) TYPE OF MEDIUM: Flexible disk (B) COMPUTER: IBM compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PROGRAM: Patentln Relay # 1.0, Version # 1.30 (EPO) (1) INFORMATION FOR SEC. ID. NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1077 base pairs (B) TYPE: nucleic acid (C) HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEC. ID NO: 1: ATGACTAAAG TCTTTAAATT GTTACTGGCA TTAGCTCTCG TTTTACCAGT TATCTCATTT 60 AGTTCTCCTG CCTCGCAAGC TGCTTCAAAT CAGCCAACTT CTAACGGACC ACAAGGCTAT 120 GCGTCAATGA ATGGAGGGAC AACCGGTGGT GCAGGCGGCC GTGTCGAATA TGCAAGCACC 180 GGAGCGCAAA TTCAGCAATT GATAGATAAT CGCAGCCGAA GTAATAACCC TGATGAACCA 240 TTAACGATT7 ATGTAAACGG AACGATTACA CAAGGAAATT CCCCACAGTC CCTTATAGAT 300 GTTAAAAATC ACCGTGGAAA AGCTCATGAA ATTAAAAACA TCTCTATTAT CGGTGTAGGA 360 ACAAATGGAG AGTTTGATGG CATTGGGATA AGACTATCAA ACGCCCATAA TATCATTATC 420 CAAAATGTAT CAATTCATCA TGTGCGAGAG GGAGAAGGCA CGGCTATTGA AGTGACAGAT 480 GAGAGTAAAA ACGTGTGGAT CGATCACAAC GAGTTTTATA GTGAATTTCC AGGTAATGGA 540 GACTCAGATT ATTACGATGG TCTCGTAGAC ATAAAAAGAA ACGCTGAATA TATTACGGTT 600 TCATGGAATA AGTTTGAGAA TCATTGGAAA ACGATGCTCG TCGGTCATAC TGATAATGCC 660 TCATTAGCGC CAGATAAAAT TACGTACCAT CACAATTATT TTAATAATCT TAATTCACGT 720 GTCCCGCTTA TTCGATACGC TGATGTCCAT ATGTTCAATA ACTATTTTAA AGACATTAAC 780 GATACAGCGA TTAACAGTCG TGTAGGGGCC CGTGTCTTTG TAGAAAACAA CTATTTTGAC 8 0 AACGTAGGAT CAGGACAAGC TGACCCAACG ACTGGTTTTA TTAAAGGGCC TGTTGGTTGG 900 TTCTATGGAA GTCCGAGTAC TGGATATTGG AATTTACGTG GAAATGTATT TGTTAATACA 960 CCGAATAGTC ATTTAAGCTC TACAACAAAC TTTACACCAC CATATAGTTA CAAAGTCCAA 1020 TCAGCTACCC AAGCTAAGTC GTCGGTTGAA CAACATTCGG GAGTAGGTGT TATCAAC 1077 (2) INFORMATION FOR SEC. ID. NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 359 amino acids (B) TYPE: amino acid (C) HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF SEQUENCE : SEC. ID NO: 2: Met Thr Lys Val Phe Lys Leu Leu Leu Ala Leu Ala Leu Val Leu Pro 1 5 10 15 Val lie Ser Phe Ser Ser Pro Ala Ser Gln Ala Ala Ser Asn Gln Pro 20 25 30 Thr Ser Asn Gly Pro Gln Gly Tyr Ala Ser Met Asn Gly Gly Thr Thr 35 40 45 Gly Gly Wing Gly Gly Arg Val Glu Tyr Ala Ser Thr Gly Wing Gln lie 50 55 60 Gln Gln Leu He Asp Asn Arg Ser Arg Ser Asn Asn Pro Asp Glu Pro 65 70 75 80 Leu Thr He Tyr Val Asn Gly Thr He Thr Gln Gly Asn Ser Pro Gln 85 90 95 Ser Leu He Asp Val Lys Asn His Arg Gly Lys Wing His Glu He Lys 100 105 110 Asn He Ser He He Gly Val Gly Thr Asn Gly Glu Phe Asp Gly He 115 120 125 Gly He Arg Leu Ser Asn Ala His Asn He He He Gln Asn Val Ser 130 135 140 He His His Val Arg Glu Gly Glu Gly Thr Ala He Glu Val Thr Asp 145 150 155 160 Glu Ser Lys Asn Val Trp He Asp His Asn Glu Phe Tyr Ser Glu Phe 165 170 175 Pro Gly Asn Gly Asp Ser Asp Tyr Tyr Asp Gly Leu Val Asp He Lys 180 185 190 Arg Asn Wing Glu Tyr He Thr Val Ser Trp Asn Lys Phe Glu Asn His 195 200 205 Trp Lys Thr Met Leu Val Gly His Thr Asp Asn Wing Ser Leu Wing Pro 210 215 220 Asp Lys He Thr Tyr His His Asn Tyr Phe Asn Asn Leu Asn Ser Arg 225 230 235 240 Val Pro Leu He Arg Tyr Wing Asp Val His Met Phe Asn Asn Tyr Phe 245 250 255 Lys Asp He Asn Asp Thr Wing He Asn Ser Arg Val Gly Wing Arg Val 260 265 270 Phe Val Glu Asn Asn Tyr Phe Asp Asn Val Gly Ser Gly Gln Wing Asp 275 280 285 Pro Thr Thr Gl 'Phe He Lys Gly Pro Val Gly Trp Phe Tyr Gly Ser 290 295 300 Pro Ser Thr Gly Tyr Trp Asn Leu Arg Gly Asn Val Phe Val Asn Thr 305 310 315 320 Pro Asn Ser His Leu Ser Ser Thr Thr Asn Phe Thr Pro Pro Tyr Ser 325 330 335 Tvr Lys Val Gln Ser Ala Thr Gln Ala Lys Ser Ser Val Glu Gln His 340 345 350 Sex Gly Val Gly Val He Asn 355 (2) INFORMATION FOR SEC. ID. NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1026 base pairs (B) TYPE: nucleic acid (C) HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANIZATION: Bacillus licheniformis - ATCC 14580 (xi) SEQUENCE DESCRIPTION: SEC. ID NO: 3: ATGAAGAAAT TAATCAGCAT CATCTTTATC TTTGTATTAG GGGTTGTCGG GTCATTGACA 60 GCGGCGGTTT CGGCAGAAGC AGCTTCTGCC TTAAACTCGG GCAAAGTAAA TCCGCTTGCC 120 GACTTCAGCT TAAAAGGCTT TGCCGCACTA AACGGCGGAA CAACGGGCGG AGAAGGCGGT 180 CAGACGGTAA CCGTAACAAC GGGAGATCAG CTGATTGCGG CATTAAAAAA TAAGAATGCA 240 AATACGCCTT TAAAAATTTA TGTCAACGGC ACCATTACAA CATCAAATAC ATCCGCATCA 300 AAGATTGACG TCAAAGACGT GTCAAACGTA TCGATTGTCG GATCAGGGAC CAAAGGGGAA 360 CTCAAAGGGA TCGGCATCAA AATATGGCGG GCCAACAACA TCATCATCCG CAACTTGAAA 420 ATTCACGAGG TCGCCTCAGG CGATAAAGAC GCGATCGGCA TTGAAGGCCC TTCTAAAAAC 480 ATTTGGGTTG ATCATAATGA GCTTTACCAC AGCCTGAACG TTGACAAAGA TTACTATGAC 540 GGATTATTTG ACGTCAAAAG AGATGCGGAA TATATTACAT TCTCTTGGAA CTATGTGCAC 600 GATGGATGGA AATCAATGCT GATGGGTTCA TCGGACAGCG ATAATTACAA CAGGACGATT 660 ACATTCCATC ATAACTGGTT TGAGAATCTG AATTCGCGTG TGCCGTCATT CCGTTTCGGA 720 - - GAAGGCCAT TTTACAACAA CTATTTCAAT AAAATCATCG ACAGCGGAAT TAATTCGAGG 780 ATGGGCGCGC GCATCAGAAT TGAGAACAAC CTCTTTGAAA ACGCCAAAGA TCCGATTGTC 840 TCTTGGTACA GCAGTTCACC GGGCTATTGG CATGTATCCA ACAACAAATT TGTAAACTCT 900 AGGGGCAGTA TGCCGACTAC CTCTACTACA ACCTATAATC CGCCATACAG CTACTCACTC 960 GACAATGTCG ACAATGTAAA ATCAATCGTC AAGCAAAATG CCGGAGTCGG CAAAATCAAT 1020 CCATAA 1026 (2) INFORMATION FOR SEC. ID. NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 341 amino acids (B) TYPE: amino acid (C) HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus licheniformis - ATCC 14580 (xi) SEQUENCE DESCRIPTION: SEC. ID NO: 4: Met Lys Lys Leu He Ser He He Phe He Phe Val Leu Gly Val Val 1 5 10 15 Gly Ser Leu Thr Ala Ala Val Ser Ala Glu Ala Ala Ser Ala Leu Asn 20 25 30 Ser Gly Lys Val Asn Pro Leu Wing ASD Phe Ser Leu Lys Gly Phe Wing 35 40"45 Wing Leu Asn Gly Gly Thr Thr Gly Gly Gly Gly Gly Gln Thr Val Thr 50 55 60 Val Thr Thr Gly Asp Gln Leu He Ala Wing Leu Lys Asn Lys Asn Wing 65 70 75 80 Asn Thr Pro Leu Lys He Tyr Val Asn Gl-y Thr He Thr Thr Ser Asn 85 90 95 Thr Ser Ala Ser Lys He Asp Val Lys Asp Val Ser Asn Val Ser He 100 105 110 Val Gly Ser Gly Thr Lys Gly Glu Leu Lys Gly He Gly He Lys He 115 120 125 Trp Arg Ala Asn A = n He He He Arg Asn Leu Lys He His Glu Val 130 135 140 Wing Ser Gly Asp Lys Asp Wing He Gly He Glu Gly Pro Ser Lys Asn 145 150 155 160 He Trp Val Aso Hi = Asn Glu Leu Tyr His Ser Leu Asn Val Asp Lys 165 170 175 Asp Tyr Tyr Asp Gly Leu Phe Asp Val Lys Arg Asp Wing Glu Tyr He 180 185 190 Thr Phe Ser Trp Asn Tyr Val His Asp Gly Trp Lys Ser Met Leu Met 195 200 205 Gly Ser Ser Aso Ser Asp Asn Tyr Asn Arg Thr He Thr Phe His His 210 215 220 Asn Trp Phe Glu Asn Leu Asn Ser Arg Val Pro Ser Phe Arg Phe Gly 225 230 235 240 Glu Gly His He Tyr Asn Asn Tyr Phe Asn Lys He He Asp Ser Gly 245 250 255 He Asn Ser Arg Met Gly Wing Arg He Arg He Glu Asn Asn Leu Phe 260 265 270 Glu Asn Wing Lys Asp Pro He Val Ser Trp Tyr Ser Be Ser Pro Gly 275 280 285 Tyr Trp His Val Ser Asn Asn Lys Phe Val Asn Ser Arg Gly Ser Met 290 295 300 Pro Thr Thr Ser Thr Thr Thr Tyr Asn Pro Pro Tyr Ser Tyr Ser Leu 305 310 315 320 Asp Asn Val Asp Asn Val Lys Ser He Val Valves Gln Asn Ala Val Val Gly 325 330 335 Gly Lys He Asn Pro 340 (2) INFORMATION FOR SEC. ID. NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1530 base pairs (B) TYPE: nucleic acid (C) HEBRA: unique (D) TOPOLOGY: l ineal (ii) TYPE OF MOLECULE: DNA (genomic) ) (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus sp. (xi) SEQUENCE DESCRIPTION: SEC. ID NO: 5: ATGATGAAGA TGAGAAAAGC ATTAAGTGTA TTAGTGATTT TCGGATTATT CGTATCTTTT 60 TTTAGTTTTG GTCATCAAGG AGCAGAAGCG GCATCATTTC. AGTCTAATAA AAATTATCAT 120 CTAGTGAATG TGAACAGTGG CAAGTACTTA GAAGTGGGGG CTGCCTCAAC AGAGAACGGT 180 GCAAATGTCC AACAATGGGA AAATACGAAT TGTCATTGTC AACAATGGCG ATTGGTGCAA 240 AATCAGGATG GTTATTATGA GATTGTAAAC CGACATAGTG GCAAAGCATT GGATGTATTT 300 GAACGTTCTT CAGCTGATGG AGCGAACATT GTACAATGGG ATTCGAATGG ACGTAGCAAT 360 CAACAATGGA CGATTCAACA AGTGGGTTCC TCTTATAAAA TAGTTAGCAG ACATAGTGGG 420 AAGGCACTCG AAGTATTTAA CCATTCTAAT CAAAATGGAG CAAATGTCGT ACAGTGGCAA 480 GATTTTGGTA ATCCGAATCA ACTTTGGAAT ATCGTCGAGG TTGGTTCAGG ACAAGCTCAC 540 GATTTCAGTA AGCCGTTGGG GTATGCCTCA ATGAATGGCG GGACCACTGG CGGTCAAGGT 600 GGACGAGTCG AATACGCGAG TACTGGCTCT CAACTACAAA AATTAATCGA TGATCGAAGT 660 CGAAGCAATA ATCCCAATCA ACCACTTACC ATTTATGTAA CTGGGAAAAT CACCCTGCAA 720 AACTCCTCTG ATGATAAAAT TGAAGTGAAA AATCATCGTG GACAAGCTCA TGAAATACGT 780 AATCTGTCTA TCATAGGTCA AGGAACAAGA GGAGAGTTTG ATGGCATTGG TTTACGATTA 840 - ATTAATGCGC ACAATGTCAT TGTGCGTAAT CTCTCCATTC ACCATGTACG AGCTGGTTCA 900 GGTGAAGGTA CATCAATTGA AGTTACTCAA GGAAGTAAGA ATATTTGGAT TGATCATAAC 960 GAATTTTATA GTCAACTGGA TGGGAATAAC AACCCTGATC TGTATGATGG TCTTGTCGAT 1020 ATTAAACGGA ATTCGGAGTA CATTACGGTC TCTTGGAACA AGTTTGAGAA TCATTGGAAA 1080 ACGATGCTCG TCGGCCATAC CGATAACGCA TCATTAGCAC CTGATAAAGT TACGTACCAC 1140 CACAACTTTT TCCACAATCT TAATTCCAGA GTTCCGTTAA TTCGATTCGC AGATGTTCAT 1200 ATGGTTAACA ACTATTTCAA AGATATTAAA GATACAGCAA TTAATAGTCG TATGGGAGCA 1260 AGAGTATTTG TAGAAAATAA CTATTTTGAG AATGTAGGAT CAGGTCAACA AGATCCGACC 1320TTAAAACTGC TGTTGGGTGG TTTTATGGTA GTTCTAGCAC TGGATATTGG 1380 AATTTAAGAG GAAATCAATT TA7TAACACA CCATCAAGCC ACTTGTCTTC CACAACGAAT 1440 TTCACACCAC CTTATCAGTT CAACGCCCAA TCCGCTCAAG ATGCAAAGCA AGCCGTTGAA 1500 CAGTTTTCGG GTGTAGGGGT TGTACAGTAG_1530_(2) INFORMATION FOR SEC. ID. NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 509 amino acids (B) TYPE: amino acid (C) HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus sp. (xi) SEQUENCE DESCRIPTION: SEC. ID NO: 6: Met Met Lys Met Arg Lys Ala Leu Ser Val Leu Val He Phe Gly Leu 1 5 10 15 Phe Val Ser Phe Phe Ser Phe Gly His Gl-n Gly Wing Glu Wing Wing Ser 20 25 30 Phe Gln Ser Asn Lys Asn Tyr His Leu Val Asn Val Asn Ser Gly Lys 35 40 45 Tyr Leu Glu Val Gly Wing Wing Ser Thr Glu A = n Gly Wing Asn Val Gln 50 55 60 Gln Trp Glu Asn Thr Asn Cys His Cys Gln Gln Trp Arg Leu Val Gln 65 70 75 80 Asn Gln Asp Gly Tyr Tyr Glu He Val Asn Arg His Ser Gly Lys Wing 85 90 95 Leu Aso Val Phe Glu Arg Ser Ser Wing Asp Gly Wing Asn He Val Gln 100 105 110 Trp A = p Ser Asn Gly Arg Ser Asn Gln Gln Trp Thr He Gln Gln Val 115 120 125 Gly Ser Ser Tyr Lys He Val Ser Arg His Ser Gly Lys Ala Leu Glu 130 135 140 Val Phe Asn hie Ser Asn Gln Asn Gly Wing A = n Val Val Gln Trp Gln 145 150 155 160 Asp Phe Gly Asn Pro Asn Gln Leu Trp Asn He Val Val Val Gly Ser 165 170 175 Gly Gln Ala His Asp Phe Ser Lys Pro Leu Gly Tyr Ala Ser Met Asr. 180 185 190 Gly Gly Thr Thr Gly Gly Gln Gly Gly Arg Val Glu Tyr Ala Ser Thr 195 200 205 Gly Ser Gln Leu Gln Lys Leu He ASD ASD Arg Ser Arg Ser Asn Asn 210 215 220 Pro Asn Gln Pro Leu Thr He Tyr Val Thr Gly Lys He Tftr Leu Gln 225 230 235 240 Asn Being As Asp Lys He Glu Val Lys Asn His Arg Gly Gln Wing 245 250 '255 His Glu He Arg Asn. Leu Be He He Gly Gln Gly Thr Arg Gly Glu 260 265 270 Phe ASP Gly He Gly Leu Arg Leu He Asn Ala His Asn Val He Val 275 280 285 Arg Asn Leu Ser He His His Val Arg Ala Gly Ser Gly Glu Gly Thr 290 295 300 Ser He Glu Val Thr Gln Gly Ser Lys Asn He Trp He Asp His Asn 305 310 315 320 Glu Phe Tyr Ser Gln Leu Asp Gly Asn A = n Asn Pro Asp Leu Tyr Asp 325 330 335 Gly Leu Val Asp He Lys Arg Asn Ser Glu Tyr He Thr Val Ser Trp 340 345 350 Asn Lys Phe Glu Asn His Trp Lys Thr Met Leu Val Gly His Thr Asp 355 360 365 Asn Ala Ser Leu Ala Pro Asp Lys Val Thr Tyr His His Asn Phe Phe 370 375 380 His Asn Leu Asn Ser Arg Val Pro Leu He Arg Phe Wing Asp Val His 385 390 - 395 400 Met Val A = n Asn Tyr Phe Lys Asp He Lys Asp Thr Wing He Asn Ser 405 410 415 Arg Met Gly Ala Arg Val Phe Val Glu Asn Asn Tyr Phe Glu Asn Val 420 425 430 Gly Ser Gly Gln Gln Asp Pro Thr Arg Gln He Lys Thr Wing Val 435 440 445 Gly Trp Phe Tyr Gly Ser Ser Thr Gly Tyr Trp Asn Leu Arg Gly 450 455 460 Asn Gln Phe He Asn Thr Pro Ser Ser His Leu Ser Ser Thr Thr Asn 465 4 0 475 480 Phe Thr Pro Pro Tyr Gln Phe Asn Wing Gln Ser Wing Gln Asp Wing Lys 485 490 495 Gln Ala Val Glu Gln Phe Ser Gly Val Gly Val Val Gln 500 505 (2) INFORMATION FOR SEC. ID. NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1047 base pairs (B) TYPE: nucleic acid (C) HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus sp. (xi) SEQUENCE DESCRIPTION: SEC. ID NO: 7: ATGGACAAAC TTTATATTGA AAAAGGAAGT GAGAGTATGA TGAGATCAAG CATCGTCAAA 60 CTAGTTGCTT TCAGTATTGT GTTTATGTTA TGGCTCGGTG TATCCTTTCA AACGGCAGAA 120 GCGAATACGC CAAATTTCAA CTTACAAGGC TTTGCCACGT TAAATGGGGG AACAACTGGT 180 GGCGCTGGTG GAGATGTAGT GACGGTTCGT ACAGGGAATG AGTTAATAAA CGCTTTGAAG 240 TCCAAAAACC CTAATCGGCC GTTAACAATT TATGTTAACG GTACGAAAC GCCTAATAAT 300 ACGTCTGATA GTAAGATCGA CATTAAGGAT GTTTCCAATG TATCGATTTT AGGGGTTGGC 360 ACAAATGGCC GATTAAACGG GATCGGTATT AAAGTATGGC GAGCGAATAA TATCATTATT 420 CGAAACTTGA CAATCCATGA AGTCCATACA GGTGATAAAG ATGCGATTAG CATGATTAGC 480 ATTGAAGGAC CATCTCGAAA CATTTGGGTT GACCATAACG AGCTTTATGC CAGCTTGAAT 540 GTTCATAAAG ATCACTATGA CGGCTTGTTT GACGTAAAGC GCGATGCTTA CAATATTACC 600 TTCTCTTGGA ATTATGTCCA TGATGGCTGG AAAGCGATGC TCATGGGGAA TTCCGATAGT 660 GATAATTATG ACCGAAACAT AACATTCCAC CATAACTACT TCAAAAACTT AAACTCTCGT 720 GTACCTGCGT ACCGTTTTGG AAAGGCGCAC TTGTTTAGCA ATTACTTTGA GAACATTTTA 780 GAAACAGGCA TCAATTCACG GATGGGAGCG GAAATGCTCG TTGAACATAA CGTTTTTGAG 840 AATGCCACCA ACCCGCTAGG ATTCTGGCAT AGCAGTCGAA CAGGTTATTG GAATGTAGCC 900 AATAACCGCT ATA7CAATAG CACGGGCAGC ATGCCGACCA- CTTCCACGAC CAATTATCGA 960 CCTCCTTACC CCTATACGGT CACACCTGTT GGTGATGTGA AATCAGTTGT CACACGTTAT 1020 GCGGGAGTTG GTGTCATCCA ACCGTAA 1047 (2) INFORMATION FOR SEC. ID. NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 348 amino acids (B) TYPE: amino acid (C) HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus sp. (xi) SEQUENCE DESCRIPTION: SEC. ID NO: 8: Met Asp Lys Leu Tyr He Glu Lys Gly Ser Glu Met Met Met Ara Ser 1 5 10 15 Ser He Val Lys Leu Val Wing Phe Ser He Val Phe Met Leu Trp Leu 20 25 30 Gly Val Ser Phe Gln Thr Wing Glu Wing Asn Thr Pro Asn Phe Asn Leu 35 40 45 Gln Gly Phe Wing Thr Leu Asn Gly Gly Thr Thr Gly Gly Wing Gly Gly 50 55 60 Asp Val Val Thr Val Arg Thr Gly Asn Glu Leu He Asn Ala Leu Lys 65 70 75 80 Ser Lys Asn Pro Asn Arg Pro Leu Thr He Tyr Val Asn Gly Thr He 85 90 95 Thr Pro Asn Asn Thr Ser Asp Ser Lys He Asp He Lys Asp Val Ser 100 105 110 Asn Val Ser He Leu Gly Val Gly Thr Asn Gly Arg Leu Asn Gly He 115 120 125 Gly He Lys Val Trp Arg Wing Asn A = n He He He Arg Asn Leu Thr 130 135 140 He His Glu Val His Thr Gly Asp Lys Asp Ala He Ser Met He Ser 145 150 155 160 He Glu Gly Pro Ser Arg Asn He Trp Val Asp His Asn Glu Leu Tyr 165 170 175 Wing Ser Leu Asn Val His Lys Asp His Tyr Asp Gly Leu Phe Asp Val 180 185 190 Lys Arg Asp Wing Tyr Asn He Thr Phe Ser TrD Asn Tyr Val His Asp 195 200 '205 Gly Trp Lys Wing Met Leu Met Gly Asn Ser Asp Ser Asp Asn Tyr Asp 210 215 220 Arg Asn He Thr Phe His His Asn Tyr Phe Lys Asn Leu Asn Ser Arg 225 230 235 240 Val Pro Wing Tyr Arg Phe Gly Lys Wing His Leu Phe Ser Asn Tyr Phe 245 250 255 Glu Asn He Leu Glu Thr Gly He Asn Ser Arg Met Gly Ala Glu Met 260 265 270 Leu Val Glu Hi = Asn Val Phe Glu Asn Wing Thr Asn Pro Leu Gly Phe 275 280 285 Trp Hi = Ser Ser Arg Thr Gly Tyr Trp Asn Val Wing Asn Asn Arg Tyr 290 295 300 He Asn Ser Thr Gly Ser Met Pro Thr Thr Ser Thr Thr Asn Tyr Arg 305 310 315 320 Pro Pro Tyr Pro Tyr Thr. Val Thr Pro Val Gly Asp Val Lys Ser Val 325 330 335 Val Thr Arg Tyr Wing Gly Val Gly Val He Gln Pro 340 345 (2) INFORMATION FOR SEC. ID. NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1008 base pairs (B) TYPE: nucleic acid (C) HEBRA: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: ADM (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus sp. (xi) SEQUENCE DESCRIPTION: SEC. ID NO: 9: ATGAGAAAAC TCTTATCGAT GATGACTGCG CTTGTACTCA TGTTTGGAAT CATGGTTGTA 60 CCTTCTATAG CCAAAGGTGA AAGCGATTCC ACTATGAATG CTGATTTTTC CATGCAAGGT 120 TTTGCGACAC TTAATGGCGG AACCACAGGA GGAGCCGGCG GGCAAACCGT AACCGTTTCT 180 ACCGGAGACG AACTGCTGGC GGCCTTGAAG AACAAAAACA GCAATACACC CCTGACGATT 240 TATGTAAACG GTACCATAAC GCCATCAAAT ACGTCCGCAA GCAAAATTGA TATTAAAGAC 300 GTAAACGATG TTTCGATCTT AGGTGTTGGC ACTCAAGGCG AAT7TAACGG CATTGGCATT 360 AAAGTATGGC GAGCCAATAA CATTATTCTC CGCAACTTGA AAATACATCA CGTCAATACA 420 GGCGACAAAG ATGCCATTAG CATTGAAGGA CCATCCAAAA ACATATGGGT TGACCACAAT B0 GAGCTCTACA ATAGTCTTGA TGTCCATAAG GATTACTACG ATGGTCTTTT TGATGTCAAA 540 CGGGACGCGG ATTACATTAC ATTCTCGTGG AATTATGTTC ATGATAGCTG GAAGAGCATG 600 CTGATGGGAT CTTCTGATTC CGATTCGTAC AACCGAAAAA TCACATTCCA CAATAACTAC 660 TTTGAAAACC TCAATTCACG TGTACCTTCC ATACGCTTTG GCGAAGCCCA CATCTTCAGC 720 AACTACTACA ATGGCATTAA TGAAACCGGC ATCAACTCCC GCATGGGGGC AAAAGTGCGC 780 ATCGAGGAAA ATCTATTTGA ACGCGCAAAC AACCCGATCG TCAGTCGCGA CAGTCGCCAA 840 GTCGGGTATT GGCACTTGAT AAACAATCAC TTTACTCAAT CAACGGGCGA AATTCCAACG 900 ACTTCAACAA TCACATATAA CCCACCTTAT TCCTATCAAG CTACTCCGGT TGGCCAAGTA 960 AAAGATGTGG TTCGTGCGAA TGCTGGTGTT GGCAAAGTAA CACCTTAA 100B (2) INFORMATION FOR SEC. ID. NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 335 amino acids (B) TYPE: amino acid (C) HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus sp. (xi) SEQUENCE DESCRITION: SEC. ID NO: 10: Met Arg Lys Leu Leu Ser Met Met Met Thr Ala Leu Val Leu Met Met Phe Gly 1 5 10 15 Met Met Val Val Pro Ser He Ala Lys Gly Glu Ser Asp Ser Thr Met 20 25 30 Asn Ala Asp Phe Ser Met Gln Gly Phe Wing Thr Leu Asn Gly Gly Thr 35 40 45 - Thr Gly Glv Wing Gly Gly Gln Thr Val Thr Val Ser Thr Gly Asp Giu 50 '55 60 Leu Leu Ala Ala Leu Lys Asn Lys Asn Ser Asn Thr Pro Leu Thr He 65 70 75 80 Tyr Val Asn Gly Thr He Thr Pro Ser Asn Thr Ser Wing Ser Lys He 85 90 95 Asp He Lys Asp Val Asn Asp Val Ser He Leu Gly Val Gly Thr Gln 100 105 110 Gly Glu Phe Asn Gly He Gly He Lys Val Trp Arg Wing Asn Asn He 115 120 125 He Leu Arg Asn Leu Lys He His His Val Asn Thr Gly Asp Lys Asp 130 135 140 Wing He Ser He Glu Gly Pro Ser Lys Asn He Trp Val Asp His Asn 145 .150 155 160 Glu Leu Tyr Asn Ser Leu Asp Val His Lys Asp Tyr Tyr Asp Gly Leu 165 170 175 Pne Asp Val Lys Arg Asp Wing Asp Tyr He Thr Phe Ser Trp Asn Tyr 180 185 190 Val Hxs Asp Ser Trp Lys Ser Met Leu Met Gly Ser Be Asp Ser Asp 195 200 205 Be Tyr Asn Arg Lys He Thr Phe His Asn Asn Tyr Phe Glu Asn Leu 210 215 220 Asn Ser Arg Val Pro Be He Arg Phe Gly Glu Wing His He Phe Ser 225 230 235 240 Asn Tyr Tyr Asn Gly He Asn Glu Thr Gly He Asn Being Arg Met Gly 245 250 255 Wing Lys Val Arg He Glu Glu Asn Leu Phe Glu Arg Wing Asn Asn Pro 260 265 270 He Val Ser Arg Asp Ser Arg Gln Val Gly Tyr Trp Kis Leu He Asn 275 280 285 Asn His Phe Tr.r Gln Ser Thr Gly Glu He Pro Thr Thr Ser Thr He 290 295 300 Thr Tyr Asn Pro Pro Tyr Ser Tyr Gln Ala Thr Pro Val Gly Gln Val 305 310 315 320 Lys Asp Val Val Arg Ala Asn Ala Gly Val Gly Lys Val Thr Gly 325 330 335 (2) INFORMATION FOR SEC. ID. NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1482 base pairs (B) TYPE: nucleic acid (C) HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEC. ID NO: 11: GCTTCTGCCT TAAACTCGGG CAAAGTAAAT CCGCTTGCCG ACTTCAGCTT AAAAGGCTTT 60 GCCGCACTAA ACGGCGGAAC AACGGGCGGA GAAGGCGGTC AGACGGTAAC CGTAACAACG 120 GGAGATCAGC TGATTGCGGC ATTAAAAAAT AAGAATGCAA ATACGCCTTT AAAAATTTAT 180 GTCAACGGCA CCATTACAAC ATCAAATACA TCCGCATCAA AGATTGACGT CAAAGACGTG 240 TCAAACGTAT CGATTGTCGG ATCAGGGACC AAAGGGGAAC TCAAAGGGAT CGGCATCAAA 300 ATATGGCGGG CCAACAACAT CATCATCCGC AACTTGAAAA TTCACGAGGT CGCCTCAGGC 360 GATAAAGACG CGATCGGCAT TGAAGGCCCT TCTAAAAACA TTTGGGTTGA TCATAATGAG 420 CTTTACCACA GCCTGAACGT TGACAAAGAT TACTATGACG GATTATTTGA CGTCAAAAGA 480 GATGCGGAAT ATATTACATT CTCTTGGAAC TATGTGCACG ATGGATGGAA ATCAATGCTG 540 ATGGGTTCAT CGGACAGCGA TAATTACAAC AGGACGATTA CATTCCATCA TAACTGGTTT 600 GAGAATCTGA ATTCGCGTGT GCCGTCATTC CGTTTCGGAG AAGGCCATAT TTACAACAAC 660 TATTTCAATA AAATCATCGA CAGCGGAATT AATTCGAGGA TGGGCGCGCG CATCAGAATT 720 ] _Q GAGAACAACC TCTTTGAAAA CGCCAAAGAT CCGATTGTCT CTTGGTACAG CAGTTCACCG 780 GGCTATTGGC ATGTATCCAA CAACAAATTT GTAAACTCTA GGGGCAGTAT GCCGACTACC 840 TCTACTACAA CCTATAATCC GCCATACAGC TACTCACTCG ACAATGTCGA CAATGTAAAA 900 TCAATCGTCA AGCAAAATGC CGGAGTCGGC AAAATCCAGC GCAGACCGCC AACACCGACC 960 CCGACTTCAC CGCCAAGCGC AAATACACCG GTATCAGGCA ATTTGAAGGT TGAATTCTAC 1020 AACAGCAATC CTTCAGATAC TACTAACTCA ATCAATCCTC AGTTCAAGGT TACTAATACC 1080 GGAAGCAGTG CAATTGATTT GTCCAAACTC ACATTGAGAT ATTATTATAC AGTAGACGGA 1140 CAGAAAGATC AGACCTTCTG GTGTGACCAT GCTGCAATAA TCGGCAGTAA CGGCAGCTAC 1200 -, .- AACGGAATTA CTTCAAATGT AAAAGGAACA TTTGTAAAAA TGAGTTCCTC AACAAATAAC 1260 GCAGACACCT ACCTTGAAAT AAGCTTTACA GGCGGAACTC TTGAACCGGG TGCACATGTT 1320 CAGATACAAG GTAGATTTGC AAAGAATGAC TGGAGTAACT ATACACAGTC AAATGACTAC 1380 CATTCAAGT CTCGTTCACA GTTTGTTGAA GGGATCAGG TAACAGCATA CTTGAACGGT 1440 GTTCTTGTAT GGGGTAAAGA ACCCGGTGGC AGTGTAGTAT AG 1482 (2) INFORMATION FOR SEC. ID. NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 493 amino acids (B) TYPE: amino acid (C) HEBRA: only 0 (°) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEC. ID NO: 12: Ala Be Ala Leu Asn Ser Gly Lys Val Asn Pro Leu Wing Asp Phe Ser 1 5 10 15 Leu Lys Gly Phe Ala Ala Leu Asn Gly Gly Thr Thr Gly Gly Glu Gly 20 25 30 - Gly Gln Thr Val Thr Val Thr Thr Gly ASD Gln Leu He Ala Ala Leu 35 40 45 Lys A = n Lys Asn Wing Asn Thr Pro Leu Lys He Tyr Val Aen Gly Thr 50 55 60 He Thr Thr Ser Asn Thr Ser Ala Ser Lys He Asp Val Lys Asp Val 65 70 75 80 Ser Asn Val Ser He Val Gly Ser Gly Thr Lys Gly Glu Leu Lys Gly 85 90 95 He Gly He Lys He Trp Arg Wing Asn Asn He He He Arg Asn Leu 100 105 110 Lys He His Glu Val Wing Ser Gly Asp Lys Asp Wing He Gly He Glu 115 120 125 Gly Pro Ser Lys Asn He Trp Val Asp His Asn Glu Leu Tyr His Ser -130 135 140 Leu Asn Val Asp Lys Asp Tyr Tyr Asp Gly Leu Phe Asp Val Lys Arg 145 150 155 160 Asp Wing Glu Tyr He Thr Phe Ser Trp Asn Tyr Val HIS Asp Gly Trp 165 170 175 Lys Ser Met Leu Met Gly Ser Ser Asp Ser Asp Asn Tyr Asn Arg Thr 180 185 190 He Thr Phe His His Asn Trp Phe Glu Asn Leu Asn Ser Arg Val Pro 195 200 205 Be Phe Arg Phe Gly Glu Gly His He Tyr Asn Asn Tyr Phe Asn Lys 210 215 220 He He Asp Ser Gly He Asn Ser Arg Met Gly Ala Arg He Arg He 225 230 235 240 Glu Asn Asn Leu Phe Glu Asn Wing Lys Asp Pro He Val Ser Trp Tyr 245 250 255 Ser Ser Pro Gly Tyr Tro His Val Ser Asn Asn Lys Phe Val Asn 260"265 270 Ser Arg Glv Ser Met Pro Thr Thr Ser Thr Thr Thr Tyr Asn Pro Pro 275 280 285 Tyr Ser Tyr Ser Leu Asp Asn Val Asp Asn Val Lys Ser He Val Lys 290 295 300 Gln Asn Wing Gly Val Gly Lys He Gln Arg Arg Pro Pro Thr Pro Thr 305 310 315 320 Pro Thr Ser Pro Pro Ser Wing Asn Thr Pro Val Ser Gly Asn Leu Lys 325 330 335 Val Glu Phe Tyr Asn Ser Asn Pro Ser Asp Thr Thr Asn Ser He Asn 340 345 350 Pro Gln Phe Lys Val Thr Asn Thr Gly Ser Ser Wing Asp Leu Ser 355 360 365 Lys Leu Thr Leu Arg Tyr Tyr Tyr Thr Val Asp Gly Gln Lys Asp Gln 370 375 380 Thr Phe Trp Cys Asp His Wing Wing He He Gly Ser Asn Gly Ser Tyr 385 390 395 400 Asn Gly He Thr Ser A = n Val Lys Gly Thr Phe Val Lys Met Ser Ser 405 410 415 Ser Thr Asn Asn Wing Asp Thr Tyr Leu Glu Be Ser Phe Thr Gly 420 425 430 Thr Leu Glu Pro Gly Wing His Val Gln He Gln Gly Arg Phe Wing Lys 435 440 445 Asn Asp Trp Ser Asn Tyr Thr Gln Be Asn Asp Tyr Be Phe Lys Ser 450 455 460 Ring Be Gln Phe Val Glu Trp Asp Gln Val Thr Ala Tyr Leu Asn Gly 465 470 475 480 Val Leu Val Trp Gly Lys Glu Pro Gly Gly Ser Val Val 485 490 (2) INFORMATION FOR SEC. ID. NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1506 base pairs (B) TYPE: nucleic acid (C) HEBRA: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: rRNA (xi) SEQUENCE DESCRIPTION: SEC. ID NO: 13: GACGAACGCD GGCGGCGUGC CUAAUACAUG CAAGÜCGAGC GGAUIIGAAGG GAGCUUGCUC 60 CCGGAUAUUA GCGGCGGACG GGUGAGUAAC ACGUGGGCAA CCUGCCCUUU AGACUGGGAU 120 AACUCCGGGA AACCGGUGCÜ "AAÜACCGGAU AACACUUUGA ACCUCCUGGU ÜCGAAGUUGA 180 AAGADGGCCü UCGUGCUAUC ACUAAAGGAU GGGCCCGCGG CGCAUUAGCU AGUUGGUAAG 240 GÜAAUGGCUU ACCAAGGCAA CGAUGCGUAG CCGACCUGAG AGGGUGAUCG GCCACACUGG 300 GACOGAGACA CGGCCCAGAC ÜCCUACGGGA GGCAGCAGUA GGGAAUCUUC CGCAAUGGAC 360 GAAAGUCUGA CGGAGCAACG CCGCGUGAGU GAGGAAGGCC -UUCGGGUCUCU AAAGCOCUGU 420 UGUGAGGGAA GAACAAGÜAU CGGUÜGAAUA AGCCGGÜACC UUGACGGUAC CUCACCAGAA 480 AGCCACGGCU AACUACGUGC CAGCAGCCGC GGUAAUACGU AGGUGGCAAG CGUUGUCCGG 540 AAUUAUUGGG CGUAAAGCGC GCGCAGGCGG CUUCUUAAGU CUGAUGUGAA AUCUCGGGGC 600 UCAACCCCGA GCGGCCAUUG GAAACUGGGG AGCUUGAGUG CAGAAGAGGA GAGUGGAAUÜ 660 CCACGUGUAG CGGUGAAAUG CGUAGAÜAUG UGGAGGAACA CCAGUGGCGA AGGCGACUCU 720 CUGGUCUGUA ACUGACGCUG AGGCGCGAAA GCGUGGGGAG CAAACAGGAU UAGAUACCCU 780 GGUAGUCCAC GCCGUAAACG AUGAGUGCUA GGUGUUAGGG GUUUCGAUGC CCGUAGUGCC 840 GAAGUAAACA CAUUAAGCAC UCCGCCUGGG GAGUACGACC GCAAGGUUGA AACUCAAAGG 900 AAUUGACGGG GACCCGCACA AGCAGUGGAG CAUGUGGUUU AAUUCGAAGC AACGCGAAGA 960 ACCUUACCAG GÜCUUGACAU CCUUUGACCA CUCUGGAGAC AGAGCUUCCC CUUCGGGGGC 1020 AAAGUGACAG GUGGUGCAUG GUUGUCGUCA GCUCGUGUCG UGAGAUGUUG GGUÜAAGUCC 1080 CGCAACGAGC GCAACCCUUG AUCUUAGUUG CCAGCAUUUA GDUGGGCACU CUAAGGUGAC 1140 UGCCGGUGAC AAACCGGAGG AAGGUGGGGA CGACGUCAAA UCAUCAUGCC CCUUAÜGACC 1200 UGGGCUACAC ACGUGCUACA AUGGAUGGUA CAAAGGGUUG CGAAGCCGCG AGGÜGAAGCC 1260 AAUCCCAUAA AGCCAUUCUC AGUUCGGAUU GCAGGCÜGCA ACÜCGCCUGC AUGAAGCCGG 1320 AAUUGCUAGU AAUCGCGGAU CAGCAUGCCG CGGUGAAUAC GÜUCCCGGGU CUUGUACACA 1380 CCGCCCGUCA CACCACGAGA GUUUGUAACA CCCGAAGUCG GUGAGGUAAC CUUUUGGAGC 1440 CAGCCGCCUA AGGÜGGGACA AAUGAÜUGGG GUGAAGUCGU AACAAGGUAG CCGUAUCGGA 1500 AGGUGC 1506 (2) INFORMATION FOR SEC. ID. NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1508 base pairs (B) TYPE: nucleic acid (C) HEBRA: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: rRNA (xi) SEQUENCE DESCRIPTION: SEC. ID NO: 14: GACGAACGCU GGCGGCGUGC CUAAOACAUG CAAGUCGAGC GGACAUUUAG GAGCUUGCUC 60 CUAAAUGUUA GCGGCGGACG GGUGAGUAAC ACGUGGGCAA CCUGCCCUGU AGACUGGGAU 120 AACAUCGAGA AAUCGGUGCU AAUACCGGAU AAUCUUGAGG AUUGCAUAAU CCDCUUGUAA 180 AAGAUGGCÜC CGGCÜAUCAC UACGGGAUGG GCCCGCGGCG CAUÜAGCUAG UUGGUAAGGÜ 240 AACGGCUUAC CAAGGCGACG AUGCGUAGCC GACCÜGAGAG GGUGAUCGGC CACACUGGGA 300 CUGAGACACG GCCCAGACUC CUACGGGAGG CAGCAGUAGG GAAUCUUCCG CAAUGGACGA 360 AAGÜCUGACG GAGCAACGCC GCGUGAGUGA UGAAGGGUUÜ CGGCUCGUAA AGCUCUGUUG 420 UUAGGGAAGA ACAAGUGCCG UUCAAAUAGG GCGGCACCUU GACGGUACCU AACCAGAAAG 480 CCACGGCUAA CUACGUGCCA GCAGCCGCGG UAAUACGUAG GÜGGCAAGCG UUGÜCCGGAA 540 UUAÜUGGGCG UAAAGCGCGC GCAGGCGGÜC UUUUAAGÜCU GAUGUGAAAU CUCGGGGCUC 600 AACCCCGAGC GGUCAUUGGA AACUGGGAGA CUUGAGUACA GAAGAGGAGA GUGGAAUUCC 660 ACGUGUAGCG GUGAAAUGCG UAGAUAUGUG GAGGAACACC AGUGGCGAAG GCGACUCUCU 720 GGUCUGUAAC UGACGCUGAG GCGCGAAAGC GUGGGGAGCA AACAGGAUUA GAUACCCUGG 780 UAGUCCACGC CGUAAACGAU GAGUGCUAGG UGUUAGGGGU UUCGAUGCCC UUAGUGCCGA 8 40 AGUUAACACA UUAAGCACUC CGCCUGGGGA GUACGACCGC AAGGUUGAAA CUCAAAGGAA 900 UUGACGGGGG CCCGCACAAG CAGUGGAGCA UGUGGUUUAA UUCGAAGCAA CGCGAAGAAC 960 CUUACCAGGU CUUGACAUCC DUAUGACCUC CCUAGAGAUA GGGAUUUCCC UUCGGGGACA 102C UAAGUGACAG GUGGUGCAUG GUUGUCGUCA GCUCGUGUCG UGAGAUGUUG GGUUAAGUCC 1080 CGCAACGAGC GCAACCCUUG AUCUUAGUUG CCAGCAUÜUA GUUGGGCACU CUAAGGUGAC 1140 UGCCGGUGAU AAACCGGAGG AAGGUGGGGA UGACGÜCAAA UCAUCAUGCC CCUUAUGACC 1200 UGGGCUACAC ACGUGCUACA AUGGAUGGUA CAAAGAGCAG CAAAACCGCG AGGUCGAGCC 1260 - - AAUCUCAUAA AGCCAÜÜCUC AGUUCGGAUU GUAGGCUGCA ACUCGCCUAC AÜGAAGCCGG 1320 AAUUGCUAGU AAUCGCGGAU CAGCAUGCCG CGGUGAAOAC GUÜCCCGGGC CDUGUACACA 1380 CCGCCCGUCA CACCACGAGA GUÜUGUAACA CCCGAAGUCG GUGGAGUAAC CCUUACGGGA 1440 GCUAGCCGCC UAAGGUGGGA CAGAUGADUG GGGUGAAGUC GUAACAAGGU AGCCGUAUCG 1500 GAAGGUGC 1508 It is noted that in relation to this date, the best method known to the applicant, to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following:

Claims (34)

1. - A pectate lyase characterized in that it comprises a first amino acid sequence consisting of seven (7) amino acid residues having the following sequence: Asn Leu Asn Ser Arg Val Pro (NLNSRVP).

2. - The pectate lyase according to claim 1, characterized in that it also comprises a second amino acid sequence consisting of six (6) amino acid residues selected from the group, consisting of the sequences Trp Val Asp His Asn Glu (WVDHNE) and Trp Asp His Asn Glu (WIDHNE).

3. - The pectate lyase according to claim 1, characterized in that it also comprises a third amino acid sequence consisting of three (3) amino acid residues having the following sequence: Ser Trp Asn (SWN).

4. A pectate lyase characterized in that it is i) a polypeptide produced by Bacillus agaradhaerens, NCIMB 40482 or DSM 8721, or by a Bacillus species having a sequence homology of 16S rDNA to Bacillus agaradhaerens, DSM 8721, or at least 99%, or ii) a polypeptide comprising an amino acid sequence as shown in positions 27-359 of SEQ. ID NO: 2, or iii) an analogue of the polypeptide defined in i) or ii) which is at least 45% homologous with said polypeptide, or iv) is derived from said polypeptide by substitution, removal or addition of one or several amino acids, with the proviso that the arginine at position 240, and optionally also the arginine at position 245, are retained and the derived polypeptide is at least 42% homologous with said polypeptide.

5. - An isolated polynucleotide molecule that encodes a polypeptide having pectate lyase activity characterized in that it is selected from the group consisting of: a) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ. ID NO: 1 from nucleotide 79 to nucleotide 1077; b) polynucleotide molecules that encode a polypeptide that is at least 45% identical to the amino acid sequence of SEQ. ID NO of amino acid residue 27 to amino acid residue 359; and c) degenerate nucleotide sequences of (a) or (b).

6. A pectate lyase characterized in that it is i) a polypeptide produced by Bacillus licheniformis, ATCC 14580, or by a Bacillus species having a sequence homology of 16S rDNA to Bacillus licheniformis, ATCC 14580, of at least 99%, or ii) a polypeptide comprising an amino acid sequence as shown in positions 28-341 of SEQ. ID NO: 4, or iii) an analogue of the polypeptide defined in i) or ii) that is at least 45% homologous with said polypeptide, or iv) is derived from said polypeptide by substitution, removal or addition of one or more amino acids, with the proviso that the arginine at position 233, and optionally also the arginine at position 238, is retained and the derived polypeptide is at least 42% homologous with said gone polypeptide.

7. - An isolated polynucleotide molecule that encodes a polypeptide having a pectate lyase activity characterized in that it is selected from the group consisting of: a) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ. ID NO: 3 from nucleotide 82 to nucleotide 1026; b) polynucleotide molecules that encode a polypeptide that is at least 45% identical to the amino acid sequence of SEQ. ID NO: from the amino acid residue 28 to the amino acid residue 341; and c) degenerate nucleotide sequences of (a) or (b).

8. - A pectate lyase characterized in that it is i) a polypeptide produced by a species of Ba c i l l s that has the 16S rDNA sequence of SEQ. ID NO: 14 or by a species of B a c i l l s s that has a sequence homology of 16 S rDNA to SEC. ID NO: 14 greater than 97.3%; ii) a polypeptide comprising an amino acid sequence as shown in positions 181-509 of SEQ. ID NO: 6, or iii) an analogue of the polypeptide defined in i) which is at least 50% homologous with said polypeptide, or iv) is derived from said polypeptide by substitution, deletion or addition of one or more amino acids, with the proviso that the arginine at position 390, and optionally also the arginine at position 395, is retained and the derived polypeptide is at least 44% homologous to said polypeptide.

9. - An isolated polynucleotide molecule that encodes a polypeptide having pectate lyase activity, characterized in that it is selected from the group consisting of: a) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ. ID NO: 5 from nucleotide 541 to nucleotide 1530; b) polynucleotide molecules that encode a polypeptide that is at least 50% identical to the amino acid sequence of SEQ. ID NO: 6 of amino acid residue 181 to amino acid residue 509; and c) degenerate nucleotide sequences of (a) or (b).

A pectate lyase characterized in that it is i) a polypeptide produced by the species of Bacillus ha lodurans, or ii) a polypeptide comprising an amino acid sequence as shown in positions 42-348 of SEQ. ID NO: 8, or iii) an analogue of the polypeptide defined in i) or ii) which is at least 45% homologous with said polypeptide, or iv) is derived from said polypeptide by substitution, deletion or addition of one or more amino acids , with the proviso that the arginine at position 240, and optionally also the arginine at position 245, is retained and the polypeptide derivative is at least 40% hsmologist with said gone polypeptide.

11. The pectate lyase according to claim 10, characterized in that the polypeptide is produced by a Bacillus sp. KJ59, DSM 12419.

12. - An isolated polynucleotide molecule that encodes a polypeptide having pectate lyase activity, characterized in that it is selected from the group consisting of: a) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ. ID NO: 7 from nucleotide 124 to nucleotide 1047; b) polynucleotide molecules that encode a polypeptide that is at least 45% identical to the amino acid sequence of SEQ. ID NO: 8 of amino acid residue 42 to amino acid residue 348: and c) degenerate nucleotide sequences of (a) or (b).

13. - A iasa pectate characterized in that it is i) a polypeptide produced by a species of Ba c i l l s that has the 16S rDNA sequence of SEC. ID NO: 13 or by a species of B a c i l l s that has a sequence homology of 16S rDNA to the SEC. ID NO: 13 greater than 98.1%; or ii) a polypeptide comprising an amino acid sequence as shown in positions 25-335 of SEQ. ID NO: 10, or iii) an analogue of the polypeptide defined in i) or ii) which is at least 45% homologous with said polypeptide, or iv) is derived from said polypeptide by substitution, removal or addition of one or more amino acids , with the proviso that the arginine at position 227, and optionally also the arginine at position 232, is retained and the derived polypeptide is at least 41% homologous with said polypeptide.

14. - An isolated polynucleotide molecule that encodes a polypeptide that has. the pectate lyase activity, characterized in that it is selected from the group consisting of: a) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ. ID NO: 9 from nucleotide 73 to nucleotide 1008; b) polynucleotide molecules that encode a polypeptide that is at least 45% identical to the amino acid sequence of SEQ. ID NO: 10 from amino acid residue 25 to amino acid residue 335; and c) degenerate nucleotide sequences of (a) or (b).

15. - The isolated oligonucleotide molecules according to claim 5, 7, 9, 11 or 14, characterized in that the polynucleotide is DNA.

16. - An expression vector characterized in that it comprises the following operably linked elements: a transcription promoter; a DNA segment selected from the group consisting of a) polynucleotide molecules that encode a polypeptide having the pectate lyase activity comprising a nucleotide sequence as shown in SEQ. ID NO: 1 from nucleotide 79 to nucleotide 1077, in SEQ. ID NO: 3 from nucleotide 82 to nucleotide 1026; in the SEC. ID NO: 5 from nucleotide 541 to nucleotide 1530; in the SEC. ID NO: 7 from nucleotide 124 to nucleotide 1047 or as shown in SEQ. ID NO: 9 from nucleotide 73 to nucleotide 1008; b) polynucleotide molecules that encode a polypeptide having pectate lyase activity that is at least -50% identical to the amino acid sequence of SEQ. ID NO: 2 from amino acid residue 27 to amino acid residue 359, SEC. ID NO: 4 from amino acid residue 28 to amino acid residue 341, of SEQ. ID NO: 6 from amino acid residue 181 to amino acid residue 509, SEC. ID NO: 8 from amino acid residue 42 to amino acid residue 348 or to the amino acid sequence of SEQ. ID NO: 10 from amino acid residue 25 to amino acid residue 335, and (c) degenerate nucleotide sequences of (a) or (b); and a transcription terminator.

17. - A cultured cell into which an expression vector has been introduced according to claim 16, characterized in that said cell expresses the polypeptide encoded by the DNA segment.

18. - An isolated polypeptide characterized in that it is selected from the group consisting of: a) polypeptide molecules having the pectate lyase activity and comprising an amino acid sequence as shown in SEQ. ID NO: 2 from residue 27 to residue 359; b) polypeptide molecules having pectate lyase activity and which are at least 45% identical to the amino acids of SEQ. ID NO: 2 from amino acid residue 27 to amino acid residue 359; c) po 1 ip molecules that have pectate lyase activity and that comprise an amino acid sequence as shown in SEQ. ID NO: from the residue to residue 241; d) polypeptide molecules having the pectate lyase activity and which are at least 45% identical to the amino acids of SEQ. ID NO: from amino acid residue 28 to amino acid residue 341; e) polypeptide molecules having pectate lyase activity and comprising an amino acid sequence as shown in SEQ. ID NO: 6 from residue 181 to residue 509; f) polypeptide molecules having pectate lyase activity and which are at least 50% identical to the amino acids of SEQ. ID NO: 6 from the amino acid residue 181 to the amino acid residue 509 g) polypeptide molecules having pectate lyase activity and comprising the amino acid sequence as shown in SEQ. ID NO: 8 from 'residue 42 to residue 348; h) polypeptide molecules having pectate lyase activity and which are at least 45% identical to the amino acids of SEQ. ID NO from amino acid residue 42 to amino acid residue 348; i) polypeptide molecules having pectate lyase activity and comprising an amino acid sequence as shown in SEQ. ID NO: 10 from residue 25 to residue 335; k) polypeptide molecules having pectate lyase activity and which are at least 45% identical to the amino acids of SEQ. ID NO: 10 from the amino acid residue 25 to the amino acid residue 335; and 1) homologous species of a), b), c), d), e), f), g), h), i) and k).

19. An enzyme preparation characterized by comprising a purified pectate lyase according to claim 1 or a purified polypeptide according to claim 18.

20. A method for producing a polypeptide having pectate lyase activity, characterized in that it comprises culturing a cell into which an expression vector has been introduced according to claim 16, whereby the cell expresses a polypeptide encoded by the segment of DNA; and recovering the polypeptide.

21. An isolated enzyme having pectate lyase activity, characterized in that the enzyme is (i) free of homologous impurities, and (ii) produced by the method according to claim 20.

22. - The preparation according to claim 19, characterized in that it also comprises one or more enzymes selected from the group consisting of proteases, cellulases (e ndo g 1 uc a na s a s), hemicellulases ß-glucanases, lipases, peroxidases, laccases, α-amylases, glucoamylases, kucinases, pectinases, reductases, oxidases, phenoloxidases, ligninases, pullulanases, ar ab inosi da sas, * mannanases, xi 1 og 1 ucanases, xylanases, pectin acetyl esterases, po 1 iga 1 act ur o na sas,. rha nmnoga 1 a c t u r ona s s, ga 1 a c t ana s s, pectin lyases, other pectate lyases, polygalacturonase, pectin methylesterases, cellobiohydrolases, transglutaminases; or mixtures thereof.

23. - A fused polypeptide characterized in that it comprises a part of the polypeptide having a pectate lyase activity, wherein the part of the polypeptide is the pectate lyase according to any of claims 1, 4, 6, 8, 10 and 13 which part of polypeptide is linked to one or more cellulose binding domains (CBD)

24. The polypeptide according to claim 23, characterized in that the CBD is obtained from the strain YS of Cl or s t r i d i um t h e rm o c e 1 1 um.

25. - The polypeptide according to claim 23, characterized in that it has the amino acid sequence of SEQ. ID NO: 12 and the mutants - or variants thereof having pectate lyase activity, with the proviso that the arginines at position 206. and 211 are conserved and the mutant or variant is at least 42% homologous with said part. of the polypeptide.

26. - A detergent composition, characterized in that it comprises the preparation of the enzyme according to claim 19 or the enzyme pectate lyase according to claim 1, 4, 6, 8, 10 or 13 and a surfactant.

27. - A process for cleaning a hard surface, characterized in that it comprises treating a hard surface with a cleaning solution containing the preparation of the enzyme according to claim 19 or the pectate lyase enzyme according to claim 1, , 6, 8, 10 or 13.

28. - A process for the treatment in machine of the fabrics, characterized in that said process comprises treating the fabrics during a washing cycle of a washing process in machine with a washing solution containing the preparation of the enzyme according to claim 19 or the pectate lyase enzyme according to claim 1, 4, 6, 8, 10 or 13.

29. A method for improving the properties of the cellulose fibers, yarns, woven or non-woven fabrics, characterized in that in said method the fibers, yarns or fabrics are treated with an effective amount of the preparation according to claim 19 or the pectate enzyme lyase according to claim 1, 2, 6, 8, 10 or 13.

30. The method according to claim 29, characterized in that the preparation of the enzyme or the enzyme is used as a washing process step.

31. A method for the degradation or modification of plant material, characterized in that in said method the plant material is treated with an effective amount according to claim 19 or the pectate lyase enzyme according to claim 1, 4, 6 , 8, 10 or 13.

32. The method according to claim 32, characterized in that the plant material is waste paper - recycling, pulps for the manufacture of paper, mechanical, or fibers subjected to a retting process.

33. - A method for the preparation of animal feed characterized by an effective amount of the preparation according to claim 19 or the pectate lyase enzyme according to claim 1, 4, 6, 8, 10 or 13 is added as an additive from animal feed to conventional animal feed ingredients.

34. - A method for processing wine or juice, characterized in that in said method the wine or juice is treated with an effective amount of the preparation according to claim 19 or an effective amount of the enzyme according to claim 1, 4, 6 , 8, 10 or 13.